Applied and Environmental Microbiology

Desulfovibrio vulgaris has been the primary pure culture sulfate reducer for developing microbial corrosion concepts. Multiple mechanisms for how it accepts electrons from Fe0 have been proposed. We investigated Fe0 oxidation with a mutant of D. vulgaris in which hydrogenase genes were deleted. The hydrogenase mutant grew as well as the parental strain with lactate as the electron donor, but unlike the parental strain was not able to grow on H2. The parental strain reduced sulfate with Fe0 as the sole electron donor, but the hydrogenase mutant did not. H2 accumulated over time in Fe0 cultures of the hydrogenase mutant and sterile controls, but not in parental strain cultures. Sulfide stimulated H2 production in uninoculated controls apparently by both reacting with Fe0 to generate H2 and facilitating electron transfer from Fe0 to H+. Parental strain supernatants did not accelerate H2 production from Fe0, ruling out a role for extracellular hydrogenases. Previously proposed electron transfer between Fe0 and D. vulgaris via soluble electron shuttles was not evident. The hydrogenase mutant did not reduce sulfate in the presence of Fe0 and either riboflavin or anthraquinone-2,6-disulfonate and these potential electron shuttles did not stimulate parental strain sulfate reduction with Fe0 as the electron donor. The results demonstrate that D. vulgaris primarily accepts electrons from Fe0 via H2 as an intermediary electron carrier. These findings clarify the interpretation of previous D. vulgaris corrosion studies and suggest that H2-mediated electron transfer is an important mechanism for iron corrosion under sulfate-reducing conditions. ImportanceMicrobial corrosion of iron in the presence of sulfate-reducing microorganisms is economically significant. There is substantial debate over how microbes accelerate iron corrosion. Tools for genetic manipulation have only been developed for a few Fe(III)-reducing and methanogenic microorganisms known to corrode iron and in each case those microbes were found to accept electrons from Fe0 via direct electron transfer. However, iron corrosion is often most intense in the presence of sulfate-reducing microbes. The finding that Desulfovibrio vulgaris relies on H2 to shuttle electrons between Fe0 and cells revives the concept, developed in some of the earliest studies on microbial corrosion, that sulfate reducers consumption of H2 is a major microbial corrosion mechanism. The results further emphasize that direct Fe0-to-microbe electron transfer has yet to be rigorously demonstrated in sulfate-reducing microbes.

Skatole (3-methylindole), a persistent and toxic N-heterocyclic aromatic pollutant, poses significant bioremediation challenges due to poorly defined biodegradation processes. This study systematically investigated the genetic determinants and metabolic mechanisms of skatole degradation in the environmentally versatile Gram-positive genus Rhodococcus. We demonstrated that skatole degradation was a broadly conserved trait across diverse Rhodococcus species, including R. aetherivorans, R. pyridinivorans, R. ruber and R. qingshengii. Genomic and functional analyses uncovered a novel flavoprotein monooxygenase (FPMO) SkaA as the initial catalyst for skatole catabolism. Heterologous expression in Escherichia coli confirmed SkaAs essential catalytic role, with high-resolution liquid chromatography-tandem mass spectrometry identifying the key product 3-methyloxindole and 3-hydroxy-3-methyloxindole. Distribution studies revealed SkaA homologs predominantly in Actinobacteria, particularly Nocardia and Rhodococcus, indicating conserved metabolic capacity for skatole transformation within this phylum. Notably, Rhodococcus strains lacking the skaA gene also retained skatole degradation activity, implying the existence of alternative genetic determinants. Crucially, phylogenetic analysis positioned SkaA as a distinct subclass within Group E FPMOs, exhibiting [≤]40% sequence identity to reported styrene/indole oxygenases. The phylogenetic segregation of skatole, indole, and styrene monooxygenases provides a predictive framework for functional annotation of Group E FPMOs. Our findings elucidate a novel skatole transformation pathway, establish Rhodococcus as environmentally versatile biocatalysts, and provide new insights into the study of Group E FPMOs. IMPORTANCESkatole is a notorious, foul-smelling compound generated by the anaerobic breakdown of tryptophan, presenting substantial risks to both the environment and public health. Despite its prevalence, the enzymatic machinery initiating its biodegradation remains poorly characterized. Our study resolves this critical gap by identifying SkaA as a functionally validated enzyme catalyzing skatoles initial oxidation step. Distribution analyses revealed SkaA homologs predominantly in Actinobacteria, particularly Rhodococcus and Nocardia. Phylogenetic analysis positioned SkaA as a novel third subclass within Group E flavoprotein monooxygenases. These breakthroughs provide molecular tools for engineering targeted bioremediation solutions and new insights into the study of Group E FPMOs.

