Journal of Bacteriology

Type IV pili (T4P) are important virulence factors that allow bacteria to adhere to and rapidly colonize their hosts. T4P are primarily composed of major pilins that undergo cycles of extension and retraction and minor pilins that initiate pilus assembly. Bacteriophages use T4P as receptors and exploit pilus dynamics to infect their hosts. Some bacteria encode pilin accessory proteins that post-translationally glycosylate major pilins to evade phage binding. TfpY is an accessory protein of unknown function that is widespread and structurally conserved among T4P-expressing bacteria. Here, we use Pseudomonas aeruginosa as a model to characterize the functional role of TfpY and its homologues in pilus assembly. TfpY expression is required for optimal pilus assembly and function; however, it does not provide phage defence, unlike previously characterized accessory proteins. TfpY can cross-complement twitching in strains expressing heterologous P. aeruginosa pilins, suggesting TfpY and its homologues play a common role in pilus assembly. We showed that TfpY likely interacts with the major pilin and specific minor pilins but is not incorporated into the pilus itself. We propose that TfpY, along with the minor pilins at the pilus tip, primes pilus assembly. We identified two unique gain-of-function mutations in T4P regulatory genes that non-specifically restore twitching in tfpY mutants by increasing levels of cAMP and expression of T4P components. This study enhances our understanding of the complex functional and regulatory relationships between pilin and accessory proteins. IMPORTANCE Type IV pili are surface filaments that enable versatile pathogens, like Pseudomonas aeruginosa , to adhere to and colonize surfaces. Pili are composed of diverse proteins called pilins, which serve as host receptors for phages. P. aeruginosa uses specific accessory proteins to glycosylate pilins to evade phage infection. Here, we show that TfpY is a conserved accessory protein that does not mediate phage defence. Instead, we propose a mechanism where TfpY facilitates efficient pilus assembly and function. A better understanding of TfpY function will provide insight into how its associated pilins have evolved to resist phage infection in the absence of post-translational modification, how some phages overcome this barrier to infection, and how this can guide the design of phage-based therapeutics.

Protein secretion is an essential cell process in bacteria, required for cell envelope biogenesis, export of virulence factors, and acquisition of nutrients, among other important functions. In the Sec secretion pathway, signal peptide-bearing precursors are recognized by the SecA ATPase and pushed across the membrane through a translocon channel made of the proteins SecY, SecE, and SecG. The Sec pathway has been extensively studied in the model organism Escherichia coli , but the Sec pathways of other bacteria such as the human pathogen Staphylococcus aureus differ in important ways from this model. Unlike in E. coli , a subset of precursors in S. aureus contains a YSIRK/GXXS (YSIRK) motif in an extended signal peptide. These proteins are secreted into the cross-wall compartment bounded by invaginating septal membranes during cell division. To gain insights into the factor(s) and mechanism(s) enabling protein secretion and spatial specificity in S. aureus , we isolated and screened a collection of temperature-sensitive ( ts ) mutants. These efforts identified at least one secA(ts ) allele as well as mutations in the secG and pepV genes. A SecA pull-down experiment identified SecDF, all ribosomal proteins, several chaperones and proteases, as well as PepV, validating the genetic screen in identifying candidate cofactors of SecA in S. aureus . IMPORTANCE All organisms use the Sec pathway for protein secretion, and key components of this pathway are essential for viability. The discovery of conditional loss-of-function mutants played an important role in defining the genetic basis of protein secretion in model organisms. In turn, the identification of Sec components facilitated mechanistic studies and revealed general rules for protein secretion but did not answer species-specific intricacies. Gram-positive bacteria, such as Staphylococcus aureus , restrict the secretion of some proteins into the septal membranes that bind their division site at mid-cell. Here, we screen a library of conditional temperature-sensitive mutants to define components of the Sec pathway of S. aureus and factors that may regulate its activity.

Bacterial spores can remain dormant for years, but they maintain the ability to recommence life through a process termed germination. Although spore germination has been reviewed many times, recent work has provided novel conceptual and molecular understandings of this important process. By using Bacillus subtilis as a model organism, here we thoroughly describe the signal transduction pathway and events that lead to spore germination, incorporating the latest findings on transcription and translation that are likely detected during germination. Then, we comprehensively review the proteins associated with germination and their respective functions. Notably, the typical germinant receptor GerA and the SpoVAF/FigP complex have been newly established as channels for ions release at early stage of germination. Moreover, given that germination is also affected by spore quality, such as molecular cargo, we collect the data about the proteins regulating sporulation to affect spore quality. Specifically, RocG-mediated glutamate catabolism during sporulation to ensure spore quality; GerE-regulated coat protein expression, and CotH-modified coat protein by phosphorylation to ensure normal coat assembly; and RNase Y-degraded RNA in newly released spores to promote dormancy. The latest progress in our understanding of these germination proteins provides valuable insights into the mechanism underlying germination.

Tuberculosis is a respiratory infection that is caused by members of the Mycobacterium tuberculosis complex, with M. tuberculosis (Mtb) being the predominant cause of the disease in humans. The approval of pretomanid and delamanid, two nitroimidazole-based compounds, for the treatment of tuberculosis encourages the development of more nitro-containing drugs that target Mtb. Similar to the nitroimidazoles, many antimycobacterial nitro-containing scaffolds are prodrugs that require reductive activation into metabolites that inhibit the growth of the pathogen. This reductive activation is mediated by mycobacterial nitroreductases, leading to the hypothesis that these nitroreductases contribute to the specificity of the nitro prodrugs for mycobacteria. In addition to their prodrug-activating activities, these nitroreductases have different native activities that support the growth of the bacteria. This review summarizes the activities of different mycobacterial nitroreductases with respect to their activation of different nitro prodrugs and highlights their physiological functions in the bacteria.

