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ABSTRACT Archaea are widespread in the environment and play fundamental roles in diverse ecosystems; however, characterization of their unique biology requires advanced tools. This is particularly challenging when characterizing gene function. Here, we generate randomly barcoded transposon libraries in the model methanogenic archaeon Methanococcus maripaludis and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions. Using our approach, we identified new genes involved in nutrient utilization and response to oxidative stress. We identified novel genes for the usage of diverse nitrogen sources in M. maripaludis including a putative regulator of alanine deamination and molybdate transporters important for nitrogen fixation. Furthermore, leveraging the fitness data, we inferred that M. maripaludis can utilize additional nitrogen sources including ʟ-glutamine, ᴅ-glucuronamide, and adenosine. Under autotrophic growth conditions, we identified a gene encoding a domain of unknown function (DUF166) that is important for fitness and hypothesize that it has an accessory role in carbon dioxide assimilation. Finally, comparing fitness costs of oxygen versus sulfite stress, we identified a previously uncharacterized class of dissimilatory sulfite reductase-like proteins (Dsr-LP; group IIId) that is important during growth in the presence of sulfite. When overexpressed, Dsr-LP conferred sulfite resistance and enabled use of sulfite as the sole sulfur source. The high-throughput approach employed here allowed for generation of a large-scale data set that can be used as a resource to further understand gene function and metabolism in the archaeal domain.IMPORTANCEArchaea are widespread in the environment, yet basic aspects of their biology remain underexplored. To address this, we apply randomly barcoded transposon libraries (RB-TnSeq) to the model archaeon Methanococcus maripaludis. RB-TnSeq coupled with high-throughput growth assays across over 100 unique conditions identified roles for previously uncharacterized genes, including several encoding proteins with domains of unknown function (DUFs). We also expand on our understanding of carbon and nitrogen metabolism and characterize a group IIId dissimilatory sulfite reductase-like protein as a functional sulfite reductase. This data set encompasses a wide range of additional conditions including stress, nitrogen fixation, amino acid supplementation, and autotrophy, thus providing an extensive data set for the archaeal community to mine for characterizing additional genes of unknown function.

ABSTRACT Eravacycline is a synthetic fluorocycline approved by the U.S. Food and Drug Administration in 2018. This study aimed to describe clinical and microbiological outcomes in addition to associated adverse effects of eravacycline used in U.S. hospitals. Real-world, observational study involving patients receiving ≥72 h of eravacycline at 19 medical centers located in all 5 regions of the United States between October 2018 and August 2022. The primary outcome was clinical success, defined as survival and absence of microbiological recurrence at 30 days from the end of eravacycline therapy and clinical improvement within 96 h of eravacycline initiation. In total, 416 patients met study criteria and were evaluated. Index culture specimens were most often isolated from the respiratory tract (24.8%, n = 103/416), wound(s) (20.9%, n = 87/416), or blood (19.5%, n = 81/416). As definitive therapy, eravacycline was most often used to treat infections caused by Enterobacterales spp. (42.3%, n = 176/416; 24.4%, n = 43/176 carbapenem-resistant), Enterococci spp. (24.0%, n = 100/416; 49.0%, 49/100 vancomycin-resistant), and Acinetobacter spp. (23.3%, n = 97/416; 47.4%, n = 46/97 carbapenem-resistant). Clinical success occurred in 75.7% of patients (n = 315/416). Thirty-nine (9.4%, n = 39/416) patients experienced a treatment emergent adverse event (TEAE) potentially related to eravacycline with the majority (51.3%, n = 20/39) being gastrointestinal intolerance. Only 27 isolates (6.5%, n = 27/416) underwent eravacycline susceptibility testing. Eravacycline is being used to treat a broad range of Gram-negative and Gram-positive bacteria in the United States including those demonstrating multidrug-resistance with consistently low reported drug-related TEAE; however, antimicrobial susceptibility testing and subsequent in vitro susceptibility data of clinical isolates was sparingly performed. IMPORTANCE The rise of multidrug-resistant (MDR) pathogens, especially MDR Gram-negatives, poses a significant challenge to clinicians and public health. These resilient bacteria have rendered many traditional antibiotics ineffective, underscoring the urgency for innovative therapeutic solutions. Eravacycline, a broad-spectrum fluorocycline tetracycline antibiotic approved by the FDA in 2018, emerges as a promising candidate, exhibiting potential against a diverse array of MDR bacteria, including Gram-negative, Gram-positive, anaerobic strains, and Mycobacterium. However, comprehensive data on its real-world application remain scarce. This retrospective cohort study, one of the largest of its kind, delves into the utilization of eravacycline across various infectious conditions in the USA during its initial 4 years post-FDA approval. Through assessing clinical, microbiological, and tolerability outcomes, the research offers pivotal insights into eravacycline’s efficacy in addressing the pressing global challenge of MDR bacterial infections.

