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Spirobacillus cienkowskii ( Spirobacillus, hereafter) is a widely distributed bacterial pathogen that has significant impacts on the population dynamics of zooplankton ( Daphnia spp .) , particularly in months when Daphnia are asexually reproducing. However, little is known about Spirobacillus' virulence, transmission mode, and dynamics. As a result, we cannot explain the dynamics of Spirobacillus epidemics in nature or use Spirobacillus as a model pathogen, despite Daphnia's tractability as a model host. Here, we work to fill these knowledge gaps experimentally. We found that Spirobacillus is among the most virulent of Daphnia pathogens, killing its host within a week and reducing host fecundity. We further found that Spirobacillus did not transmit horizontally among hosts unless the host died or was destroyed (i.e., it is an "obligate killer"). In experiments aimed at quantifying the dynamics of horizontal transmission among asexually reproducing Daphnia , we demonstrated that Spirobacillus transmits poorly in the laboratory. In mesocosms, Spirobacillus failed to generate epidemics; in experiments wherein individual Daphnia were exposed, Spirobacillus' transmission success was low. In the (limited) set of conditions we considered, Spirobacillus' transmission success did not change with host density or pathogen dose and declined following environmental incubation. Finally, we conducted a field survey of Spirobacillus' prevalence within egg cases (ephippia) made by sexually reproducing Daphnia . We found Spirobacillus DNA in ~40% of ephippia, suggesting that, in addition to transmitting horizontally among asexually reproducing Daphnia , Spirobacillus may transmit vertically from sexually reproducing Daphnia . Our work fills critical gaps in the biology of Spirobacillus and illuminates new hypotheses vis-à-vis its life history., Importance: Spirobacillus cienkowskii is a bacterial pathogen of zooplankton, first described in the 19 th century and recently placed in a new family of bacteria, the Silvanigrellaceae . Spirobacillus causes large epidemics in lake zooplankton populations and increases the probability that zooplankton will be eaten by predators. However, little is known about how Spirobacillus transmits among hosts, to what extent it reduces host survival and reproduction (i.e., how virulent it is), and what role virulence plays in Spirobacillus ' life cycle. Here, we experimentally quantified Spirobacillus' virulence and showed that Spirobacillus must kill its host to transmit horizontally. We also found evidence that Spirobacillus may transmit vertically via Daphnia' s seed-like egg sacks. Our work will help scientists to (i) understand Spirobacillus epidemics, (ii) use Spirobacillus as a model pathogen for the study of host-parasite interactions, and (iii) better understand the unusual group of bacteria to which Spirobacillus belongs., Competing Interests: The authors declare no conflict of interest.

It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, extracellular beta-lactamases produced by resistant cells that subsequently degrade penicillin and related antibiotics allow neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show in multiple bacterial species that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface-grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss was still observed. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated.IMPORTANCEPlasmids are routinely used in microbiology as readouts of cell biology or tools to manipulate cell function. Central to these studies is the assumption that all cells in an experiment contain the plasmid. Plasmid maintenance in a host cell typically depends on a plasmid-encoded antibiotic resistance marker, which provides a selective advantage when the plasmid-containing cell is grown in the presence of antibiotic. Here, we find that growth of plasmid-containing bacteria on a surface and to a lesser extent in liquid culture in the presence of three distinct antibiotic families leads to the evolution of a significant number of plasmid-free cells, which rely on the resistance mechanisms of the plasmid-containing cells. This process generates a heterogenous population of plasmid-free and plasmid-containing bacteria, an outcome which could confound further experimentation., Competing Interests: The authors declare no conflict of interest.

Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods.IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs., Competing Interests: RUH is managing member of AquaVitas, LLC and founder of the ASU non-profit project OneWaterOneHealth operating in the same intellectual space. LPS is employed by Ceres Nanosciences.

Extracellular electron transfer is a process by which bacterial cells can exchange electrons with a redox-active material located outside of the cell. In Shewanella oneidensis , this process is natively used to facilitate respiration using extracellular electron acceptors such as Fe(III) or an anode. Previously, it was demonstrated that this process can be used to drive the microbial electrosynthesis (MES) of 2,3-butanediol (2,3-BDO) in S. oneidensis exogenously expressing butanediol dehydrogenase (BDH). Electrons taken into the cell from a cathode are used to generate NADH, which in turn is used to reduce acetoin to 2,3-BDO via BDH. However, generating NADH via electron uptake from a cathode is energetically unfavorable, so NADH dehydrogenases couple the reaction to proton motive force. We therefore need to maintain the proton gradient across the membrane to sustain NADH production. This work explores accomplishing this task by bidirectional electron transfer, where electrons provided by the cathode go to both NADH formation and oxygen (O 2 ) reduction by oxidases. We show that oxidases use trace dissolved oxygen in a microaerobic bioelectrical chemical system (BES), and the translocation of protons across the membrane during O 2 reduction supports 2,3-BDO generation. Interestingly, this process is inhibited by high levels of dissolved oxygen in this system. In an aerated BES, O 2 molecules react with the strong reductant (cathode) to form reactive oxygen species, resulting in cell death.IMPORTANCEMicrobial electrosynthesis (MES) is increasingly employed for the generation of specialty chemicals, such as biofuels, bioplastics, and cancer therapeutics. For these systems to be viable for industrial scale-up, it is important to understand the energetic requirements of the bacteria to mitigate unnecessary costs. This work demonstrates sustained production of an industrially relevant chemical driven by a cathode. Additionally, it optimizes a previously published system by removing any requirement for phototrophic energy, thereby removing the additional cost of providing a light source. We also demonstrate the severe impact of oxygen intrusion into bioelectrochemical systems, offering insight to future researchers aiming to work in an anaerobic environment. These studies provide insight into both the thermodynamics of electrosynthesis and the importance of the bioelectrochemical systems' design., Competing Interests: The authors declare no conflict of interest.

This study investigates migration phenotypes of 265 Escherichia coli soil isolates from the Buffalo River basin in Minnesota, USA. Migration rates on semisolid tryptone swim plates ranged from nonmotile to 190% of the migration rate of a highly motile E. coli K-12 strain. The nonmotile isolate, LGE0550, had mutations in flagellar and chemotaxis genes, including two IS 3 elements in the flagellin-encoding gene fliC . A genome-wide association study (GWAS), associating the migration rates with genetic variants in specific genes, yielded two metabolic variants ( rygD - serA and metR - metE ) with previous implications in chemotaxis. As a novel way of confirming GWAS results, we used minimal medium swim plates to confirm the associations. Other variants in metabolic genes and genes that are associated with biofilm were positively or negatively associated with migration rates. A determination of growth phenotypes on Biolog EcoPlates yielded differential growth for the 10 tested isolates on d-malic acid, putrescine, and d-xylose, all of which are important in the soil environment. IMPORTANCE E. coli is a Gram-negative, facultative anaerobic bacterium whose life cycle includes extra host environments in addition to human, animal, and plant hosts. The bacterium has the genomic capability of being motile. In this context, the significance of this study is severalfold: (i) the great diversity of migration phenotypes that we observed within our isolate collection supports previous (G. NandaKafle, A. A. Christie, S. Vilain, and V. S. Brözel, Front Microbiol 9:762, 2018, https://doi.org/10.3389/fmicb.2018.00762; Y. Somorin, F. Abram, F. Brennan, and C. O'Byrne, Appl Environ Microbiol 82:4628-4640, 2016, https://doi.org/10.1128/AEM.01175-16) ideas of soil promoting phenotypic heterogeneity, (ii) such heterogeneity may facilitate bacterial growth in the many different soil niches, and (iii) such heterogeneity may enable the bacteria to interact with human, animal, and plant hosts.

Traditional antibiotics target essential cellular components or metabolic pathways conserved in both pathogenic and nonpathogenic bacteria. Unfortunately, long-term antibiotic use often leads to antibiotic resistance and disruption of the overall microbiota. In this work, we identified a phenylamino acetamide compound, named 187R, that strongly inhibited the expression of the type III secretion system (T3SS) encoding genes and the secretion of the T3SS effector proteins in Pseudomonas aeruginosa. T3SS is an important virulence factor, as T3SS-deficient strains of P. aeruginosa are greatly attenuated in virulence. We further showed that 187R had no effect on bacterial growth, implying a reduced selective pressure for the development of resistance. 187R-mediated repression of T3SS was dependent on ExsA, the master regulator of T3SS in P. aeruginosa. The impact of 187R on the host-associated microbial community was also tested using the Arabidopsis thaliana phyllosphere as a model. Both culture-independent (Illumina sequencing) and culture-dependent (Biolog) methods showed that the application of 187R had little impact on the composition and function of microbial community compared to the antibiotic streptomycin. Together, these results suggested that compounds that target virulence factors could serve as an alternative strategy for disease management caused by bacterial pathogens. IMPORTANCE New antimicrobial therapies are urgently needed, since antibiotic resistance in human pathogens has become one of the world's most urgent public health problems. Antivirulence therapy has been considered a promising alternative for the management of infectious diseases, as antivirulence compounds target only the virulence factors instead of the growth of bacteria, and they are therefore unlikely to affect commensal microorganisms. However, the impacts of antivirulence compounds on the host microbiota are not well understood. We report a potent synthetic inhibitor of the P. aeruginosa T3SS, 187R, and its effect on the host microbiota of Arabidopsis . Both culture-independent (Illumina sequencing) and culture-dependent (Biolog) methods showed that the impacts of the antivirulence compound on the composition and function of host microbiota were limited. These results suggest that antivirulence compounds can be a potential alternative method to antibiotics.

