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ABSTRACT Grapes have been cultivated for wine production for millennia. Wine production involves a complex biochemical process where sugars in grape must are converted into alcohol and other compounds by microbial fermentation, primarily by the yeast Saccharomyces cerevisiae. Commercially available S. cerevisiae strains are often used in winemaking, but indigenous (native) strains are gaining attention for their potential to contribute unique flavors. Recent advancements in high-throughput DNA sequencing have revolutionized our understanding of microbial communities during wine fermentation. Indeed, transcriptomic analysis of S. cerevisiae during wine fermentation has revealed a core gene expression program and provided insights into how this yeast adapts to fermentation conditions. Here, we assessed how the age of vines impacts the grape fungal microbiome and used transcriptomics to characterize microbial functions in grape must fermented with commercial and native S. cerevisiae. We discovered that ~130-year-old Zinfandel vines harbor higher fungal loads on their grapes compared to 20-year-old Zinfandel vines, but fungal diversity is similar. Additionally, a comparison of inoculated and uninoculated fermentations showed distinct fungal dynamics, with uninoculated fermentations harboring the yeasts Metschnikowia and Pichia. Transcriptomic analysis revealed significant differences in gene expression between fermentations inoculated and not inoculated with a commercial S. cerevisiae strain. Genes related to metabolism, stress response, and cell adhesion were differentially expressed, indicating varied functionality of S. cerevisiae in these fermentations. These findings provide insights into S. cerevisiae function during fermentation and highlight the potential for indigenous yeast to contribute to wine diversity.IMPORTANCEUnderstanding microbial functions during wine fermentation, particularly the role of Saccharomyces cerevisiae, is crucial for enhancing wine quality. While commercially available S. cerevisiae strains are commonly used, indigenous strains can offer unique flavors, potentially reflecting vineyard terroir. By leveraging high-throughput DNA sequencing and transcriptomic analysis, we explored the impact of vine age on the grape mycobiome and characterized microbial functions during grape fermentation. Our findings revealed that older vines harbor higher fungal loads, but fungal diversity remains similar across vine ages. Additionally, uninoculated fermentations exhibited diverse fungal dynamics, including the beneficial wine yeasts Metschnikowia and Pichia. Transcriptomic analysis uncovered significant differences in S. cerevisiae gene expression between inoculated and uninoculated fermentations, highlighting the potential of indigenous yeast to enhance wine diversity and inform winemaking practices.

ABSTRACT Mycobacteroides abscessus (MAB) causes lung infections in people with cystic fibrosis (pwCF), and infecting strains show significant genetic variability both between and within individuals. MAB isolates can be divided into dominant clonal clusters (DCCs) or non-clustering groups and can present as smooth or rough colonies on agar plates. Both DCCs and the rough colony morphology have been linked to increased pathogenicity, but the mechanisms are unclear. This study explored the genomes of MAB isolates collected from individuals within the CF@LANTA CF center along with publicly available genomes to identify genes associated with more pathogenic MAB DCCs. Sixty-eight isolates from 26 CF individuals colonized by MAB were morphotyped and sequenced, with almost half of these isolates being members of DCC group 1 (DCC1). While lung function was not significantly impacted by colonization with DCC1 or rough isolates, 102 genes were specifically associated with DCC1 isolates. These genes were enriched for functions in sulfur-based DNA modification, DNA integration, and phenylacetic acid (PAA) catabolism. PAA is produced by the human gut microbiota and found throughout the human body. We show that strains containing PAA metabolic genes allow MAB to use PAA as a sole carbon and energy source. Although the benefits of PAA metabolic genes and other enriched pathways remain unclear, these findings highlight genes associated with emerging MAB CF strains.IMPORTANCEA primary challenge in treating bacterial infections is the wide spectrum of disease and genetic variability across bacterial strains. This is particularly evident in Mycobacteroides abscessus (MAB), an emerging pathogen affecting people with cystic fibrosis (pwCF). MAB exhibits significant genetic diversity both within and between individuals. However, seven dominant circulating clones (DCCs) have emerged as the major cause of human infections, demonstrating increased pathogenicity. Understanding the mechanisms underlying this increased pathogenicity and the associated genetic factors is crucial for developing novel treatment strategies. Our findings reveal that specific genes are associated with the DCC1 isolate of MAB, many of which are implicated in antimicrobial susceptibility or virulence.

ABSTRACT Understanding the molecular mechanisms governing microbial interactions is crucial for unraveling the complexities of microbial communities and their ecological impacts. Here, we employed a two-species model system comprising the oral bacteria Aggregatibacter actinomycetemcomitans and Streptococcus gordonii to investigate how synergistic and antagonistic interactions between microbes impact their resilience to environmental change and invasion by other microbes. We used an in vitro colony biofilm model and focused on two S. gordonii-produced extracellular molecules, L-lactate and H2O2, which are known to impact fitness of this dual-species community. While the ability of A. actinomycetemcomitans to cross-feed on S. gordonii-produced L-lactate enhanced its fitness during co-culture, this function showed little impact on the ability of co-cultures to resist environmental change. In fact, the ability of A. actinomycetemcomitans to catabolize L-lactate may be detrimental in the presence of tetracycline, highlighting the complexity of interactions under antimicrobial stress. Furthermore, H2O2, known for its antimicrobial properties, had negative impacts on both species in our model system. However, H2O2 production by S. gordonii enhanced A. actinomycetemcomitans tolerance to tetracycline, suggesting a protective role under antibiotic pressure. Finally, S. gordonii significantly inhibited the bacterium Serratia marcescens from invading in vitro biofilms, but this inhibition was lost during co-culture with A. actinomycetemcomitans and in a murine abscess model, where S. gordonii actually promoted S. marcescens invasion. These data indicate that microbial interactions can impact fitness of a bacterial community upon exposure to stresses, but these impacts are highly environment dependent.IMPORTANCEMicrobial interactions are critical modulators of the emergence of microbial communities and their functions. However, how these interactions impact the fitness of microbes in established communities upon exposure to environmental stresses is poorly understood. Here, we utilized a two-species community consisting of Aggregatibacter actinomycetemcomitans and Streptococcus gordonii to examine the impact of synergistic and antagonistic interactions on microbial resilience to environmental fluctuations and susceptibility to microbial invasion. We focused on the S. gordonii-produced extracellular molecules, L-lactate and H2O2, which have been shown to mediate interactions between these two microbes. We discovered that seemingly beneficial functions, such as A. actinomycetemcomitans cross-feeding on S. gordonii-produced L-Lactate, can paradoxically exacerbate vulnerabilities, such as susceptibility to antibiotics. Moreover, our data highlight the context-dependent nature of microbial interactions, emphasizing that a seemingly potent antimicrobial, such as H2O2, can have both synergistic and antagonistic effects on a microbial community dependent on the environment.

ABSTRACTThe ESKAPEE pathogen Pseudomonas aeruginosa is a common cause of chronic wound and cystic fibrosis lung infections, as well as acute burn and nosocomial infections. Many of these infections are recalcitrant to conventional antibiotic therapies due to both traditional antibiotic resistance mechanisms and antimicrobial tolerance. Recent successes with bacteriophage (phage) therapy to treat chronic human P. aeruginosa infections have led to a renewed interest in isolating and characterizing new P. aeruginosa phages. Here, we isolated and characterized a new lytic phage (termed PIP, pili-infecting phage) capable of infecting P. aeruginosa PA14. PIP is a tailed phage with an icosahedral head and flexible tail containing a genome that is 57,462 bp in length. Phylogenetic analysis reveals that PIP belongs to the subfamily Queuovirinae and genus Nipunavirus but is highly divergent in gene content from known Nipunaviruses. By isolating and characterizing a P. aeruginosa strain that spontaneously evolved resistance to PIP, we show that the receptor for PIP is Type IV pili. In summary, we isolated a new P. aeruginosa phage species with a unique genome, thus increasing the diversity of phages known to infect this important human pathogen.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa causes both acute and chronic human infections. These infections are notoriously difficult to treat due to both antibiotic resistance and antibiotic tolerance. The increasing frequency of antibiotic failure in P. aeruginosa infections has led scientists to explore other treatment options, including bacteriophage (phage) therapy. To this end, there has been a significant effort to identify new Pseudomonas phages. Here, we isolated and characterized a bacteriophage (termed PIP, pili-infecting phage) that infects P. aeruginosa PA14. Examination of the PIP genome revealed that this phage represents a new species in the subclass Queuovirinae. The isolation and characterization of spontaneous PA14 mutants that are resistant to PIP infection revealed Type IV pili as the PIP receptor. Ultimately, this study characterizes a new species of Pseudomonas phage, thus enhancing the known diversity of phages that infect this important pathogen.

ABSTRACT Bacteria rely on extracellular chemical cues to sense their environment, interact with one another, and shape their chemical landscape. Understanding this biochemistry can help us elucidate the mechanisms by which bacterial interactions impact community function. By applying mass spectrometry to mono- and co-cultures of the oral pathogen Aggregatibacter actinomycetemcomitans and the oral commensal Streptococcus gordonii, we identified hundreds of extracellular small molecules produced by these bacteria. We discovered that A. actinomycetemcomitans secretes millimolar amounts of glutathione and identified a five-gene operon (called gttABCDE) that is important for maximum secretion. The metabolomics data set generated in this study provides a valuable data set for unraveling the importance of individual molecules for mediating polymicrobial interactions in the oral cavity. IMPORTANCE Microbes produce a large array of extracellular molecules, which serve as signals and cues to promote polymicrobial interactions and alter the function of microbial communities. This has been particularly well studied in the human oral microbiome, where key metabolites have been shown to impact both health and disease. Here, we used an untargeted mass spectrometry approach to comprehensively assess the extracellular metabolome of the pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii during mono- and co-culture. We generated and made publicly available a metabolomic data set that includes hundreds of potential metabolites and leveraged this data set to identify an operon important for glutathione secretion in A. actinomycetemcomitans.