Lignin is a plant heteropolymer composed of phenolic subunits. Because of its heterogeneity and recalcitrance, the development of efficient methods for its valorization still remains an open challenge. One approach to utilize lignin is its chemical deconstruction into mixtures of monomeric phenolic compounds followed by biological funneling into a single product. Novosphingobium aromaticivorans DSM12444 has been previously engineered to produce 2-pyrone-4,6-dicarboxylic acid (PDC) from depolymerized lignin by simultaneously metabolizing multiple aromatics through convergent routes involving the intermediates 3-methoxygallic acid (3-MGA) and protocatechuic acid (PCA). We investigated enzymes predicted to be responsible for O-demethylation and oxidative aromatic ring opening, two critical reactions involved in the metabolism of phenolics compounds by N. aromaticivorans. The results showed the involvement of DesA in O-demethylation of syringic and vanillic acids, LigM in O-demethylation of vanillic acid and 3-MGA, and a new O-demethylase, DmtS, in the conversion of 3-MGA into gallic acid (GA). In addition, we found that LigAB was the main aromatic ring opening dioxygenase involved in 3-MGA, PCA, and GA metabolism, and that a previously uncharacterized dioxygenase, LigAB2, had high activity with GA. Our results indicate a metabolic route not previously identified in N. aromaticivorans that involves O-demethylation of 3-MGA to GA. We predict this pathway channels [~]15% of the carbon flow from syringic acid, with the rest following ring opening of 3-MGA. The new knowledge obtained in this study allowed for the creation of an improved engineered strain for the funneling of aromatic compounds that exhibits stoichiometric conversion of syringic acid into PDC. IMPORTANCEFor lignocellulosic biorefineries to effectively contribute to reduction of fossil fuel use, they need to become efficient at producing chemicals from all major components of plant biomass. Making products from lignin will require engineering microorganisms to funnel multiple phenolic compounds to the chemicals of interest, and N. aromaticivorans is a promising chassis for this technology. The ability of N. aromaticivorans to efficiently and simultaneously degrade many phenolic compounds may be linked to having functionally redundant aromatic degradation pathways and enzymes with broad substrate specificity. A detailed knowledge of aromatic degradation pathways is thus essential to identify genetic engineering targets to maximize product yields. Furthermore, knowledge of enzyme substrate specificity is critical to redirect flow of carbon to desired pathways. This study described an uncharacterized pathway in N. aromaticivorans and the enzymes that participate in this pathway, allowing the engineering of an improved strain for production of PDC from lignin.

Microbacterium deferre sp. nov. A1-JK is a metabolically versatile Gram-positive bacterium isolated from the oxic-anoxic interface of freshwater sediment, inhabited by cable bacteria. M. deferre A1-JK could simultaneously reduce oxygen and Fe(III), challenging the traditional view of microbial Fe(III)-reduction as a strictly anaerobic process. Electrochemical studies revealed extracellular electron transfer (EET) metabolism facilitated by soluble flavin shuttles, identified via HPLC. Genomic analyses uncovered EET pathways involving cytochrome FccA and flavin reductase FmnA. Its metabolic versatility also allowed for weak electroactivity in alkaline (pH 9-10) and saline conditions (0-4% NaCl). The ability to reduce Fe(III), in the presence of atmospheric oxygen highlights its adaptability to dynamic sediment environments with fluctuating oxygen and Fe(III) gradients. These findings underscore the metabolic versatility of M. deferre A1-JK at oxic-anoxic interfaces, providing insights into the coexistence of aerobic and anaerobic processes in a single cell. ImportanceMicrobacterium deferre A1-JK is a newly discovered gram-positive bacterium that employs flavin-mediated extracellular electron transfer to respire minerals and electrodes. Associated with cable bacteria in freshwater sediments, M. deferre A1-JK was able to simultaneously reduce oxygen and Fe(III). This makes it highly adaptable to changing environments, such as those found in (cable-bacteria-containing) sediments with fluctuating oxygen and Fe concentrations. The metabolic versatility of M. deferre A1-JK offers opportunities to fundamentally revise our understanding of microbial metabolism and nutrient cycling models that strictly separate aerobic and anaerobic metabolisms.