ABSTRACT Phage therapy has gained attention due to the spread of antibiotic-resistant bacteria and narrow pipeline of novel antibiotics. Phage cocktails are hypothesized to slow the overall development of resistance by challenging the bacteria with more than one phage. Here, we have used a combination of plate-, planktonic-, and biofilm-based screening assays to try to identify phage-antibiotic combinations that will eradicate preformed biofilms of Staphylococcus aureus strains that are otherwise difficult to kill. We have focused on methicillin-resistant S aureus (MRSA) strains and their daptomycin-nonsusceptible vancomycin-intermediate (DNS-VISA) derivatives to understand whether the phage-antibiotic interactions are altered by the changes associated with evolution from MRSA to DNS-VISA (which is known to occur in patients receiving antibiotic therapy). We evaluated the host range and cross-resistance patterns of five obligately lytic S. aureus myophages to select a three-phage cocktail. We screened these phages for their activity against 24-h bead biofilms and found that biofilms of two strains, D712 (DNS-VISA) and 8014 (MRSA), were the most resistant to killing by single phages. Specifically, even initial phage concentrations of 107 PFU per well could not prevent visible regrowth of bacteria from the treated biofilms. However, when we treated biofilms of the same two strains with phage-antibiotic combinations, we prevented bacterial regrowth when using up to 4 orders of magnitude less phage and antibiotic concentrations that were lower than our measured minimum biofilm inhibitory concentration. We did not see a consistent association between phage activity and the evolution of DNS-VISA genotypes in this small number of bacterial strains. IMPORTANCE The extracellular polymeric matrix of biofilms presents an impediment to antibiotic diffusion, facilitating the emergence of multidrug-resistant populations. While most phage cocktails are designed for the planktonic state of bacteria, it is important to take the biofilm mode of growth (the predominant mode of bacterial growth in nature) into consideration, as it is unclear how interactions between any specific phage and its bacterial hosts will depend on the physical properties of the growth environment. In addition, the extent of bacterial sensitivity to any given phage may vary from the planktonic to the biofilm state. Therefore, phage-containing treatments targeting biofilm infections such as catheters and prosthetic joint material may not be merely based on host range characteristics. Our results open avenues to new questions regarding phage-antibiotic treatment efficiency in the eradication of topologically structured biofilm settings and the extent of eradication efficacy relative to the single agents in biofilm populations.

ABSTRACT Forty-six patients were treated with eravacycline (ERV) for Acinetobacter baumannii infections, where 69.5% of isolates were carbapenem resistant (CRAB). Infections were primarily pulmonary (58.3%), and most patients received combination therapy (84.4%). The median (IQR) ERV duration was 6.9 days (5.1 to 11.1). Thirty-day mortality was 23.9% in the cohort and 21.9% in CRAB patients. One patient experienced an ERV-possible adverse event. IMPORTANCE Acinetobacter baumannii, particularly when carbapenem resistant (CRAB), is one of the most challenging pathogens in the health care setting. This is complicated by the fact that there is no consensus guideline regarding management of A. baumannii infections. However, the recent Infectious Diseases Society of America guidelines for treatment of resistant Gram-negative infections provided expert recommendations for CRAB management. The panel suggest using minocycline among tetracycline derivatives rather than eravacycline (ERV) until sufficient clinical data are available. Therefore, we present the largest multicenter real-world cohort in patients treated with ERV for A. baumannii, where the majority of isolates were CRAB (69.5%). Our analysis demonstrate that patients treated with ERV-based regimens achieved a 30-day mortality of 23.9% and had a low incidence of ERV-possible adverse events (2.1%). This study is important as it fills the gap in the literature regarding the use of a novel tetracycline (i.e., ERV) in the treatment of this challenging health care infection.

