Search

Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds (p-coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p-coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+p-coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).IMPORTANCEA newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

Oysters play an important role in coastal ecology and are a globally popular seafood source. However, their filter-feeding lifestyle enables coastal pathogens, toxins, and pollutants to accumulate in their tissues, potentially endangering human health. While pathogen concentrations in coastal waters are often linked to environmental conditions and runoff events, these do not always correlate with pathogen concentrations in oysters. Additional factors related to the microbial ecology of pathogenic bacteria and their relationship with oyster hosts likely play a role in accumulation but are poorly understood. In this study, we investigated whether microbial communities in water and oysters were linked to accumulation of Vibrio parahaemolyticus, Vibrio vulnificus, or fecal indicator bacteria. Site-specific environmental conditions significantly influenced microbial communities and potential pathogen concentrations in water. Oyster microbial communities, however, exhibited less variability in microbial community diversity and accumulation of target bacteria overall and were less impacted by environmental differences between sites. Instead, changes in specific microbial taxa in oyster and water samples, particularly in oyster digestive glands, were linked to elevated levels of potential pathogens. For example, increased levels of V. parahaemolyticus were associated with higher relative abundances of cyanobacteria, which could represent an environmental vector for Vibrio spp. transport, and with decreased relative abundance of Mycoplasma and other key members of the oyster digestive gland microbiota. These findings suggest that host and microbial factors, in addition to environmental variables, may influence pathogen accumulation in oysters. IMPORTANCE Bacteria in the marine environment cause thousands of human illnesses annually. Bivalves are a popular seafood source and are important in coastal ecology, but their ability to concentrate pathogens from the water can cause human illness, threatening seafood safety and security. To predict and prevent disease, it is critical to understand what causes pathogenic bacteria to accumulate in bivalves. In this study, we examined how environmental factors and host and water microbial communities were linked to potential human pathogen accumulation in oysters. Oyster microbial communities were more stable than water communities, and both contained the highest concentrations of Vibrio parahaemolyticus at sites with warmer temperatures and lower salinities. High oyster V. parahaemolyticus concentrations corresponded with abundant cyanobacteria, a potential vector for transmission, and a decrease in potentially beneficial oyster microbes. Our study suggests that poorly understood factors, including host and water microbiota, likely play a role in pathogen distribution and pathogen transmission.

Transcription factors (TFs) bind to the promoters of target genes to regulate gene expression in response to different stimuli. The functions and regulatory mechanisms of transcription factors (TFs) in Verticillium dahliae are, however, still largely unclear. This study showed that a C2H2-type zinc finger TF, VdCf2 (V. dahliae chorion transcription factor 2), plays key roles in V. dahliae growth, melanin production, and virulence. Transcriptome sequencing analysis showed that VdCf2 was involved in the regulation of expression of genes encoding secreted proteins, pathogen-host interaction (PHI) homologs, TFs, and G protein-coupled receptors (GPCRs). Furthermore, VdCf2 positively regulated the expression of VdPevD1 (VDAG_02735), a previously reported virulence factor. VdCf2 thus regulates the expression of several pathogenicity-related genes that also contribute to virulence in V. dahliae. VdCf2 also inhibited the transcription of the Vd276-280 gene cluster and interacted with two members encoding proteins (VDAG_07276 and VDAG_07278) in the gene cluster. IMPORTANCE Verticillium dahliae is an important soilborne phytopathogen which can ruinously attack numerous host plants and cause significant economic losses. Transcription factors (TFs) were reported to be involved in various biological processes, such as hyphal growth and virulence of pathogenic fungi. However, the functions and regulatory mechanisms of TFs in V. dahliae remain largely unclear. In this study, we identified a new transcription factor, VdCf2 (V. dahliae chorion transcription factor 2), based on previous transcriptome data, which participates in growth, melanin production, and virulence of V. dahliae. We provide evidence that VdCf2 regulates the expression of the pathogenicity-related gene VdPevD1 (VDAG_02735) and Vd276-280 gene cluster. VdCf2 also interacts with VDAG_07276 and VDAG_07278 in this gene cluster based on a yeast two-hybrid and bimolecular fluorescence complementation assay. These results revealed the regulatory mechanisms of a pivotal pathogenicity-related transcription factor, VdCf2 in V. dahliae.

Xylella fastidiosa is an insect-transmitted bacterial plant pathogen found across the Americas and, more recently, worldwide. X. fastidiosa infects plants of at least 563 species belonging to 82 botanical families. While the species X. fastidiosa infects many plants, particular strains have increased plant specificity. Understanding the molecular underpinnings of plant host specificity in X. fastidiosa is vital for predicting host shifts and epidemics. While there may exist multiple genetic determinants of host range in X. fastidiosa, the drivers of the unique relationships between X. fastidiosa and its hosts should be elucidated. Our objective with this study was to predict the ancestral plant hosts of this pathogen using phylogenetic and genomic methods based on a large data set of pathogen whole-genome data from agricultural hosts. We used genomic data to construct maximum-likelihood (ML) phylogenetic trees of subsets of the core and pan-genomes. With those trees, we ran ML ancestral state reconstructions of plant host at two taxonomic scales (genus and multiorder clades). Both the core and pan-genomes were informative in terms of predicting ancestral host state, giving new insight into the history of the plant hosts of X. fastidiosa. Subsequently, gene gain and loss in the pan-genome were found to be significantly correlated with plant host through genes that had statistically significant associations with particular hosts. IMPORTANCE Xylella fastidiosa is a globally important bacterial plant pathogen with many hosts; however, the underpinnings of host specificity are not known. This paper contains important findings about the usage of phylogenetics to understand the history of host specificity in this bacterial species, as well as convergent evolution in the pan-genome. There are strong signals of historical host range that give us insights into the history of this pathogen and its various invasions. The data from this paper are relevant in making decisions for quarantine and eradication, as they show the historical trends of host switching, which can help us predict likely future host shifts. We also demonstrate that using multilocus sequence type (MLST) genes in this system, which is still a commonly used process for policymaking, does not reconstruct the same phylogenetic topology as whole-genome data.

Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop-out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers. IMPORTANCE Understanding the genetic basis of P. putida's diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen. This study provides functional evidence linking hundreds of genes to their roles in the metabolism of nitrogenous compounds, and provides an interactive tool for visualizing these data. We further characterize several aminotransferases, lactamases, and regulators, which are of particular interest for metabolic engineering.

Enterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Commensal strains of E. faecium can confer beneficial effects on host physiology and immunity, but antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient gene-editing methods. To address these limitations, here, we report that the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Notably, the expression of RecT in combination with clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scarless single-stranded DNA recombineering to generate specific gene-editing mutants in E. faecium. Moreover, we demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic-selectable markers to generate gene deletion mutants. As a further proof of principle, we use CRISPR-Cas9-mediated recombineering to knock out both sortase A genes in E. faecium for downstream functional characterization. The general RecT-mediated recombineering methods described here should significantly enhance genetic studies of E. faecium and other closely related species for functional and mechanistic studies. IMPORTANCE Enterococcus faecium is widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Nevertheless, commensal Enterococcus strains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. This functional dichotomy of Enterococcus species and strains highlights the need for in-depth studies to discover and characterize the genetic components underlying its diverse activities. However, current genetic engineering methods in E. faecium still require passive homologous recombination from plasmid DNA. This involves the successful cloning of multiple homologous fragments into a plasmid, introducing the plasmid into E. faecium, and screening for double-crossover events that can collectively take up to multiple weeks to perform. To alleviate these challenges, we show that RecT recombinase enables the rapid and efficient integration of mutagenic DNA templates to generate substitutions, deletions, and insertions in the genomic DNA of E. faecium. These improved recombineering methods should facilitate functional and mechanistic studies of Enterococcus.

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed gene expression levels of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression was observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi.IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that allow fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species—aspen, pine, and spruce—under various culture conditions. We found that F. pinicola is able to modify gene expression (transcription levels) across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This study provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates.