Antibiotic-resistant bacteria and the spread of antibiotic resistance genes (ARGs) pose a serious risk to human and veterinary health. While many studies focus on the movement of live antibiotic-resistant bacteria to the environment, it is unclear whether extracellular ARGs (eARGs) from dead cells can transfer to live bacteria to facilitate the evolution of antibiotic resistance in nature. Here, we use eARGs from dead, antibiotic-resistant Pseudomonas stutzeri cells to track the movement of eARGs to live P. stutzeri cells via natural transformation, a mechanism of horizontal gene transfer involving the genomic integration of eARGs. In sterile, antibiotic-free agricultural soil, we manipulated the eARG concentration, soil moisture, and proximity to eARGs. We found that transformation occurred in soils inoculated with just 0.25 μg of eDNA g -1 soil, indicating that even low concentrations of soil eDNA can facilitate transformation (previous estimates suggested ∼2 to 40 μg eDNA g -1 soil). When eDNA was increased to 5 μg g -1 soil, there was a 5-fold increase in the number of antibiotic-resistant P. stutzeri cells. We found that eARGs were transformed under soil moistures typical of terrestrial systems (5 to 30% gravimetric water content) but inhibited at very high soil moistures (>30%). Overall, this work demonstrates that dead bacteria and their eARGs are an overlooked path to antibiotic resistance. More generally, the spread of eARGs in antibiotic-free soil suggests that transformation allows genetic variants to establish in the absence of antibiotic selection and that the soil environment plays a critical role in regulating transformation. IMPORTANCE Bacterial death can release eARGs into the environment. Agricultural soils can contain upwards of 10 9 ARGs g -1 soil, which may facilitate the movement of eARGs from dead to live bacteria through a mechanism of horizontal gene transfer called natural transformation. Here, we track the spread of eARGs from dead, antibiotic-resistant Pseudomonas stutzeri cells to live antibiotic-susceptible P. stutzeri cells in sterile agricultural soil. Transformation increased with the abundance of eARGs and occurred in soils ranging from 5 to 40% gravimetric soil moisture but was lowest in wet soils (>30%). Transformants appeared in soil after 24 h and persisted for up to 15 days even when eDNA concentrations were only a fraction of those found in field soils. Overall, our results show that natural transformation allows eARGs to spread and persist in antibiotic-free soils and that the biological activity of eDNA after bacterial death makes environmental eARGs a public health concern.

Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infections. Cattle are an important STEC reservoir, although little is known about specific pathogen traits that impact persistence in the farm environment. Hence, we sought to evaluate STEC isolates recovered from beef cattle in a single herd in Michigan. To do this, we collected fecal grabs from 26 cattle and resampled 13 of these animals at 3 additional visits over a 3-month period. In all, 66 STEC isolates were recovered for genomics and biofilm quantification using crystal violet assays. The STEC population was diverse, representing seven serotypes, including O157:H7, O26:H11, and O103:H2, which are commonly associated with human infections. Although a core genome analysis of 2,933 genes grouped isolates into clusters based on serogroups, some isolates within each cluster had variable biofilm levels and virulence gene profiles. Most (77.8%; n  = 49) isolates harbored stx 2a , while 38 (57.5%) isolates formed strong biofilms. Isolates belonging to the predominant serogroup O6 ( n  = 36; 54.5%) were more likely to form strong biofilms, persistently colonize multiple cattle, and be acquired over time. A high-quality single nucleotide polymorphism (SNP) analysis of 33 O6 isolates detected between 0 and 13 single nucleotide polymorphism (SNP) differences between strains, indicating that highly similar strain types were persisting in this herd. Similar findings were observed for other persistent serogroups, although key genes were found to differ among strong and weak biofilm producers. Together, these data highlight the diversity and persistent nature of some STEC types in this important food animal reservoir. IMPORTANCE Food animal reservoirs contribute to Shiga toxin-producing Escherichia coli (STEC) evolution via the acquisition of horizontally acquired elements like Shiga toxin bacteriophages that enhance pathogenicity. In cattle, persistent fecal shedding of STEC contributes to contamination of beef and dairy products and to crops being exposed to contaminated water systems. Hence, identifying factors important for STEC persistence is critical. This longitudinal study enhances our understanding of the genetic diversity of STEC types circulating in a cattle herd and identifies genotypic and phenotypic traits associated with persistence. Key findings demonstrate that multiple STEC types readily persist in and are transmitted across cattle in a shared environment. These dynamics also enhance the persistence of virulence genes that can be transferred between bacterial hosts, resulting in the emergence of novel STEC strain types. Understanding how pathogens persist and diversify in reservoirs is important for guiding new preharvest prevention strategies aimed at reducing foodborne transmission to humans., (Copyright © 2020 American Society for Microbiology.)

Gastrointestinal (GI) or gut microbiotas play essential roles in host development and physiology. These roles are influenced partly by the microbial community composition. During early developmental stages, the ecological processes underlying the assembly and successional changes in host GI community composition are influenced by numerous factors, including dispersal from the surrounding environment, age-dependent changes in the gut environment, and changes in dietary regimes. However, the relative importance of these factors to the gut microbiota is not well understood. We examined the effects of environmental (diet and water sources) and host early ontogenetic development on the diversity of and the compositional changes in the gut microbiota of a primitive teleost fish, the lake sturgeon ( Acipenser fulvescens ), based on massively parallel sequencing of the 16S rRNA gene. Fish larvae were raised in environments that differed in water source (stream versus filtered groundwater) and diet (supplemented versus nonsupplemented Artemia fish). We quantified the gut microbial community structure at three stages (prefeeding and 1 and 2 weeks after exogenous feeding began). The diversity declined and the community composition differed significantly among stages; however, only modest differences associated with dietary or water source treatments were documented. Many taxa present in the gut were over- or underrepresented relative to neutral expectations in each sampling period. The findings indicate dynamic relationships between the gut microbiota composition and host gastrointestinal physiology, with comparatively smaller influences being associated with the rearing environments. Neutral models of community assembly could not be rejected, but selectivity associated with microbe-host GI tract interactions through early ontogenetic stages was evident. The results have implications for sturgeon conservation and aquaculture production specifically and applications of microbe-based management in teleost fish generally. IMPORTANCE We quantified the effects of environment (diet and water sources) and host early ontogenetic development on the diversity of and compositional changes in gut microbial communities based on massively parallel sequencing of the 16S rRNA genes from the GI tracts of larval lake sturgeon ( Acipenser fulvescens ). The gut microbial community diversity declined and the community composition differed significantly among ontogenetic stages; however, only modest differences associated with dietary or water source treatments were documented. Selectivity associated with microbe-host GI tract interactions through early ontogenetic stages was evident. The results have implications for lake sturgeon and early larval ecology and survival in their natural habitat and for conservation and aquaculture production specifically, as well as applications of microbe-based management in teleost fish generally., (Copyright © 2020 American Society for Microbiology.)

Biological nitrogen fixation can be an important source of nitrogen in tropical forests that serve as a major CO 2 sink. Extensive deforestation of the Amazon is known to influence microbial communities and the biogeochemical cycles they mediate. However, it is unknown how diazotrophs (nitrogen-fixing microorganisms) respond to deforestation and subsequent ecosystem conversion to agriculture, as well as whether they can recover in secondary forests that are established after agriculture is abandoned. To address these knowledge gaps, we combined a spatially explicit sampling approach with high-throughput sequencing of nifH genes. The main objectives were to assess the functional distance decay relationship of the diazotrophic bacterial community in a tropical forest ecosystem and to quantify the roles of various factors that drive the observed changes in the diazotrophic community structure. We observed an increase in local diazotrophic diversity (α-diversity) with a decrease in community turnover (β-diversity), associated with a shift in diazotrophic community structure as a result of the forest-to-pasture conversion. Both diazotrophic community turnover and structure showed signs of recovery in secondary forests. Changes in the diazotrophic community were primarily driven by the change in land use rather than differences in geochemical characteristics or geographic distances. The diazotroph communities in secondary forests resembled those in primary forests, suggesting that at least partial recovery of diazotrophs is possible following agricultural abandonment. IMPORTANCE The Amazon region is a major tropical forest region that is being deforested at an alarming rate to create space for cattle ranching and agriculture. Diazotrophs (nitrogen-fixing microorganisms) play an important role in supplying soil N for plant growth in tropical forests. It is unknown how diazotrophs respond to deforestation and whether they can recover in secondary forests that establish after agriculture is abandoned. Using high-throughput sequencing of nifH genes, we characterized the response of diazotrophs' β-diversity and identified major drivers of changes in diazotrophs from forest-to-pasture and pasture-to-secondary-forest conversions. Studying the impact of land use change on diazotrophs is important for a better understanding of the impact of deforestation on tropical forest ecosystem functioning, and our results on the potential recovery of diazotrophs in secondary forests imply the possible restoration of ecosystem functions in secondary forests., (Copyright © 2020 American Society for Microbiology.)