Nitrification by heterotrophic microorganisms is an important part of the nitrogen cycle in the environment. The enzyme responsible for the core function of heterotrophic nitrification is pyruvic oxime dioxygenase (POD). POD is a non-heme, Fe(II)-dependent enzyme that catalyzes the dioxygenation of pyruvic oxime to produce pyruvate and nitrite. To analyze the catalytic mechanism of POD, the crystal structure of POD from Alcaligenes faecalis (AfPOD) was determined at 1.76 Å resolution. The enzyme is a homotetramer, and the subunit structure is homologous to those of class II aldolases, in particular, a zinc-dependent L-fuculose-1-phosphate aldolase. The active site of the subunit is located at the bottom of a cleft formed with an adjacent subunit. The iron ion at the active site is coordinated by three histidines and three water molecules in an octahedral geometry. The putative oxygen tunnel was connected between the active site and the central cavity of the tetramer. The N-terminal region of AfPOD, which is essential for catalytic activity, is disordered in the crystal. Structure prediction with AlphaFold2 combined with mutational experiments suggested that the disordered N-terminal region adopts an α-helix conformation and participates in the formation of the active site. The catalytic mechanism of the dioxygenase reaction by POD is discussed on the basis of the molecular docking model. IMPORTANCE Our knowledge of nitrification has increased considerably in recent decades with the discovery of new nitrifying microorganisms and the characterization of their biochemical processes. Some heterotrophic bacteria and fungi are known to show nitrification activities, but the molecular mechanisms have been poorly understood. Here, we performed a structural characterization of pyruvic oxime dioxygenase (POD), a key enzyme in heterotrophic nitrification that produces nitrite from ammonia using pyruvic oxime as an intermediate. Structural and enzymatic analyses revealed that POD is a unique dioxygenase with features such as an aldolase backbone, an N-terminal α-helix, and an oxygen tunnel. Our results provide insights not only into the molecular mechanisms but also into the design of specific inhibitors of heterotrophic nitrification.

Ubiquitous in nature, biofilms provide stability in a fluctuating environment and provide protection from stressors. Biofilms formed in industrial processes are exceedingly problematic and costly. While biofilms of sulfate-reducing bacteria in the environment are often beneficial because of their capacity to remove toxic metals from water, in industrial pipelines, these biofilms cause a major economic impact due to their involvement in metal and concrete corrosion. The mechanisms by which biofilms of sulfate-reducing bacteria form, however, are not well understood. Our previous work identified two proteins, named by their gene loci DVU1012 and DVU1545, as adhesins in the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough. Both proteins are localized to the cell surface and the presence of at least one of the proteins, with either being sufficient, is necessary for biofilm formation to occur. In this study, differences in cell attachment and early biofilm formation in single deletion mutants of these adhesins were identified. Cells lacking DVU1012 had a different attachment strategy from wild-type (WT) and ΔDVU1545 cells, more often attaching as single cells than aggregates, which indicated that DVU1012 was more important for cell-to-cell attachment. ΔDVU1545 cells had increased cell attachment compared to WT cells when grown in static cultures. To date, comparisons of the D. vulgaris Hildenborough have been made to the large adhesion protein system in environmental pseudomonads. Yet, we and others have shown distinct mechanistic differences in the systems. We propose to name these proteins in D. vulgaris Hildenborough biofilm formation system to facilitate comparisons. IMPORTANCE Biofilms of sulfate-reducing bacteria contribute to biocorrosion, costing the United States hundreds of millions of dollars annually. In contrast, these biofilms can be used to bioremediate toxic heavy metals and to generate bioelectricity. As one of the most abundant groups of organisms on Earth, it is pertinent to better understand mechanistically how the biofilms of sulfate-reducing bacteria form so we may use this knowledge to help in efforts to mitigate biocorrosion, to promote bioremediation, and to produce clean energy. This study shows that the absence of either one of two biofilm adhesins impacts surface colonization by a sulfate-reducing bacterium, and that these two biofilm adhesins differ in their effect on cell attachment compared to other well-documented bacteria such as Pseudomonas species.

The ability to treat infections is threatened by the rapid emergence of antibiotic resistance among pathogenic microbes. Therefore, new antimicrobials are needed. Here we evaluate mannitol-1-phosphate 5-dehydrogenase (MtlD) as a potential new drug target. In many bacteria, mannitol is transported into the cell and phosphorylated by MtlA, the EIICBA component of a phosphoenolpyruvate-dependent sugar phosphotransferase system. MtlD catalyzes the conversion of mannitol-1-phosphate (Mtl-1P) to fructose-6-phosphate, which enters the glycolytic pathway. Mutants lacking mtlD are sensitive to mannitol due to accumulation of Mtl-1P. Here, we constructed mtlD mutants in four different bacterial species ( Cronobacter sakazakii , Pseudomonas aeruginosa, five serovars of Salmonella enterica , and three strains of Escherichia coli ), confirming and quantifying their mannitol sensitivity. The quantification of mannitol sensitivity in vitro was complicated by an inoculum effect and a resumption of growth following mannitol intoxication. The rate of resumption at different mannitol concentrations and cell population densities is fairly constant and reveals what is likely an intoxication processing rate. Provision of mannitol in drinking water, or by intraperitoneal injection, dramatically attenuates infection of a Salmonella enterica serovar Typhimurium mtlD mutant in mouse models of both gastroenteritis and systemic infection. Using CC003/Unc mice, we find that a mtlD mutant of Salmonella enterica serovar Typhi is also attenuated by provision of mannitol in drinking water. Therefore, we postulate that MtlD could be a valuable new therapeutic target. IMPORTANCE The ability to treat infections is threatened by the rapid emergence of antibiotic resistance. Mannitol is a polyol used in human medicine and the food industry. During catabolism of mannitol, many bacteria transport mannitol across the inner membrane forming the toxic intermediate mannitol-1-phosphate (Mtl-1P). Mtl-1P must be processed by mannitol dehydrogenase (MtlD) or it accumulates intracellularly, causing growth attenuation. We test and confirm here that mtlD mutants of Escherichia coli (including UPEC, and EHEC), Salmonella (including serovars Typhi, and Paratyphi A, B, and C), Cronobacter , and Pseudomonas experience mannitol sensitivity in vitro . Furthermore, providing mannitol in drinking water can alleviate both gastrointestinal and systemic Salmonella infections in mice. This suggests that inhibition of MtlD could be a viable antimicrobial strategy.