ABSTRACT Hard-wired within the genome of a pathogen is the information regarding factors responsible for its pathogenicity. Advances in functional genomics, particularly gene expression analysis, have made possible genome-wide interrogation of a pathogen to decipher pathogenicity-associated genes. Standard protocols assess differential expression of genes during pathogenesis; however, often a conservative approach is taken, which requires expression fold change above an arbitrarily defined threshold and a statistical significance test to infer a gene as differentially expressing. This renders a high-confidence set of differentially expressed genes in pathogenesis, however, at the cost of numerous false negatives. To circumvent this problem and comprehensively catalog pathogenicity-associated genes, we have developed a novel pipeline that uses standard protocol in combination with gene co-expression network of a pathogen constructed using publicly available RNA-Seq data sets. We assessed the efficacy of this pipeline on Pseudomonas aeruginosa PAO1, a model bacterial pathogen, highlighting the power of our network-based approach in discovering novel genes or pathways associated with the pathogenesis, or antibiotic resistance of this strain. Complementing standard protocol with a gene network-based method thus elevated the ability to identify pathogenicity-associated genes in P. aeruginosa PAO1.IMPORTANCEWe present here a new systems-level approach to decipher genetic factors and biological pathways associated with virulence and/or antibiotic treatment of bacterial pathogens. The power of this approach was demonstrated by application to a well-studied pathogen Pseudomonas aeruginosa PAO1. Our gene co-expression network-based approach unraveled known and unknown genes and their networks associated with pathogenicity in P. aeruginosa PAO1. The systems-level investigation of P. aeruginosa PAO1 helped identify putative pathogenicity and resistance-associated genetic factors that could not otherwise be detected by conventional approaches of differential gene expression analysis. The network-based analysis uncovered modules that harbor genes not previously reported by several original studies on P. aeruginosa virulence and resistance. These could potentially act as molecular determinants of P. aeruginosa PAO1 pathogenicity and responses to antibiotics.

IntroductionProteus mirabilis is a key pathobiont in catheter-associated urinary tract infections (CA-UTIs), which is well known to form crystalline biofilms that occlude catheters. Urease activity alkylates urine through the release of ammonia, consequentially resulting in higher levels of Mg2+ and Ca2+ and formation of crystals. In this study, we showed that N-acetyl cysteine (NAC), a thiol antioxidant, is a potent urease inhibitor that prevents crystalline biofilm formation.MethodsTo quantify urease activity, Berthelot’s method was done on bacterial extracts treated with NAC. We also used an in vitro catheterised glass bladder model to study the effect of NAC treatment on catheter occlusion and biofilm encrustation in P. mirabilis infections. Inductively-coupled plasma mass spectrometry (ICP-MS) was performed on catheter samples to decipher elemental profiles.ResultsNAC inhibits urease activity of clinical P. mirabilis isolates at concentrations as low as 1 mM, independent of bacterial killing. The study also showed that NAC is bacteriostatic on P. mirabilis, and inhibited biofilm formation and catheter occlusion in an in vitro. A significant 4-8log10 reduction in viable bacteria was observed in catheters infected in this model. Additionally, biofilms in NAC treated catheters displayed a depletion of calcium, magnesium, or phosphates (>10 fold reduction), thus confirming the absence of any urease activity in the presence of NAC. Interestingly, we also showed that not only is NAC anti-inflammatory in bladder epithelial cells (BECs), but that it mutes its inflammatory response to urease and P. mirabilis infection by reducing the production of IL-6, IL-8 and IL-1b.DiscussionUsing biochemical, microbiological and immunological techniques, this study displays the functionality of NAC in preventing catheter occlusion by inhibiting urease activity. The study also highlights NAC as a strong anti-inflammatory antibiofilm agent that can target both bacterial and host factors in the treatment of CA-UTIs.

The facultative intracellular pathogen Listeria monocytogenes, like many related Firmicutes, uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in cbpB, the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for cbpB-dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while cbpB was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Listeria monocytogenes IMPORTANCE Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Listeria monocytogenes Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.

ABSTRACT Pathogenic microbial ecosystems are often polymicrobial, and interbacterial interactions drive emergent properties of these communities. In the oral cavity, Streptococcus gordonii is a foundational species in the development of plaque biofilms, which can contribute to periodontal disease and, after gaining access to the bloodstream, target remote sites such as heart valves. Here, we used a transposon sequencing (Tn-Seq) library of S. gordonii to identify genes that influence fitness in a murine abscess model, both as a monoinfection and as a coinfection with an oral partner species, Porphyromonas gingivalis. In the context of a monoinfection, conditionally essential genes were widely distributed among functional pathways. Coinfection with P. gingivalis almost completely changed the nature of in vivo gene essentiality. Community-dependent essential (CoDE) genes under the coinfection condition were primarily related to DNA replication, transcription, and translation, indicating that robust growth and replication are required to survive with P. gingivalis in vivo. Interestingly, a group of genes in an operon encoding streptococcal receptor polysaccharide (RPS) were associated with decreased fitness of S. gordonii in a coinfection with P. gingivalis. Individual deletion of two of these genes (SGO_2020 and SGO_2024) resulted in the loss of RPS production by S. gordonii and increased susceptibility to killing by neutrophils. P. gingivalis protected the RPS mutants by inhibiting neutrophil recruitment, degranulation, and neutrophil extracellular trap (NET) formation. These results provide insight into genes and functions that are important for S. gordonii survival in vivo and the nature of polymicrobial synergy with P. gingivalis. Furthermore, we show that RPS-mediated immune protection in S. gordonii is dispensable and detrimental in the presence of a synergistic partner species that can interfere with neutrophil killing mechanisms. IMPORTANCE Bacteria responsible for diseases originating at oral mucosal membranes assemble into polymicrobial communities. However, we know little regarding the fitness determinants of the organisms that initiate community formation. Here, we show that the extracellular polysaccharide of Streptococcus gordonii, while important for streptococcal survival as a monoinfection, is detrimental to survival in the context of a coinfection with Porphyromonas gingivalis. We found that the presence of P. gingivalis compensates for immune protective functions of extracellular polysaccharide, rendering production unnecessary. The results show that fitness determinants of bacteria in communities differ substantially from those of individual species in isolation. Furthermore, constituents of communities can undertake activities that relieve the burden of energetically costly biosynthetic reactions on partner species.

ABSTRACT Our understanding of how bacterial pathogens colonize and persist during human infection has been hampered by the limited characterization of bacterial physiology during infection and a research bias toward in vitro, fast-growing bacteria. Recent research has begun to address these gaps in knowledge by directly quantifying bacterial mRNA levels during human infection, with the goal of assessing microbial community function at the infection site. However, mRNA levels are not always predictive of protein levels, which are the primary functional units of a cell. Here, we used carefully controlled chemostat experiments to examine the relationship between mRNA and protein levels across four growth rates in the bacterial pathogen Pseudomonas aeruginosa. We found a genome-wide positive correlation between mRNA and protein abundances across all growth rates, with genes required for P. aeruginosa viability having stronger correlations than nonessential genes. We developed a statistical method to identify genes whose mRNA abundances poorly predict protein abundances and calculated an RNA-to-protein (RTP) conversion factor to improve mRNA predictions of protein levels. The application of the RTP conversion factor to publicly available transcriptome data sets was highly robust, enabling the more accurate prediction of P. aeruginosa protein levels across strains and growth conditions. Finally, the RTP conversion factor was applied to P. aeruginosa human cystic fibrosis (CF) infection transcriptomes to provide greater insights into the functionality of this bacterium in the CF lung. This study addresses a critical problem in infection microbiology by providing a framework for enhancing the functional interpretation of bacterial human infection transcriptome data. IMPORTANCE Our understanding of bacterial physiology during human infection is limited by the difficulty in assessing bacterial function at the infection site. Recent studies have begun to address this question by quantifying bacterial mRNA levels in human-derived samples using transcriptomics. One challenge for these studies is the poor predictivity of mRNA for protein levels for some genes. Here, we addressed this challenge by measuring the transcriptomes and proteomes of P. aeruginosa grown at four growth rates. Our results revealed that the growth rate does not impact the genome-wide correlation of mRNA and protein levels. We used statistical methods to identify the genes for which mRNA and protein were poorly correlated and developed an RNA-to-protein (RTP) conversion factor that improved the predictivity of protein levels across strains and growth conditions. Our results provide new insights into mRNA-protein correlations and tools to enhance our understanding of bacterial physiology from transcriptome data.