Soil extracellular enzymes play a significant role in the N mineralization process. However, few studies have documented the linkage between enzyme activity and the microbial community that performs the function. This study examined the effects of inorganic and organic N fertilization on soil microbial communities and their N mineralization functions over 4 years. Soils were collected from silage corn field plots with four contrasting N treatments: control (no additional N), ammonium sulfate (AS; 100 and 200 kg of N ha -1 ), and compost (200 kg of N ha -1 ). Illumina amplicon sequencing was used to comprehensively assess the overall bacterial community (16S rRNA genes), bacterial ureolytic community ( ureC ), and bacterial chitinolytic community ( chiA ). Selected genes involved in N mineralization were also examined using quantitative real-time PCR and metagenomics. Enzymes (and marker genes) included protease ( npr and sub ), chitinase ( chiA ), urease ( ureC ), and arginase ( rocF ). Compost significantly increased diversity of overall bacterial communities even after one application, while ammonium fertilizers had no influence on the overall bacterial communities over four seasons. Bacterial ureolytic and chitinolytic communities were significantly changed by N fertilization. Compost treatment strongly elevated soil enzyme activities after 4 years of repeated application. Functional gene abundances were not significantly affected by N treatments, and they were not correlated with corresponding enzyme activities. N mineralization genes were recovered from soil metagenomes based on a gene-targeted assembly. Understanding how the structure and function of soil microbial communities involved with N mineralization change in response to fertilization practices may indicate suitable agricultural management practices that improve ecosystem services while reducing negative environmental consequences. IMPORTANCE Agricultural N management practices influence the enzymatic activities involved in N mineralization. However, specific enzyme activities do not identify the microbial species directly involved in the measured process, leaving the link between the composition of the microbial community and the production of key enzymes poorly understood. In this study, the application of high-throughput sequencing, real-time PCR, and metagenomics shed light on how the abundance and diversity of microorganisms involved in N mineralization respond to N management. We suggest that N fertilization has significantly changed bacterial ureolytic and chitinolytic communities., (Copyright © 2020 American Society for Microbiology.)

The brown planthopper Nilaparvata lugens (Hemiptera) is a major pest of rice crops in Asia. Artificial transinfections of Wolbachia have recently been used for reducing host impacts, but transinfections have not yet been undertaken with another important endosymbiont, Cardinium This endosymbiont can manipulate the reproduction of hosts through phenotypes such as cytoplasmic incompatibility (CI), which is strong in the related white-backed planthopper, Sogatella furcifera (Hemiptera). Here, we stably infected N. lugens with Cardinium from S. furcifera and showed that it exhibits perfect maternal transmission in N. lugens The density of Cardinium varied across developmental stages and tissues of the transinfected host. Cardinium did not induce strong CI in N. lugens , likely due to its low density in testicles. The infection did decrease fecundity and hatching rate in the transinfected host, but a decrease in fecundity was not apparent when transinfected females mated with Wolbachia -infected males. The experiments show the feasibility of transferring Cardinium endosymbionts across hosts, but the deleterious effects of Cardinium on N. lugens limit its potential to spread in wild populations of N. lugens in the absence of strong CI. IMPORTANCE In this study we established a Cardinium -infected N. lugens line that possessed complete maternal transmission. Cardinium had a widespread distribution in tissues of N. lugens , and this infection decreased the fecundity and hatching rate of the host. Our findings emphasize the feasibility of transinfection of Cardinium in insects, which expands the range of endosymbionts that could be manipulated for pest control., (Copyright © 2020 American Society for Microbiology.)

Vibrio cholerae is a Gram-negative bacterial pathogen that causes the disease cholera, which affects nearly 1 million people each year. In between outbreaks, V. cholerae resides in fresh and salt water environments, where it is able to persist through changes in temperature, oxygen, and salinity. One key characteristic that promotes environmental persistence of V. cholerae is the ability to form multicellular communities, called biofilms, that often adhere to biotic and abiotic sources. Biofilm formation in V. cholerae is positively regulated by the dinucleotide second messenger cyclic dimeric GMP (c-di-GMP). While most research on the c-di-GMP regulon has focused on biofilm formation or motility, we hypothesized that the c-di-GMP signaling network encompassed a larger set of effector functions than reported. We found that high intracellular c-di-GMP increased catalase activity ∼4-fold relative to strains with unaltered c-di-GMP. Genetic studies demonstrated that c-di-GMP mediated catalase activity was due to increased expression of the catalase-encoding gene katB Moreover, c-di-GMP mediated regulation of catalase activity and katB expression required the c-di-GMP dependent transcription factors VpsT and VpsR. Lastly, we found that high c-di-GMP increased survival after H 2 O 2 challenge in a katB -, vpsR -, and vpsT -dependent manner. Our results indicate that antioxidant production is regulated by c-di-GMP uncovering a new node in the growing VpsT and VpsR c-di-GMP signaling network of V. cholerae IMPORTANCE As a result of infection with V. cholerae , patients become dehydrated, leading to death if not properly treated. The aquatic environment is the natural reservoir for V. cholerae , where it can survive alterations in temperature, salinity, and oxygen. The second messenger molecule c-di-GMP is an important signal regulating host and aquatic environmental persistence because it controls whether V. cholerae will form a biofilm or disperse through flagellar motility. In this work, we demonstrate another function of c-di-GMP in V. cholerae biology: promoting tolerance to the reactive oxygen species H 2 O 2 through the differential regulation of catalase expression. Our results suggest a mechanism where c-di-GMP simultaneously controls biofilm formation and antioxidant production, which could promote persistence in human and marine environments., (Copyright © 2019 American Society for Microbiology.)

Toxin-antitoxin (TA) systems are genetic elements composed of a protein toxin and a counteracting antitoxin that is either a noncoding RNA or protein. In type I TA systems, the antitoxin is a noncoding small RNA (sRNA) that base pairs with the cognate toxin mRNA interfering with its translation. Although type I TA systems have been extensively studied in Escherichia coli and a few human or animal bacterial pathogens, they have not been characterized in plant-pathogenic bacteria. In this study, we characterized a chromosomal locus in the plant pathogen Erwinia amylovora Ea1189 that is homologous to the hok-sok type I TA system previously identified in the Enterobacteriaceae -restricted plasmid R1. Phylogenetic analysis indicated that the chromosomal location of the hok-sok locus is, thus far, unique to E. amylovora We demonstrated that ectopic overexpression of hok is highly toxic to E. amylovora and that the sRNA sok reversed the toxicity of hok through mok , a reading frame presumably translationally coupled with hok We also identified the region that is essential for maintenance of the main toxicity of Hok. Through a hok-sok deletion mutant (Ea1189Δ hok-sok ), we determined the contribution of the hok-sok locus to cellular growth, micromorphology, and catalase activity. Combined, our findings indicate that the hok-sok TA system, besides being potentially self-toxic, provides fitness advantages to E. amylovora IMPORTANCE Bacterial toxin-antitoxin systems have received great attention because of their potential as targets for antimicrobial development and as tools for biotechnology. Erwinia amylovora , the causal agent of fire blight disease on pome fruit trees, is a major plant-pathogenic bacterium. In this study, we identified and functionally characterized a unique chromosomally encoded hok-sok toxin-antitoxin system in E. amylovora that resembles the plasmid-encoded copies of this system in other Enterobacteriaceae This study of a type I toxin-antitoxin system in a plant-pathogenic bacterium provides the basis to further understand the involvement of toxin-antitoxin systems during infection by a plant-pathogenic bacterium. The new linkage between the hok-sok toxin-antitoxin system and the catalase-mediated oxidative stress response leads to additional considerations of targeting this system for antimicrobial development., (Copyright © 2019 American Society for Microbiology.)

Free-living nitrogen fixation (FLNF) in the rhizosphere, or N fixation by heterotrophic bacteria living on/near root surfaces, is ubiquitous and a significant source of N in some terrestrial systems. FLNF is also of interest in crop production as an alternative to chemical fertilizer, potentially reducing production costs and ameliorating negative environmental impacts of fertilizer N additions. Despite this interest, a mechanistic understanding of controls (e.g., carbon, oxygen, nitrogen, and nutrient availability) on FLNF in the rhizosphere is lacking but necessary. FLNF is distinct from and occurs under more diverse and dynamic conditions than symbiotic N fixation; therefore, predicting FLNF rates and understanding controls on FLNF has proven difficult. This has led to large gaps in our understanding of FLNF, and studies aimed at identifying controls on FLNF are needed. Here, we provide a mechanistic overview of FLNF, including how various controls may influence FLNF in the rhizosphere in comparison with symbiotic N fixation occurring in plant nodules where environmental conditions are moderated by the plant. We apply this knowledge to a real-world example, the bioenergy crop switchgrass ( Panicum virgatum ), to provide context of how FLNF may function in a managed system. We also highlight future challenges to assessing FLNF and understanding how FLNF functions in the environment and significantly contributes to plant N availability and productivity., (Copyright © 2019 American Society for Microbiology.)