Chlamydia pneumoniae is an obligate intracellular bacterium of eukaryotic cells characterized by a unique biphasic life cycle; its biosynthesis and replication must occur within a cytoplasmic vacuole or inclusion. Certain inclusion membrane proteins have been demonstrated to mediate the interactions between intra-inclusion chlamydial organisms and the host cell. It has been demonstrated previously that the C. pneumoniae -encoded Cpn0308 localizes to the inclusion membrane; however, its function remains unknown. In the current study, a yeast two-hybrid assay was conducted to screen Cpn0308 as a bait against a HeLa cell cDNA library, revealing its binding to the host protein acyl-coenzyme A binding domain-containing 3 (ACBD3). The interaction between Cpn0308 and ACBD3 was confirmed through co-immunoprecipitation and GST (Glutathione S-transferase) pull-down assays. The two proteins were also co-localized in HeLa cells co-expressing Cpn0308 and ACBD3, as well as in C. pneumoniae -infected cells, as observed under confocal fluorescence microscopy. Given that ACBD3 plays a crucial role in maintaining host cell lipid homeostasis and its Golgi dynamic domain is responsible for interacting with Cpn0308, we hypothesize that the Cpn0308-ACBD3 interaction may facilitate C. pneumoniae ’s acquisition of host lipids, thereby benefiting chlamydial survival. This study lays a foundation for further elucidating the mechanisms of Cpn0308-mediated C. pneumoniae pathogenesis. IMPORTANCE The biosynthesis and replication of Chlamydia pneumoniae ( Cpn ) must occur within the cytoplasmic vacuoles or inclusions of host cells. Inclusion bodies play a crucial role in mediating the interactions between Cpn and host cells. Cpn0308 is localized to the inclusion membrane; however, its function is unknown. In this study, Cpn0308 was found to bind to host protein acyl-coenzyme A binding domain-containing 3 (ACBD3) through some standard approaches. Co-localization of the two proteins was observed in both original HeLa cells and Cpn-infected HeLa cells. ACBD3 plays a significant role in maintaining lipid homeostasis in host cells; we speculate that the Cpn0308-ACBD3 interaction may facilitate the acquisition of host lipids by C. pneumoniae , thereby enhancing chlamydial survival.

Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the expression of dozens of genes, many of which encode shared products, called “public goods.” P. aeruginosa possesses two complete acyl-homoserine lactone (AHL) QS circuits: the LasR-I and RhlR-I systems. Canonically, these systems are hierarchically organized: RhlR-I activity depends on LasR-I activation. However, in contrast to laboratory strains, isolates from people with cystic fibrosis can engage in AHL QS using only the transcription factor RhlR. In these isolates, RhlR regulates AHL QS and the production of secreted public goods, such as the exoprotease elastase, which are accessible to both producing and non-producing cells. When P. aeruginosa strains that use LasR to regulate elastase production are grown on casein as the sole carbon and energy source, LasR-null mutant “cheaters” commonly arise in populations due to a selective growth advantage. We asked if these social dynamics might differ in “RhlR cooperators”: populations that use RhlR, not LasR, to regulate public goods. We passaged RhlR cooperators from several genetic backgrounds in casein broth. We found that cheaters emerged among most RhlR cooperators. However, in one isolate background, E90, RhlR-null mutants were dramatically outcompeted by RhlR cooperators. In this background, the mechanism by which RhlR mutants are outcompeted by RhlR cooperators is AHL-dependent and occurs in stationary phase but is not the same as previously described “policing” mechanisms. Our data suggest that cheating, or the lack thereof, does not explain the lack of RhlR mutants observed in most infection environments. IMPORTANCE Quorum sensing (QS) mutants arise in a variety of populations of bacteria, but mutants of the gene encoding the transcription factor RhlR in Pseudomonas aeruginosa appear to be infrequent. Our work provides insight on the mechanisms through which RhlR-mediated cooperation is maintained in a LasR-null population of P. aeruginosa . Characterizing the selective pressure(s) that disfavor mutations from occurring in RhlR may enhance our understanding of P. aeruginosa evolution in chronic infections and potentially guide the development of therapeutics targeting the RhlR-I QS circuit.