Xenophagy is a selective macroautophagic process that protects the host cytosol by entrapping and delivering microbes to a degradative compartment. Both noncanonical autophagic pathways and xenophagy are activated by microbes during infection, but the relative importance and function of these distinct processes are not clear. In this study, we used bacterial and host mutants to dissect the contribution of autophagic processes responsible for bacterial growth restriction of Listeria monocytogenesL. monocytogenes is a facultative intracellular pathogen that escapes from phagosomes, grows in the host cytosol, and avoids autophagy by expressing three determinants of pathogenesis: two secreted phospholipases C (PLCs; PlcA and PlcB) and a surface protein (ActA). We found that shortly after phagocytosis, wild-type (WT) L. monocytogenes escaped from a noncanonical autophagic process that targets damaged vacuoles. During this process, the autophagy marker LC3 localized to single-membrane phagosomes independently of the ULK complex, which is required for initiation of macroautophagy. However, growth restriction of bacteria lacking PlcA, PlcB, and ActA required FIP200 and TBK1, both involved in the engulfment of microbes by xenophagy. Time-lapse video microscopy revealed that deposition of LC3 on L. monocytogenes-containing vacuoles via noncanonical autophagy had no apparent role in restricting bacterial growth and that, upon access to the host cytosol, WT L. monocytogenes utilized PLCs and ActA to avoid subsequent xenophagy. In conclusion, although noncanonical autophagy targets phagosomes, xenophagy was required to restrict the growth of L. monocytogenes, an intracellular pathogen that damages the entry vacuole.

ABSTRACT Diverse bacterial volatile compounds alter bacterial stress responses and physiology, but their contribution to population dynamics in polymicrobial communities is not well known. In this study, we showed that airborne volatile hydrogen cyanide (HCN) produced by a wide range of Pseudomonas aeruginosa clinical strains leads to at-a-distance in vitro inhibition of the growth of a wide array of Staphylococcus aureus strains. We determined that low-oxygen environments not only enhance P. aeruginosa HCN production but also increase S. aureus sensitivity to HCN, which impacts P. aeruginosa-S. aureus competition in microaerobic in vitro mixed biofilms as well as in an in vitro cystic fibrosis lung sputum medium. Consistently, we demonstrated that production of HCN by P. aeruginosa controls S. aureus growth in a mouse model of airways coinfected by P. aeruginosa and S. aureus. Our study therefore demonstrates that P. aeruginosa HCN contributes to local and distant airborne competition against S. aureus and potentially other HCN-sensitive bacteria in contexts relevant to cystic fibrosis and other polymicrobial infectious diseases. IMPORTANCE Airborne volatile compounds produced by bacteria are often only considered attractive or repulsive scents, but they also directly contribute to bacterial physiology. Here, we showed that volatile hydrogen cyanide (HCN) released by a wide range of Pseudomonas aeruginosa strains controls Staphylococcus aureus growth in low-oxygen in vitro biofilms or aggregates and in vivo lung environments. These results are of pathophysiological relevance, since lungs of cystic fibrosis patients are known to present microaerobic areas and to be commonly associated with the presence of S. aureus and P. aeruginosa in polymicrobial communities. Our study therefore provides insights into how a bacterial volatile compound can contribute to the exclusion of S. aureus and other HCN-sensitive competitors from P. aeruginosa ecological niches. It opens new perspectives for the management or monitoring of P. aeruginosa infections in lower-lung airway infections and other polymicrobial disease contexts.

Upon entry into the host cell cytosol, the facultative intracellular pathogen Listeria monocytogenes coordinates the expression of numerous essential virulence factors by allosteric binding of glutathione (GSH) to the Crp-Fnr family transcriptional regulator PrfA. Here, we report that robust virulence gene expression can be recapitulated by growing bacteria in a synthetic medium containing GSH or other chemical reducing agents. Bacteria grown under these conditions were 45-fold more virulent in an acute murine infection model and conferred greater immunity to a subsequent lethal challenge than bacteria grown in conventional media. During cultivation in vitro, PrfA activation was completely dependent on the intracellular levels of GSH, as a glutathione synthase mutant (ΔgshF) was activated by exogenous GSH but not reducing agents. PrfA activation was repressed in a synthetic medium supplemented with oligopeptides, but the repression was relieved by stimulation of the stringent response. These data suggest that cytosolic L. monocytogenes interprets a combination of metabolic and redox cues as a signal to initiate robust virulence gene expression in vivoIMPORTANCE Intracellular pathogens are responsible for much of the worldwide morbidity and mortality from infectious diseases. These pathogens have evolved various strategies to proliferate within individual cells of the host and avoid the host immune response. Through cellular invasion or the use of specialized secretion machinery, all intracellular pathogens must access the host cell cytosol to establish their replicative niches. Determining how these pathogens sense and respond to the intracellular compartment to establish a successful infection is critical to our basic understanding of the pathogenesis of each organism and for the rational design of therapeutic interventions. Listeria monocytogenes is a model intracellular pathogen with robust in vitro and in vivo infection models. Studies of the host-sensing and downstream signaling mechanisms evolved by L. monocytogenes often describe themes of pathogenesis that are broadly applicable to less tractable pathogens. Here, we describe how bacteria use external redox states as a cue to activate virulence.

Cyclic diadenosine monophosphate (c-di-AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall-active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the β-lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl-CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100-fold increase in virulence of the ΔdacA mutant. PycA is inhibited by c-di-AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down-stream enzymes suppressed ΔdacA phenotypes. These data suggested that c-di-AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild-type bacteria.

ABSTRACT The oral microbiota is enormously diverse, with over 700 microbial species identified across individuals that play a vital role in the health of our mouth and our overall well-being. In addition, as oral diseases such as caries (cavities) and periodontitis (gum disease) are mediated through interspecies microbial interactions, this community serves as an important model system to study the complexity and dynamics of polymicrobial interactions. Here, we review historical and recent progress in our understanding of the oral microbiome, highlighting how oral microbiome research has significantly contributed to our understanding of microbial communities, with broad implications in polymicrobial diseases and across microbial community ecology. Further, we explore innovations and challenges associated with analyzing polymicrobial systems and suggest future directions of study. Finally, we provide a conceptual framework to systematically study microbial interactions within complex communities, not limited to the oral microbiota.

Listeria monocytogenes is an environmental saprophyte and facultative intracellular bacterial pathogen with a well-defined life-cycle that involves escape from a phagosome, rapid cytosolic growth, and ActA-dependent cell-to-cell spread, all of which are dependent on the master transcriptional regulator PrfA. The environmental cues that lead to temporal and spatial control of L. monocytogenes virulence gene expression are poorly understood. In this study, we took advantage of the robust up-regulation of ActA that occurs intracellularly and expressed Cre recombinase from the actA promoter and 5' untranslated region in a strain in which loxP sites flanked essential genes, so that activation of actA led to bacterial death. Upon screening for transposon mutants that survived intracellularly, six genes were identified as necessary for ActA expression. Strikingly, most of the genes, including gshF, spxA1, yjbH, and ohrA, are predicted to play important roles in bacterial redox regulation. The mutants identified in the genetic selection fell into three broad categories: (1) those that failed to reach the cytosolic compartment; (2) mutants that entered the cytosol, but failed to activate the master virulence regulator PrfA; and (3) mutants that entered the cytosol and activated transcription of actA, but failed to synthesize it. The identification of mutants defective in vacuolar escape suggests that up-regulation of ActA occurs in the host cytosol and not the vacuole. Moreover, these results provide evidence for two non-redundant cytosolic cues; the first results in allosteric activation of PrfA via increased glutathione levels and transcriptional activation of actA while the second results in translational activation of actA and requires yjbH. Although the precise host cues have not yet been identified, we suggest that intracellular redox stress occurs as a consequence of both host and pathogen remodeling their metabolism upon infection.

Cyclic di-adenosine monophosphate (c-di-AMP) is a widely distributed second messenger that appears to be essential in multiple bacterial species, including the Gram-positive facultative intracellular pathogen Listeria monocytogenes. In this study, the only L. monocytogenes diadenylate cyclase gene, dacA, was deleted using a Cre-lox system activated during infection of cultured macrophages. All ΔdacA strains recovered from infected cells harbored one or more suppressor mutations that allowed growth in the absence of c-di-AMP. Suppressor mutations in the synthase domain of the bi-functional (p)ppGpp synthase/hydrolase led to reduced (p)ppGpp levels. A genetic assay confirmed that dacA was essential in wild-type but not strains lacking all three (p)ppGpp synthases. Further genetic analysis suggested that c-di-AMP was essential because accumulated (p)ppGpp altered GTP concentrations, thereby inactivating the pleiotropic transcriptional regulator CodY. We propose that c-di-AMP is conditionally essential for metabolic changes that occur in growth in rich medium and host cells but not minimal medium.

Intracellular pathogens are responsible for much of the world-wide morbidity and mortality due to infectious diseases. To colonize their hosts successfully, pathogens must sense their environment and regulate virulence gene expression appropriately. Accordingly, on entry into mammalian cells, the facultative intracellular bacterial pathogen Listeria monocytogenes remodels its transcriptional program by activating the master virulence regulator PrfA. Here we show that bacterial and host-derived glutathione are required to activate PrfA. In this study a genetic selection led to the identification of a bacterial mutant in glutathione synthase that exhibited reduced virulence gene expression and was attenuated 150-fold in mice. Genome sequencing of suppressor mutants that arose spontaneously in vivo revealed a single nucleotide change in prfA that locks the protein in the active conformation (PrfA*) and completely bypassed the requirement for glutathione during infection. Biochemical and genetic studies support a model in which glutathione-dependent PrfA activation is mediated by allosteric binding of glutathione to PrfA. Whereas glutathione and other low-molecular-weight thiols have important roles in redox homeostasis in all forms of life, here we demonstrate that glutathione represents a critical signalling molecule that activates the virulence of an intracellular pathogen.