Flavobacterium psychrophilum , the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), causes significant economic losses in salmonid aquaculture, particularly in rainbow trout ( Oncorhynchus mykiss ). Prior studies have used multilocus sequence typing (MLST) to examine genetic heterogeneity within F. psychrophilum At present, however, its population structure in North America is incompletely understood, as only 107 isolates have been genotyped. Herein, MLST was used to investigate the genetic diversity of an additional 314 North American F. psychrophilum isolates that were recovered from ten fish host species from 20 U.S. states and 1 Canadian province over nearly four decades. These isolates were placed into 66 sequence types (STs), 47 of which were novel, increasing the number of clonal complexes (CCs) in North America from 7 to 12. Newly identified CCs were diverse in terms of host association, distribution, and association with disease. The largest F. psychrophilum CC identified was CC-ST10, within which 10 novel genotypes were discovered, most of which came from O. mykiss experiencing BCWD. This discovery, among others, provides evidence for the hypothesis that ST10 (i.e., the founding ST of CC-ST10) originated in North America. Furthermore, ST275 (in CC-ST10) was recovered from wild/feral adult steelhead and marks the first recovery of CC-ST10 from wild/feral fish in North America. Analyses also revealed that at the allele level, the diversification of F. psychrophilum in North America is driven three times more frequently by recombination than random nucleic acid mutation, possibly indicating how new phenotypes emerge within this species. IMPORTANCE Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which cause substantial losses in farmed fish populations worldwide. To better prevent and control BCWD and RTFS outbreaks, we sought to characterize the genetic diversity of several hundred F. psychrophilum isolates that were recovered from diseased fish across North America. Results highlighted multiple F. psychrophilum genetic strains that appear to play an important role in disease events in North American aquaculture facilities and suggest that the practice of trading fish eggs has led to the continental and transcontinental spread of this bacterium. The knowledge generated herein will be invaluable toward guiding the development of future disease prevention techniques., (Copyright © 2019 American Society for Microbiology.)

Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger molecule that is an important virulence regulator in the plant pathogen Erwinia amylovora Intracellular levels of c-di-GMP are modulated by diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP and by phosphodiesterase (PDE) enzymes that degrade c-di-GMP. The regulatory role of the PDE enzymes in E. amylovora has not been determined. Using a combination of single, double, and triple deletion mutants, we determined the effects of each of the four putative PDE-encoding genes ( pdeA , pdeB , pdeC , and edcA ) in E. amylovora on cellular processes related to virulence. Our results indicate that pdeA and pdeC are the two phosphodiesterases most active in virulence regulation in E. amylovora Ea1189. The deletion of pdeC resulted in a measurably significant increase in the intracellular pool of c-di-GMP, and the highest intracellular concentrations of c-di-GMP were observed in the Ea1189 Δ pdeAC and Ea1189 Δ pdeABC mutants. The regulation of virulence traits due to the deletion of the pde genes showed two patterns. A stronger regulatory effect was observed on amylovoran production and biofilm formation, where both Ea1189 Δ pdeA and Ea1189 Δ pdeC mutants exhibited significant increases in these two phenotypes in vitro In contrast, the deletion of two or more pde genes was required to affect motility and virulence phenotypes. Our results indicate a functional redundancy among the pde genes in E. amylovora for certain traits and indicate that the intracellular degradation of c-di-GMP is mainly regulated by pdeA and pdeC , but they also suggest a role for pdeB in regulating motility and virulence. IMPORTANCE Precise control of the expression of virulence genes is essential for successful infection of apple hosts by the fire blight pathogen, Erwinia amylovora The presence and buildup of a signaling molecule called cyclic di-GMP enables the expression and function of some virulence determinants in E. amylovora , such as amylovoran production and biofilm formation. However, other determinants, such as those for motility and the type III secretion system, are expressed and functional when cyclic di-GMP is absent. Here, we report studies of enzymes called phosphodiesterases, which function in the degradation of cyclic di-GMP. We show the importance of these enzymes in virulence gene regulation and the ability of E. amylovora to cause plant disease., (Copyright © 2018 American Society for Microbiology.)

Several studies monitoring alterations in the community structure upon resistant starch (RS) interventions are available, although comprehensive function-based analyses are lacking. Recently, a multiomics approach based on 16S rRNA gene sequencing, metaproteomics, and metabolomics on fecal samples from individuals subjected to high and low doses of type 2 RS (RS2; 48 g and 3 g/2,500 kcal, respectively, daily for 2 weeks) in a crossover intervention experiment was performed. In the present study, we did pathway-based metagenomic analyses on samples from a subset of individuals ( n = 12) from that study to obtain additional detailed insights into the functional structure at high resolution during RS2 intervention. A mechanistic framework based on obtained results is proposed where primary degradation was governed by Firmicutes , with Ruminococcus bromii as a major taxon involved, providing fermentation substrates and increased acetate concentrations for the growth of various major butyrate producers exhibiting the enzyme butyryl-coenzyme A (CoA):acetate CoA-transferase. H 2 -scavenging sulfite reducers and acetogens concurrently increased. Individual responses of gut microbiota were noted, where seven of the 12 participants displayed all features of the outlined pattern, whereas four individuals showed mixed behavior and one subject was unresponsive. Intervention order did not affect the outcome, emphasizing a constant substrate supply for maintaining specific functional communities. IMPORTANCE Manipulation of gut microbiota is increasingly recognized as a promising approach to reduce various noncommunicable diseases, such as obesity and type 2 diabetes. Specific dietary supplements, including resistant starches (RS), are often a focus, yet comprehensive insights into functional responses of microbiota are largely lacking. Furthermore, unresponsiveness in certain individuals is poorly understood. Our data indicate that distinct parts of microbiota work jointly to degrade RS and successively form health-promoting fermentation end products. It highlights the need to consider both primary degraders and specific more-downstream-acting bacterial groups in order to achieve desired intervention outcomes. The gained insights will assist the design of personalized treatment strategies based on an individual's microbiota., (Copyright © 2018 American Society for Microbiology.)

Quorum sensing (QS) is a form of bacterial chemical communication that regulates cellular phenotypes, including certain cooperative behaviors, in response to environmental and demographic changes. Despite the existence of proposed mechanisms that stabilize QS against defector exploitation, it is unclear if or how QS cooperators can proliferate in some model systems in populations mostly consisting of defectors. We predicted that growth in fragmented subpopulations could allow QS cooperators to invade a QS defector population. This could occur despite cooperators having lower relative fitnesses than defectors due to favored weighting of genotypes that produce larger populations of bacteria. Mixed metapopulations of Vibrio QS-proficient or unconditional cooperators and QS defectors were diluted and fragmented into isolated subpopulations in an environment that requires QS-regulated public good production to achieve larger population yields. Under these conditions, we observed global invasions of both cooperator genotypes into populations composed of primarily defectors. This spatially dependent increase in cooperator frequency was replicated for QS cooperators when mixed populations were competed in soft agar motility plates under conditions that allowed cooperators to disperse and outcompete defectors at the population edge, despite being less motile in isolation than defectors. These competition results show that the coordinated growth and dispersal of QS cooperators to additional resources is heavily favored in comparison to unconditional cooperation, and that dispersal of cooperators by motility into new environments, examined here in laboratory populations, constitutes a key mechanism for maintaining QS-regulated cooperation in the face of defection. IMPORTANCE Behaviors that are cooperative in nature are at risk of exploitation by cheating and are thus difficult to maintain by natural selection alone. While bacterial cell-cell communication, known as quorum sensing (QS), can stabilize microbial cooperative behaviors and is widespread in Vibrio species, it is unclear how QS can increase the frequency of cooperative strains in the presence of defectors without additional mechanisms. In this study, we demonstrate under multiple conditions that QS-mediated cooperation can increase in populations of Vibrio strains when cells experience narrow population bottlenecks or disperse from defectors. This occurred for both conditional cooperation mediated by QS and for unconditional cooperation, although conditional cooperators were better able to stabilize cooperation over a much wider range of conditions. Thus, we observed that population structuring allowed for assortment of competing genotypes and promoted cooperation via kin selection in microbes in a QS-dependent manner., (Copyright © 2018 American Society for Microbiology.)