Vibrio parahaemolyticus is the most common cause of seafood-borne illness reported in the United States. The draft genomes of 132 North American clinical and oyster V. parahaemolyticus isolates were sequenced to investigate their phylogenetic and biogeographic relationships. The majority of oyster isolate sequence types (STs) were from a single harvest location; however, four were identified from multiple locations. There was population structure along the Gulf and Atlantic Coasts of North America, with what seemed to be a hub of genetic variability along the Gulf Coast, with some of the same STs occurring along the Atlantic Coast and one shared between the coastal waters of the Gulf and those of Washington State. Phylogenetic analyses found nine well-supported clades. Two clades were composed of isolates from both clinical and oyster sources. Four were composed of isolates entirely from clinical sources, and three were entirely from oyster sources. Each single-source clade consisted of one ST. Some human isolates lack tdh, trh, and some type III secretion system (T3SS) genes, which are established virulence genes of V. parahaemolyticus Thus, these genes are not essential for pathogenicity. However, isolates in the monophyletic groups from clinical sources were enriched in several categories of genes compared to those from monophyletic groups of oyster isolates. These functional categories include cell signaling, transport, and metabolism. The identification of genes in these functional categories provides a basis for future in-depth pathogenicity investigations of V. parahaemolyticusIMPORTANCEVibrio parahaemolyticus is the most common cause of seafood-borne illness reported in the United States and is frequently associated with shellfish consumption. This study contributes to our knowledge of the biogeography and functional genomics of this species around North America. STs shared between the Gulf Coast and the Atlantic seaboard as well as Pacific waters suggest possible transport via oceanic currents or large shipping vessels. STs frequently isolated from humans but rarely, if ever, isolated from the environment are likely more competitive in the human gut than other STs. This could be due to additional functional capabilities in areas such as cell signaling, transport, and metabolism, which may give these isolates an advantage in novel nutrient-replete environments such as the human gut.

With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB-Tn-Seq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA). Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yields.IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired.

Microbial interactions abound in natural ecosystems and shape community structure and function. Substantial attention has been given to cataloging mechanisms by which microbes interact, but there is a limited understanding of the genetic landscapes that promote or hinder microbial interactions. We previously developed a mutualistic coculture pairing Escherichia coli and Rhodopseudomonas palustris, wherein E. coli provides carbon to R. palustris in the form of glucose fermentation products and R. palustris fixes N2 gas and provides nitrogen to E. coli in the form of NH4+ The stable coexistence and reproducible trends exhibited by this coculture make it ideal for interrogating the genetic underpinnings of a cross-feeding mutualism. Here, we used random barcode transposon sequencing (RB-TnSeq) to conduct a genome-wide search for E. coli genes that influence fitness during cooperative growth with R. palustris RB-TnSeq revealed hundreds of genes that increased or decreased E. coli fitness in a mutualism-dependent manner. Some identified genes were involved in nitrogen sensing and assimilation, as expected given the coculture design. The other identified genes were involved in diverse cellular processes, including energy production and cell wall and membrane biogenesis. In addition, we discovered unexpected purine cross-feeding from R. palustris to E. coli, with coculture rescuing growth of an E. coli purine auxotroph. Our data provide insight into the genes and gene networks that can influence a cross-feeding mutualism and underscore that microbial interactions are not necessarily predictable a prioriIMPORTANCE Microbial communities impact life on Earth in profound ways, including driving global nutrient cycles and influencing human health and disease. These community functions depend on the interactions that resident microbes have with the environment and each other. Thus, identifying genes that influence these interactions will aid the management of natural communities and the use of microbial consortia as biotechnology. Here, we identified genes that influenced Escherichia coli fitness during cooperative growth with a mutualistic partner, Rhodopseudomonas palustris Although this mutualism centers on the bidirectional exchange of essential carbon and nitrogen, E. coli fitness was positively and negatively affected by genes involved in diverse cellular processes. Furthermore, we discovered an unexpected purine cross-feeding interaction. These results contribute knowledge on the genetic foundation of a microbial cross-feeding interaction and highlight that unanticipated interactions can occur even within engineered microbial communities.

Obligate aerobic organisms rely on a functional electron transport chain for energy conservation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. We here investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the single or double knockout mutant strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants, including quantitative physiological experiments, transcript analysis, proteomics, and enzyme activity assays revealed distinct responses to type 2 and type 1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely by rerouting of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart metabolic engineering efforts.IMPORTANCE While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. This high degree of metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse toward a host with a controlled and efficient supply of redox cofactors for product synthesis.

Microorganisms that release plant-available phosphate from natural soil phosphate stores may serve as biological alternatives to costly and environmentally damaging phosphate fertilizers. To explore this possibility, we engineered a collection of root bacteria to release plant-available orthophosphate from phytate, an abundant phosphate source in many soils. We identified 82 phylogenetically diverse phytase genes, refactored their sequences for optimal expression in Proteobacteria, and then synthesized and engineered them into the genomes of three root-colonizing bacteria. Liquid culture assays revealed 41 engineered strains with high levels of phytate hydrolysis. Among these, we identified 12 strains across three bacterial hosts that confer a growth advantage on the model plant Arabidopsis thaliana when phytate is the sole phosphate source. These data demonstrate that DNA synthesis approaches can be used to generate plant-associated strains with novel phosphate-solubilizing capabilities.IMPORTANCE Phosphate fertilizers are essential for high-yield agriculture yet are costly and environmentally damaging. Microbes that release soluble phosphate from naturally occurring sources in the soil are appealing, as they may reduce the need for such fertilizers. In this study, we used synthetic biology approaches to create a collection of engineered root-associated microbes with the ability to release phosphate from phytate. We demonstrate that these strains improve plant growth under phosphorus-limited conditions. This represents a first step in the development of phosphate-mining bacteria for future use in crop systems.

Xylella fastidiosa is an economically important bacterial plant pathogen. With insights gained from 72 genomes, this study investigated differences among the three main subspecies, which have allopatric origins: X. fastidiosa subsp. fastidiosa, multiplex, and pauca The origin of recombinogenic X. fastidiosa subsp. morus and sandyi was also assessed. The evolutionary rate of the 622 genes of the species core genome was estimated at the scale of an X. fastidiosa subsp. pauca subclade (7.62 × 10-7 substitutions per site per year), which was subsequently used to estimate divergence time for the subspecies and introduction events. The study characterized genes present in the accessory genome of each of the three subspecies and investigated the core genome to detect genes potentially under positive selection. Recombination is recognized to be the major driver of diversity in X. fastidiosa, potentially facilitating shifts to novel plant hosts. The relative effect of recombination in comparison to point mutation was calculated (r/m = 2.259). Evidence of recombination was uncovered in the core genome alignment; X. fastidiosa subsp. fastidiosa in the United States was less prone to recombination, with an average of 3.22 of the 622 core genes identified as recombining regions, whereas a specific clade of X. fastidiosa subsp. multiplex was found to have on average 9.60 recombining genes, 93.2% of which originated from X. fastidiosa subsp. fastidiosa Interestingly, for X. fastidiosa subsp. morus, which was initially thought to be the outcome of genome-wide recombination between X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex, intersubspecies homologous recombination levels reached 15.30% in the core genome. Finally, there is evidence of X. fastidiosa subsp. pauca strains from citrus containing genetic elements acquired from strains infecting coffee plants as well as genetic elements from both X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex In summary, our data provide new insights into the evolution and epidemiology of this plant pathogen.IMPORTANCEXylella fastidiosa is an important vector-borne plant pathogen. We used a set of 72 genomes that constitutes the largest assembled data set for this bacterial species so far to investigate genetic relationships and the impact of recombination on phylogenetic clades and to compare genome content at the subspecies level, and we used a molecular dating approach to infer the evolutionary rate of X. fastidiosa The results demonstrate that recombination is important in shaping the genomes of X. fastidiosa and that each of the main subspecies is under different selective pressures. We hope insights from this study will improve our understanding of X. fastidiosa evolution and biology.