ABSTRACT Bacterial biofilms are difficult to eradicate and can complicate many infections by forming on tissues and medical devices. Phage+antibiotic combinations (PAC) may be more active on biofilms than either type of agent alone, but it is difficult to predict which PAC regimens will be reliably effective. To establish a method for screening PAC combinations against Staphylococcus aureus biofilms, we conducted biofilm time-kill analyses (TKA) using various combinations of phage Sb-1 with clinically relevant antibiotics. We determined the activity of PAC against biofilm versus planktonic bacteria and investigated the emergence of resistance during (24 h) exposure to PAC. As expected, fewer treatment regimens were effective against biofilm than planktonic bacteria. In experiments with isogenic strain pairs, we also saw less activity of PACs against DNS-VISA mutants versus their respective parentals. The most effective treatment against both biofilm and planktonic bacteria was the phage+daptomycin+ceftaroline regimen, which met our stringent definition of bactericidal activity (>3 log10 CFU/mL reduction). With the VISA-DNS strain 8015 and DNS strain 684, we detected anti-biofilm synergy between Sb-1 and DAP in the phage+daptomycin regimen (>2 log10 CFU/mL reduction versus best single agent). We did not observe any bacterial resensitization to antibiotics following treatment, but phage resistance was avoided after exposure to PAC regimens for all tested strains. The release of bacterial membrane vesicles tended to be either unaffected or reduced by the various treatment regimens. Interestingly, phage yields from certain biofilm experiments were greater than from similar planktonic experiments, suggesting that Sb-1 might be more efficiently propagated on biofilm. IMPORTANCE Biofilm-associated multidrug-resistant infections pose significant challenges for antibiotic therapy. The extracellular polymeric matrix of biofilms presents an impediment for antibiotic diffusion, facilitating the emergence of multidrug-resistant populations. Some bacteriophages (phages) can move across the biofilm matrix, degrade it, and support antibiotic penetration. However, little is known about how phages and their hosts interact in the biofilm environment or how different phage+antibiotic combinations (PACs) impact biofilms in comparison to the planktonic state of bacteria, though scattered data suggest that phage+antibiotic synergy occurs more readily under biofilm-like conditions. Our results demonstrated that phage Sb-1 can infect MRSA strains both in biofilm and planktonic states and suggested PAC regimens worthy of further investigation as adjuncts to antibiotics.

ABSTRACT Methanocaldococcus sp. strain FS406-22, a hyperthermophilic methanogen, fixes nitrogen with a minimal set of known nif genes. Only four structural nif genes, nifH, nifD, nifK, and nifE, are present in a cluster, and a nifB homolog is present elsewhere in the genome. nifN, essential for the final synthesis of the iron-molybdenum cofactor of nitrogenase in well-characterized diazotrophs, is absent from FS406-22. In addition, FS406-22 encodes four novel hypothetical proteins, and a ferredoxin, in the nif cluster. Here, we develop a set of genetic tools for FS406-22 and test the functionality of genes in the nif cluster by making markerless in-frame deletion mutations. Deletion of the gene for one hypothetical protein, designated Hp4, delayed the initiation of diazotrophic growth and decreased the growth rate, an effect we confirmed by genetic complementation. NifE also appeared to play a role in diazotrophic growth, and the encoding of Hp4 and NifE in a single operon suggested they may work together in some way in the synthesis of the nitrogenase cofactor. No role could be discerned for any of the other hypothetical proteins, nor for the ferredoxin, despite the presence of these genes in a variety of related organisms. Possible pathways and evolutionary scenarios for the synthesis of the nitrogenase cofactor in an organism that lacks nifN are discussed. IMPORTANCE Methanocaldococcus has been considered a model genus, but genetic tools have not been forthcoming until recently. Here, we develop and illustrate the utility of positive selection with either of two selective agents (simvastatin and neomycin), negative selection, generation of markerless in-frame deletion mutations, and genetic complementation. These genetic tools should be useful for a variety of related species. We address the question of the minimal set of nif genes, which has implications for how nitrogen fixation evolved.

ABSTRACT Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes.

ABSTRACT A central pursuit of microbial ecology is to accurately model changes in microbial community composition in response to environmental factors. This goal requires a thorough understanding of the drivers of variability in microbial populations. However, most microbial ecology studies focus on the effects of environmental factors on mean population abundances, rather than on population variability. Here, we imposed several experimental disturbances upon periphyton communities and analyzed the variability of populations within disturbed communities compared with those in undisturbed communities. We analyzed both the bacterial and the diatom communities in the periphyton under nine different disturbance regimes, including regimes that contained multiple disturbances. We found several similarities in the responses of the two communities to disturbance; all significant treatment effects showed that populations became less variable as the result of environmental disturbances. Furthermore, multiple disturbances to these communities were often interactive, meaning that the effects of two disturbances could not have been predicted from studying single disturbances in isolation. These results suggest that environmental factors had repeatable effects on populations within microbial communities, thereby creating communities that were more similar as a result of disturbances. These experiments add to the predictive framework of microbial ecology by quantifying variability in microbial populations and by demonstrating that disturbances can place consistent constraints on the abundance of microbial populations. Although models will never be fully predictive due to stochastic forces, these results indicate that environmental stressors may increase the ability of models to capture microbial community dynamics because of their consistent effects on microbial populations. IMPORTANCE There are many reasons why microbial community composition is difficult to model. For example, the high diversity and high rate of change of these communities make it challenging to identify causes of community turnover. Furthermore, the processes that shape community composition can be either deterministic, which cause communities to converge upon similar compositions, or stochastic, which increase variability in community composition. However, modeling microbial community composition is possible only if microbes show repeatable responses to extrinsic forcing. In this study, we hypothesized that environmental stress acts as a deterministic force that shapes microbial community composition. Other studies have investigated if disturbances can alter microbial community composition, but relatively few studies ask about the repeatability of the effects of disturbances. Mechanistic models implicitly assume that communities show consistent responses to stressors; here, we define and quantify microbial variability to test this assumption. Author Video: An author video summary of this article is available.