Natural transformation (NT) is a conserved mechanism of horizontal gene transfer in bacterial species. During this process, DNA is taken up into the cytoplasm where it can be integrated into the host genome by homologous recombination. We have previously shown that some cytoplasmic exonucleases inhibit NT by degrading ingested DNA prior to its successful recombination. However, one exonuclease, SbcB, counterintuitively promotes NT in Vibrio cholerae . Here, through a systematic analysis of the distinct steps of NT, we show that SbcB acts downstream of DNA uptake into the cytoplasm, but upstream of recombinational branch migration. Through mutational analysis, we show that SbcB promotes NT in a manner that does not rely on its exonuclease activity. Finally, we provide genetic evidence that SbcB directly interacts with the primary bacterial recombinase, RecA. Together, these data advance our molecular understanding of horizontal gene transfer in V. cholerae and reveal that SbcB promotes homologous recombination during NT in a manner that does not rely on its canonical exonuclease activity. IMPORTANCE Horizontal gene transfer by natural transformation contributes to the spread of antibiotic resistance and virulence factors in bacterial species. Here, we study how one protein, SbcB, helps facilitate this process in the facultative bacterial pathogen Vibrio cholerae . SbcB is a well-known for its exonuclease activity (i.e., the ability to degrade the ends of linear DNA). Through this study, we uncover that while SbcB is important for natural transformation, it does not facilitate this process using its exonuclease activity. Thus, this work helps further our understanding of the molecular events required for this conserved evolutionary process and uncovers a function for SbcB beyond its canonical exonuclease activity.

An outer membrane (OM) is the hallmark feature that is often used to distinguish “Gram-negative” bacteria. Our understanding of how the OM is built rests largely on studies of Escherichia coli . In that organism—and seemingly in all species of the Proteobacterial phyla—the essential pathways that assemble the OM each rely on one or more lipoproteins that have been trafficked to the OM. Hence, the lipoprotein trafficking pathway appeared to be foundational for the ability of these bacteria to build their OM. However, such a notion now appears to be misguided. New phylogenetic analyses now show us that lipoprotein trafficking was likely the very last of the essential OM assembly systems to have evolved. The emergence of lipoprotein trafficking must have been a powerful innovation for the ancestors of Proteobacteria, given how it assumed such a central place in OM biogenesis. In this minireview, we broadly discuss the biosynthesis and trafficking of lipoproteins and ponder why the newest OM assembly system (lipoprotein trafficking) has become so key to building the Proteobacterial OM. We examine the diversity among lipoprotein trafficking systems, noting uniting commonalities and highlighting key differences. Current novel antibiotic development is targeted against a small subset of Proteobacterial species that cause severe human diseases; several inhibitors of lipoprotein biosynthesis and OM trafficking have been recently reported that may become new antibiotics. Understanding the diversity in lipoprotein trafficking may yield selective new antibiotics that preferentially kill important human pathogens while sparing species of normal healthy flora.

The methylation of ε-amino groups in protein lysine residues is an important posttranslational modification in eukaryotes. This modification plays a pivotal role in the regulation of diverse biological processes, including epigenetics, transcriptional control, and cellular signaling. Recent research has begun to reveal the potential role of methylation in modulating bacterial immune evasion and adherence to host cells. In this study, we analyzed the cell surface proteins of the toluene-degrading bacterium Acinetobacter sp. Tol 5 by label-free liquid chromatography‒mass spectrometry and found multiple lysine methylation in its trimeric autotransporter adhesin (TAA), AtaA. Over 130 lysine residues of AtaA, consisting of 3,630 amino acids and containing 234 lysine residues, were methylated. We identified that the outer membrane protein lysine methyltransferase (OM PKMT) of Tol 5, KmtA, specifically methylates the lysine residues of AtaA. In the KmtA-deficient mutant, most lysine methylations on AtaA were absent, indicating that KmtA is responsible for the methylation of multiple lysine residues throughout AtaA. Bioinformatic analysis revealed that the OM PKMT genes were widely distributed among Gram-negative bacteria, including pathogens with TAAs that promote infectivity, such as Burkholderia mallei and Haemophilus influenzae . Although KmtA has sequence similarities to the OM PKMTs of Rickettsia involved in infectivity, KmtA-like PKMTs formed a distinct cluster from those of the Rickettsia type according to the clustering analysis, suggesting that they are new types of OM PKMTs. Furthermore, the deletion of Tol 5 KmtA led to an increase in AtaA on the cell surface and enhanced bacterial adhesion, resulting in slower growth. IMPORTANCE Lysine methylation has been underexplored in prokaryotes, and information on it is limited to some pathogens. Our finding is the second case of multiple lysine methylation of bacterial outer membrane (OM) proteins, following that of OmpB of Rickettsia . The newly found target of methylation, AtaA, a trimeric autotransporter adhesin family protein of Acinetobacter sp. Tol 5 isolated from activated sludge, extends our understanding of OM protein methylation to non-pathogenic environmental strains. The newly identified enzyme KmtA shows higher specificity than rickettsial lysin methylases, protein lysine methyltransferases, and methylates more lysine residues of the target, which raises interest in the mechanism underlying its biological specificity. The widespread presence of KmtA-like PKMTs throughout Gram-negative bacteria suggests that lysine methylation functions more extensively in bacterial physiology than previously recognized.