The long process of the assimilatory sulfate-reduction consists of sulfate reduction to H2S and then H2S assimilation for cysteine synthesis. However, recent studies indicate that both H2S and zero valent sulfur (S0) are produced during dissimilatory sulfate reduction process, which leads to the question whether S0 also is involved in assimilatory sulfate-reduction. Herein, phylogenetic analysis grouped assimilatory sulfite reductases into five clusters and we re-examined four representatives from two major clusters. These enzymes produced both S0 and H2S from sulfite. However, S0 was more like a direct product, while H2S was just a derivative of S0 via chemical reduction. Phylogenetic analysis of cysteine synthases grouped them into six clusters, and we re-examined six representatives from two major clusters. These enzymes used both glutathione persulfide (GSSH, a common intracellular compound containing a S0 atom) and H2S as substrates to synthesize cysteine, but preferred to use GSSH. These findings indicated that S0, other than H2S, is the key intermediate during the process of inorganic sulfur converting to organic sulfur. ImportanceSulfate reduction is an important link in the global sulfur cycle. Assimilatory sulfate-reduction consists of sulfate-reduction to H2S and then H2S assimilation for cysteine synthesis. This concept was founded in 1950s and has not been challenged until today. In this study, we re-examined four representative assimilatory sulfite reductases (aSiRs) and found that these enzymes directly reduced sulfite to zero valent sulfur (S0). A small portion of produced S0 was reduced to H2S via aSiR-independent chemical reactions. Further, we re-examined six representative cysteine synthases and found that these enzymes used S0 derivative compounds (thiosulfate and glutathione persulfide) as substrates to synthesize cysteine. Thus, S0 is the key intermediate of assimilatory sulfate reduction process, other than H2S.

Sulfate-reducing microorganisms extensively contribute to the corrosion of ferrous metal infrastructure. There is substantial debate over their corrosion mechanisms. We investigated Fe0 corrosion with Desulfovibrio vulgaris, the sulfate reducer most often employed in corrosion studies. Cultures were grown with both lactate and Fe0 as potential electron donors to replicate the common environmental condition in which organic substrates help fuel the growth of corrosive microbes. Fe0 was corroded in cultures of a D. vulgaris hydrogenase-deficient mutant with the 1:1 correspondence between Fe0 loss and H2 accumulation expected for Fe0 oxidation coupled to H+ reduction to H2. This result and the extent of sulfate reduction indicated that D. vulgaris was not capable of direct Fe0-to-microbe electron transfer even though it was provided with a supplementary energy source in the presence of abundant ferrous sulfide. Corrosion in the hydrogenase-deficient mutant cultures was greater than in sterile controls, demonstrating the H2 removal was not necessary for the enhanced corrosion observed in the presence of microbes. The parental H2-consuming strain corroded more Fe0 than the mutant strain, which could be attributed to H2 oxidation coupled to sulfate reduction producing sulfide that further stimulated Fe0 oxidation. The results suggest that H2 consumption is not necessary for microbially enhanced corrosion, but H2 oxidation can indirectly promote corrosion by increasing sulfide generation from sulfate reduction. The finding that, D. vulgaris was incapable of direct electron uptake from Fe0 reaffirms that direct metal-to-microbe electron transfer has yet to be rigorously described in sulfate-reducing microbes. Impact StatementThe economic impact of microbial corrosion of iron-containing metals is substantial. A better understanding of how microbes accelerate corrosion is expected to lead to the development of methods to prevent corrosion. The results presented here refute the suggestion, frequently made in the microbiology literature, that microbial H2 uptake stimulates Fe0 corrosion. Also refuted, are previous claims that Desulfovibrio vulgaris is capable of directly extracting electrons from Fe0. The results are consistent with the concept that sulfide produced by sulfate-reducers promotes Fe0 oxidation with the production of H2. The results illustrate that appropriate mutants can provide rigor to corrosion mechanism studies.