The accumulation of petroleum-based plastic waste has become a major issue for the environment. A sustainable and biodegradable solution can be found in Polyhydroxyalkanoates (PHAs), a microbially produced biopolymer. An analysis of the global phylogenetic and ecological distribution of potential PHA producing bacteria and archaea was carried out by mining a global genome repository for PHA synthase (PhaC), a key enzyme involved in PHA biosynthesis. Bacteria from the phylum Actinobacteria were found to contain the PhaC Class II genotype which produces medium-chain length PHAs, a physiology until now only found within a few Pseudomonas species. Further, several PhaC genotypes were discovered within Thaumarchaeota, an archaeal phylum with poly-extremophiles and the ability to efficiently use CO2 as a carbon source, a significant ecological group which have thus far been little studied for PHA production. Bacterial and archaeal PhaC genotypes were also observed in high salinity and alkalinity conditions, as well as high-temperature geothermal ecosystems. These genome mining efforts uncovered previously unknown candidate taxa for biopolymer production, as well as microbes from environmental niches with properties that could potentially improve PHA production. This in silico study provides valuable insights into unique PHA producing candidates, supporting future bioprospecting efforts toward better targeted and relevant taxa to further enhance the diversity of exploitable PHA production systems.

Lake Magic is an extremely acidic, hypersaline lake found in Western Australia, with the highest concentrations of aluminum and silica in the world. Previous studies of Lake Magic diversity have revealed that the lake hosts acid- and halotolerant bacterial and fungal species. However, they have not canvassed microbial population dynamics across flooding, evapo-concentration and desiccation stages. In this study, we used amplicon sequencing and potential function prediction on sediment and salt mat samples. We observed that the bacterial and fungal diversity in Lake Magic is strongly driven by carbon, temperature, pH and salt concentrations at the different stages of the lake. We also saw that the fungal diversity decreased as the environmental conditions became more extreme. However, prokaryotic diversity was very dynamic and bacteria dominated archaeal species, both in abundance and diversity, perhaps because bacteria better tolerate the extreme variation in conditions. Bacterial species diversity was the highest during early flooding stage and decreased during more stressful conditions. We observed an increase in acid tolerant and halotolerant species in the sediment, involved in functions such as sulfur and iron metabolism, i.e., species involved in buffering the external environment. Thus, due to activity within the microbial community, the environmental conditions in the sediment do not change to the same degree as conditions in the salt mat, resulting in the sediment becoming a safe haven for microbes, which are able to thrive during the extreme conditions of the evapo-concentration and desiccation stages.

Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection.

Bacterial antibiotic resistance has increased in recent decades, raising concerns in hospital and community settings. Novel, innovative strategies are needed to eradicate bacteria, particularly within biofilms, and diminish the likelihood of recurrence. In this study, we investigated whether glutathione (GSH) can act as a biofilm disruptor, and enhance antibiotic effectiveness against various bacterial pathogens. Biological levels (10 mM) of GSH did not have a significant effect in inhibiting growth or disrupting the biofilm in four out of six species tested. However, exposure to 30 mM GSH showed >50% decrease in growth for all bacterial species, with almost 100% inhibition of Streptococcus pyogenes and an average of 94–52% inhibition for Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive S. aureus (MSSA) and multi-drug resistant Acinetobacter baumannii (MRAB) isolates, respectively. Klebsiella pneumoniae and Enterobacter sp. isolates were however, highly resistant to 30 mM GSH. With respect to biofilm viability, all species exhibited a >50% decrease in viability with 30 mM GSH, with confocal imaging showing considerable change in the biofilm architecture of MRAB isolates. The mechanism of GSH-mediated biofilm disruption is possibly due to a concentration-dependent increase in GSH acidity that triggers cleaving of the matrix components. Enzymatic treatment of MRAB revealed that eDNA and polysaccharides are essential for biofilm stability and eDNA removal enhanced amikacin efficiency. Combination of GSH, amikacin and DNase-I showed the greatest reduction in MRAB biofilm viability. Additionally, GSH alone and in combination with amikacin fostered human fibroblast cell (HFF-1) growth and confluence while inhibiting MRAB adhesion and colonization.

Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis, and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism.

The effect of different cropping systems on CO2 fixation by soil microorganisms was studied by comparing soils from three exemplary cropping systems: continuous cropping of paddy rice (rice-rice), rotation of paddy rice and rapeseed (rice-rapeseed), and rotated cropping of rapeseed and corn (rapeseed-corn). Soils from different cropping systems were continuously labeling with 14C-CO2 for 110 days. The CO2-fixing bacterial communities were investigated by analyzing the cbbL gene encoding ribulose-1,5-bisphosphate carboxylase oxygenase (RubisCO). Abundance, diversity and activity of cbbL-carrying bacteria were analyzed by quantitative PCR, cbbL clone libraries and enzyme assays. After 110 days incubation, substantial amounts of 14C-CO2 were incorporated into soil organic carbon (14C-SOC) and microbial organic carbon (14C-MBC). Rice-rice rotated soil showed stronger incorporation rates when looking at 14C-SOC and 14C-MBC contents. These differences in incorporation rates were also reflected by RubisCO activities. 14C-MBC, cbbL gene abundances and RubisCO activity were found to correlate significantly with 14C-SOC, indicating cbbL-carrying bacteria to be key players for CO2 fixation in these soils. The analysis of clone libraries revealed distinct cbbL-carrying bacterial communities for the individual soils analyzed. Most of the identified operational taxonomic units (OTU) were related to Nitrobacter hamburgensis, Methylibium petroleiphilum, Rhodoblastus acidophilus, Bradyrhizobium, Cupriavidus metallidurans, Rubrivivax, Burkholderia, stappia and Thiobacillus thiophilus. OTUs related to Rubrivivax gelatinosus were specific for rice-rice soil. OTUs linked to Methylibium petroleiphilum were exclusively found in rice-rapeseed soil. Observed differences could be linked to differences in soil parameters such as SOC. We conclude that the long-term application of cropping systems alters underlying soil parameters, which in turn selects for distinct autotrophic communities.

Soil carbon (C) storage is dependent upon the complex dynamics of fresh and native organic matter cycling, which are regulated by plant and soil-microbial activities. A fundamental challenge exists to link microbial biodiversity with plant-soil C cycling processes to elucidate the underlying mechanisms regulating soil carbon. To address this, we contrasted vegetated grassland soils with bare soils, which had been plant-free for 3 years, using stable isotope (13C) labelled substrate assays and molecular analyses of bacterial communities. Vegetated soils had higher C and N contents, biomass, and substrate-specific respiration rates. Conversely, following substrate addition unlabelled, native soil C cycling was accelerated in bare soil and retarded in vegetated soil; indicative of differential priming effects. Functional differences were reflected in bacterial biodiversity with Alphaproteobacteria and Acidobacteria dominating vegetated and bare soils respectively. Significant isotopic enrichment of soil RNA was found after substrate addition and rates varied according to substrate type. However assimilation was independent of plant presence which, in contrast to large differences in 13CO2 respiration rates, indicated greater substrate C use efficiency in bare, Acidobacteria-dominated soils. Stable isotope probing revealed most community members had utilised substrates with little evidence for competitive outgrowth of sub-populations. Our findings support theories on how plant-mediated soil resource availability affects the turnover of different pools of soil carbon, and we further identify a potential role of soil microbial biodiversity. Specifically we conclude that emerging theories on the life histories of dominant soil taxa can be invoked to explain the changes in soil carbon cycling linked to resource availability, and that there is a strong case for considering microbial biodiversity in future studies investigating the turnover of different pools of soil carbon.

ABSTRACT Pseudomonas aeruginosa and Staphylococcus aureus are two of the most common coinfecting bacteria in human infections, including the cystic fibrosis (CF) lung. There is emerging evidence that coinfection with these microbes enhances disease severity and antimicrobial tolerance through direct interactions. However, one of the challenges to studying microbial interactions relevant to human infection is the lack of experimental models with the versatility to investigate complex interaction dynamics while maintaining biological relevance. Here, we developed a model based on an in vitro medium that mimics human CF lung secretions (synthetic CF sputum medium [SCFM2]) and allows time-resolved assessment of fitness and community spatial structure at the micrometer scale. Our results reveal that P. aeruginosa and S. aureus coexist as spatially structured communities in SCFM2 under static growth conditions, with S. aureus enriched at a distance of 3.5 μm from P. aeruginosa. Multispecies aggregates were rare, and aggregate (biofilm) sizes resembled those in human CF sputum. Elimination of P. aeruginosa’s ability to produce the antistaphylococcal small molecule HQNO (2-heptyl-4-hydroxyquinoline N-oxide) had no effect on bacterial fitness but altered the spatial structure of the community by increasing the distance of S. aureus from P. aeruginosa to 7.6 μm. Lastly, we show that coculture with P. aeruginosa sensitizes S. aureus to killing by the antibiotic tobramycin compared to monoculture growth despite HQNO enhancing tolerance during coculture. Our findings reveal that SCFM2 is a powerful model for studying P. aeruginosa and S. aureus and that HQNO alters S. aureus biogeography and antibiotic susceptibility without affecting fitness. IMPORTANCE Many human infections result from the action of multispecies bacterial communities. Within these communities, bacteria have been proposed to directly interact via physical and chemical means, resulting in increased disease and antimicrobial tolerance. One of the challenges to studying multispecies infections is the lack of robust, infection-relevant model systems with the ability to study these interactions through time with micrometer-scale precision. Here, we developed a versatile in vitro model for studying the interactions between Pseudomonas aeruginosa and Staphylococcus aureus, two bacteria that commonly coexist in human infections. Using this model along with high-resolution, single-cell microscopy, we showed that P. aeruginosa and S. aureus form communities that are spatially structured at the micrometer scale, controlled in part by the production of an antimicrobial by P. aeruginosa. In addition, we provide evidence that this antimicrobial enhances S. aureus tolerance to an aminoglycoside antibiotic only during coculture.