MutT proteins are Nudix hydrolases characterized by the presence of a Nudix box, GX5EX7REUXEEXGU, where U is a bulky hydrophobic residue and X is any residue. Major MutT proteins hydrolyze 8-oxo-(d)GTP (8-oxo-GTP or 8-oxo-dGTP) to the corresponding 8-oxo-(d)GMP, preventing their incorporation into nucleic acids. Mycobacterial MutT1 comprises an N-terminal domain (NTD) harboring the Nudix box motif, and a C-terminal domain (CTD) harboring the RHG histidine phosphatase motif. Interestingly, unlike other MutTs, the MutT1 hydrolyses the mutagenic 8-oxo-(d)GTP to the corresponding 8-oxo-(d)GDP. Nucleoside diphosphate kinase (NDK), a conserved protein, carries out reversible conversion of (d)NDPs to (d)NTPs through phospho-NDK (NDK- Pi ) intermediate. Recently, we showed that NDK- Pi converts 8-oxo-dGDP to 8-oxo-dGTP and escalates A to C mutations in a MutT-deficient Escherichia coli . We now show that both Mycobacterium tuberculosis MutT1 and Mycobacterium smegmatis MutT1, through their NTD (Nudix hydrolase motifs) function as protein phosphatase to regulate the levels of NDK- Pi and prevent it from catalyzing conversion of (d)NDPs to (d)NTPs (including conversion of 8-oxo-dGDP to 8-oxo-dGTP). To corroborate this function, we show that Msm MutT1 decreases A to C mutations in E. coli under the conditions of Eco NDK overexpression. IMPORTANCE MutT proteins, having a Nudix box domain, hydrolyze the mutagenic 8-oxo-dGTP to 8-oxo-dGMP. However, mycobacterial MutT (MutT1) comprises an N-terminal domain (NTD) harboring a Nudix box, and a C-terminal domain (CTD) harboring an RHG histidine phosphatase. Unlike other MutTs, mycobacterial MutT1 hydrolyses 8-oxo-dGTP to 8-oxo-dGDP. Nucleoside diphosphate kinase (NDK), a conserved protein, converts 8-oxo-dGDP to 8-oxo-dGTP through phospho-NDK (NDK- Pi ) intermediate and escalates A to C mutations. Here, we show that the mycobacterial MutT1 is unprecedented in that its NTD (Nudix box), functions as protein phosphatase to regulate NDK- Pi levels and prevents it from converting dNDPs to dNTPs (including 8-oxo-dGDP to 8-oxo-dGTP conversion). In addition, mycobacterial MutT1 decreases A to C mutations in Escherichia coli under the conditions of NDK overexpression.

Modifications of transfer RNA (tRNA) have been shown to play critical roles in the biogenesis, metabolism, structural stability, and function of RNA molecules, and the specific modifications of nucleobases with sulfur atoms in tRNA are present in prokaryotes and eukaryotes. The s ² group of s ² U34 stabilizes anticodon structure, confers ribosome-binding ability to tRNA, and improves reading frame maintenance. In particular, specific enzymes catalyze the biosynthesis of sulfur-containing nucleosides of s ² U34, such as the L-cysteine desulfurase IscS and the tRNA thiouridylase MnmA in Escherichia coli . Until recently, the mechanism of sulfur transfer in E. coli was considered to involve persulfide chemistry; however, a newly proposed mechanism suggests the involvement of a [4Fe–4S] cluster bound to MnmA. This review provides a critical appraisal of recent evidence for [4Fe–4S]-dependent or [4Fe–4S]-independent tRNA thiolation in 2-thiouridine formation.

The outer membrane (OM) of Gram-negative bacteria is the outermost layer of the cell and serves as permeability barrier against environmental toxins, including antibiotics. The OM is built by several pathways that transport and assemble lipids and proteins into the OM. Since the OM is an essential organelle for the cell, envelope stress responses (ESRs) continuously monitor its assembly to preserve viability if defects arise. While ESRs have been extensively characterized in Escherichia coli , they are generally narrowly conserved. Lipoprotein trafficking to the OM via the “Lol” pathway is a linchpin for all OM assembly pathways. In E. coli , defects in this essential process are sensed when the sensor OM lipoprotein NlpE activates the CpxAR two-component system. Distantly related Acinetobacter baumannii encodes an NlpE homolog but lacks any Cpx homolog; how OM lipoprotein stress might be sensed and mitigated in these bacteria is therefore unclear. Here, we used CRISPRi to transiently induce defects in OM lipoprotein synthesis (targeting lgt and lnt ) or trafficking (targeting lolA ) in A. baumannii . We defined the transcriptional response to blocks in OM lipoprotein biogenesis. After scrutinizing candidate ESRs, we identified the BfmRS two-component systems as specifically critical for preserving A. baumannii viability during stress in OM lipoprotein biogenesis. Surprisingly, A. baumannii NlpE played no role in combatting OM lipoprotein stress. Our study identifies an A. baumannii ESR for OM lipoprotein biogenesis defects that acts in a distinct mechanism, not involving the NlpE sensor lipoprotein. IMPORTANCE As the cell’s surface, the outer membrane (OM) of bacteria, such as Acinetobacter baumannii , is continuously under assault from the environment or host. OM integrity is needed for cell survival, and envelope stress responses (ESRs) act to detect and repair any defects. ESRs are well-defined in Escherichia coli but are poorly conserved. We sought to identify an ESR for the essential process of OM lipoprotein biogenesis in A. baumannii . We found that the BfmRS two-component system performs this function and does so without relying on its NlpE sensor homolog, suggesting a novel mechanism of stress sensing is involved in A. baumannii . Our work identifies a key cellular role for BfmRS.