Besides an industrial pollutant, 2,4-dinitrophenol (DNP) has been used illegally as a weight loss drug that had claimed human lives. Little is known about the metabolism of DNP, particularly among Gram-negative bacteria. In this study, two non-contiguous genetic loci of Paraburkholderia (formerly Burkholderia) sp. strain KU-46 genome were identified and four key initial genes (dnpA, dnpB, and dnpC1C2) were characterized to provide molecular and biochemical evidence for the degradation of DNP via the formation of 4-nitrophenol (NP), a pathway that is unique among DNP utilizing bacteria. Reverse transcription PCR analysis indicated that the dnpA gene encoding the initial hydride transferase (28 kDa), and the dnpB gene encoding a nitrite-eliminating enzyme (33 kDa), are inducible by DNP and the two genes are organized in an operon. Purified DnpA and DnpB from overexpression clones in Escherichia coli effected the transformation of DNP to NP via the formation of hydride-Meisenheimer complex of DNP. The function of DnpB appears new since all homologs of DnpB sequences in the protein database are annotated as putative nitrate ABC transporter substrate-binding proteins. The gene cluster responsible for the degradation of DNP after NP formation was designated dnpC1C2DXFER. DnpC1 and DnpC2 were functionally characterized as the respective FAD reductase and oxygenase components of the two-component NP monooxygenase. Both NP and 4-nitrocatechol were shown to be substrates, producing hydroquinone and hydroxyquinol, respectively. Elucidation of the hqdA1A2BCD gene cluster allows the delineation of the final degradation pathway of hydroquinone to {beta}-ketoadipate prior to its entry to the tricarboxylic acid cycle.\n\nImportanceThis study fills a gap in our knowledge and understanding of the genetic basis and biochemical pathway for the degradation of 2,4-dinitrophenol (DNP) in Gram-negative bacteria, represented by the prototypical Paraburkholderia sp. strain KU-46 that metabolizes DNP through the formation of 4-nitrophenol, a pathway unseen by other DNP utilizers. The newly cloned genes could serve as DNA probes in biomonitoring as well as finding application in new biocatalyst development to access green chemicals. By and large, knowledge of the diverse strategies used by microorganisms to degrade DNP will contribute to the development of bioremediation solutions since DNP is an industrial pollutant used widely in the chemical industry for the synthesis of pesticides, insecticides, sulfur dyes, wood preservatives, and explosives, etc. (119 words)

2,4-Dichlorophenoxyacetic acid (2,4-D) is an herbicide commonly used in aquatic and terrestrial environments that is degraded by bacteria through the TFD pathway. Previous work has relied on culture-based methods to develop primers for qPCR analysis of the gene cassette in environmental samples. In this study, we combined molecular and genomic approaches to examine the accuracy of established tfdA qPCR primers on environmental samples and update the phylogeny of tfdA genes detected in bacterial genomes. We found most putative 2,4-D degraders are within the Proteobacteria but also found several novel degraders including members of the phyla Candidatus Rokubacteria and Candidatus Eremiobacteraeota. In silico analysis of established primers showed potential amplification of < 5% of putative degrader sequences but 52-100% of experimentally verified degraders when allowing for three and one mismatches between template and primer sequences, respectively. Overall, our work expands the diversity of putative 2,4-D degraders and demonstrates the limitations of culture-based tools for investigating functional diversity of microorganisms in the environment. ImportanceCultivation-based methods can misrepresent the diversity of environmental microorganisms. Our work showcases one example of how culture-based development of molecular tools underestimates the full spectrum of 2,4-D degrading microorganisms. Accurately identifying microorganisms with 2,4-D degradation potential is crucial for understanding the biodegradation potential of a commonly used herbicide across terrestrial, aquatic, and subsurface environments. Additionally, this work reinforces well-documented pitfalls associated with relying on cultured representatives when constructing primers and the challenges of translating findings from a few cultured representatives to understudied or unknown microorganisms in complex environments.

Most non-model Vibrio species lack the genetic tools needed for targeted mutagenesis, which limits the ability to functionally characterize newly identified pathways. To address this challenge, we present here efficient, robust methods for genetically manipulating Vibrio species that rely on RecA-mediated homologous recombination and two well-characterized counterselection methods, galactokinase (galK) 2-Deoxy-D-galactose (DOG-2) toxicity and the rpsLR/rpsLS streptomycin susceptibility system, both of which are active across a broad range of Vibrio species. We further characterized two genus-specific conserved promoters capable of driving high-level ectopic expression across all tested species. These promoters were incorporated into two broadly applicable, conjugatively transferable suicide backbones designed to facilitate double homologous recombination. Using these systems, we successfully disrupted polar flagellar motility in multiple Vibrio species and introduced extensive modifications to both flagellar and secretory pathways in V. diazotrophicus. Notably, although the galK system exhibited broader applicability, the rpsL system proved to be more efficient in cases where a streptomycin resistant strain could be generated. We also developed two mobilizable replicative backbones that express pH-stable fluorescent proteins for use within the genus. Collectively, these tools expand the genetic toolkit available for both gene disruption and heterologous gene expression in non-model members of the Vibrionaceae. ImportanceMembers of the Vibrionaceae are not only among the most abundant and ecologically influential microorganisms in marine ecosystems, but they also represent major drivers of disease across a wide range of hosts, including humans. However, identifying the genetic determinants of Vibrionaceae pathogenesis has remained challenging due to their halophilic growth requirements, restriction enzyme profiles, and resistance to expressing foreign proteins. Canonical counterselection pathways are largely ineffective in these species, which underscores the need for novel and efficient genetic tools to advance functional studies. This study adapts two strategies previously successful in other non-model organisms for use within the Vibrio genus and its close relatives. These methods therefore represent an essential step toward overcoming long-standing genetic barriers in Vibrio species and provide a framework to expand our understanding of biology in this important bacterial genus.