Recent experimental and bioinformatic advances enable the recovery of genomes belonging to yet-uncultured microbial lineages directly from environmental samples. Here, we report on the recovery and characterization of single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) representing candidate phylum LCP-89, previously defined based on 16S rRNA gene sequences. Analysis of LCP-89 genomes recovered from Zodletone Spring, an anoxic spring in Oklahoma, predicts slow-growing, rod-shaped organisms. LCP-89 genomes contain genes for cell wall lipopolysaccharide (LPS) production but lack the entire machinery for peptidoglycan biosynthesis, suggesting an atypical cell wall structure. The genomes, however, encode S-layer homology domain-containing proteins, as well as machinery for the biosynthesis of CMP-legionaminate, inferring the possession of an S-layer glycoprotein. A nearly complete chemotaxis machinery coupled to the absence of flagellar synthesis and assembly genes argues for the utilization of alternative types of motility. A strict anaerobic lifestyle is predicted, with dual respiratory (nitrite ammonification) and fermentative capacities. Predicted substrates include a wide range of sugars and sugar alcohols and a few amino acids. The capability of rhamnose metabolism is confirmed by the identification of bacterial microcompartment genes to sequester the toxic intermediates generated. Comparative genomic analysis identified differences in oxygen sensitivities, respiratory capabilities, substrate utilization preferences, and fermentation end products between LCP-89 genomes and those belonging to its four sister phyla (Calditrichota, SM32-31, AABM5-125-24, and KSB1) within the broader FCB (Fibrobacteres-Chlorobi-Bacteroidetes) superphylum. Our results provide a detailed characterization of members of the candidate division LCP-89 and highlight the importance of reconciling 16S rRNA-based and genome-based phylogenies.IMPORTANCE Our understanding of the metabolic capacities, physiological preferences, and ecological roles of yet-uncultured microbial phyla is expanding rapidly. Two distinct approaches are currently being utilized for characterizing microbial communities in nature: amplicon-based 16S rRNA gene surveys for community characterization and metagenomics/single-cell genomics for detailed metabolic reconstruction. The occurrence of multiple yet-uncultured bacterial phyla has been documented using 16S rRNA surveys, and obtaining genome representatives of these yet-uncultured lineages is critical to our understanding of the role of yet-uncultured organisms in nature. This study provides a genomics-based analysis highlighting the structural features and metabolic capacities of a yet-uncultured bacterial phylum (LCP-89) previously identified in 16S rRNA surveys for which no prior genomes have been described. Our analysis identifies several interesting structural features for members of this phylum, e.g., lack of peptidoglycan biosynthetic machinery and the ability to form bacterial microcompartments. Predicted metabolic capabilities include degradation of a wide range of sugars, anaerobic respiratory capacity, and fermentative capacities. In addition to the detailed structural and metabolic analysis provided for candidate division LCP-89, this effort represents an additional step toward a unified scheme for microbial taxonomy by reconciling 16S rRNA gene-based and genomics-based taxonomic outlines.

Many strains of the spirochete Leptospira interrogans serovar Pomona express the osmotically inducible sphingomyelinase gene sph2 at much higher levels than strains from other serovars. We developed a new green fluorescent protein (GFP) reporter plasmid to examine sph2 gene expression determinants. The vector enables the fusion of the test promoter to the ribosome-binding site and coding region of gfp We fused the sph2 promoters from the L. interrogans serovar Lai strain 56601 and from the L. interrogans serovar Pomona strain LC82-25 to gfp to examine the molecular determinants of differential sph2 expression between the two strains. Similar to what was observed with the native sph2 genes, the introduction of the plasmids into the Lai 56601 strain resulted in near background levels of gfp expression from the Lai sph2 promoter, while the expression from the Pomona sph2 promoter was high. The expression of both fusions increased at physiologic levels of osmolarity achieved by adding sodium chloride to the culture medium. We examined the role of a 17-bp upstream element found in all L. interrogans strains expressing low basal levels of sph2 and missing from Pomona strains that express sph2 at high levels. When the 17-bp sequence present upstream of the Lai sph2 promoter was deleted or scrambled, the fusion expression increased substantially. Conversely, the insertion of the 17-bp sequence upstream of the Pomona sph2 promoter diminished fusion expression. In contrast, the removal of an insertion sequence-like element that is found only in the Pomona sph2 upstream sequence had no effect on the expression from the Pomona sph2 fusion in the Lai strain. These findings demonstrate the utility of the gfp reporter plasmid in analyzing gene expression in L. interrogansIMPORTANCE Genetic tools are needed to examine gene expression in the pathogen Leptospira interrogans We developed a reporter plasmid that replicates in L. interrogans with green fluorescent protein (GFP) as the readout of promoter activity. We demonstrated an application of the new reporter plasmid by identifying an upstream element responsible for the poor basal expression of the sph2 sphingomyelinase gene in an L. interrogans serovar Lai strain. This new tool is useful for the discovery of the molecular determinants of L. interrogans gene expression.

Magnetosomes are complex bacterial organelles that serve as model systems for studying bacterial cell biology, biomineralization, and global iron cycling. Magnetosome biogenesis is primarily studied in two closely related Alphaproteobacteria of the genus Magnetospirillum that form cubooctahedral-shaped magnetite crystals within a lipid membrane. However, chemically and structurally distinct magnetic particles have been found in physiologically and phylogenetically diverse bacteria. Due to a lack of molecular genetic tools, the mechanistic diversity of magnetosome formation remains poorly understood. Desulfovibrio magneticus RS-1 is an anaerobic sulfate-reducing deltaproteobacterium that forms bullet-shaped magnetite crystals. A recent forward genetic screen identified 10 genes in the conserved magnetosome gene island of D. magneticus that are essential for its magnetic phenotype. However, this screen likely missed mutants with defects in crystal size, shape, and arrangement. Reverse genetics to target the remaining putative magnetosome genes using standard genetic methods of suicide vector integration have not been feasible due to the low transconjugation efficiency. Here, we present a reverse genetic method for targeted mutagenesis in D. magneticus using a replicative plasmid. To test this method, we generated a mutant resistant to 5-fluorouracil by making a markerless deletion of the upp gene that encodes uracil phosphoribosyltransferase. We also used this method for targeted marker exchange mutagenesis by replacing kupM, a gene identified in our previous screen as a magnetosome formation factor, with a streptomycin resistance cassette. Overall, our results show that targeted mutagenesis using a replicative plasmid is effective in D. magneticus and may also be applied to other genetically recalcitrant bacteria.IMPORTANCE Magnetotactic bacteria (MTB) are a group of organisms that form intracellular nanometer-scale magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membranes, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization and are potential platforms for biotechnological applications. Due to a lack of genetic tools and unculturable representatives, the mechanisms of magnetosome formation in phylogenetically deeply branching MTB remain unknown. These MTB contain elongated bullet-/tooth-shaped magnetite and greigite crystals that likely form in a manner distinct from that of the cubooctahedral-shaped magnetite crystals of the genetically tractable MTB within the Alphaproteobacteria Here, we present a method for genome editing in Desulfovibrio magneticus RS-1, a cultured representative of the deeply branching MTB of the class Deltaproteobacteria This marks a crucial step in developing D. magneticus as a model for studying diverse mechanisms of magnetic particle formation by MTB.

Historically, piscine francisellosis in various warm-, temperate-, and cold-water fish hosts has been attributed to Francisella noatunensis From 2015 to 2016, an undescribed Francisella sp. was recovered during mortality events in cultured spotted rose snapper (Lutjanus guttatus) off the Pacific coast of Central America. Despite high mortality and emaciation, limited gross findings were observed in affected fish. Histological examination revealed multifocal granulomatous lesions, with the presence of numerous small, pleomorphic coccobacilli, predominantly in the peritoneum, spleen, kidneys, liver, pancreas, heart, and intestine. Sequencing of an ∼1,400-bp fragment of the 16S rRNA gene demonstrated these isolates to be most similar (99.9% identity) to Francisella sp. isolate TX077308 cultured from seawater in the Gulf of Mexico, while sharing