ABSTRACT Bacteria alter their local chemical environment through both consumption and the production of a variety of molecules, ultimately shaping the local ecology. Molecular oxygen (O2) is a key metabolite that affects the physiology and behavior of virtually all bacteria, and its consumption often results in O2 gradients within sessile bacterial communities (biofilms). O2 plays a critical role in several bacterial phenotypes, including antibiotic tolerance; however, our understanding of O2 levels within and surrounding biofilms has been hampered by the difficulties in measuring O2 levels in real-time for extended durations and at the micron scale. Here, we developed electrochemical methodology based on scanning electrochemical microscopy to quantify the O2 gradients present above a Pseudomonas aeruginosa biofilm. These results reveal that a biofilm produces a hypoxic zone that extends hundreds of microns from the biofilm surface within minutes and that the biofilm consumes O2 at a maximum rate. Treating the biofilm with levels of the antibiotic ciprofloxacin that kill 99% of the bacteria did not affect the O2 gradient, indicating that the biofilm is highly resilient to antimicrobial treatment in regard to O2 consumption. IMPORTANCE O2 is a fundamental environmental metabolite that affects all life on earth. While toxic to many microbes and obligately required by others, those that have appropriate physiological responses survive and can even benefit from various levels of O2, particularly in biofilm communities. Although most studies have focused on measuring O2 within biofilms, little is known about O2 gradients surrounding biofilms. Here, we developed electrochemical methodology based on scanning electrochemical microscopy to measure the O2 gradients surrounding biofilms in real time on the micron scale. Our results reveal that P. aeruginosa biofilms produce a hypoxic zone that can extend hundreds of microns from the biofilm surface and that this gradient remains even after the addition of antibiotic concentrations that eradicated 99% of viable cells. Our results provide a high resolution of the O2 gradients produced by P. aeruginosa biofilms and reveal sustained O2 consumption in the presence of antibiotics.

ABSTRACT The facultative intracellular pathogen Listeria monocytogenes, like many related Firmicutes, uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in cbpB, the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for cbpB-dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while cbpB was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Listeria monocytogenes. IMPORTANCE Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Listeria monocytogenes. Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.

ABSTRACT Laboratory models are a cornerstone of modern microbiology, but the accuracy of these models has not been systematically evaluated. As a result, researchers often choose models based on intuition or incomplete data. We propose a general quantitative framework to assess model accuracy from RNA sequencing data and use this framework to evaluate models of Pseudomonas aeruginosa cystic fibrosis (CF) lung infection. We found that an in vitro synthetic CF sputum medium model and a CF airway epithelial cell model had the highest genome-wide accuracy but underperformed on distinct functional categories, including porins and polyamine biosynthesis for the synthetic sputum medium and protein synthesis for the epithelial cell model. We identified 211 “elusive” genes that were not mimicked in a reference strain grown in any laboratory model but found that many were captured by using a clinical isolate. These methods provide researchers with an evidence-based foundation to select and improve laboratory models. IMPORTANCE Laboratory models have become a cornerstone of modern microbiology. However, the accuracy of even the most commonly used models has never been evaluated. Here, we propose a quantitative framework based on gene expression data to evaluate model performance and apply it to models of Pseudomonas aeruginosa cystic fibrosis lung infection. We discovered that these models captured different aspects of P. aeruginosa infection physiology, and we identify which functional categories are and are not captured by each model. These methods will provide researchers with a solid basis to choose among laboratory models depending on the scientific question of interest and will help improve existing experimental models.

ABSTRACT Laboratory models have been invaluable for the field of microbiology for over 100 years and have provided key insights into core aspects of bacterial physiology such as regulation and metabolism. However, it is important to identify the extent to which these models recapitulate bacterial physiology within a human infection environment. Here, we performed transcriptomics (RNA-seq), focusing on the physiology of the prominent pathogen Staphylococcus aureus in situ in human cystic fibrosis (CF) infection. Through principal-component and hierarchal clustering analyses, we found remarkable conservation in S. aureus gene expression in the CF lung despite differences in the patient clinic, clinical status, age, and therapeutic regimen. We used a machine learning approach to identify an S. aureus transcriptomic signature of 32 genes that can reliably distinguish between S. aureus transcriptomes in the CF lung and in vitro. The majority of these genes were involved in virulence and metabolism and were used to improve a common CF infection model. Collectively, these results advance our knowledge of S. aureus physiology during human CF lung infection and demonstrate how in vitro models can be improved to better capture bacterial physiology in infection. IMPORTANCE Although bacteria have been studied in infection for over 100 years, the majority of these studies have utilized laboratory and animal models that often have unknown relevance to the human infections they are meant to represent. A primary challenge has been to assess bacterial physiology in the human host. To address this challenge, we performed transcriptomics of S. aureus during human cystic fibrosis (CF) lung infection. Using a machine learning framework, we defined a “human CF lung transcriptome signature” that primarily included genes involved in metabolism and virulence. In addition, we were able to apply our findings to improve an in vitro model of CF infection. Understanding bacterial gene expression within human infection is a critical step toward the development of improved laboratory models and new therapeutics.

Biocrusts are aggregated crusts that exist on the soil surface of arid environments. They are complex microbial communities comprised of cyanobacteria, lichens, mosses, algae and fungi. Recently, biocrusts have gained significant attention due to their ubiquitous distribution and likely important ecological roles, including soil stabilization, soil moisture retention, carbon (C) and nitrogen (N) fixation, as well as microbial engineers for semi-arid ecosystem restoration. Here, we collected three co-occurring types of biocrust (Cyanobacterial crust, Crustose lichen, and Foliose lichen) and their underlying soil from arid zones within Western Australia. Bacterial microbiome composition was determined through 16S rRNA gene amplicon sequencing to assess the extent of microbiome selection within the crusts versus underlying soil and biogeochemical measures performed to determine whether the crusts had significant impact upon the underlying soil for nutrient input. We determined that the bacterial communities of native biocrusts are distinct from those in their underlying soil, where dominant bacterial taxa differed according to crust morphologies. δ15N revealed that N-fixation appeared most evident in Foliose lichen crust (1.73 ± 1.04‰). Consequently, depending upon the crust type, biocrusts contained higher concentrations of organic C (2 to 50 times), total N (4 to 16 times) and available ammonium (2 to 4 times), though this enrichment did not extend to the soils underneath them. These findings demonstrate that biocrust communities are seemingly islands of biological activity in an arid landscape, uniquely different from their surrounding and underlying soil.

ABSTRACT Opportunistic pathogens establishing new infections experience strong selection to adapt, often favoring mutants that persist. Capturing this initial dynamic is critical for identifying the first adaptations that drive pathogenesis. Here we used a porcine full-thickness burn wound model of chronic infection to study the evolutionary dynamics of diverse Pseudomonas aeruginosa infections. Wounds were infected with a mixed community of six P. aeruginosa strains, including the model PA14 strain (PA14-1), and biopsies taken at 3, 14, and 28 days postinfection. Hyperbiofilm-forming rugose small-colony variants (RSCVs) were the earliest and predominant phenotypic variant. These variants were detected on day 3 and persisted, with the majority evolved from PA14-1. Whole-genome sequencing of PA14-1 RSCV isolates revealed driver mutations exclusively in the wsp pathway, conferring hyperbiofilm phenotypes. Several of the wsp mutant RSCVs also acquired CRISPR-Cas adaptive immunity to prophages isolated from the P. aeruginosa wound isolate (B23-2) that was also present in the inoculum. These observations emphasize the importance of interstrain dynamics and the role of lysogenic phages in the survival of an invading pathogen. Rather than being a side effect of chronicity, the rapid rise of RSCVs in wounds is evidence of positive selection on the Wsp chemosensory system to produce mutants with elevated biofilm formation capacity. We predict that RSCVs provide a level of phenotypic diversity to the infecting bacterial community and are common, early adaptations during infections. This would likely have significant consequences for clinical outcomes. IMPORTANCE Bacteria adapt to infections by evolving variants that are more fit and persistent. These recalcitrant variants are typically observed in chronic infections. However, it is unclear when and why these variants evolve. To address these questions, we used a porcine chronic wound model to study the evolutionary dynamics of Pseudomonas aeruginosa in a mixed-strain infection. We isolated hyperbiofilm variants that persisted early in the infection. Interstrain interactions were also observed, where adapted variants acquired CRISPR-mediated immunity to phages. We show that when initiating infection, P. aeruginosa experiences strong positive selection for hyperbiofilm phenotypes produced by mutants of a single chemosensory system, the Wsp pathway. We predict that hyperbiofilm variants are early adaptations to infection and that interstrain interactions may influence bacterial burden and infection outcomes.