The microbial communities inhabiting food production environments are distinguished from those of other built environments in their capacity to influence food quality and safety, impacting consumer health. However, how indoor environmental conditions drive the bacterial and fungal communities of food production facilities remains largely unknown. In this study of five commercial food production facilities, we employed remote wireless sensors paired with marker-gene amplicon sequencing (bacterial 16S rRNA genes and fungal ITS) of processing equipment and non-processing built environment surfaces (N=2,329) to profile spatial and longitudinal changes in bacterial and fungal communities, and their association with indoor climate. We identify multiple associations between indoor environmental conditions and microbial community structure, demonstrating the role of the indoor environment in shaping microbial communities on food processing and non-processing surfaces. This suggests that indoor climate could be manipulated to rationally modify surface communities, with the aim of enhancing food quality and safety.

Microbial communities of small freshwater bodies are poorly understood. Four ponds in Knox County, Ohio, were sampled over two years to investigate the relationship between the microbial taxa profiles, antibiotic resistance genes (ARGs), and environmental factors such as pH and tannin concentrations. For each site, microbial communities were collected by filtration and metagenomes were analyzed by short-read sequencing. Taxa profiles were predicted by the Kraken2/Bracken pipelines. Bacterial taxa with high abundance in these ponds included Betaproteobacteria (Polynucleobacter and Methylopumilus) and Actinobacteria (Planktophila, Nanopelagicus, and Mycolicibacterium). One pond, a former quarry with elevated pH, showed high prevalence of Cyanobacteria with a seasonal shift from Synechococcus to Planktothrix in the fall. Planktothrix increase was associated with acidification. ARGs were quantified using the ShortBRED pipeline to detect and quantify hits to a marker set derived from the Comprehensive Antibiotic Resistance Database (CARD). The top two ARGs with the largest marker hits encode components of a Stenotrophomonas drug efflux pump powered by proton-motive force (smeABC) and a mycobacterial global regulator that activates a drug pump and other cell defenses (mtrA). Pump function and global activation of transcription incur large energy expenditures, whose fitness cost may increase at high external pH where the cells proton-motive force is diminished. The smeABC and mtrA prevalence showed a modest correlation with acidifying conditions (low pH and high tannins) which contribute a large transmembrane pH difference to the proton-motive force, thus increasing the cells energy available for pump function and global gene expression. IMPORTANCECompared to rivers and lakes, pond microbial ecosystems are understudied despite close contact with agriculture and recreation. Environmental microbes offer health benefits as well as hazards for human contact. Small water bodies may act as reservoirs for drug-resistant organisms and transfer of antibiotic resistance genes. Yet, the public is rarely aware of the potential for exposure to ARG-carrying organisms in recreational water bodies. Little is known about the capacity for freshwater microbial communities to remediate drug pollution and which biochemical factors may select against antibiotic resistance genes. This study analyzes the bacterial taxa composition and ARG prevalence including possible influence of factors such as pH and tannic acid levels.