ABSTRACT Diverse bacteria, including several Pseudomonas species, produce a class of redox-active metabolites called phenazines that impact different cell types in nature and disease. Phenazines can affect microbial communities in both positive and negative ways, where their presence is correlated with decreased species richness and diversity. However, little is known about how the concentration of phenazines is modulated in situ and what this may mean for the fitness of members of the community. Through culturing of phenazine-degrading mycobacteria, genome sequencing, comparative genomics, and molecular analysis, we identified several conserved genes that are important for the degradation of three Pseudomonas-derived phenazines: phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), and pyocyanin (PYO). PCA can be used as the sole carbon source for growth by these organisms. Deletion of several genes in Mycobacterium fortuitum abolishes the degradation phenotype, and expression of two genes in a heterologous host confers the ability to degrade PCN and PYO. In cocultures with phenazine producers, phenazine degraders alter the abundance of different phenazine types. Not only does degradation support mycobacterial catabolism, but also it provides protection to bacteria that would otherwise be inhibited by the toxicity of PYO. Collectively, these results serve as a reminder that microbial metabolites can be actively modified and degraded and that these turnover processes must be considered when the fate and impact of such compounds in any environment are being assessed. IMPORTANCE Phenazine production by Pseudomonas spp. can shape microbial communities in a variety of environments ranging from the cystic fibrosis lung to the rhizosphere of dryland crops. For example, in the rhizosphere, phenazines can protect plants from infection by pathogenic fungi. The redox activity of phenazines underpins their antibiotic activity, as well as providing pseudomonads with important physiological benefits. Our discovery that soil mycobacteria can catabolize phenazines and thereby protect other organisms against phenazine toxicity suggests that phenazine degradation may influence turnover in situ. The identification of genes involved in the degradation of phenazines opens the door to monitoring turnover in diverse environments, an essential process to consider when one is attempting to understand or control communities influenced by phenazines.

ABSTRACT Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. IMPORTANCE P. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance.

Serial dilution and plate counting is often taught in courses for both microbiology and allied health students. Lecture examples and examination questions addressing how the method is used can sometimes be contrived: artificial data sets may have little or no meaning other than to have students perform a calculation. Here we provide a set of activities employing data sets acquired from the primary literature. Our objective was to have the students think critically about a real scenario in which serial dilution and plate count was used. Each activity requires students to read a paragraph describing the study, predict the results, perform the appropriate calculations, and then evaluate the results in light of their predictions. To test the efficacy of these activities, a pretest quiz was given to approximately 100 students in an allied health/general microbiology course. After a lecture on how microbes are enumerated, students were given a different quiz. The class was then divided randomly into groups of three or four students and assigned one of the activities. A postactivity quiz was also administered. Approximately two weeks later, a serial dilution/plate count question was used on an examination and served as a final posttest. Standardized learning gains were calculated for the quiz administered after each learning activity. Even though learning gains were significantly higher after the lecture, there was also a significant improvement between the lecture and the activity. Using an exercise based on an authentic set of data significantly improved student learning gains, and is a useful practice for teaching microbiology.

ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas) system is an adaptive immune system present in many archaea and bacteria. CRISPR/Cas systems are incredibly diverse, and there is increasing evidence of CRISPR/Cas systems playing a role in cellular functions distinct from phage immunity. Previously, our laboratory reported one such alternate function in which the type 1-F CRISPR/Cas system of the opportunistic pathogen Pseudomonas aeruginosa strain UCBPP-PA14 (abbreviated as P. aeruginosa PA14) inhibits both biofilm formation and swarming motility when the bacterium is lysogenized by the bacteriophage DMS3. In this study, we demonstrated that the presence of just the DMS3 protospacer and the protospacer-adjacent motif (PAM) on the P. aeruginosa genome is necessary and sufficient for this CRISPR-dependent loss of these group behaviors, with no requirement of additional DMS3 sequences. We also demonstrated that the interaction of the CRISPR system with the DMS3 protospacer induces expression of SOS-regulated phage-related genes, including the well-characterized pyocin operon, through the activity of the nuclease Cas3 and subsequent RecA activation. Furthermore, our data suggest that expression of the phage-related genes results in bacterial cell death on a surface due to the inability of the CRISPR-engaged strain to downregulate phage-related gene expression, while these phage-related genes have minimal impact on growth and viability under planktonic conditions. Deletion of the phage-related genes restores biofilm formation and swarming motility while still maintaining a functional CRISPR/Cas system, demonstrating that the loss of these group behaviors is an indirect effect of CRISPR self-targeting. IMPORTANCE The various CRISPR/Cas systems found in both archaea and bacteria are incredibly diverse, and advances in understanding the complex mechanisms of these varied systems has not only increased our knowledge of host-virus interplay but has also led to a major advancement in genetic engineering. Recently, increasing evidence suggested that bacteria can co-opt the CRISPR system for functions besides adaptive immunity to phage infection. This study examined one such alternative function, and this report describes the mechanism of type 1-F CRISPR-dependent loss of the biofilm and swarming in the medically relevant opportunistic pathogen Pseudomonas aeruginosa. Since both biofilm formation and swarming motility are important in the virulence of P. aeruginosa, a full understanding of how the CRISPR system can regulate such group behaviors is fundamental to developing new therapeutics.

ABSTRACT Hydrogenotrophic methanogenic Archaea require reduced ferredoxin as an anaplerotic source of electrons for methanogenesis. H2 oxidation by the hydrogenase Eha provides these electrons, consistent with an H2 requirement for growth. Here we report the identification of alternative pathways of ferredoxin reduction in Methanococcus maripaludis that operate independently of Eha to stimulate methanogenesis. A suppressor mutation that increased expression of the glycolytic enzyme glyceraldehyde-3-phosphate:ferredoxin oxidoreductase resulted in a strain capable of H2-independent ferredoxin reduction and growth with formate as the sole electron donor. In this background, it was possible to eliminate all seven hydrogenases of M. maripaludis. Alternatively, carbon monoxide oxidation by carbon monoxide dehydrogenase could also generate reduced ferredoxin that feeds into methanogenesis. In either case, the reduced ferredoxin generated was inefficient at stimulating methanogenesis, resulting in a slow growth phenotype. As methanogenesis is limited by the availability of reduced ferredoxin under these conditions, other electron donors, such as reduced coenzyme F420, should be abundant. Indeed, when F420-reducing hydrogenase was reintroduced into the hydrogenase-free mutant, the equilibrium of H2 production via an F420-dependent formate:H2 lyase activity shifted markedly toward H2 compared to the wild type. IMPORTANCE Hydrogenotrophic methanogens are thought to require H2 as a substrate for growth and methanogenesis. Here we show alternative pathways in methanogenic metabolism that alleviate this H2 requirement and demonstrate, for the first time, a hydrogenotrophic methanogen that is capable of growth in the complete absence of H2. The demonstration of alternative pathways in methanogenic metabolism suggests that this important group of organisms is metabolically more versatile than previously thought.

Bacteriophage infection has profound effects on bacterial biology. Clustered regular interspaced short palindromic repeats (CRISPRs) and cas (CRISPR-associated) genes are found in most archaea and many bacteria and have been reported to play a role in resistance to bacteriophage infection. We observed that lysogenic infection of Pseudomonas aeruginosa PAl4 with bacteriophage DMS3 inhibits biofilm formation and swarming motility, both important bacterial group behaviors. This inhibition requires the CRISPR region in the host. Mutation or deletion of five of the six cas genes and one of the two CRISPRs in this region restored biofilm formation and swarming to DMS3 lysogenized strains. Our observations suggest a role for CRISPR regions in modifying the effects of lysogeny on P. aeruginosa.

A study was conducted on the hydrogen sulfide (H2S) production and fermentative gas production by Salmonella typhimurium. The findings suggest that in addition to linking oxidation with phosphorylation, F0F1 ATP synthase plays an important part in the proton movement accompanying certain anaerobic reductions and oxidations. Moreover, ATP synthase mutants are also found to be defective in the production of both H2S and fermentative gas.