Leptothrix ochracea is known for producing large volumes of iron oxyhydroxide sheaths that alter wetland biogeochemistry. For over a century, these delicate structures have fascinated microbiologists and geoscientists. Because L. ochracea still resists long-term in vitro culture, the debate regarding its metabolic classification dates back to 1885. We developed a novel culturing technique for L. ochracea using in situ natural waters and coupled this with single-cell genomics and nanoscale secondary-ion mass spectrophotometry (nanoSIMS) to probe L. ochracea's physiology. In microslide cultures L. ochracea doubled every 5.7 h and had an absolute growth requirement for ferrous iron, the genomic capacity for iron oxidation, and a branched electron transport chain with cytochromes putatively involved in lithotrophic iron oxidation. Additionally, its genome encoded several electron transport chain proteins, including a molybdopterin alternative complex III (ACIII), a cytochrome bd oxidase reductase, and several terminal oxidase genes. L. ochracea contained two key autotrophic proteins in the Calvin-Benson-Bassham cycle, a form II ribulose bisphosphate carboxylase, and a phosphoribulose kinase. L. ochracea also assimilated bicarbonate, although calculations suggest that bicarbonate assimilation is a small fraction of its total carbon assimilation. Finally, L. ochracea's fundamental physiology is a hybrid of those of the chemolithotrophic Gallionella-type iron-oxidizing bacteria and the sheathed, heterotrophic filamentous metal-oxidizing bacteria of the Leptothrix-Sphaerotilus genera. This allows L. ochracea to inhabit a unique niche within the neutrophilic iron seeps.IMPORTANCELeptothrix ochracea was one of three groups of organisms that Sergei Winogradsky used in the 1880s to develop his hypothesis on chemolithotrophy. L. ochracea continues to resist cultivation and appears to have an absolute requirement for organic-rich waters, suggesting that its true physiology remains unknown. Further, L. ochracea is an ecological engineer; a few L. ochracea cells can generate prodigious volumes of iron oxyhydroxides, changing the ecosystem's geochemistry and ecology. Therefore, to determine L. ochracea's basic physiology, we employed new single-cell techniques to demonstrate that L. ochracea oxidizes iron to generate energy and, despite having predicted genes for autotrophic growth, assimilates a fraction of the total CO2 that autotrophs do. Although not a true chemolithoautotroph, L. ochracea's physiological strategy allows it to be flexible and to extensively colonize iron-rich wetlands.

Escherichia coli serotype O157:H7 is a zoonotic food- and waterborne bacterial pathogen that causes a high hospitalization rate and can cause life-threatening complications. Increasingly, E. coli O157:H7 infections appear to originate from fresh produce. Ruminants, such as cattle, are a prominent reservoir of E. coli O157:H7 in the United States. California is one of the most agriculturally productive regions in the world for fresh produce, beef, and milk. The close proximity of fresh produce and cattle presents food safety challenges on a uniquely large scale. We performed a survey of E. coli O157:H7 on 20 farms in California to observe the regional diversity and prevalence of E. coli O157:H7. Isolates were obtained from enrichment cultures of cow feces. Some farms were sampled on two dates. Genomes from isolates were sequenced to determine their relatedness and pathogenic potential. E. coli O157:H7 was isolated from approximately half of the farms. The point prevalence of E. coli O157:H7 on farms was highly variable, ranging from zero to nearly 90%. Within farms, generally one or a few lineages were found, even when the rate of isolation was high. On farms with high isolation rates, a single clonal lineage accounted for most of the isolates. Farms that were visited months after the first visit might have had the same lineages of E. coli O157:H7. Strains of E. coli O157:H7 may be persistent for months on farms.IMPORTANCE This survey of 20 cow-calf operations from different regions of California provides an in depth look at resident Escherichia coli O157:H7 populations at the molecular level. E. coli O157:H7 is found to have a highly variable prevalence, and with whole-genome sequencing, high prevalences in herds were found to be due to a single lineage shed from multiple cows. Few repeat lineages were found between farms in this area; therefore, we predict that E. coli O157:H7 has significant diversity in this area beyond what is detected in this survey. All isolates from this study were found to have pathogenic potential based on the presence of key virulence gene sequences. This represents a novel insight into pathogen diversity within a single subtype and will inform future attempts to survey regional pathogen populations.

Multidrug-resistant (MDR) Salmonella enterica can be spread from cattle to humans through direct contact with animals shedding Salmonella as well as through the food chain, making MDR Salmonella a serious threat to human health. The objective of this study was to use whole-genome sequencing to compare antimicrobial-resistant (AMR) Salmonella enterica serovars Typhimurium, Newport, and Dublin isolated from dairy cattle and humans in Washington State and New York State at the genotypic and phenotypic levels. A total of 90 isolates were selected for the study (37 S Typhimurium, 32 S Newport, and 21 S Dublin isolates). All isolates were tested for phenotypic antibiotic resistance to 12 drugs using Kirby-Bauer disk diffusion. AMR genes were detected in the assembled genome of each isolate using nucleotide BLAST and ARG-ANNOT. Genotypic prediction of phenotypic resistance resulted in a mean sensitivity of 97.2 and specificity of 85.2. Sulfamethoxazole-trimethoprim resistance was observed only in human isolates (P < 0.05), while resistance to quinolones and fluoroquinolones was observed only in 6 S Typhimurium isolates from humans in Washington State. S Newport isolates showed a high degree of AMR profile similarity, regardless of source. S Dublin isolates from New York State differed from those from Washington State based on the presence/absence of plasmid replicons, as well as phenotypic AMR susceptibility/nonsusceptibility (P < 0.05). The results of this study suggest that distinct factors may contribute to the emergence and dispersal of AMR S. enterica in humans and farm animals in different regions.IMPORTANCE The use of antibiotics in food-producing animals has been hypothesized to select for AMR Salmonella enterica and associated AMR determinants, which can be transferred to humans through different routes. Previous studies have sought to assess the degree to which AMR livestock- and human-associated Salmonella strains overlap, as well as the spatial distribution of Salmonella's associated AMR determinants, but have often been limited by the degree of resolution at which isolates can be compared. Here, a comparative genomics study of livestock- and human-associated Salmonella strains from different regions of the United States shows that while many AMR genes and phenotypes were confined to human isolates, overlaps between the resistomes of bovine and human-associated Salmonella isolates were observed on numerous occasions, particularly for S Newport. We have also shown that whole-genome sequencing can be used to reliably predict phenotypic resistance across Salmonella isolated from bovine sources.

The aim of this study is to obtain a systems-level understanding of the interactions between Dehalococcoides and corrinoid-supplying microorganisms by analyzing community structures and functional compositions, activities, and dynamics in trichloroethene (TCE)-dechlorinating enrichments. Metagenomes and metatranscriptomes of the dechlorinating enrichments with and without exogenous cobalamin were compared. Seven putative draft genomes were binned from the metagenomes. At an early stage (2 days), more transcripts of genes in the Veillonellaceae bin-genome were detected in the metatranscriptome of the enrichment without exogenous cobalamin than in the one with the addition of cobalamin. Among these genes, sporulation-related genes exhibited the highest differential expression when cobalamin was not added, suggesting a possible release route of corrinoids from corrinoid producers. Other differentially expressed genes include those involved in energy conservation and nutrient transport (including cobalt transport). The most highly expressed corrinoid de novo biosynthesis pathway was also assigned to the Veillonellaceae bin-genome. Targeted quantitative PCR (qPCR) analyses confirmed higher transcript abundances of those corrinoid biosynthesis genes in the enrichment without exogenous cobalamin than in the enrichment with cobalamin. Furthermore, the corrinoid salvaging and modification pathway of Dehalococcoides was upregulated in response to the cobalamin stress. This study provides important insights into the microbial interactions and roles played by members of dechlorinating communities under cobalamin-limited conditions.IMPORTANCE The key chloroethene-dechlorinating bacterium Dehalococcoides mccartyi is a cobalamin auxotroph, thus acquiring corrinoids from other community members. Therefore, it is important to investigate the microbe-microbe interactions between Dehalococcoides and the corrinoid-providing microorganisms in a community. This study provides systems-level information, i.e., taxonomic and functional compositions and dynamics of the supportive microorganisms in dechlorinating communities under different cobalamin conditions. The findings shed light on the important roles of Veillonellaceae species in the communities compared to other coexisting community members in producing and providing corrinoids for Dehalococcoides species under cobalamin-limited conditions.