Wooden boards are essential tools in cheese ripening and there are accumulating observations suggesting their antimicrobial effect against foodborne bacterial pathogens, such as Listeria monocytogenes. However, poor bacterial recovery of bacteria from wood can confound quantification of pathogen burdens. To assess L. monocytogenes survival on wooden cheese boards, we applied a disruptive grinding method and tracked native board-associated bacterial counts as controls. Our data revealed that L. monocytogenes declines on clean zones of wooden boards, but can replicate on areas where there is suitable cheese. Our microbiota analysis revealed diverse bacterial communities on wooden board surfaces, with a prominent presence of Brevibacterium, Brachybacterium, and Staphylococcus genera. We further identified seven bacterial species that inhibit L. monocytogenes, belonging to Bacillus, Staphylococcus, and Serratia phyla, as well as Lactococcus lactis. We focused on a Bacillus safensis isolate as a novel biocontrol agent candidate, and found it to potently inhibit L. monocytogenes via secreted antimicrobial factors. Our genomic, bioinformatic, and biochemical analyses indicate that those factors are likely antimicrobial peptides encoded by multiple biosynthetic gene clusters, several of which are unique to B. safensis and have not been characterized. A sub-inhibitory concentration of B. safensis supernatant induces a significant down-regulation of prophage elements and up-regulation antimicrobial stress response in L. monocytogenes. Taken together, our findings indicate that the wooden board microbiota is a rich source of antimicrobial-producing bacteria with potential applications in foodborne pathogen control strategies. IMPORTANCEDespite stringent food safety measures, L. monocytogenes foodborne outbreaks remain frequent with high hospitalization and mortality rates. Removal of L. monocytogenes from food processing environments is extremely challenging, because this pathogen is ubiquitous and encodes a wide array of stress response mechanisms that enable it to thrive under harsh conditions. Our study found that clean wooden boards used in cheese ripening inhibit L. monocytogenes, causing a noticeable decline in pathogen population following surface inoculation. Bacterial communities on wooden cheese boards are rich and diverse, and harbor many species that produce antimicrobial compounds against L. monocytogenes, with the example of a new B. safensis isolate. Therefore, wooden board microbiota is a promising source for future antimicrobial discovery efforts.

The freshwater chemolithoautotrophic Gram-negative bacterium Sideroxydans lithotrophicus ES-1 oxidizes Fe(II) at the cell surface. In this organism, it is proposed that the monoheme cytochrome MtoD from the Mto pathway transfer electrons across the periplasm to an inner membrane NapC/NirT family tetraheme cytochrome encoded by Slit_2495, for which we propose the name ImoA (inner membrane oxidoreductase). ImoA has been proposed to function as the quinone reductase, receiving electrons from iron oxidizing extracellular electron uptake pathway to reduce the quinone pool. In this study, ImoA was cloned on a pBAD plasmid vector and overexpressed in Escherichia coli. Biochemical and spectroscopic characterization of the purified ImoA reveals that this 26.5 kDa cytochrome contains one high-spin and three low-spin hemes. Our data show that ImoA can function as a quinol oxidase and is able to functionally replace CymA, a related NapC/NirT family tetraheme cytochrome required for anaerobic respiration of a wide range of substrates by Shewanella oneidensis. We demonstrate that ImoA can transfer electrons to different periplasmic proteins from S. oneidensis including STC and FccA, but in a manner that is distinct from that of CymA. Phylogenetic analysis shows that ImoA is clustered closer to NirT sequences than to CymA. This study suggests that ImoA functions as a quinol oxidase in S. oneidensis and raises questions about the directionality and/or reversibility of electron flow through the Mto pathway in S. lithotrophicus ES-1. ImportanceFe(II)-oxidizing bacteria play an important role in the biogeochemical cycling of iron, representing a promising class of organisms for the development of novel biotechnological processes, including bioelectrosynthesis. These organisms perform extracellular electron transfer, taking up electrons from Fe(II) outside of the cell, possibly through a porin-cytochrome complex in the outer membrane. The electrons are then transferred to the quinone pool in the inner membrane via periplasmic and inner membrane electron transfer proteins. In this paper, we produced and characterized the NapC/NirT family tetraheme cytochrome ImoA, encoded by Slit_2495, an inner membrane protein from the Gram-negative Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1, proposed to be involved in extracellular electron transfer to the quinone pool. We show that ImoA may function instead as a quinol oxidase. The obtained insights represent the first step in understanding mechanisms of electron flow in S. lithotrophicus ES-1 and may lead towards practical biotechnological applications of Fe(II)-oxidizing bacteria.