Treatment of food-producing animals with antimicrobial drugs (AMD) is controversial because of concerns regarding promotion of antimicrobial resistance (AMR). To investigate this concern, resistance genes in metagenomic bovine fecal samples during a clinical trial were analyzed to assess the impacts of treatment on beef feedlot cattle resistomes. Four groups of cattle were exposed, using a 2-by-2 factorial design, to different regimens of antimicrobial treatment. Injections of ceftiofur crystalline-free acid (a third-generation cephalosporin) were used to treat all cattle in treatment pens or only a single animal, and either chlortetracycline was included in the feed of all cattle in a pen or the feed was untreated. On days 0 and 26, respectively, pre- and posttrial fecal samples were collected, and resistance genes were characterized using shotgun metagenomics. Treatment with ceftiofur was not associated with changes to β-lactam resistance genes. However, cattle fed chlortetracycline had a significant increase in relative abundance of tetracycline resistance genes. There was also an increase of an AMR class not administered during the study, which is a possible indicator of coselection of resistance genes. Samples analyzed in this study had previously been evaluated by culture characterization ( Escherichia coli and Salmonella ) and quantitative PCR (qPCR) of metagenomic fecal DNA, which allowed comparison of results with this study. In the majority of samples, genes that were selectively enriched through culture and qPCR were not identified through shotgun metagenomic sequencing in this study, suggesting that changes previously documented did not reflect changes affecting the majority of bacterial genetic elements found in the predominant fecal resistome. IMPORTANCE Despite significant concerns about public health implications of AMR in relation to use of AMD in food animals, there are many unknowns about the long- and short-term impact of common uses of AMD for treatment, control, and prevention of disease. Additionally, questions commonly arise regarding how to best measure and quantify AMR genes in relation to public health risks and how to determine which genes are most important. These data provide an introductory view of the utility of using shotgun metagenomic sequencing data as an outcome for clinical trials evaluating the impact of using AMD in food animals., (Copyright © 2018 Weinroth et al.)

Shewanella oneidensis MR-1 is a metal-reducing bacterium with the ability to utilize many different terminal electron acceptors, including oxygen and solid-metal oxides. Both metal oxide reduction and aerobic respiration have been studied extensively in this organism. However, electron transport chain processes upstream of the terminal oxidoreductases have been relatively understudied in this organism, especially electron transfer from NADH to respiratory quinones. Genome annotation indicates that S. oneidensis MR-1 encodes four NADH dehydrogenases, a proton-translocating dehydrogenase (Nuo), two sodium ion-translocating dehydrogenases (Nqr1 and Nqr2), and an "uncoupling" dehydrogenase (Ndh), but none of these complexes have been studied. Therefore, we conducted a study specifically focused on the effects of individual NADH dehydrogenase knockouts in S. oneidensis MR-1. We observed that two of the single-mutant strains, the Δ nuoN and Δ nqrF1 mutants, exhibited significant growth defects compared with the wild type. However, the defects were minor and only apparent under certain growth conditions. Further testing of the Δ nuoN Δ nqrF1 double-mutant strain yielded no growth in minimal medium under oxic conditions, indicating that Nuo and Nqr1 have overlapping functions, but at least one is necessary for aerobic growth. Coutilization of proton- and sodium ion-dependent energetics has important implications for the growth of this organism in environments with varied pH and salinity, including microbial electrochemical systems. IMPORTANCE Bacteria utilize a wide variety of metabolic pathways that allow them to take advantage of different energy sources, and to do so with varied efficiency. The efficiency of a metabolic process determines the growth yield of an organism, or the amount of biomass it produces per amount of substrate consumed. This parameter has important implications in biotechnology and wastewater treatment, where low growth yields are often preferred to minimize the production of microbial biomass. In this study, we investigated respiratory pathways containing NADH dehydrogenases with varied efficiency (i.e., the number of ions translocated per NADH oxidized) in the metal-reducing bacterium Shewanella oneidensis MR-1. We observed that two different respiratory pathways are used concurrently, and at least one pathway must be functional for growth under oxic conditions., (Copyright © 2018 Duhl et al.)

A site in Oak Ridge, TN, USA, has sediments that contain >3% iron oxides and is contaminated with uranium (U). The U(VI) was bioreduced to U(IV) and immobilized in situ through intermittent injections of ethanol. It then was allowed to reoxidize via the invasion of low-pH (3.6 to 4.0), high-nitrate (up to 200 mM) groundwater back into the reduced zone for 1,383 days. To examine the biogeochemical response, high-throughput sequencing and network analysis were applied to characterize bacterial population shifts, as well as cooccurrence and coexclusion patterns among microbial communities. A paired t test indicated no significant changes of α-diversity for the bioactive wells. However, both nonmetric multidimensional scaling and analysis of similarity confirmed a significant distinction in the overall composition of the bacterial communities between the bioreduced and the reoxidized sediments. The top 20 major genera accounted for >70% of the cumulative contribution to the dissimilarity in the bacterial communities before and after the groundwater invasion. Castellaniella had the largest dissimilarity contribution (17.7%). For the bioactive wells, the abundance of the U(VI)-reducing genera Geothrix , Desulfovibrio , Ferribacterium , and Geobacter decreased significantly, whereas the denitrifying Acidovorax abundance increased significantly after groundwater invasion. Additionally, seven genera, i.e., Castellaniella , Ignavibacterium , Simplicispira , Rhizomicrobium , Acidobacteria Gp1, Acidobacteria Gp14, and Acidobacteria Gp23, were significant indicators of bioactive wells in the reoxidation stage. Canonical correspondence analysis indicated that nitrate, manganese, and pH affected mostly the U(VI)-reducing genera and indicator genera. Cooccurrence patterns among microbial taxa suggested the presence of taxa sharing similar ecological niches or mutualism/commensalism/synergism interactions. IMPORTANCE High-throughput sequencing technology in combination with a network analysis approach were used to investigate the stabilization of uranium and the corresponding dynamics of bacterial communities under field conditions with regard to the heterogeneity and complexity of the subsurface over the long term. The study also examined diversity and microbial community composition shift, the common genera, and indicator genera before and after long-term contaminated-groundwater invasion and the relationship between the target functional community structure and environmental factors. Additionally, deciphering cooccurrence and coexclusion patterns among microbial taxa and environmental parameters could help predict potential biotic interactions (cooperation/competition), shared physiologies, or habitat affinities, thus, improving our understanding of ecological niches occupied by certain specific species. These findings offer new insights into compositions of and associations among bacterial communities and serve as a foundation for future bioreduction implementation and monitoring efforts applied to uranium-contaminated sites., (Copyright © 2018 American Society for Microbiology.)

Recently, methanotrophic and methylotrophic bacteria were found to utilize rare earth elements (REEs). To monitor the REE content in culture media of these bacteria, we have developed a rapid screening method using the Arsenazo III (AS III) dye for spectrophotometric REE detection in the low μM (0.1 to 10 μM) range. We designed this assay to follow La III and Eu III depletion from the culture medium by the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum strain SolV. The assay can also be modified to screen the uptake of other REEs, such as Pr III , or to monitor the depletion of La III from growth media in neutrophilic methylotrophs such as Methylobacterium extorquens strain AM1. The AS III assay presents a convenient and fast detection method for REE levels in culture media and is a sensitive alternative to inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption spectroscopy (AAS). IMPORTANCE REE-dependent bacterial metabolism is a quickly emerging field, and while the importance of REEs for both methanotrophic and methylotrophic bacteria is now firmly established, many important questions, such as how these insoluble elements are taken up into cells, are still unanswered. Here, an Arsenazo III dye-based assay has been developed for fast, specific, and sensitive determination of REE content in different culture media. This assay presents a useful tool for optimizing cultivation protocols, as well as for routine REE monitoring during bacterial growth without the need for specialized analytical instrumentation. Furthermore, this assay has the potential to promote the discovery of other REE-dependent microorganisms and can help to elucidate the mechanisms for acquisition of REEs by methanotrophic and methylotrophic bacteria., (Copyright © 2018 Hogendoorn et al.)

The most common practice in studying and cataloguing prokaryotic diversity involves the grouping of sequences into operational taxonomic units (OTUs) at the 97% 16S rRNA gene sequence identity level, often using partial gene sequences, such as PCR-generated amplicons. Due to the high sequence conservation of rRNA genes, organisms belonging to closely related yet distinct species may be grouped under the same OTU. However, it remains unclear how much diversity has been underestimated by this practice. To address this question, we compared the OTUs of genomes defined at the 97% or 98.5% 16S rRNA gene identity level against OTUs of the same genomes defined at the 95% whole-genome average nucleotide identity (ANI), which is a much more accurate proxy for species. Our results show that OTUs resulting from a 98.5% 16S rRNA gene identity cutoff are more accurate than 97% compared to 95% ANI (90.5% versus 89.9% accuracy) but indistinguishable from any other threshold in the 98.29 to 98.78% range. Even with the more stringent thresholds, however, the 16S rRNA gene-based approach commonly underestimates the number of OTUs by ∼12%, on average, compared to the ANI-based approach (∼14% underestimation when using the 97% identity threshold). More importantly, the degree of underestimation can become 50% or more for certain taxa, such as the genera Pseudomonas , Burkholderia , Escherichia , Campylobacter , and Citrobacter These results provide a quantitative view of the degree of underestimation of extant prokaryotic diversity by 16S rRNA gene-defined OTUs and suggest that genomic resolution is often necessary. IMPORTANCE Species diversity is one of the most fundamental pieces of information for community ecology and conservational biology. Therefore, employing accurate proxies for what a species or the unit of diversity is are cornerstones for a large set of microbial ecology and diversity studies. The most common proxies currently used rely on the clustering of 16S rRNA gene sequences at some threshold of nucleotide identity, typically 97% or 98.5%. Here, we explore how well this strategy reflects the more accurate whole-genome-based proxies and determine the frequency with which the high conservation of 16S rRNA sequences masks substantial species-level diversity., (Copyright © 2018 American Society for Microbiology.)