Bacterial aggregates are observed in both natural and artificial environments. In the context of disease, aggregates have been isolated from chronic and acute infections. Pseudomonas aeruginosa ( Pa ) aggregates contribute significantly to chronic infections, particularly in the lungs of people with cystic fibrosis (CF). Unlike the large biofilm structures observed in vitro , Pa in CF sputum forms smaller aggregates (~10–1,000 cells), and the mechanisms behind their formation remain underexplored. This study aims to identify genes essential and unique to Pa aggregate formation in a synthetic CF sputum media (SCFM2). We cultured Pa strain PAO1 in SCFM2 and LB, both with and without mucin, and used RNA sequencing (RNA-seq) to identify differentially expressed genes. The presence of mucin revealed 13 significantly differentially expressed (DE) genes, predominantly downregulated, with 40% encoding hypothetical proteins unique to aggregates. Using high-resolution microscopy, we assessed the ability of mutants to form aggregates. Notably, no mutant exhibited a completely planktonic phenotype. Instead, we identified multiple spatial phenotypes described as “normal,” “entropic,” or “impaired.” Entropic mutants displayed tightly packed, raft-like structures, while impaired mutants had loosely packed cells. Predictive modeling linked the prioritized genes to metabolic shifts, iron acquisition, surface modification, and quorum sensing. Co-culture experiments with wild-type PAO1 revealed further spatial heterogeneity and the ability to “rescue” some mutant phenotypes, suggesting cooperative interactions during growth. This study enhances our understanding of Pa aggregate biology, specifically the genes and pathways unique to aggregation in CF-like environments. Importantly, it provides insights for developing therapeutic strategies targeting aggregate-specific pathways. IMPORTANCE This study identifies genes essential for the formation of Pseudomonas aeruginosa (Pa) aggregates in cystic fibrosis (CF) sputum, filling a critical gap in understanding their specific biology. Using a synthetic CF sputum model (SCFM2) and RNA sequencing, 13 key genes were identified, whose disruption led to distinct spatial phenotypes observed through high-resolution microscopy. The addition of wild-type cells either rescued the mutant phenotype or increased spatial heterogeneity, suggesting cooperative interactions are involved in aggregate formation. This research advances our knowledge of Pa aggregate biology, particularly the unique genes and pathways involved in CF-like environments, offering valuable insights for developing targeted therapeutic strategies against aggregate-specific pathways.

Outer membrane vesicles (OMVs) are small membrane enclosed sacs released from bacteria which serve as carriers of biomolecules that shape interactions with the surrounding environment. The periodontal pathogen, Porphyromonas gingivalis , is a prolific OMV producer. Here, we investigated how the structure of lipid A, a core outer membrane molecule, influences P. gingivalis OMV production, OMV-dependent TLR4 activation, and biofilm formation. We examined mutant strains of P. gingivalis 33277 deficient for enzymes that alter lipid A phosphorylation and acylation status. The lipid A C4′-phosphatase ( lpxF )-deficient strain and strains bearing inactivating point mutations in the LpxF active site displayed markedly reduced OMV production relative to WT. In contrast, strains deficient for either the lipid A C1-phosphatase ( lpxE ) or the lipid A deacylase (PGN_1123; lpxZ ) genes did not display alterations in OMV abundance compared to WT. These data indicate that lipid A C4′-phosphate removal is required for typical OMV formation. In addition, OMVs produced by ΔlpxF and ΔlpxZ strains, possessing only penta-acylated lipid A, stimulated robust TLR4 activation, whereas OMVs obtained from WT and ΔlpxE strains, containing predominantly tetra-acylated lipid A, did not. Hence, lipid A remodeling modulates the capacity of OMVs to engage host TLR4-dependent immunity. Finally, we demonstrate an inverse relationship between OMV abundance and biofilm density, with the ∆lpxF mutants forming denser biofilms than either WT, ΔlpxE , or ΔlpxZ strains. Therefore, OMVs may also contribute to pathogenesis by regulating biofilm formation and dispersal. IMPORTANCE Porphyromonas gingivalis is a bacterium strongly associated with periodontitis. P. gingivalis exports lipids, proteins, and other biomolecules that contribute to the bacterium’s ability to persist in inflammatory conditions encountered during disease. These biomolecules are exported through several mechanisms, including via outer membrane vesicles (OMVs). Despite their ubiquity, the mechanisms that drive outer membrane vesicle production vary among bacteria and are not fully understood. In this study, we report that C4′ dephosphorylation of lipid A, a major outer membrane molecule, is required for robust outer membrane vesicle production and biological function in P. gingivalis . This finding adds to the growing body of evidence that lipid A structure is an important factor in outer membrane vesicle biogenesis in diverse bacterial species.

Pseudomonas aeruginosa have a versatile metabolism; they can adapt to many stressors, including limited oxygen and nutrient availability. This versatility is especially important within a biofilm where multiple microenvironments are present. As a facultative anaerobe, P. aeruginosa can survive under anaerobic conditions utilizing denitrification. This process produces nitric oxide (NO) which has been shown to result in cell elongation. However, the molecular mechanism underlying this phenotype is poorly understood. Our laboratory has previously shown that NosP is a NO-sensitive hemoprotein that works with the histidine kinase NahK to regulate biofilm formation in P. aeruginosa . In this study, we identify NahK as a novel regulator of denitrification under anaerobic conditions. Under anaerobic conditions, deletion of nahK leads to a reduction of growth coupled with reduced transcriptional expression and activity of the denitrification reductases. Furthermore, during stationary phase under anaerobic conditions, Δ nahK does not exhibit cell elongation, which is characteristic of P. aeruginosa . We determine the loss of cell elongation is due to changes in NO accumulation in Δ nahK . We further provide evidence that NahK may regulate denitrification through modification of RsmA levels. IMPORTANCE Pseudomonas aeruginosa is an opportunistic multi-drug resistance pathogen that is associated with hospital-acquired infections. P. aeruginosa is highly virulent, in part due to its versatile metabolism and ability to form biofilms. Therefore, better understanding of the molecular mechanisms that regulate these processes should lead to new therapeutics to treat P. aeruginosa infections. The histidine kinase NahK has been previously shown to be involved in both nitric oxide (NO) signaling and quorum sensing through RsmA. The data presented here demonstrate that NahK is responsive to NO produced during denitrification to regulate cell morphology. Understanding the role of NahK in metabolism under anaerobic conditions has larger implications in determining its role in a heterogeneous metabolic environment such as a biofilm.