Human infection with Francisella tularensis, a potentially lethal bacterial pathogen, typically occurs after exposure to contaminated water, soil, food, or an infected animal. While F. tularensis can persist in environmental sources over long periods of time, the genetic requirements that permit its long-term viability are not understood. To address this question, we developed a laboratory model for persistence of F. tularensis in fresh water, finding that viable cells could be recovered for 3 - 8 weeks after incubation at 4{degrees}C. Using this model, we took an unbiased, transposon insertion sequencing approach to identify genes critical for this persistence of F. tularensis cells. We found that mutants in mpl, a gene encoding murein peptide ligase, are defective for persistence in fresh water. Previous studies had identified mpl as critical for intramacrophage survival. Murein peptide ligase plays a role in peptidoglycan recycling, suggesting that F. tularensis uses this enzyme to maintain cell wall integrity during hypoosmotic and intramacrophage stress conditions. Our results highlight the importance of understanding how bacterial cell envelopes have evolved and adapted to maintain their integrity in a variety of stress conditions.

Enzymes that degrade PET and other plastics are being discovered with increasing pace, and engineering approaches further increase their activity. Most proposed applications for such enzymes require their purification, enabling the regeneration of monomeric subunits suitable for the production of new plastic. However, large amounts of plastics exist as environmental contaminants, and are unlikely to be suitable for standard recycling. Such waste may still be suitable for bioremediation using enzymatic activities, albeit without the economic incentives of a full recycling process. Here we develop a delivery system for PET hydrolases, based on engineered yeast biofilms that grow and secrete enzymes directly on the substrate to be degraded. The system does not require any enzyme purification steps, and the biofilms at least partially assimilate the degradation products into biomass. Our work provides proof-of-principle that demonstrates the potential of biofilm-based approaches.

Shewanella oneidensis MR-1 is a model electroactive bacterium whose extracellular electron transfer (EET) pathway includes a sequential network of c-type cytochromes that span the inner membrane, periplasm, and outer membrane. While electrochemical studies revealed the critical role of outer-membrane cytochromes in mediating both outward EET from cells to external surfaces and lateral biofilm conduction across cells, the specific functional role of the periplasmic cytochromes in these processes is less understood. Dissecting the contributions of the periplasmic components has been challenged by the complexity of the periplasmic cytochrome network and the inherent variability of native biofilms, which confounds the electrochemical comparison of cytochrome mutants. Here, we overcome these limitations with a synthetic biology approach combining targeted deletion of genes encoding key periplasmic cytochromes with light-induced biofilm patterning to create uniform, geometrically defined biofilms on electrodes for robust electrochemical comparisons. Voltammetric measurements of patterned S. oneidensis mutant biofilms confirmed the essential role of periplasmic cytochromes in facilitating outward EET, a contribution that becomes apparent when flavins are present to accelerate interfacial electron transfer between outer-membrane cytochromes and the electrode. In contrast to this crucial role in routing outward EET across the periplasm, electrochemical gating measurements of lateral biofilm conductivity revealed that the periplasmic cytochromes do not contribute to long-distance electron transport along cellular layers bridging electrodes. These findings provide new insights into the role of periplasmic cytochromes in S. oneidensis, and distinguish their contributions to routing outward EET across the cell envelope versus biofilm conductivity. ImportanceMicrobes capable of extracellular electron transfer (EET) are central to global biogeochemical cycles and emerging bioelectrochemical technologies. In the important model EET bacterium Shewanella oneidensis MR-1, the outer-membrane components that interface with external surfaces are well-characterized. However, the functional role of the periplasmic components linking the inner and outer membranes has remained obscured by the complex network of multiple cytochromes and biofilm heterogeneity limiting precise comparisons across mutants. By combining light-induced biofilm patterning with electrochemical analysis, we successfully revealed the specific contributions of periplasmic cytochromes: these components are essential for facilitating the outward EET across the cell envelope but do not impact lateral long-distance electron transport across the biofilm. The results refine our understanding of extracellular respiration and provide design rules for engineering living electronic materials.

Environmental monitoring of bacteria using phage-based biosensors has been widely developed for many different species. However, there are only a few available methods to detect specific bacteriophages in raw environmental samples. In this work, we developed a simple and efficient assay to rapidly monitor the phage content of a given sample. The assay is based on the bistable expression of the Salmonella enterica opvAB operon. Under regular growth conditions, opvAB is only expressed by a small fraction of the bacterial subpopulation. In the OpvABON subpopulation, synthesis of the OpvA and OpvB products shortens the O-antigen in the lipopolysaccharide and confers resistance to phages that use LPS as a receptor. As a consequence, the OpvABON subpopulation is selected in the presence of such phages. Using an opvAB::gfp fusion, we could monitor LPS-binding phages in various media, including raw water samples. To enlarge our phage-biosensor panoply, we also developed several coliphage biosensors that proved efficient to detect LPS- as well as protein-binding coliphages. Moreover, the combination of these tools allows to identify what is the bacterial receptor triggering phage infection. The opvAB::gfp biosensor thus comes in different flavours to efficiently detect a wide range of bacteriophages and identify the type of receptor they recognize. ImportanceDetection and accurate counting of bacteriophages, the viruses that specifically infect bacteria, from environmental samples still constitutes a challenge for those interested in isolating and characterizing bacteriophages for ecological or biotechnological purposes. This work provides a simple and accurate method based on the bi-stable expression of genes that confer resistance to certain classes of bacteriophages in different bacterial models. It paves the way for future development of highly efficient phage biosensors that can discriminate among several receptor-binding phages and that could be declined in many more versions. In a context where phage ecology, research, and therapy are flourishing again, it becomes essential to possess simple and efficient tools for phage detection.