Mining of mineral resources substantially alters both the above and below-ground soil ecosystem, which then requires rehabilitation back to a pre-mining state. For belowground rehabilitation, recovery of the soil microbiome to a state which can support key biogeochemical cycles, and effective plant colonization is usually required. One solution proposed has been to translate microbial inocula from agricultural systems to mine rehabilitation scenarios, as a means of reconditioning the soil microbiome for planting. Here, we experimentally determine both the aboveground plant fitness outcomes and belowground soil microbiome effects of a commercially available soil microbial inocula (SMI). We analyzed treatment effects at four levels of complexity; no SMI addition control, Nitrogen addition alone, SMI addition and SMI plus Nitrogen addition over a 12-week period. Our culture independent analyses indicated that SMIs had a differential response over the 12-week incubation period, where only a small number of the consortium members persisted in the semi-arid ecosystem, and generated variable plant fitness responses, likely due to plant-microbiome physiological mismatching and low survival rates of many of the SMI constituents. We suggest that new developments in custom-made SMIs to increase rehabilitation success in mine site restoration are required, primarily based upon the need for SMIs to be ecologically adapted to both the prevailing edaphic conditions and a wide range of plant species likely to be encountered.

ABSTRACT Over half of adults experience gingivitis, a mild yet treatable form of periodontal disease caused by the overgrowth of oral microbes. Left untreated, gingivitis can progress to a more severe and irreversible disease, most commonly chronic periodontitis. While periodontal diseases are associated with a shift in the oral microbiota composition, it remains unclear how this shift impacts microbiota function early in disease progression. Here, we analyzed the transition from health to gingivitis through both 16S v4-v5 rRNA amplicon and metatranscriptome sequencing of subgingival plaque samples from individuals undergoing an experimental gingivitis treatment. Beta-diversity analysis of 16S rRNA reveals that samples cluster based on disease severity and patient but not by oral hygiene status. Significant shifts in the abundance of several genera occurred during disease transition, suggesting a dysbiosis due to development of gingivitis. Comparing taxonomic abundance with transcriptomic activity revealed concordance of bacterial diversity composition between the two quantification assays in samples originating from both healthy and diseased teeth. Metatranscriptome sequencing analysis indicates that during the early stages of transition to gingivitis, a number of virulence-related transcripts were significantly differentially expressed in individual and across pooled patient samples. Upregulated genes include those involved in proteolytic and nucleolytic processes, while expression levels of those involved in surface structure assembly and other general virulence functions leading to colonization or adaptation within the host are more dynamic. These findings help characterize the transition from health to periodontal disease and identify genes associated with early disease. IMPORTANCE Although more than 50% of adults have some form of periodontal disease, there remains a significant gap in our understanding of its underlying cause. We initiated this study in order to better characterize the progression from oral health to disease. We first analyzed changes in the abundances of specific microorganisms in dental plaque collected from teeth during health and gingivitis, the mildest form of periodontal disease. We found that the clinical score of disease and patient from whom the sample originated but not tooth brushing are significantly correlated with microbial community composition. While a number of virulence-related gene transcripts are differentially expressed in gingivitis samples relative to health, not all are increased, suggesting that the overall activity of the microbiota is dynamic during disease transition. Better understanding of which microbes are present and their function during early periodontal disease can potentially lead to more targeted prophylactic approaches to prevent disease progression.

ABSTRACT Microbial communities are shaped by interactions among their constituent members. Some Gram-negative bacteria employ type VI secretion systems (T6SSs) to inject protein toxins into neighboring cells. These interactions have been theorized to affect the composition of host-associated microbiomes, but the role of T6SSs in the evolution of gut communities is not well understood. We report the discovery of two T6SSs and numerous T6SS-associated Rhs toxins within the gut bacteria of honey bees and bumble bees. We sequenced the genomes of 28 strains of Snodgrassella alvi, a characteristic bee gut microbe, and found tremendous variability in their Rhs toxin complements: altogether, these strains appear to encode hundreds of unique toxins. Some toxins are shared with Gilliamella apicola, a coresident gut symbiont, implicating horizontal gene transfer as a source of toxin diversity in the bee gut. We use data from a transposon mutagenesis screen to identify toxins with antibacterial function in the bee gut and validate the function and specificity of a subset of these toxin and immunity genes in Escherichia coli. Using transcriptome sequencing, we demonstrate that S. alvi T6SSs and associated toxins are upregulated in the gut environment. We find that S. alvi Rhs loci have a conserved architecture, consistent with the C-terminal displacement model of toxin diversification, with Rhs toxins, toxin fragments, and cognate immunity genes that are expressed and confer strong fitness effects in vivo. Our findings of T6SS activity and Rhs toxin diversity suggest that T6SS-mediated competition may be an important driver of coevolution within the bee gut microbiota. IMPORTANCE The structure and composition of host-associated bacterial communities are of broad interest, because these communities affect host health. Bees have a simple, conserved gut microbiota, which provides an opportunity to explore interactions between species that have coevolved within their host over millions of years. This study examined the role of type VI secretion systems (T6SSs)—protein complexes used to deliver toxic proteins into bacterial competitors—within the bee gut microbiota. We identified two T6SSs and diverse T6SS-associated toxins in bacterial strains from bees. Expression of these genes is increased in bacteria in the bee gut, and toxin and immunity genes demonstrate antibacterial and protective functions, respectively, when expressed in Escherichia coli. Our results suggest that coevolution among bacterial species in the bee gut has favored toxin diversification and maintenance of T6SS machinery, and demonstrate the importance of antagonistic interactions within host-associated microbial communities.

ABSTRACT Upon entry into the host cell cytosol, the facultative intracellular pathogen Listeria monocytogenes coordinates the expression of numerous essential virulence factors by allosteric binding of glutathione (GSH) to the Crp-Fnr family transcriptional regulator PrfA. Here, we report that robust virulence gene expression can be recapitulated by growing bacteria in a synthetic medium containing GSH or other chemical reducing agents. Bacteria grown under these conditions were 45-fold more virulent in an acute murine infection model and conferred greater immunity to a subsequent lethal challenge than bacteria grown in conventional media. During cultivation in vitro, PrfA activation was completely dependent on the intracellular levels of GSH, as a glutathione synthase mutant (ΔgshF) was activated by exogenous GSH but not reducing agents. PrfA activation was repressed in a synthetic medium supplemented with oligopeptides, but the repression was relieved by stimulation of the stringent response. These data suggest that cytosolic L. monocytogenes interprets a combination of metabolic and redox cues as a signal to initiate robust virulence gene expression in vivo. IMPORTANCE Intracellular pathogens are responsible for much of the worldwide morbidity and mortality from infectious diseases. These pathogens have evolved various strategies to proliferate within individual cells of the host and avoid the host immune response. Through cellular invasion or the use of specialized secretion machinery, all intracellular pathogens must access the host cell cytosol to establish their replicative niches. Determining how these pathogens sense and respond to the intracellular compartment to establish a successful infection is critical to our basic understanding of the pathogenesis of each organism and for the rational design of therapeutic interventions. Listeria monocytogenes is a model intracellular pathogen with robust in vitro and in vivo infection models. Studies of the host-sensing and downstream signaling mechanisms evolved by L. monocytogenes often describe themes of pathogenesis that are broadly applicable to less tractable pathogens. Here, we describe how bacteria use external redox states as a cue to activate virulence.

ABSTRACT Environmental factors relating to soil pH are important regulators of bacterial taxonomic biodiversity, yet it remains unclear if such drivers affect community functional potential. To address this, we applied whole-genome metagenomics to eight geographically distributed soils at opposing ends of a landscape soil pH gradient (where “low-pH” is ~pH 4.3 and “high-pH” is ~pH 8.3) and evaluated functional differences with respect to functionally annotated genes. First, differences in taxonomic and functional diversity between the two pH categories were assessed with respect to alpha diversity (mean sample richness) and gamma diversity (total richness pooled for each pH category). Low-pH soils, also exhibiting higher organic matter and moisture, consistently had lower taxonomic alpha and gamma diversity, but this was not apparent in assessments of functional alpha and gamma diversity. However, coherent changes in the relative abundances of annotated genes between low- and high-pH soils were identified; with strong multivariate clustering of samples according to pH independent of geography. Assessment of indicator genes revealed that the acidic organic-rich soils possessed a greater abundance of cation efflux pumps, C and N direct fixation systems, and fermentation pathways, indicating adaptations to both acidity and anaerobiosis. Conversely, high-pH soils possessed more direct transporter-mediated mechanisms for organic C and N substrate acquisition. These findings highlight the distinctive physiological adaptations required for bacteria to survive in soils of various nutrient availability and edaphic conditions and more generally indicate that bacterial functional versatility with respect to functional gene annotations may not be constrained by taxonomy. IMPORTANCE Over a set of soil samples spanning Britain, the widely reported reductions in bacterial taxonomic richness at low pH were found not to be accompanied by significant reductions in the richness of functional genes. However, consistent changes in the abundance of related functional genes were observed, characteristic of differential ecological and nutrient acquisition strategies between high-pH mineral soils and low-pH organic anaerobic soils. Our assessment at opposing ends of a soil gradient encapsulates the limits of functional diversity in temperate climates and identifies key pathways that may serve as indicators for soil element cycling and C storage processes in other soil systems. To this end, we make available a data set identifying functional indicators of the different soils; as well as raw sequences, which given the geographic scale of our sampling should be of value in future studies assessing novel genetic diversity of a wide range of soil functional attributes.