Legionella pneumophila is an environmentally acquired pathogen that causes respiratory disease in humans. While the discovery of L. pneumophila is relatively recent compared to other bacterial pathogens, over the past 50 years, L. pneumophila has emerged as a powerhouse for studying host-pathogen interactions. In its natural habitat of fresh water, L. pneumophila interacts with a diverse array of protozoan hosts and readily evolve to expand their host range. This has led to the accumulation of the most extensive arsenal of secreted virulence factors described for a bacterial pathogen and their ability to infect humans. Within amoebae and human alveolar macrophages, the bacteria replicate within specialized membrane-bound compartments, establishing L. pneumophila as a model for studying intracellular vacuolar pathogens. In contrast, the virulence factors required for intracellular replication are specifically tailored to individual host cells types, allowing the pathogen to adapt to variation between disparate niches. The broad host range of this pathogen, combined with the extensive diversity and genome plasticity across the Legionella genus, has thus established this bacterium as an archetype to interrogate pathogen evolution, functional genomics, and ecology. In this review, we highlight the features of Legionella that establish them as a versatile model organism, new paradigms in bacteriology and bacterial pathogenesis resulting from the study of Legionella, as well as current and future questions that will undoubtedly expand our understanding of the complex and intricate biology of the microbial world.

Bacterial microcompartments (BMCs) are self-assembled protein structures often utilized by bacteria as a modular metabolic unit, enabling the catalysis and utilization of less common carbon and nitrogen sources within a self-contained compartment. The ethanolamine (EA) utilization (eut) BMC has been widely demonstrated in enteropathogens, such as Salmonella enterica, and current research is exploring its activity in the commensal species that populate the human gut. Escherichia coli Nissle 1917 (EcN) is a strong colonizer and probiotic in gut microbial communities and has been used extensively for microbiome engineering. In this study, the utilization of ethanolamine as a sole carbon source and the formation of the eut BMC in EcN were demonstrated through growth assays and visualization with transmission electron microscopy. Subsequently, flux balance analysis was used to further investigate the metabolic activity of this pathway. It was found that not only is the utilization of the eut BMC for the degradation of EA as a carbon source in EcN comparable with that of Salmonella enterica but also that ammonium is released into solution as a byproduct in EcN but not in S. enterica. Control of EA-dependent growth was demonstrated using different concentrations of the operon inducer, vitamin B12. We show that vitamin B12-dependent EA utilization as the sole carbon source enables growth in EcN, and demonstrate the concurrent formation of the BMC shell and inducible control of the eut operon. IMPORTANCE The human gut is a complex environment of different bacterial species, nutrient sources, and changing conditions that are essential for human health. An imbalance can allow for the emergence of opportunistic pathogens. Bacterial microcompartments (BMCs) are utilized by bacteria to metabolize less common nutrients, conferring a growth advantage. Although widely studied in enteropathogens, there is limited research on BMC activity in commensal species. We demonstrate the formation of the eut BMC and utilization of ethanolamine as a carbon source in the human gut commensal Escherichia coli Nissle 1917 (EcN). Additionally, we found increased ammonium production when EcN utilized ethanolamine but did not see the same in Salmonella enterica, highlighting potential differences in how these species affect the wider microbial community.

Vancomycin-resistant enterococci (VRE) are important pathogens in hospitalized patients; however, the factors involved in VRE colonization of hospitalized patients are not well characterized. Bacteriocins provide a competitive advantage to enterococci in experimental models of colonization, but little is known about bacteriocin content in samples derived from humans and even less is known about their dynamics in the clinical setting. To identify bacteriocins which may be relevant in the transmission of VRE, we present a systematic analysis of bacteriocin content in the genomes of 2,248 patient-derived E. faecium isolates collected over a 6-year period from a single hospital system. We used computational methods to broadly search for bacteriocin structural genes and a functional assay to look for phenotypes consistent with bacteriocin expression. We identified homology to 15 different bacteriocins, with 2 having a high presence in this clinical cohort. Bacteriocin 43 (bac43) was found in a total of 58% of isolates, increasing from 8% to 91% presence over the 6-year collection period. There was little genetic variation in the bac43 structural or immunity genes across isolates. The enterocin A structural gene was found in 98% of isolates, but only 0.3% of isolates had an intact enterocin A gene cluster and displayed a bacteriocin-producing phenotype. This study presents a wide survey of bacteriocins from hospital isolates and identified bac43 as highly conserved, increasing in prevalence, and phenotypically functional. This makes bac43 an interesting target for future investigation for a potential role in E. faecium transmission. IMPORTANCE While enterococci are a normal inhabitant of the human gut, vancomycin-resistant E. faecalis and E. faecium are urgent public health threats responsible for hospital-associated infections. Bacteriocins are ribosomally synthesized antimicrobial proteins and are commonly used by bacteria to provide a competitive advantage in polymicrobial environments. Bacteriocins have the potential to be used by E. faecium to invade and dominate the human gut leading to a greater propensity for transmission. In this work, we explore bacteriocin content in a defined clinically derived population of E. faecium using both genetic and phenotypic studies. We show that one highly active bacteriocin is increasing in prevalence over time and demonstrates great potential relevance to E. faecium transmission.