Niche partitioning and sequence evolution drive genomic and phenotypic divergence, which ultimately leads to bacterial diversification. This study investigated the genomic composition of two Shewanella baltica clades previously identified through multilocus sequencing typing and recovered from the redox transition zone in the central Baltic Sea. Comparative genomic analysis revealed significantly higher interclade than intraclade genomic dissimilarity and that a subset of genes present in clade A were associated with potential adaptation to respiration of sulfur compounds present in the redox transition zone. The transcriptomic divergence between two representative strains of clades A and D, OS185 and OS195, was also characterized and revealed marked regulatory differences. We found that both the transcriptional divergence of shared genes and expression of strain-specific genes led to differences in regulatory patterns between strains that correlate with environmental redox niches. For instance, under anoxic conditions of respiratory nitrate ammonification, OS185-the strain isolated from a nitrate-rich environment-upregulated nearly twice the number of shared genes upregulated by OS195-the strain isolated from an H 2 S-containing anoxic environment. Conversely, OS195 showed stronger induction of strain-specific genes, especially those associated with sulfur compound respiration, under thiosulfate-reducing conditions. A positive association between the level of transcriptional divergence and the level of sequence divergence for shared genes was also noted. Our results provide further support for the hypothesis that genomic changes impacting transcriptional regulation play an important role in the diversification of ecologically distinct populations. IMPORTANCE This study examined potential mechanisms through which co-occurring Shewanella baltica strains diversified to form ecologically distinct populations. At the time of isolation, the strains studied composed the major fraction of culturable nitrate-reducing communities in the Baltica Sea. Analysis of genomic content of 13 S. baltica strains from two clades representing different ecotypes demonstrated that one clade specifically possesses a number of genes that could favor successful adaptation to respire sulfur compounds in the portion of the water column from which these strains were isolated. In addition, transcriptional profiling of fully sequenced strains representative of these two clades, OS185 and OS195, under oxygen-, nitrate-, and thiosulfate-respiring conditions demonstrated that the strains exhibit relatively similar transcriptional responses during aerobic growth but more-distinct transcriptional responses under nitrate- and thiosulfate-respiring conditions. Results from this study provide insights into how genomic and gene regulatory diversification together impacted the redox specialization of the S. baltica strains., (Copyright © 2018 American Society for Microbiology.)

Cyanobacteria are emerging as alternative crop species for the production of fuels, chemicals, and biomass. Yet, the success of these microbes depends on the development of cost-effective technologies that permit scaled cultivation and cell harvesting. Here, we investigate the feasibility of engineering cell morphology to improve biomass recovery and decrease energetic costs associated with lysing cyanobacterial cells. Specifically, we modify the levels of Min system proteins in Synechococcus elongatus PCC 7942. The Min system has established functions in controlling cell division by regulating the assembly of FtsZ, a tubulin-like protein required for defining the bacterial division plane. We show that altering the expression of two FtsZ-regulatory proteins, MinC and Cdv3, enables control over cell morphology by disrupting FtsZ localization and cell division without preventing continued cell growth. By varying the expression of these proteins, we can tune the lengths of cyanobacterial cells across a broad dynamic range, anywhere from an ∼20% increased length (relative to the wild type) to near-millimeter lengths. Highly elongated cells exhibit increased rates of sedimentation under low centrifugal forces or by gravity-assisted settling. Furthermore, hyperelongated cells are also more susceptible to lysis through the application of mild physical stress. Collectively, these results demonstrate a novel approach toward decreasing harvesting and processing costs associated with mass cyanobacterial cultivation by altering morphology at the cellular level. IMPORTANCE We show that the cell length of a model cyanobacterial species can be programmed by rationally manipulating the expression of protein factors that suppress cell division. In some instances, we can increase the size of these cells to near-millimeter lengths with this approach. The resulting elongated cells have favorable properties with regard to cell harvesting and lysis. Furthermore, cells treated in this manner continue to grow rapidly at time scales similar to those of uninduced controls. To our knowledge, this is the first reported example of engineering the cell morphology of cyanobacteria or algae to make them more compatible with downstream processing steps that present economic barriers to their use as alternative crop species. Therefore, our results are a promising proof-of-principle for the use of morphology engineering to increase the cost-effectiveness of the mass cultivation of cyanobacteria for various sustainability initiatives., (Copyright © 2017 American Society for Microbiology.)