ABSTRACT Chronic polymicrobial infections are associated with increased virulence compared to monospecies infections. However, our understanding of microbial dynamics during polymicrobial infection is limited. A recent study by Limoli and colleagues (D. H. Limoli, G. B. Whitfield, T. Kitao, M. L. Ivey, M. R. Davis, Jr., et al., mBio 8:e00186-17, 2017, https://doi.org/10.1128/mBio.00186-17 ) provides insight into a mechanism that may contribute to the coexistence of Pseudomonas aeruginosa and Staphylococcus aureus in the cystic fibrosis (CF) lung. CF lung infections have frequently been used to investigate microbial interactions due to both the complex polymicrobial community and chronic nature of these infections. The hypothesis of Limoli et al. is that the conversion of P. aeruginosa to its mucoidy phenotype during chronic CF infection promotes coexistence by diminishing its ability to kill S. aureus. Highlighting a new facet of microbial interaction between two species that are traditionally thought of as competitors, this study provides a platform for studying community assembly in a relevant infection setting.

ABSTRACT The microbial communities inhabiting chronic infections are often composed of spatially organized micrometer-sized, highly dense aggregates. It has recently been hypothesized that aggregates are responsible for the high tolerance of chronic infections to host immune functions and antimicrobial therapies. Little is currently known regarding the mechanisms controlling aggregate formation and antimicrobial tolerance primarily because of the lack of robust, biologically relevant experimental systems that promote natural aggregate formation. Here, we developed an in vitro model based on chronic Pseudomonas aeruginosa infection of the cystic fibrosis (CF) lung. This model utilizes a synthetic sputum medium that readily promotes the formation of P. aeruginosa aggregates with sizes similar to those observed in human CF lung tissue. Using high-resolution imaging, we exploited this model to elucidate the life history of P. aeruginosa and the mechanisms that this bacterium utilizes to tolerate antimicrobials, specifically, bacteriophage. In the early stages of growth in synthetic sputum, planktonic cells form aggregates that increase in size over time by expansion. In later growth, migrant cells disperse from aggregates and colonize new areas, seeding new aggregates. When added simultaneously with phage, P. aeruginosa was readily killed and aggregates were unable to form. When added after initial aggregate formation, phage were unable to eliminate all of the aggregates because of exopolysaccharide production; however, seeding of new aggregates by dispersed migrants was inhibited. We propose a model in which aggregates provide a mechanism that allows P. aeruginosa to tolerate phage therapy during chronic infection without the need for genetic mutation. IMPORTANCE Bacteria in chronic infections often reside in communities composed of micrometer-sized, highly dense aggregates. A primary challenge for studying aggregates has been the lack of laboratory systems that promote natural aggregate formation in relevant environments. Here, we developed a growth medium that mimics chronic lung infection and promotes natural aggregate formation by the bacterium Pseudomonas aeruginosa. High-resolution, single-cell microscopy allowed us to characterize P. aeruginosa’s life history—seeding, aggregate formation, and dispersal—in this medium. Our results reveal that this bacterium readily forms aggregates that release migrants to colonize new areas. We also show that aggregates allow P. aeruginosa to tolerate therapeutic bacteriophage addition, although this treatment limits P. aeruginosa dissemination by targeting migrants.

ABSTRACT Environmental conditions affect bacterial behavior and can greatly influence the course of an infection. However, the environmental cues that elicit bacterial responses in specific infection sites are relatively unknown. Pseudomonas aeruginosa is ubiquitous in nature and typically innocuous. However, it is also one of the most prevalent causes of fatal sepsis in burn wound patients. The aim of this study was to determine the impact of environmental factors, specifically the availability of arginine, on the pathogenesis of P. aeruginosa in burn wound infections. Comparison of burned versus noninjured tissue revealed that l-arginine (l-Arg) was significantly depleted in burn wounds as a consequence of elevated arginase produced by myeloid-derived suppressor cells. We also observed that l-Arg was a potent chemoattractant for P. aeruginosa, and while low concentrations of l-Arg increased P. aeruginosa’s swimming motility, high concentrations resulted in diminished swimming. Based on these observations, we tested whether the administration of exogenous l-Arg into the burn wound could attenuate the virulence of P. aeruginosa in thermally injured mice. Administration of l-Arg resulted in decreased P. aeruginosa spread and sepsis and increased animal survival. Taken together, these data demonstrate that the availability of environmental arginine greatly influences the virulence of P. aeruginosa in vivo and may represent a promising phenotype-modulating tool for future therapeutic avenues. IMPORTANCE Despite our growing understanding of the pathophysiology of burn wounds and the evolution of techniques and practices to manage infections, sepsis remains a significant medical concern for burn patients. P. aeruginosa continues to be a leader among all causes of bacteremic infections due to its tendency to cause complications in immunocompromised patients and its ubiquitous presence in the hospital setting. With the unforgiving emergence of multidrug-resistant strains, it is critical that alternative strategies to control or prevent septic infections in burn patients be developed in parallel with novel antimicrobial agents. In this study, we observed that administration of l-Arg significantly reduced bacterial spread and sepsis in burned mice infected with P. aeruginosa. Given the safety of l-Arg in high doses and its potential wound-healing benefits, this conditionally essential amino acid may represent a useful tool to modulate bacterial behavior in vivo and prevent sepsis in burn patients.

ABSTRACT Bacteria rarely inhabit infection sites alone, instead residing in diverse, multispecies communities. Despite this fact, bacterial pathogenesis studies primarily focus on monoculture infections, overlooking how community interactions influence the course of disease. In this study, we used global mutant fitness profiling (transposon sequencing [Tn-seq]) to determine the genetic requirements for the pathogenic bacterium Aggregatibacter actinomycetemcomitans to cause disease when coinfecting with the commensal bacterium Streptococcus gordonii. Our results show that S. gordonii extensively alters A. actinomycetemcomitans requirements for virulence factors and biosynthetic pathways during infection. In addition, we discovered that the presence of S. gordonii enhances the bioavailability of oxygen during infection, allowing A. actinomycetemcomitans to shift from a primarily fermentative to a respiratory metabolism that enhances its growth yields and persistence. Mechanistically, respiratory metabolism enhances the fitness of A. actinomycetemcomitans in vivo by increasing ATP yields via central metabolism and creating a proton motive force. Our results reveal that, similar to cross-feeding, where one species provides another species with a nutrient, commensal bacteria can also provide electron acceptors that promote the respiratory growth and fitness of pathogens in vivo, an interaction that we term cross-respiration. IMPORTANCE Commensal bacteria can enhance the virulence of pathogens in mixed-species infections. However, knowledge of the mechanisms underlying this clinically relevant phenomenon is lacking. To bridge this gap, we comprehensively determined the genes a pathogen needs to establish coinfection with a commensal. Our findings show that the metabolism of the pathogen is low-energy-yielding in monoinfection, but in coinfection, the commensal improves the fitness of the pathogen by increasing the bioavailability of oxygen, thereby shifting the pathogen toward a high-energy-yielding metabolism. Similar to cross-feeding, this interaction, which we term cross-respiration, illustrates that commensal bacteria can provide electron acceptors that enhance the virulence of pathogens during infection.

ABSTRACT Antimicrobial-resistant bacteria pose a serious threat in the clinic. This is particularly true for opportunistic pathogens that possess high intrinsic resistance. Though many studies have focused on understanding the acquisition of bacterial resistance upon exposure to antimicrobials, the mechanisms controlling intrinsic resistance are not well understood. In this study, we subjected the model opportunistic superbug Pseudomonas aeruginosa to 14 antimicrobials under highly controlled conditions and assessed its response using expression- and fitness-based genomic approaches. Our results reveal that gene expression changes and mutant fitness in response to sub-MIC antimicrobials do not correlate on a genomewide scale, indicating that gene expression is not a good predictor of fitness determinants. In general, fewer fitness determinants were identified for antiseptics and disinfectants than for antibiotics. Analysis of gene expression and fitness data together allowed the prediction of antagonistic interactions between antimicrobials and insight into the molecular mechanisms controlling these interactions. IMPORTANCE Infections involving multidrug-resistant pathogens are difficult to treat because the therapeutic options are limited. These infections impose a significant financial burden on infected patients and on health care systems. Despite years of antimicrobial resistance research, we lack a comprehensive understanding of the intrinsic mechanisms controlling antimicrobial resistance. This work uses two fine-scale genomic approaches to identify genetic loci important for antimicrobial resistance of the opportunistic pathogen Pseudomonas aeruginosa. Our results reveal that antibiotics have more resistance determinants than antiseptics/disinfectants and that gene expression upon exposure to antimicrobials is not a good predictor of these resistance determinants. In addition, we show that when used together, genomewide gene expression and fitness profiling can provide mechanistic insights into multidrug resistance mechanisms.

ABSTRACT Cells within biofilms exhibit physiological heterogeneity, in part because of chemical gradients existing within these spatially structured communities. Previous work has examined how chemical gradients develop in large biofilms containing >108 cells. However, many bacterial communities in nature are composed of small, densely packed aggregates of cells (≤105 bacteria). Using a gelatin-based three-dimensional (3D) printing strategy, we confined the bacterium Pseudomonas aeruginosa within picoliter-sized 3D “microtraps” that are permeable to nutrients, waste products, and other bioactive small molecules. We show that as a single bacterium grows into a maximally dense (1012 cells ml−1) clonal population, a localized depletion of oxygen develops when it reaches a critical aggregate size of ~55 pl. Collectively, these data demonstrate that chemical and phenotypic heterogeneity exists on the micrometer scale within small aggregate populations. IMPORTANCE Before developing into large, complex communities, microbes initially cluster into aggregates, and it is unclear if chemical heterogeneity exists in these ubiquitous micrometer-scale aggregates. We chose to examine oxygen availability within an aggregate since oxygen concentration impacts a number of important bacterial processes, including metabolism, social behaviors, virulence, and antibiotic resistance. By determining that oxygen availability can vary within aggregates containing ≤105 bacteria, we establish that physiological heterogeneity exists within P. aeruginosa aggregates, suggesting that such heterogeneity frequently exists in many naturally occurring small populations.