This study developed RNA-based predictive models describing the survival of Vibrio parahaemolyticus in Eastern oysters (Crassostrea virginica) during storage at 0, 4, and 10°C. Postharvested oysters were inoculated with a cocktail of five V. parahaemolyticus strains and were then stored at 0, 4, and 10°C for 21 or 11 days. A real-time reverse transcription-PCR (RT-PCR) assay targeting expression of the tlh gene was used to evaluate the number of surviving V. parahaemolyticus cells, which was then used to establish primary molecular models (MMs). Before construction of the MMs, consistent expression levels of the tlh gene at 0, 4, and 10°C were confirmed, and this gene was used to monitor the survival of the total V. parahaemolyticus cells. In addition, the tdh and trh genes were used for monitoring the survival of virulent V. parahaemolyticus Traditional models (TMs) were built based on data collected using a plate counting method. From the MMs, V. parahaemolyticus populations had decreased 0.493, 0.362, and 0.238 log10 CFU/g by the end of storage at 0, 4, and 10°C, respectively. Rates of reduction of V. parahaemolyticus shown in the TMs were 2.109, 1.579, and 0.894 log10 CFU/g for storage at 0, 4, and 10°C, respectively. Bacterial inactivation rates (IRs) estimated with the TMs (-0.245, -0.152, and -0.121 log10 CFU/day, respectively) were higher than those estimated with the MMs (-0.134, -0.0887, and -0.0732 log10 CFU/day, respectively) for storage at 0, 4, and 10°C. Higher viable V. parahaemolyticus numbers were predicted using the MMs than using the TMs. On the basis of this study, RNA-based predictive MMs are the more accurate and reliable models and can prevent false-negative results compared to TMs.IMPORTANCE One important method for validating postharvest techniques and for monitoring the behavior of V. parahaemolyticus is to establish predictive models. Unfortunately, previous predictive models established based on plate counting methods or on DNA-based PCR can underestimate or overestimate the number of surviving cells. This study developed and validated RNA-based molecular predictive models to describe the survival of V. parahaemolyticus in oysters during low-temperature storage (0, 4, and 10°C). The RNA-based predictive models show the advantage of being able to count all of the culturable, nonculturable, and stressed cells. By using primers targeting the tlh gene and pathogenesis-associated genes (tdh and trh), real-time RT-PCR can evaluate the total surviving V. parahaemolyticus population as well as differentiate the pathogenic ones from the total population. Reliable and accurate predictive models are very important for conducting risk assessment and management of pathogens in food.

Campylobacter is the leading cause of human gastroenteritis worldwide. Wild birds, including American crows, are abundant in urban, suburban, and agricultural settings and are likely zoonotic vectors of Campylobacter Their proximity to humans and livestock increases the potential spreading of Campylobacter via crows between the environment, livestock, and humans. However, no studies have definitively demonstrated that crows are a vector for pathogenic Campylobacter We used genomics to evaluate the zoonotic and pathogenic potential of Campylobacter from crows to other animals with 184 isolates obtained from crows, chickens, cows, sheep, goats, humans, and nonhuman primates. Whole-genome analysis uncovered two distinct clades of Campylobacter jejuni genotypes; the first contained genotypes found only in crows, while a second genotype contained "generalist" genomes that were isolated from multiple host species, including isolates implicated in human disease, primate gastroenteritis, and livestock abortion. Two major β-lactamase genes were observed frequently in these genomes (oxa-184, 55%, and oxa-61, 29%), where oxa-184 was associated only with crows and oxa-61 was associated with generalists. Mutations in gyrA, indicative of fluoroquinolone resistance, were observed in 14% of the isolates. Tetracycline resistance (tetO) was present in 22% of the isolates, yet it occurred in 91% of the abortion isolates. Virulence genes were distributed throughout the genomes; however, cdtC alleles recapitulated the crow-only and generalist clades. A specific cdtC allele was associated with abortion in livestock and was concomitant with tetO These findings indicate that crows harboring a generalist C. jejuni genotype may act as a vector for the zoonotic transmission of Campylobacter IMPORTANCE: This study examined the link between public health and the genomic variation of Campylobacter in relation to disease in humans, primates, and livestock. Use of large-scale whole-genome sequencing enabled population-level assessment to find new genes that are linked to livestock disease. With 184 Campylobacter genomes, we assessed virulence traits, antibiotic resistance susceptibility, and the potential for zoonotic transfer to observe that there is a "generalist" genotype that may move between host species.

UnlabelledHuman norovirus (HuNoV) is a leading cause of foodborne diseases worldwide. High-pressure processing (HPP) is one of the most promising nonthermal technologies for the decontamination of viral pathogens in foods. However, the survival of HuNoVs after HPP is poorly understood because these viruses cannot be propagated in vitro In this study, we estimated the survival of different HuNoV strains within genogroup II (GII) after HPP treatment using viral receptor-binding ability as an indicator. Four HuNoV strains (one GII genotype 1 [GII.1] strain, two GII.4 strains, and one GII.6 strain) were treated at high pressures ranging from 200 to 600 MPa. After treatment, the intact viral particles were captured by porcine gastric mucin-conjugated magnetic beads (PGM-MBs) that contained histo-blood group antigens, the functional receptors for HuNoVs. The genomic RNA copies of the captured HuNoVs were quantified by real-time reverse transcriptase PCR (RT-PCR). Two GII.4 HuNoVs had similar sensitivities to HPP. The resistance of HuNoV strains against HPP ranked as follows: GII.1 > GII.6 > GII.4, with GII.4 being the most sensitive. Evaluation of temperature and matrix effects on HPP-mediated inactivation of HuNoV GII.4, GII.1, and GII.6 strains showed that HuNoV was more easily inactivated at lower temperatures and at a neutral pH. In addition, phosphate-buffered saline (PBS) and minimal essential medium (MEM) can provide protective effects against HuNoV inactivation compared to H2O. Collectively, this study demonstrated that (i) different HuNoV strains within GII exhibited different sensitivities to high pressure, and (ii) HPP is capable of inactivating HuNoV GII strains by optimizing pressure parameters.ImportanceHuman norovirus (HuNoV) is a leading cause of foodborne disease worldwide. Noroviruses are highly diverse, both antigenically and genetically. Genogroup II (GII) contains the majority of HuNoVs, with GII genotype 4 (GII.4) being the most prevalent. Recently, GII.1 and GII.6 have emerged and caused many outbreaks worldwide. However, the survival of these GII HuNoVs is poorly understood because they are uncultivable in vitro Using a novel receptor-binding assay conjugated with real-time RT-PCR, we found that GII HuNoVs had variable susceptibilities to high-pressure processing (HPP), which is one of the most promising food-processing technologies. The resistance of HuNoV strains to HPP ranked as follows: GII.1 > GII.6 > GII.4. This study highlights the ability of HPP to inactivate HuNoV and the need to optimize processing conditions based on HuNoV strain variability and sample matrix.

UnlabelledStaphylococcus aureus is an important pathogen of humans and animals. We genome sequenced 90 S. aureus isolates from The Gambia: 46 isolates from invasive disease in humans, 13 human carriage isolates, and 31 monkey carriage isolates. We inferred multiple anthroponotic transmissions of S. aureus from humans to green monkeys (Chlorocebus sabaeus) in The Gambia over different time scales. We report a novel monkey-associated clade of S. aureus that emerged from a human-to-monkey switch estimated to have occurred 2,700 years ago. Adaptation of this lineage to the monkey host is accompanied by the loss of phage-carrying genes that are known to play an important role in human colonization. We also report recent anthroponotic transmission of the well-characterized human lineages sequence type 6 (ST6) and ST15 to monkeys, probably because of steadily increasing encroachment of humans into the monkeys' habitat. Although we have found no evidence of transmission of S. aureus from monkeys to humans, as the two species come into ever-closer contact, there might be an increased risk of additional interspecies exchanges of potential pathogens.ImportanceThe population structures of Staphylococcus aureus in humans and monkeys in sub-Saharan Africa have been previously described using multilocus sequence typing (MLST). However, these data lack the power to accurately infer details regarding the origin and maintenance of new adaptive lineages. Here, we describe the use of whole-genome sequencing to detect transmission of S. aureus between humans and nonhuman primates and to document the genetic changes accompanying host adaptation. We note that human-to-monkey switches tend to be more common than the reverse and that a novel monkey-associated clade is likely to have emerged from such a switch approximately 2,700 years ago. Moreover, analysis of the accessory genome provides important clues as to the genetic changes underpinning host adaptation and, in particular, shows that human-to-monkey switches tend to be associated with the loss of genes known to confer adaptation to the human host.

UnlabelledEfficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1 We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an efficient strategy for yeast metabolic engineering.ImportanceIn order to enable rapid and efficient fermentation of cellulosic hydrolysates by engineered yeast, we delve into the limiting factors of cellobiose fermentation by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1). Through laboratory evolution, we isolated mutant strains capable of fermenting cellobiose much faster than a parental strain. Genome sequencing of the fast cellobiose-fermenting mutant reveals that there are massive amplifications of cdt-1 and gh1-1 in the yeast genome. We also found positive and quantitative relationships between the rates of cellobiose consumption and the copy numbers of cdt-1 and gh1-1 in the evolved strains. Our results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step for efficient cellobiose fermentation. We demonstrate the feasibility of optimizing not only heterologous metabolic pathways in yeast through laboratory evolution but also on-demand gene amplification in yeast, which can be broadly applicable for metabolic engineering.

In natural and artificial aquatic environments, population structures and dynamics of photosynthetic microbes are heavily influenced by the grazing activity of protistan predators. Understanding the molecular factors that affect predation is critical for controlling toxic cyanobacterial blooms and maintaining cyanobacterial biomass production ponds for generating biofuels and other bioproducts. We previously demonstrated that impairment of the synthesis or transport of the O-antigen component of lipopolysaccharide (LPS) enables resistance to amoebal grazing in the model predator-prey system consisting of the heterolobosean amoeba HGG1 and the cyanobacterium Synechococcus elongates PCC 7942 (R. S. Simkovsky et al., Proc Natl Acad Sci U S A 109:16678-16683, 2012,http://dx.doi.org/10.1073/pnas.1214904109). In this study, we used this model system to identify additional gene products involved in the synthesis of O antigen, the ligation of O antigen to the lipid A-core conjugated molecule (including a novel ligase gene), the generation of GDP-fucose, and the incorporation of sugars into the lipid A core oligosaccharide ofS. elongatus Knockout of any of these genes enables resistance to HGG1, and of these, only disruption of the genes involved in synthesis or incorporation of GDP-fucose into the lipid A-core molecule impairs growth. Because these LPS synthesis genes are well conserved across the diverse range of cyanobacteria, they enable a broader understanding of the structure and synthesis of cyanobacterial LPS and represent mutational targets for generating resistance to amoebal grazers in novel biomass production strains.

Extremely thermoacidophilic Crenarchaeota belonging to the order Sulfolobales flourish in hot acidic habitats that are strongly oxidizing. The pH extremes of these habitats, however, often exceed the acid tolerance of type species and strains. Here, adaptive laboratory evolution was used over a 3-year period to test whether such organisms harbor additional thermoacidophilic capacity. Three distinct cell lines derived from a single type species were subjected to high-temperature serial passage while culture acidity was gradually increased. A 178-fold increase in thermoacidophily was achieved after 29 increments of shifted culture pH resulting in growth at pH 0.8 and 80°C. These strains were named super-acid-resistant Crenarchaeota (SARC). Mathematical modeling using growth parameters predicted the limits of acid resistance, while genome resequencing and transcriptome resequencing were conducted for insight into mechanisms responsible for the evolved trait. Among the mutations that were detected, a set of eight nonsynonymous changes may explain the heritability of increased acid resistance despite an unexpected lack of transposition. Four multigene components of the SARC transcriptome implicated oxidative stress as a primary challenge accompanying growth at acid extremes. These components included accelerated membrane biogenesis, induction of the mer operon, and an increased capacity for the generation of energy and reductant.

Shotgun metagenomic sequencing does not depend on gene-targeted primers or PCR amplification; thus, it is not affected by primer bias or chimeras. However, searching rRNA genes from large shotgun Illumina data sets is computationally expensive, and no approach exists for unsupervised community analysis of small-subunit (SSU) rRNA gene fragments retrieved from shotgun data. We present a pipeline, SSUsearch, to achieve the faster identification of short-subunit rRNA gene fragments and enabled unsupervised community analysis with shotgun data. It also includes classification and copy number correction, and the output can be used by traditional amplicon analysis platforms. Shotgun metagenome data using this pipeline yielded higher diversity estimates than amplicon data but retained the grouping of samples in ordination analyses. We applied this pipeline to soil samples with paired shotgun and amplicon data and confirmed bias against Verrucomicrobia in a commonly used V6-V8 primer set, as well as discovering likely bias against Actinobacteria and for Verrucomicrobia in a commonly used V4 primer set. This pipeline can utilize all variable regions in SSU rRNA and also can be applied to large-subunit (LSU) rRNA genes for confirmation of community structure. The pipeline can scale to handle large amounts of soil metagenomic data (5 Gb memory and 5 central processing unit hours to process 38 Gb [1 lane] of trimmed Illumina HiSeq2500 data) and is freely available at https://github.com/dib-lab/SSUsearch under a BSD license.

In the next decades, the increasing material and energetic demand to support population growth and higher standards of living will amplify the current pressures on ecosystems and will call for greater investments in infrastructures and modern technologies. A valid approach to overcome such future challenges is the employment of sustainable bio-based technologies that explore the metabolic richness of microorganisms. Collectively, the metabolic capabilities of Chloroflexota , spanning aerobic and anaerobic conditions, thermophilic adaptability, anoxygenic photosynthesis, and utilization of toxic compounds as electron acceptors, underscore the phylum's resilience and ecological significance. These diverse metabolic strategies, driven by the interplay between temperature, oxygen availability, and energy metabolism, exemplify the complex adaptations that enabled Chloroflexota to colonize a wide range of ecological niches. In demonstrating the metabolic richness of the Chloroflexota phylum, specific members exemplify the diverse capabilities of these microorganisms: Chloroflexus aurantiacus showcases adaptability through its thermophilic and phototrophic growth, whereas members of the Anaerolineae class are known for their role in the degradation of complex organic compounds, contributing significantly to the carbon cycle in anaerobic environments, highlighting the phylum's potential for biotechnological exploitation in varying environmental conditions. In this context, the metabolic diversity of Chloroflexota must be considered a promising asset for a large range of applications. Currently, this bacterial phylum is organized into eight classes possessing different metabolic strategies to survive and thrive in a wide variety of extreme environments. This review correlates the ecological role of Chloroflexota in such environments with the potential application of their metabolisms in biotechnological approaches., Competing Interests: The authors declare no conflict of interest.

The role of the food delivery matrix in probiotic performance in the intestine is not well understood. Because probiotics are often provided to consumers in dairy products, we investigated the contributions of milk to the health-benefiting performance of Lactobacillus casei BL23 in a dextran sulfate sodium (DSS)-induced murine model of ulcerative colitis. L. casei BL23 protected against the development of colitis when ingested in milk but not in a nutrient-free buffer simulating consumption as a nutritional supplement. Consumption of (acidified) milk alone also provided some protection against weight loss and intestinal inflammation but was not as effective as L. casei and milk in combination. In contrast, L. casei mutants deficient in DltD (lipoteichoic acid d-alanine transfer protein) or RecA (recombinase A) were unable to protect against DSS-induced colitis, even when consumed in the presence of milk. Mice fed either L. casei or milk contained reduced quantities of colonic proinflammatory cytokines, indicating that the L. casei DltD(-) and RecA(-) mutants as well as L. casei BL23 in nutrient-free buffer were effective at modulating immune responses. However, there was not a direct correlation between colitis and quantities of these cytokines at the time of sacrifice. Identification of the cecal microbiota by 16S rRNA gene sequencing showed that L. casei in milk enriched for Comamonadaceae and Bifidobacteriaceae; however, the consumption of neither L. casei nor milk resulted in the restoration of the microbiota to resemble that of healthy animals. These findings strongly indicate that probiotic strain efficacy can be influenced by the food/supplement delivery matrix.

Bartonella infection among cats from shelters can pose a health risk to adopters. Bartonella henselae is the most common species, with B. clarridgeiae and B. koehlerae being less common. The lower rates of infection by the latter species may reflect their rarity or an inefficiency of culture techniques. To assess the incidence of infection, blood cultures, serology, and PCR testing were performed on 193 kittens (6 to 17 weeks old) and 158 young adult cats (5 to 12 months old) from a modern regional shelter. Classical B. henselae culture medium was compared to a medium supplemented with insect cell growth factors. Bartonella colonies were isolated from 115 (32.8%) animals, including 50 (25.9%) kittens and 65 (41.1%) young adults. Therefore, young adults were twice as likely to be culture positive as kittens. Enhanced culture methods did not improve either the isolation rate or species profile. B. henselae was isolated from 40 kittens and 55 young adults, while B. clarridgeiae was cultured from 10 animals in each group. B. koehlerae was detected in one young adult by PCR only. B. henselae genotype II was more commonly isolated from young adults, and genotype I was more frequently isolated from kittens. Kittens were 4.7 times more likely to have a very high bacterial load than young adults. A significantly higher incidence of bacteremia in the fall and winter than in the spring and summer was observed. Bartonella antibodies were detected in 10% (19/193) of kittens and 46.2% (73/158) of young adults, with culture-positive kittens being 9.4 times more likely to be seronegative than young adults.