Communication has been suggested as a mechanism to stabilize cooperation. In bacteria, chemical communication, termed quorum sensing (QS), has been hypothesized to fill this role, and extracellular public goods are often induced by QS at high cell densities. Here we show, with the bacterium Vibrio harveyi, that QS provides strong resistance against invasion of a QS defector strain by maximizing the cellular growth rate at low cell densities while achieving maximum productivity through protease upregulation at high cell densities. In contrast, QS mutants that act as defectors or unconditional cooperators maximize either the growth rate or the growth yield, respectively, and thus are less fit than the wild-type QS strain. Our findings provide experimental evidence that regulation mediated by microbial communication can optimize growth strategies and stabilize cooperative phenotypes by preventing defector invasion, even under well-mixed conditions. This effect is due to a combination of responsiveness to environmental conditions provided by QS, lowering of competitive costs when QS is not induced, and pleiotropic constraints imposed on defectors that do not perform QS., Importance: Cooperation is a fundamental problem for evolutionary biology to explain. Conditional participation through phenotypic plasticity driven by communication is a potential solution to this dilemma. Thus, among bacteria, QS has been proposed to be a proximate stabilizing mechanism for cooperative behaviors. Here, we empirically demonstrate that QS in V. harveyi prevents cheating and subsequent invasion by nonproducing defectors by maximizing the growth rate at low cell densities and the growth yield at high cell densities, whereas an unconditional cooperator is rapidly driven to extinction by defectors. Our findings provide experimental evidence that QS regulation prevents the invasion of cooperative populations by QS defectors even under unstructured conditions, and they strongly support the role of communication in bacteria as a mechanism that stabilizes cooperative traits., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that can cause hemorrhagic colitis and hemolytic-uremic syndrome. Cattle are the primary reservoir for STEC, and food or water contaminated with cattle feces is the most common source of infections in humans. Consequently, we conducted a cross-sectional study of 1,096 cattle in six dairy herds (n = 718 animals) and five beef herds (n = 378 animals) in the summers of 2011 and 2012 to identify epidemiological factors associated with shedding. Fecal samples were obtained from each animal and cultured for STEC. Multivariate analyses were performed to identify risk factors associated with STEC positivity. The prevalence of STEC was higher in beef cattle (21%) than dairy cattle (13%) (odds ratio [OR], 1.76; 95% confidence interval [CI], 1.25, 2.47), with considerable variation occurring across herds (range, 6% to 54%). Dairy cattle were significantly more likely to shed STEC when the average temperature was >28.9°C 1 to 5 days prior to sampling (OR, 2.5; 95% CI, 1.25, 4.91), during their first lactation (OR, 1.8; 95% CI, 1.1, 2.8), and when they were <30 days in milk (OR, 3.9; 95% CI, 2.1, 7.2). These data suggest that the stress or the negative energy balance associated with lactation may result in increased STEC shedding frequencies in Michigan during the warm summer months. Future prevention strategies aimed at reducing stress during lactation or isolating high-risk animals could be implemented to reduce herd-level shedding levels and avoid transmission of STEC to susceptible animals and people., Importance: STEC shedding frequencies vary considerably across cattle herds in Michigan, and the shedding frequency of strains belonging to non-O157 serotypes far exceeds the shedding frequency of O157 strains, which is congruent with human infections in the state. Dairy cattle sampled at higher temperatures, in their first lactation, and early in the milk production stage were significantly more likely to shed STEC, which could be due to stress or a negative energy balance. Future studies should focus on the isolation of high-risk animals to decrease herd shedding levels and the potential for contamination of the food supply., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: This paper examines the recovery of the enteric adenovirus human adenovirus 40 (HAdV 40) by cross-flow ultrafiltration and interprets recovery values in terms of physicochemical interactions of virions during sample concentration. Prior to ultrafiltration, membranes were either blocked by exposure to calf serum (CS) or coated with a polyelectrolyte multilayer (PEM). HAdV 40 is a hydrophobic virus with a point of zero charge between pH 4.0 and pH 4.3. In accordance with predictions from the extended Derjaguin-Landau-Verwey-Overbeek theory, the preelution recovery of HAdV (rpre) from deionized water was higher with PEM-coated membranes (rpre (PEM) = 74.8% ± 9.7%) than with CS-blocked membranes (rpre (CS) = 54.1% ± 6.2%). With either membrane type, the total virion recovery after elution (rpost) was high for both deionized water (rpost (PEM) = 99.5% ± 6.6% and rpost (CS) = 98.8% ± 7.7%) and tap water (rpost (PEM) = 89% ± 15% and rpost (CS) = 93.7% ± 6.9%). The nearly 100% recoveries suggest that the polyanion (sodium polyphosphate) and surfactant (Tween 80) in the eluent disrupt electrostatic and hydrophobic interactions between the virion and the membrane. Addition of EDTA to the eluent greatly improved the elution efficacy (rpost (CS) = 88.6% ± 4.3% and rpost (PEM) = 87.0% ± 6.9%) with surface water, even when the organic carbon concentration in the water was high (9.4 ± 0.1 mg/liter). EDTA likely disrupts cation bridging between virions and particles in the feed water matrix or the fouling layer on the membrane surface. For complex water matrices, the eluent composition is the most important factor for achieving high virion recovery., Importance: Herein we present the results of a comprehensive physicochemical characterization of HAdV 40, an important human pathogen. The data on HAdV 40 surface properties enabled rigorous modeling to gain an understanding of the energetics of virion-virion and virion-filter interactions. Cross-flow filtration for concentration and recovery of HAdV 40 was evaluated, with postelution recoveries from ultrapure water (99%), tap water (∼91%), and high-carbon-content surface water (∼84%) being demonstrated. These results are significant because of the very low adenovirus recoveries that have been reported, to date, for other methods. The recovery data were interpreted in terms of specific interactions, and the eluent composition was designed accordingly to maximize HAdV 40 recovery., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The use of a multilocus sequence typing (MLST) technique has identified the intraspecific genetic diversity of U.S. Flavobacterium psychrophilum, an important pathogen of salmonids worldwide. Prior to this analysis, little U.S. F. psychrophilum genetic information was known; this is of importance when considering targeted control strategies, including vaccine development. Herein, MLST was used to investigate the genetic diversity of 96 F. psychrophilum isolates recovered from rainbow trout (Oncorhynchus mykiss), coho salmon (Oncorhynchus kisutch), and Chinook salmon (Oncorhynchus tshawytscha) that originated from nine U.S. states. The isolates fell into 34 distinct sequence types (STs) that clustered in 5 clonal complexes (CCs) (n = 63) or were singletons (n = 33). The distribution of STs varied spatially, by host species, and in association with mortality events. Several STs (i.e., ST9, ST10, ST30, and ST78) were found in multiple states, whereas the remaining STs were localized to single states. With the exception of ST256, which was recovered from rainbow trout and Chinook salmon, all STs were found to infect a single host species. Isolates that were collected during bacterial cold water disease outbreaks most frequently belonged to CC-ST10 (e.g., ST10 and ST78). Collectively, the results of this study clearly demonstrate the genetic diversity of F. psychrophilum within the United States and identify STs of clinical significance. Although the majority of STs described herein were novel, some (e.g., ST9, ST10, ST13, ST30, and ST31) were previously recovered on other continents, which demonstrates the transcontinental distribution of F. psychrophilum genotypes., Importance: Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS) and is an important bacterial pathogen of wild and farmed salmonids worldwide. These infections are responsible for large economic losses globally, yet the genetic diversity of this pathogen remains to be fully investigated. Previous studies have identified the genetic diversity of this pathogen in other main aquaculture regions; however, little effort has been focused on the United States. In this context, this study aims to examine the genetic diversity of F. psychrophilum from the United States, as this region remains important in salmonid aquaculture., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Ecological understanding of disease risk, emergence, and dynamics and of the efficacy of control strategies relies heavily on efficient tools for microorganism identification and characterization. Misdetection, such as the misclassification of infected hosts as healthy, can strongly bias estimates of disease prevalence and lead to inaccurate conclusions. In natural plant ecosystems, interest in assessing microbial dynamics is increasing exponentially, but guidelines for detection of microorganisms in wild plants remain limited, particularly so for plant viruses. To address this gap, we explored issues and solutions associated with virus detection by serological and molecular methods in noncrop plant species as applied to the globally important Barley yellow dwarf virus PAV (Luteoviridae), which infects wild native plants as well as crops. With enzyme-linked immunosorbent assays (ELISA), we demonstrate how virus detection in a perennial wild plant species may be much greater in stems than in leaves, although leaves are most commonly sampled, and may also vary among tillers within an individual, thereby highlighting the importance of designing effective sampling strategies. With reverse transcription-PCR (RT-PCR), we demonstrate how inhibitors in tissues of perennial wild hosts can suppress virus detection but can be overcome with methods and products that improve isolation and amplification of nucleic acids. These examples demonstrate the paramount importance of testing and validating survey designs and virus detection methods for noncrop plant communities to ensure accurate ecological surveys and reliable assumptions about virus dynamics in wild hosts., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes (vcrA, bvcA, and tceA) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Bacterial microcompartments (BMCs) are proteinaceous organelles encapsulating enzymes that catalyze sequential reactions of metabolic pathways. BMCs are phylogenetically widespread; however, only a few BMCs have been experimentally characterized. Among them are the carboxysomes and the propanediol- and ethanolamine-utilizing microcompartments, which play diverse metabolic and ecological roles. The substrate of a BMC is defined by its signature enzyme. In catabolic BMCs, this enzyme typically generates an aldehyde. Recently, it was shown that the most prevalent signature enzymes encoded by BMC loci are glycyl radical enzymes, yet little is known about the function of these BMCs. Here we characterize the glycyl radical enzyme-associated microcompartment (GRM) loci using a combination of bioinformatic analyses and active-site and structural modeling to show that the GRMs comprise five subtypes. We predict distinct functions for the GRMs, including the degradation of choline, propanediol, and fuculose phosphate. This is the first family of BMCs for which identification of the signature enzyme is insufficient for predicting function. The distinct GRM functions are also reflected in differences in shell composition and apparently different assembly pathways. The GRMs are the counterparts of the vitamin B12-dependent propanediol- and ethanolamine-utilizing BMCs, which are frequently associated with virulence. This study provides a comprehensive foundation for experimental investigations of the diverse roles of GRMs. Understanding this plasticity of function within a single BMC family, including characterization of differences in permeability and assembly, can inform approaches to BMC bioengineering and the design of therapeutics., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Temporal changes in the distribution of Salmonella subtypes in livestock populations may have important impacts on human health. The first objective of this research was to determine the within-farm changes in the population of subtypes of Salmonella on Michigan dairy farms that were sampled longitudinally in 2000-2001 and again in 2009. The second objective was to determine the yearly frequency (2001 through 2012) of reported human illnesses in Michigan associated with the same subtypes. Comparable sampling techniques were used to collect fecal and environmental samples from the same 18 Michigan dairy farms in 2000-2001 and 2009. Serotypes, multilocus sequence types (STs), and pulsed-field gel electrophoresis (PFGE) banding patterns were identified for isolates from 6 farms where >1 Salmonella isolate was recovered in both 2000-2001 and 2009. The distribution of STs was significantly different between time frames (P < 0.05); only two of 31 PFGE patterns were identified in both time frames, and each was recovered from the same farm in each time frame. Previously reported within-farm decreases in the frequency of multidrug-resistant (MDR) Salmonella were due to recovery of MDR subtypes of S. enterica serotypes Senftenberg and Typhimurium in 2000-2001 and genetically distinct, pansusceptible subtypes of the same serotypes in 2009. The annual frequency of human illnesses between 2001 and 2012 with a PFGE pattern matching a bovine strain decreased for patterns recovered from dairy farms in 2000-2001 and increased for patterns recovered in 2009. These data suggest important changes in the population of Salmonella on dairy farms and in the frequency of human illnesses associated with cattle-derived subtypes., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The sequencing chips and kits of the Ion Torrent Personal Genome Machine (PGM), which employs semiconductor technology to measure pH changes in polymerization events, have recently been upgraded. The quality of PGM sequences has not been reassessed, and results have not been compared in the context of a gene-targeted microbial ecology study. To address this, we compared sequence profiles across available PGM chips and chemistries and with 454 pyrosequencing data by determining error types and rates and diazotrophic community structures. The PGM was then used to assess differences in nifH-harboring bacterial community structure among four corn-based cropping systems. Using our suggested filters from mock community analyses, the overall error rates were 0.62, 0.36, and 0.39% per base for chips 318 and 314 with the 400-bp kit and chip 318 with the Hi-Q chemistry, respectively. Compared with the 400-bp kit, the Hi-Q kit reduced indel rates by 28 to 59% and produced one to seven times more reads acceptable for downstream analyses. The PGM produced higher frameshift rates than pyrosequencing that were corrected by the RDP FrameBot tool. Significant differences among platforms were identified, although the diversity indices and overall site-based conclusions remained similar. For the cropping system analyses, a total of 6,182 unique NifH operational taxonomic units at 5% amino acid dissimilarity were obtained. The current crop type, as well as the crop rotation history, significantly influenced the composition of the soil diazotrophic community detected., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A pilot-scale field experiment demonstrated that a one-time amendment of emulsified vegetable oil (EVO) reduced groundwater U(VI) concentrations for 1 year in a fast-flowing aquifer. However, little is known about how EVO amendment stimulates the functional gene composition, structure, and dynamics of groundwater microbial communities toward prolonged U(VI) reduction. In this study, we hypothesized that EVO amendment would shift the functional gene composition and structure of groundwater microbial communities and stimulate key functional genes/groups involved in EVO biodegradation and reduction of electron acceptors in the aquifer. To test these hypotheses, groundwater microbial communities after EVO amendment were analyzed using a comprehensive functional gene microarray. Our results showed that EVO amendment stimulated sequential shifts in the functional composition and structure of groundwater microbial communities. Particularly, the relative abundance of key functional genes/groups involved in EVO biodegradation and the reduction of NO3 (-), Mn(IV), Fe(III), U(VI), and SO4 (2-) significantly increased, especially during the active U(VI) reduction period. The relative abundance for some of these key functional genes/groups remained elevated over 9 months. Montel tests suggested that the dynamics in the abundance, composition, and structure of these key functional genes/groups were significantly correlated with groundwater concentrations of acetate, NO3 (-), Mn(II), Fe(II), U(VI), and SO4 (2-). Our results suggest that EVO amendment stimulated dynamic succession of key functional microbial communities. This study improves our understanding of the composition, structure, and function changes needed for groundwater microbial communities to sustain a long-term U(VI) reduction., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Flavobacteria (members of the family Flavobacteriaceae) dominate the bacterial community in the Anopheles mosquito midgut. One such commensal, Elizabethkingia anophelis, is closely associated with Anopheles mosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements of E. anophelis have not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation of E. anophelis, including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function in E. anophelis. A flavobacterial expression system based on the promoter PompA was integrated into the E. anophelis chromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-tagged E. anophelis associated with mosquitoes at successive developmental stages and propagated in Anopheles gambiae and Anopheles stephensi but not in Aedes triseriatus mosquitoes. Feeding NanoLuc-tagged cells to A. gambiae and A. stephensi in the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected in Aedes triseriatus mosquitoes under the same conditions. Of the initial E. anophelis cells provided to larvae, 23%, 71%, and 85% were digested in A. stephensi, A. gambiae, and Aedes triseriatus, respectively, demonstrating that E. anophelis adapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells in A. stephensi significantly increased when arginine was added to a sugar diet, showing it to be an important amino acid for E. anophelis. Animal erythrocytes promoted E. anophelis growth in vivo and in vitro, indicating that this bacterium could obtain nutrients by participating in erythrocyte lysis in the mosquito midgut., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The suitability of traditional microbial indicators (i.e., Escherichia coli and enterococci) has been challenged due to the lack of correlation with pathogens and evidence of possible regrowth in the natural environment. In this study, the relationships between alternative microbial indicators of potential human fecal contamination (Bacteroides thetaiotaomicron, Methanobrevibacter smithii, human polyomaviruses [HPyVs], and F+ and somatic coliphages) and pathogens (Salmonella spp., Pseudomonas aeruginosa, rotavirus, astrovirus, norovirus GI, norovirus GII, and adenovirus) were compared with those of traditional microbial indicators, as well as environmental parameters (temperature, conductivity, salinity, pH, dissolved oxygen, total organic carbon, total suspended solids, turbidity, total nitrogen, and total phosphorus). Water samples were collected from surface waters of urban catchments in Singapore. Salmonella and P. aeruginosa had significant positive correlations with most of the microbial indicators, especially E. coli and enterococci. Norovirus GII showed moderately strong positive correlations with most of the microbial indicators, except for HPyVs and coliphages. In general, high geometric means and significant correlations between human-specific markers and pathogens suggest the possibility of sewage contamination in some areas. The simultaneous detection of human-specific markers (i.e., B. thetaiotaomicron, M. smithii, and HPyVs) with E. coli and enterococcus supports the likelihood of recent fecal contamination, since the human-specific markers are unable to regrow in natural surface waters. Multiple-linear-regression results further confirm that the inclusion of M. smithii and HPyVs, together with traditional indicators, would better predict the occurrence of pathogens. Further study is needed to determine the applicability of such models to different geographical locations and environmental conditions., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