ABSTRACT The human microbiome plays important roles in health, but when disrupted, these same indigenous microbes can cause disease. The composition of the microbiome changes during the transition from health to disease; however, these changes are often not conserved among patients. Since microbiome-associated diseases like periodontitis cause similar patient symptoms despite interpatient variability in microbial community composition, we hypothesized that human-associated microbial communities undergo conserved changes in metabolism during disease. Here, we used patient-matched healthy and diseased samples to compare gene expression of 160,000 genes in healthy and diseased periodontal communities. We show that health- and disease-associated communities exhibit defined differences in metabolism that are conserved between patients. In contrast, the metabolic gene expression of individual species was highly variable between patients. These results demonstrate that despite high interpatient variability in microbial composition, disease-associated communities display conserved metabolic profiles that are generally accomplished by a patient-specific cohort of microbes. IMPORTANCE The human microbiome project has shown that shifts in our microbiota are associated with many diseases, including obesity, Crohn’s disease, diabetes, and periodontitis. While changes in microbial populations are apparent during these diseases, the species associated with each disease can vary from patient to patient. Taking into account this interpatient variability, we hypothesized that specific microbiota-associated diseases would be marked by conserved microbial community behaviors. Here, we use gene expression analyses of patient-matched healthy and diseased human periodontal plaque to show that microbial communities have highly conserved metabolic gene expression profiles, whereas individual species within the community do not. Furthermore, disease-associated communities exhibit conserved changes in metabolic and virulence gene expression.

ABSTRACT Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection. IMPORTANCE Listeria monocytogenes is a Gram-positive intracellular pathogen and the causative agent of the food-borne illness listeriosis. Upon infection, L. monocytogenes stimulates expression of IFN-β and coregulated genes dependent upon host detection of a secreted bacterial signaling nucleotide, c-di-AMP. Using a forward genetic screen for mutants that induced high levels of host IFN-β expression, we identified a c-di-AMP phosphodiesterase, PdeA, that degrades c-di-AMP. Here we characterize L. monocytogenes mutants that express enhanced or diminished levels of c-di-AMP. Decreased c-di-AMP levels by conditional depletion of the diadenylate cyclase (DacA) or overexpression of PdeA attenuated bacterial growth and led to bacteriolysis, suggesting that its production is essential for viability and may regulate cell wall metabolism. Mutants lacking PdeA had a distinct transcriptional profile, which may provide insight into additional roles for the molecule. This work demonstrates that c-di-AMP is a critical signaling molecule required for bacterial replication, cell wall stability, and pathogenicity.

ABSTRACT Natural transformation by competent bacteria is a primary means of horizontal gene transfer; however, evidence that competence drives bacterial diversity and evolution has remained elusive. To test this theory, we used a retrospective comparative genomic approach to analyze the evolutionary history of Aggregatibacter actinomycetemcomitans, a bacterial species with both competent and noncompetent sister strains. Through comparative genomic analyses, we reveal that competence is evolutionarily linked to genomic diversity and speciation. Competence loss occurs frequently during evolution and is followed by the loss of clustered regularly interspaced short palindromic repeats (CRISPRs), bacterial adaptive immune systems that protect against parasitic DNA. Relative to noncompetent strains, competent bacteria have larger genomes containing multiple rearrangements. In contrast, noncompetent bacterial genomes are extremely stable but paradoxically susceptible to infective DNA elements, which contribute to noncompetent strain genetic diversity. Moreover, incomplete noncompetent strain CRISPR immune systems are enriched for self-targeting elements, which suggests that the CRISPRs have been co-opted for bacterial gene regulation, similar to eukaryotic microRNAs derived from the antiviral RNA interference pathway. IMPORTANCE The human microbiome is rich with thousands of diverse bacterial species. One mechanism driving this diversity is horizontal gene transfer by natural transformation, whereby naturally competent bacteria take up environmental DNA and incorporate new genes into their genomes. Competence is theorized to accelerate evolution; however, attempts to test this theory have proved difficult. Through genetic analyses of the human periodontal pathogen Aggregatibacter actinomycetemcomitans, we have discovered an evolutionary connection between competence systems promoting gene acquisition and CRISPRs (clustered regularly interspaced short palindromic repeats), adaptive immune systems that protect bacteria against genetic parasites. We show that competent A. actinomycetemcomitans strains have numerous redundant CRISPR immune systems, while noncompetent bacteria have lost their CRISPR immune systems because of inactivating mutations. Together, the evolutionary data linking the evolution of competence and CRISPRs reveals unique mechanisms promoting genetic heterogeneity and the rise of new bacterial species, providing insight into complex mechanisms underlying bacterial diversity in the human body.

ABSTRACT Gram-negative bacteria naturally produce outer membrane vesicles (OMVs) that arise through bulging and pinching off of the outer membrane. OMVs have several biological functions for bacteria, most notably as trafficking vehicles for toxins, antimicrobials, and signaling molecules. While their biological roles are now appreciated, the mechanism of OMV formation has not been fully elucidated. We recently demonstrated that the signaling molecule 2-heptyl-3-hydroxy-4-quinolone (PQS) is required for OMV biogenesis in P. aeruginosa. We hypothesized that PQS stimulates OMV formation through direct interaction with the outer leaflet of the outer membrane. To test this hypothesis, we employed a red blood cell (RBC) model that has been used extensively to study small-molecule–membrane interactions. Our results revealed that addition of PQS to RBCs induced membrane curvature, resulting in the formation of membrane spicules (spikes), consistent with small molecules that are inserted stably into the outer leaflet of the membrane. Radiotracer experiments demonstrated that sufficient PQS was inserted into the membrane to account for this curvature and that curvature induction was specific to PQS structure. These data suggest that a low rate of interleaflet flip-flop forces PQS to accumulate in and expand the outer leaflet relative to the inner leaflet, thus inducing membrane curvature. In support of PQS-mediated outer leaflet expansion, the PQS effect was antagonized by chlorpromazine, a molecule known to be preferentially inserted into the inner leaflet. Based on these data, we propose a bilayer-couple model to describe P. aeruginosa OMV biogenesis and suggest that this is a general mechanism for bacterial OMV formation. IMPORTANCE Despite the ubiquity and importance of outer membrane vesicle (OMV) production in Gram-negative bacteria, the molecular details of OMV biogenesis are not fully understood. Early experiments showed that 2-heptyl-3-hydroxy-4-quinolone (PQS) induces OMV formation through physical interaction with the membrane but did not elucidate the mechanism. The present study demonstrates that PQS specifically and reversibly promotes blebbing of model membranes dependent upon the same properties that are required for OMV formation in P. aeruginosa. These results are consistent with a mechanism where expansion of the outer leaflet relative to the inner leaflet induces localized membrane curvature. This “bilayer-couple” model can account for OMV formation under all conditions and is easily generalized to other Gram-negative bacteria. The model therefore raises the possibility of a universal paradigm for vesicle production in prokaryotes with features strikingly different from what is known in eukaryotes.

ABSTRACT Bacteria are social organisms that display distinct behaviors/phenotypes when present in groups. These behaviors include the abilities to construct antibiotic-resistant sessile biofilm communities and to communicate with small signaling molecules (quorum sensing [QS]). Our understanding of biofilms and QS arises primarily from in vitro studies of bacterial communities containing large numbers of cells, often greater than 108 bacteria; however, in nature, bacteria often reside in dense clusters (aggregates) consisting of significantly fewer cells. Indeed, bacterial clusters containing 101 to 105 cells are important for transmission of many bacterial pathogens. Here, we describe a versatile strategy for conducting mechanistic studies to interrogate the molecular processes controlling antibiotic resistance and QS-mediated virulence factor production in high-density bacterial clusters. This strategy involves enclosing a single bacterium within three-dimensional picoliter-scale microcavities (referred to as bacterial “lobster traps”) defined by walls that are permeable to nutrients, waste products, and other bioactive small molecules. Within these traps, bacteria divide normally into extremely dense (1012 cells/ml) clonal populations with final population sizes similar to that observed in naturally occurring bacterial clusters. Using these traps, we provide strong evidence that within low-cell-number/high-density bacterial clusters, QS is modulated not only by bacterial density but also by population size and flow rate of the surrounding medium. We also demonstrate that antibiotic resistance develops as cell density increases, with as few as ~150 confined bacteria exhibiting an antibiotic-resistant phenotype similar to biofilm bacteria. Together, these findings provide key insights into clinically relevant phenotypes in low-cell-number/high-density bacterial populations. IMPORTANCE Prokaryotes are social organisms capable of coordinated group behaviors, including the abilities to construct antibiotic-resistant biofilms and to communicate with small signaling molecules (quorum sensing [QS]). While there has been significant effort devoted to understanding biofilm formation and QS, few studies have examined these processes in high-density/low-cell-number populations. Such studies have clinical significance, as many infections are initiated by small bacterial populations (