The mission of the United States Culture Collection Network (USCCN; http://usccn.org) is "to facilitate the safe and responsible utilization of microbial resources for research, education, industry, medicine, and agriculture for the betterment of human kind." Microbial culture collections are a key component of life science research, biotechnology, and emerging global biobased economies. Representatives and users of several microbial culture collections from the United States and Europe gathered at the University of California, Davis, to discuss how collections of microorganisms can better serve users and stakeholders and to showcase existing resources available in public culture collections.

The nutritional status of pregnant women is vital for healthy outcomes and is a concern for a large proportion of the world's population. The role of the microbiota in pregnancy and nutrition is a promising new area of study with potential health ramifications. In many African countries, maternal and infant death and morbidity are associated with malnutrition. Here, we assess the influence of probiotic yogurt containing Lactobacillus rhamnosus GR-1, supplemented with Moringa plant as a source of micronutrients, on the health and oral, gut, vaginal, and milk microbiotas of 56 pregnant women in Tanzania. In an open-label study design, 26 subjects received yogurt daily, and 30 were untreated during the last two trimesters and for 1 month after birth. Samples were analyzed using 16S rRNA gene sequencing, and dietary recalls were recorded. Women initially categorized as nourished or undernourished consumed similar calories and macronutrients, which may explain why there was no difference in the microbiota at any body site. Consumption of yogurt increased the relative abundance of Bifidobacterium and decreased Enterobacteriaceae in the newborn feces but had no effect on the mother's microbiota at any body site. The microbiota of the oral cavity and GI tract remained stable over pregnancy, but the vaginal microbiota showed a significant increase in diversity leading up to and after birth. In summary, daily micronutrient-supplemented probiotic yogurt provides a safe, affordable food for pregnant women in rural Tanzania, and the resultant improvement in the gut microbial profile of infants is worthy of further study.

Terpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5 sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of a dxs deletion in Escherichia coli grown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-type E. coli yajO gene, annotated as a putative xylose reductase, or via various mutations in the native ribB gene. In vitro analysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway in E. coli for production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

Cyanobacteria are photosynthetic bacteria that are currently being developed as biological production platforms. They derive energy from light and carbon from atmospheric carbon dioxide, and some species can fix atmospheric nitrogen. One advantage of developing cyanobacteria for renewable production of biofuels and other biological products is that they are amenable to genetic manipulation, facilitating bioengineering and synthetic biology. To expand the currently available genetic toolkit, we have demonstrated the utility of synthetic theophylline-responsive riboswitches for effective regulation of gene expression in four diverse species of cyanobacteria, including two recent isolates. We evaluated a set of six riboswitches driving the expression of a yellow fluorescent protein reporter in Synechococcus elongatus PCC 7942, Leptolyngbya sp. strain BL0902, Anabaena sp. strain PCC 7120, and Synechocystis sp. strain WHSyn. We demonstrated that riboswitches can offer regulation of gene expression superior to that of the commonly used isopropyl-β-d-thiogalactopyranoside induction of a lacI(q)-Ptrc promoter system. We also showed that expression of the toxic protein SacB can be effectively regulated, demonstrating utility for riboswitch regulation of proteins that are detrimental to biomass accumulation. Taken together, the results of this work demonstrate the utility and ease of use of riboswitches in the context of genetic engineering and synthetic biology in diverse cyanobacteria, which will facilitate the development of algal biotechnology.

The management and control of mosquito vectors of human disease currently rely primarily on chemical insecticides. However, larvicidal treatments can be effective, and if based on biological insecticides, they can also ameliorate the risk posed to human health by chemical insecticides. The aerobic bacteria Bacillus thuringiensis and Lysinibacillus sphaericus have been used for vector control for a number of decades. But a more cost-effective use would be an anaerobic bacterium because of the ease with which these can be cultured. More recently, the anaerobic bacterium Clostridium bifermentans subsp. malaysia has been reported to have high mosquitocidal activity, and a number of proteins were identified as potentially mosquitocidal. However, the cloned proteins showed no mosquitocidal activity. We show here that four toxins encoded by the Cry operon, Cry16A, Cry17A, Cbm17.1, and Cbm17.2, are all required for toxicity, and these toxins collectively show remarkable selectivity for Aedes rather than Anopheles mosquitoes, even though C. bifermentans subsp. malaysia is more toxic to Anopheles. Hence, toxins that target Anopheles are different from those expressed by the Cry operon.

Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

Homologous recombination plays an important role in the structuring of genetic variation of many bacteria; however, its importance in adaptive evolution is not well established. We investigated the association of intersubspecific homologous recombination (IHR) with the shift to a novel host (mulberry) by the plant-pathogenic bacterium Xylella fastidiosa. Mulberry leaf scorch was identified about 25 years ago in native red mulberry in the eastern United States and has spread to introduced white mulberry in California. Comparing a sequence of 8 genes (4,706 bp) from 21 mulberry-type isolates to published data (352 isolates representing all subspecies), we confirmed previous indications that the mulberry isolates define a group distinct from the 4 subspecies, and we propose naming the taxon X. fastidiosa subsp. morus. The ancestry of its gene sequences was mixed, with 4 derived from X. fastidiosa subsp. fastidiosa (introduced from Central America), 3 from X. fastidiosa subsp. multiplex (considered native to the United States), and 1 chimeric, demonstrating that this group originated by large-scale IHR. The very low within-type genetic variation (0.08% site polymorphism), plus the apparent inability of native X. fastidiosa subsp. multiplex to infect mulberry, suggests that this host shift was achieved after strong selection acted on genetic variants created by IHR. Sequence data indicate that a single ancestral IHR event gave rise not only to X. fastidiosa subsp. morus but also to the X. fastidiosa subsp. multiplex recombinant group which infects several hosts but is the only type naturally infecting blueberry, thus implicating this IHR in the invasion of at least two novel native hosts, mulberry and blueberry.

Human noroviruses (HuNoVs) are the major cause of epidemic nonbacterial gastroenteritis. Although quantitative (real-time) reverse transcription-PCR (qRT-PCR) is widely used for detecting HuNoVs, it only detects the presence of viral RNA and does not indicate viral infectivity. Human blood group antigens (HBGAs) have been identified as receptors/co-receptors for both HuNoVs and Tulane virus (TV) and are crucial for viral infection. We propose that viral infectivity can be evaluated with a molecular assay based on receptor-captured viruses. In this study, we employed TV as an HuNoV surrogate to validate the HBGA-based capture qRT-PCR method against the 50% tissue culture infectious dose (TCID50) method. We employed type B HBGA on an immuno-well module to concentrate TV, followed by amplification of the captured viral genome by in situ qRT-PCR. We first demonstrated that this in situ capture qRT-PCR (ISC-qRT-PCR) method could effectively concentrate and detect TV. We then treated TV under either partial or full inactivation conditions and measured the remaining infectivity by ISC-qRT-PCR and a tissue culture-based amplification method (TCID50). We found that the ISC-qRT-PCR method could be used to evaluate virus inactivation deriving from damage to the capsid and study interactions between the capsid and viral receptor. Heat, chlorine, and ethanol treatment primarily affect the capsid structure, which in turns affects the ability of the capsid to bind to viral receptors. Inactivation of the virus by these methods could be reflected by the ISC-qRT-PCR method and confirmed by TCID50 assay. However, the loss of the infectivity caused by damage to the viral genome (such as that from UV irradiation) could not be effectively reflected by this method. Despite this limitation, the ISC-qRT-PCR provides an alternative approach to determine inactivation of Tulane virus. A particular advantage of the ISC-qRT-PCR method is that it is also a faster and easier method to effectively recover and detect the viruses, as there is no need to extract viral RNA or to transfer the captured virus from magnetic beads to PCR tubes for further amplification. Therefore, ISC-qRT-PCR can be easily adapted for use in automated systems for multiple samples.