White-tailed deer are an important reservoir for pathogens that can contribute a large portion of microbial pollution in fragmented agricultural and forest landscapes. The scarcity of experimental data on survival of microorganisms in and release from deer feces makes prediction of their fate and transport less reliable and development of efficient strategies for environment protection more difficult. The goal of this study was to estimate parameters for modeling Escherichia coli survival in and release from deer (Odocoileus virginianus) feces. Our objectives were as follows: (i) to measure survival of E. coli in deer pellets at different temperatures, (ii) to measure kinetics of E. coli release from deer pellets at different rainfall intensities, and (iii) to estimate parameters of models describing survival and release of microorganisms from deer feces. Laboratory experiments were conducted to study E. coli survival in deer pellets at three temperatures and to estimate parameters of Chick's exponential model with temperature correction based on the Arrhenius equation. Kinetics of E. coli release from deer pellets were measured at two rainfall intensities and used to derive the parameters of Bradford-Schijven model of bacterial release. The results showed that parameters of the survival and release models obtained for E. coli in this study substantially differed from those obtained by using other source materials, e.g., feces of domestic animals and manures. This emphasizes the necessity of comprehensive studies of survival of naturally occurring populations of microorganisms in and release from wildlife animal feces in order to achieve better predictions of microbial fate and transport in fragmented agricultural and forest landscapes., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Biofilms formed by dissimilatory metal reducers are of interest to develop permeable biobarriers for the immobilization of soluble contaminants such as uranium. Here we show that biofilms of the model uranium-reducing bacterium Geobacter sulfurreducens immobilized substantially more U(VI) than planktonic cells and did so for longer periods of time, reductively precipitating it to a mononuclear U(IV) phase involving carbon ligands. The biofilms also tolerated high and otherwise toxic concentrations (up to 5 mM) of uranium, consistent with a respiratory strategy that also protected the cells from uranium toxicity. The enhanced ability of the biofilms to immobilize uranium correlated only partially with the biofilm biomass and thickness and depended greatly on the area of the biofilm exposed to the soluble contaminant. In contrast, uranium reduction depended on the expression of Geobacter conductive pili and, to a lesser extent, on the presence of the c cytochrome OmcZ in the biofilm matrix. The results support a model in which the electroactive biofilm matrix immobilizes and reduces the uranium in the top stratum. This mechanism prevents the permeation and mineralization of uranium in the cell envelope, thereby preserving essential cellular functions and enhancing the catalytic capacity of Geobacter cells to reduce uranium. Hence, the biofilms provide cells with a physically and chemically protected environment for the sustained immobilization and reduction of uranium that is of interest for the development of improved strategies for the in situ bioremediation of environments impacted by uranium contamination., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Shiga toxin-producing Escherichia coli (STEC) infections are a critical public health concern because they can cause severe clinical outcomes, such as hemolytic uremic syndrome, in humans. Determining the presence or absence of virulence genes is essential in assessing the potential pathogenicity of STEC strains. Currently, there is limited information about the virulence genes carried by swine STEC strains; therefore, this study was conducted to examine the presence and absence of 69 virulence genes in STEC strains recovered previously from finishing swine in a longitudinal study. A subset of STEC strains was analyzed by pulsed-field gel electrophoresis (PFGE) to examine their genetic relatedness. Swine STEC strains (n = 150) were analyzed by the use of a high-throughput real-time PCR array system, which included 69 virulence gene targets. Three major pathotypes consisted of 16 different combinations of virulence gene profiles, and serotypes were determined in the swine STEC strains. The majority of the swine STEC strains (n = 120) belonged to serotype O59:H21 and carried the same virulence gene profile, which consisted of 9 virulence genes: stx2e, iha, ecs1763, lpfAO113, estIa (STa), ehaA, paa, terE, and ureD. The eae, nleF, and nleH1-2 genes were detected in one swine STEC strain (O49:H21). Other genes encoding adhesins, including iha, were identified (n = 149). The PFGE results demonstrated that swine STEC strains from pigs raised in the same finishing barn were closely related. Our results revealed diverse virulence gene contents among the members of the swine STEC population and enhance understanding of the dynamics of transmission of STEC strains among pigs housed in the same barn., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.