In response to starvation, endospore-forming bacteria differentiate into stress-resistant spores that can remain dormant for years yet rapidly germinate and resume growth when nutrients become available. To identify uncharacterized factors involved in the exit from dormancy, we performed a transposon-sequencing screen taking advantage of the loss of spore heat resistance that accompanies germination. We reasoned that transposon insertions that impair but do not block germination will lose resistance more slowly than wild type after exposure to nutrients and will therefore survive heat treatment. Using this approach, we identified most of the known germination genes and several new ones. We report an initial characterization of 15 of these genes and a more detailed analysis of one (ymaF). Spores lacking ymaF (renamed gerY) are impaired in germination in response to both L-alanine and L-asparagine, D-glucose, D-fructose, and K⁺. GerY is a soluble protein synthesized under σE control in the mother cell. A YFP-GerY fusion localizes around the developing and mature spore in a manner that depends on CotE and SafA, indicating that it is a component of the spore coat. Coat proteins encoded by the gerP operon and gerT are also required for efficient germination, and we show that spores lacking two or all three of these loci have more severe defects in the exit from dormancy. Our data are consistent with a model in which GerY, GerT, and the GerP proteins are required for efficient transit of nutrients through the coat to access the germination receptors, but each acts independently in this process. IMPORTANCE Pathogens in the orders Bacillales and Clostridiales resist sterilization by differentiating into stress-resistant spores. Spores are metabolically inactive and can remain dormant for decades, yet upon exposure to nutrients, they rapidly resume growth, causing food spoilage, food-borne illness, or life-threatening disease. The exit from dormancy, called germination, is a key target in combating these important pathogens. Here, we report a high-throughput genetic screen using transposon sequencing to identify novel germination factors that ensure the efficient exit from dormancy. We identify several new factors and characterize one in greater detail. This factor, renamed GerY, is part of the proteinaceous coat that encapsulates the dormant spore. Our data suggest that GerY enables efficient transit of nutrients through the coat to trigger germination.

Pseudomonas aeruginosa is a common opportunistic pathogen and a model organism for studying bacterial sociality. A social behavior of P. aeruginosa that is critical for its success as a pathogen is its ability to form protective biofilms. Many of P. aeruginosa’s social phenotypes are regulated by quorum sensing—a type of cell-cell communication that allows bacteria to respond to population density. Although biofilm formation is known to be affected by quorum sensing, evidence for direct regulation of biofilm production by quorum regulators has remained elusive. In this work, we show that production of the major biofilm matrix polysaccharide Psl in P. aeruginosa PAO1 is regulated by the quorum regulators LasR and RhlR in stationary-phase cultures. Secretion of Psl into the culture medium requires LasR, RhlR, and the quorum signal molecules N-3-oxo-dodecanoyl-homoserine lactone and N-butanoyl homoserine lactone. Psl production in strains unable to synthesize the homoserine lactone signals can be restored by exogenous introduction of the signal molecules. We found that LasR and RhlR perform different roles in the regulation of Psl production: LasR acts at the promoter of the psl operon and activates transcription of the Psl biosynthetic genes, while RhlR activates translation of the psl transcripts. This work contributes to our understanding of the overlapping but distinct functions of the Las and Rhl quorum-sensing systems and implicates both in the direct regulation of biofilm matrix production. IMPORTANCE Pseudomonas aeruginosa biofilms are responsible for many treatment-resistant infections in humans. Many cooperative behaviors in P. aeruginosa are controlled by quorum sensing, but evidence for a direct role of quorum sensing in the regulation of biofilm matrix production has been scant. In this work, we show that the Las and Rhl quorum-sensing systems have distinct roles in regulating production of the matrix polysaccharide Psl and that this regulation happens at the level of transcription (Las) and translation (Rhl) of the psl operon. These findings deepen our understanding of overlapping functions of Las and Rhl quorum sensing and the complex regulation of biofilm development in P. aeruginosa.

ATP, most widely known as the primary energy source for numerous cellular processes, also exhibits the characteristics of a biological hydrotrope. The viable but nonculturable (VBNC) and persister states are two prevalent dormant phenotypes employed by bacteria to survive challenging environments, both of which are associated with low metabolic activity. Here, we investigate the intracellular ATP concentration of individual VBNC and persister cells using a sensitive ATP biosensor QUEEN-7μ and reveal that both types of cells possess a lower intracellular ATP concentration than culturable and sensitive cells, although there is a certain overlap in the intracellular ATP concentrations between antibiotic-sensitive cells and persisters. Moreover, we successfully separated VBNC cells from culturable cells using fluorescence-activated cell sorting based on the intracellular ATP concentration threshold of 12.5 µM. Using an enriched VBNC cell population, we confirm that the precipitation of proteins involved in key biological processes promotes VBNC cell formation. Notably, using green light-illuminated proteorhodopsin (PR), we demonstrate that VBNC cells can be effectively resuscitated by elevating their intracellular ATP concentration. These findings highlight the crucial role of intracellular ATP concentration in the regulation of bacterial cell fate and provide new insights into the formation of VBNC and persister cells. IMPORTANCE The viable but nonculturable (VBNC) and persister states are two dormant phenotypes employed by bacteria to counter stressful conditions and play a crucial role in chronic and recurrent bacterial infections. However, the lack of precise detection methods poses significant threats to public health. Our study reveals lower intracellular ATP concentrations in these states and establishes an ATP threshold for distinguishing VBNC from culturable cells. Remarkably, we revive VBNC cells by elevating their intracellular ATP levels. This echoes recent eukaryotic studies where modulating metabolism impacts outcomes like osteoarthritis treatment and lifespan extension in Caenorhabditis elegans. Our findings underscore the crucial role of intracellular ATP levels in governing bacterial fate, emphasizing ATP manipulation as a potential strategy to steer bacterial behavior.