Enterococcus faecium NRRL B-2354 is a surrogate microorganism used in place of pathogens for validation of thermal processing technologies and systems. We evaluated the safety of strain NRRL B-2354 based on its genomic and functional characteristics. The genome of E. faecium NRRL B-2354 was sequenced and found to comprise a 2,635,572-bp chromosome and a 214,319-bp megaplasmid. A total of 2,639 coding sequences were identified, including 45 genes unique to this strain. Hierarchical clustering of the NRRL B-2354 genome with 126 other E. faecium genomes as well as pbp5 locus comparisons and multilocus sequence typing (MLST) showed that the genotype of this strain is most similar to commensal, or community-associated, strains of this species. E. faecium NRRL B-2354 lacks antibiotic resistance genes, and both NRRL B-2354 and its clonal relative ATCC 8459 are sensitive to clinically relevant antibiotics. This organism also lacks, or contains nonfunctional copies of, enterococcal virulence genes including acm, cyl, the ebp operon, esp, gelE, hyl, IS16, and associated phenotypes. It does contain scm, sagA, efaA, and pilA, although either these genes were not expressed or their roles in enterococcal virulence are not well understood. Compared with the clinical strains TX0082 and 1,231,502, E. faecium NRRL B-2354 was more resistant to acidic conditions (pH 2.4) and high temperatures (60°C) and was able to grow in 8% ethanol. These findings support the continued use of E. faecium NRRL B-2354 in thermal process validation of food products.

The bacterial pathogen Xylella fastidiosa infects xylem and causes disease in many plant species in the Americas. Different subspecies of this bacterium and different genotypes within subspecies infect different plant hosts, but the genetics of host adaptation are unknown. Here we examined the hypothesis that the introduction of novel genetic variation via intersubspecific homologous recombination (IHR) facilitates host shifts. We investigated IHR in 33 X. fastidiosa subsp. multiplex isolates previously identified as recombinant based on 8 loci (7 multilocus sequence typing [MLST] loci plus 1 locus). We found significant evidence of introgression from X. fastidiosa subsp. fastidiosa in 4 of the loci and, using published data, evidence of IHR in 6 of 9 additional loci. Our data showed that IHR regions in 2 of the 4 loci were inconsistent (12 mismatches) with X. fastidiosa subsp. fastidiosa alleles found in the United States but consistent with alleles from Central America. The other two loci were consistent with alleles from both regions. We propose that the recombinant forms all originated via genomewide recombination of one X. fastidiosa subsp. multiplex ancestor with one X. fastidiosa subsp. fastidiosa donor from Central America that was introduced into the United States but subsequently disappeared. Using all of the available data, 5 plant hosts of the recombinant types were identified, 3 of which also supported non-IHR X. fastidiosa subsp. multiplex, but 2 were unique to recombinant types from blueberry (7 isolates from Georgia, 3 from Florida); and blackberry (1 each from Florida and North Carolina), strongly supporting the hypothesis that IHR facilitated a host shift to blueberry and possibly blackberry.

Certain strains of Enterococcus faecium and Enterococcus faecalis contribute beneficially to animal health and food production, while others are associated with nosocomial infections. To determine whether there are structural and functional genomic features that are distinct between nonclinical (NC) and clinical (CL) strains of those species, we analyzed the genomes of 31 E. faecium and 38 E. faecalis strains. Hierarchical clustering of 7,017 orthologs found in the E. faecium pangenome revealed that NC strains clustered into two clades and are distinct from CL strains. NC E. faecium genomes are significantly smaller than CL genomes, and this difference was partly explained by significantly fewer mobile genetic elements (ME), virulence factors (VF), and antibiotic resistance (AR) genes. E. faecium ortholog comparisons identified 68 and 153 genes that are enriched for NC and CL strains, respectively. Proximity analysis showed that CL-enriched loci, and not NC-enriched loci, are more frequently colocalized on the genome with ME. In CL genomes, AR genes are also colocalized with ME, and VF are more frequently associated with CL-enriched loci. Genes in 23 functional groups are also differentially enriched between NC and CL E. faecium genomes. In contrast, differences were not observed between NC and CL E. faecalis genomes despite their having larger genomes than E. faecium. Our findings show that unlike E. faecalis, NC and CL E. faecium strains are equipped with distinct structural and functional genomic features indicative of adaptation to different environments.

The combination of ecological diversity with genetic and experimental tractability makes Drosophila a powerful model for the study of animal-associated microbial communities. Despite the known importance of yeasts in Drosophila physiology, behavior, and fitness, most recent work has focused on Drosophila-bacterial interactions. In order to get a more complete understanding of the Drosophila microbiome, we characterized the yeast communities associated with different Drosophila species collected around the world. We focused on the phylum Ascomycota because it constitutes the vast majority of the Drosophila-associated yeasts. Our sampling strategy allowed us to compare the distribution and structure of the yeast and bacterial communities in the same host populations. We show that yeast communities are dominated by a small number of abundant taxa, that the same yeast lineages are associated with different host species and populations, and that host diet has a greater effect than host species on yeast community composition. These patterns closely parallel those observed in Drosophila bacterial communities. However, we do not detect a significant correlation between the yeast and bacterial communities of the same host populations. Comparative analysis of different symbiont groups provides a more comprehensive picture of host-microbe interactions. Future work on the role of symbiont communities in animal physiology, ecological adaptation, and evolution would benefit from a similarly holistic approach.

The horizontal transfer of genes by mobile genetic elements such as plasmids and phages can accelerate genome diversification of Vibrio spp., affecting their physiology, pathogenicity, and ecological character. In this study, sequence analysis of three plasmids from Vibrio spp. previously isolated from salt marsh sediment revealed the remarkable diversity of these elements. Plasmids p0908 (81.4 kb), p23023 (52.5 kb), and p09022 (31.0 kb) had a predicted 99, 64, and 32 protein-coding sequences and G+C contents of 49.2%, 44.7%, and 42.4%, respectively. A phylogenetic tree based on concatenation of the host 16S rRNA and rpoA nucleotide sequences indicated p23023 and p09022 were isolated from strains most closely related to V. mediterranei and V. campbellii, respectively, while the host of p0908 forms a clade with V. fluvialis and V. furnissii. Many predicted proteins had amino acid identities to proteins of previously characterized phages and plasmids (24 to 94%). Predicted proteins with similarity to chromosomally encoded proteins included RecA, a nucleoid-associated protein (NdpA), a type IV helicase (UvrD), and multiple hypothetical proteins. Plasmid p0908 had striking similarity to enterobacteria phage P1, sharing genetic organization and amino acid identity for 23 predicted proteins. This study provides evidence of genetic exchange between Vibrio plasmids, phages, and chromosomes among diverse Vibrio spp.

Here we report associations between secondary metabolite production and phylogenetically distinct but closely related marine actinomycete species belonging to the genus Salinispora. The pattern emerged in a study that included global collection sites, and it indicates that secondary metabolite production can be a species-specific, phenotypic trait associated with broadly distributed bacterial populations. Associations between actinomycete phylotype and chemotype revealed an effective, diversity-based approach to natural product discovery that contradicts the conventional wisdom that secondary metabolite production is strain specific. The structural diversity of the metabolites observed, coupled with gene probing and phylogenetic analyses, implicates lateral gene transfer as a source of the biosynthetic genes responsible for compound production. These results conform to a model of selection-driven pathway fixation occurring subsequent to gene acquisition and provide a rare example in which demonstrable physiological traits have been correlated to the fine-scale phylogenetic architecture of an environmental bacterial community.

Importance: The viability of probiotics in the human gastrointestinal tract is important, as some reports indicate that the health benefits of live bacteria are greater than those of dead ones. Therefore, the higher the viability of the probiotic strain, the better it may be. However, probiotic strains lose their viability due to gastrointestinal stress such as gastric acid and bile. This study provides an example of the use of co-culture or pH-controlled monoculture, which uses more stringent conditions (lower pH) than normal monoculture to produce probiotic strains that are more resistant to gastrointestinal stress. In addition, co-cultured beverages showed higher viability of the probiotic strain in the human gastrointestinal tract than monocultured beverages in our human study., Competing Interests: The authors declare no conflict of interest.