Bacterial detoxification of mycotoxins has the potential to offer a low-cost solution to ensure that feed and food commodities contaminated by fungal growth become safe to consume. Among bacteria, Rhodococcus species are of particular interest because they can be metabolically versatile, non-pathogenic, and environment-friendly. However, the native response of Rhodococcus environmental isolates appears inadequate for current detoxification needs. By analyzing the detoxification of aflatoxin by two Rhodococcus species: R. pyridinivorans and R. erythropolis, we examine important features of the dynamics that could guide future optimization of bacterial detoxification. Our results for Rhodococcus species suggest that detoxification happens through a regulated process of secreting extracellular enzymes. We show that enzyme fatigue in the presence of the toxin determines the lifetime of the enzyme and limits the overall detoxification performance of these species. Additionally, we show that the regulation of enzyme production can be both species- and environment-dependent. Overall, our quantitative approach reveals that enzyme fatigue is a major determinant of overall detoxification and needs to be accounted for in assessing the performance of detoxification by live cells or cell-free filtrates.

The emergence of multidrug-resistant Vibrio parahaemolyticus poses a severe threat to mariculture sustainability, highlighting the urgent need for eco-friendly antimicrobial agents. In this study, we demonstrated that L-Cysteine (L-Cys) functions as an inhibitor of V. parahaemolyticus, with a minimum inhibitory concentration of 7.5 mM. Microscopic observation and viability assays revealed that L-Cys compromises bacterial membrane integrity, ultimately leading to cell death. Further investigation indicated that the antibacterial effect is primarily attributed to the intracellular production of hydrogen sulfide (H2S) generated by L-Cys metabolism. Transcriptomic and biochemical analyses showed that L-Cys induced metabolic reprogramming by suppressing fatty acid {beta}-oxidation, one-carbon metabolism, and antioxidant enzymes. This disruption of redox homeostasis results in accelerated accumulation of reactive oxygen species (ROS). In addition to its antibacterial effect, L-Cys also effectively reduced the virulence factor of bacterial motility. Finally, L-Cys demonstrated broad-spectrum antimicrobial activity against other pathogenic Vibrio species, including V. alginolyticus and V. anguillarum. Our findings suggest that L-Cys is a promising antimicrobial agent inducing ROS to mediate membrane disruption, with the advantages of cost-effectiveness and environmental safety for controlling vibriosis. ImportanceVibrio parahaemolyticus is a significant pathogen in aquaculture and a common cause of seafood-borne gastroenteritis worldwide. The increasing prevalence of antibiotic-resistant strains of this bacterium highlights the need for alternative control agents. This study shows that L-cysteine (L-Cys) inhibits the growth of V. parahaemolyticus. Our data indicate that L-Cys is metabolized to produce hydrogen sulfide, which contributes to the accumulation of ROS and disrupts bacterial membrane integrity. Additionally, L-Cys reduces bacterial motility and shows inhibitory effects against other Vibrio species. These findings suggest that L-Cys may represent a useful agent for managing Vibrio infections in aquaculture settings.

Extracellular electron transfer (EET) 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 microbial electrosynthesis 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 O2 reduction by oxidases. We show that oxidases use trace dissolved oxygen in a microaerobic bioelectrical chemical systems (BES), and the translocation of protons across the membrane during O2 reduction supports 2,3-BDO generation. Interestingly, this process is inhibited by high levels of dissolved oxygen in this system. In an aerated BES, O2 molecules react with the strong reductant (cathode) to form reactive oxygen species, resulting in cell death. ImportanceMicrobial electrosynthesis 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 bioelectrochemical systems design.