Journal of Bacteriology最新文献

FlhF, an SRP GTPase, regulates the polarity and flagellation patterns in various bacteria, including Pseudomonas aeruginosa. FlhF is a multidomain protein that comprises three domains: B, N, and G, or the GTPase domain. However, the roles of different domains in the assembly and maintenance of flagella in this clinically important bacterium remain unclear. In this study, we report that the C-terminal GTPase domain plays a critical role in maintaining the polar localization of FlhF. Complementation assays using truncated constructs show that the flagellar assembly depends on both the B- and N-domains. In addition, GTP binding by FlhF regulates its interaction with FliG, a flagellar C-ring protein that facilitates flagellar basal body assembly in P. aeruginosa.IMPORTANCEPathogenic bacteria rely on flagellar motility for infection and colonization. FlhF, a member of the SRP GTPase family, is a key factor regulating flagellation patterns in various bacteria. In this study, we investigated the domain-wise role of FlhF in P. aeruginosa, an ESKAPE pathogen that uses a single flagellum for its virulence, biofilm formation, and pathogenesis. Our study reveals the role of FlhF in the assembly and spatial regulation of flagella in P. aeruginosa. In addition, we examined the interaction of FlhF with the flagellar ring protein. This study provides insights into the molecular mechanism underlying polar assembly of flagella, thus offering a targeted approach for controlling infection by this pathogen.

Members of the genus Pseudomonas synthesize diverse natural products that contribute to their versatility in free-living and host-associated lifestyles. Here, we characterized the in vivo functions of the nitroimidazole antibiotic azomycin in the genus Pseudomonas. We found that genes with similarity to azomycin biosynthesis genes rohPQRST are prevalent within the Pseudomonas syringae species complex and rarely present in the Pseudomonas fluorescens species complex. Azomycin production was detectable in culture by biocontrol strains Pseudomonas spp. DF41 and CMR5a. Pseudomonas sp. DF41 exhibited anti-oomycete activity that was lost in a ∆rohPQRST mutant. Purified azomycin was sufficient to kill the oomycete pathogens Aphanomyces and Phytophthora. Although DF41 has been studied for its role in biocontrol of plant pathogens, we found that azomycin exhibited phytotoxicity against Pisum sativum (pea) plants at similar concentrations to those that inhibited oomycetes. However, consistent with its use as a biocontrol agent, Pseudomonas sp. DF41 only produced azomycin in planta when pea plants were infected with the oomycete pathogen Aphanomyces euteiches. Our findings suggest dual roles for azomycin in Pseudomonas, functioning both as a biocontrol agent of oomycete pathogens, as well as a phytotoxic molecule with a potential role in plant virulence.IMPORTANCEWhile many natural products are studied for their roles in the treatment of plant or human disease, the ecological functions of natural products are understudied. We found that an antibiotic, azomycin, is produced by Pseudomonas species and has toxicity against both plants and oomycete pathogens. Our findings suggest a complex ecological role of azomycin production by Pseudomonas in both the amelioration and exacerbation of plant disease.

Isoniazid (INH) is a first-line drug for treating drug-susceptible tuberculosis. The genome of Mycobacterium smegmatis encodes two cAMP receptor proteins (Crp), MSMEG_6189 (Crp1) and MSMEG_0539 (Crp2). The deletion of crp1 markedly increased susceptibility to INH, whereas the deletion of crp2 had no effect. In contrast, susceptibility to rifampicin was unchanged in the Δcrp1 mutant. Gene expression analysis revealed strong upregulation of katG1, which encodes the INH-activating catalase-peroxidase, and an almost complete abolishment of ahpC expression, which encodes alkyl hydroperoxide reductase and contributes to INH resistance, in the Δcrp1 mutant. The mutant also exhibited elevated intracellular reactive oxygen species (ROS) levels and reduced respiration. These elevated ROS levels likely resulted from alterations in the respiratory electron transport chain, including reduced levels of the cytochrome bcc₁ complex, increased expression of the nuo and sdh1 operons (encoding type I NADH dehydrogenase and succinate dehydrogenase, respectively), and the loss of bd quinol oxidase induction under conditions of diminished bcc₁ complex levels. ROS accumulation, in turn, appears to inactivate the FurA repressor, leading to the induction of katG1 expression. Collectively, these findings suggest that the loss of Crp1 increases INH susceptibility through multiple mechanisms that promote INH activation, including ROS-mediated induction of katG1 and increased intracellular ROS levels. Furthermore, the absence of ahpC expression in the Δcrp1 mutant further contributes to its increased INH susceptibility.

Importance: This study revealed that the cAMP receptor protein, Crp1, plays a role in controlling key genes involved in both INH activation (katG1) and resistance (ahpC) in Mycobacterium smegmatis. Inactivation of Crp1 was shown to cause an increase in cellular ROS levels, which likely results from a bottleneck of electron flow at the cytochrome bcc₁ complex in the electron transport chain and the abolishment of bd quinol oxidase induction under conditions of reduced bcc1 complex levels. The resulting increase in ROS, combined with the simultaneous loss of ahpC expression, renders the bacterium significantly more vulnerable to INH. These findings provide deeper insights into the interplay among oxidative phosphorylation, ROS generation, and INH susceptibility.

Clostridioides difficile is a common cause of acute gastrointestinal (GI) inflammation in mammals, which can have detrimental effects on host health. C. difficile-associated disease requires the secretion of high-molecular-weight toxins after colonization of the GI tract. The molecular mechanisms of GI colonization by C. difficile include potential interactions with host cells and the mucus layer formed from secreted mucin glycoproteins. C. difficile associates with the mucus layer in vivo and will associate with both epithelial cells and mucosal surfaces in vitro. Previously, we found a substantial defect in binding to mucosal surfaces for mutants of the major flagellar subunit, fliC, while mutation of the major subunit of type IV pili, pilA1, showed increased adhesion. To elucidate the mechanisms by which C. difficile interacts with ex vivo mucosal surfaces, we have measured swimming motility, mucosal adhesion, and levels of flagellation by transmission electron microscopy for mutants of flagellar and T4P genes in C. difficile R20291. We discovered that the pilA1 mutant showed increased flagellation, while decreases in flagellation were found for fliC, fliD, and flg-Δ3 OFF (a phase-locked mutant with low transcription of the F3 flagellar operon), which were associated with both low swimming motility and low adhesion to mucosal surfaces. However, the reversed flg-Δ3 ON mutant showed increased flagellation without a significant increase in adhesion. We also found that the fliC mutant was defective in binding to mucus-secreting HT-29 MTX cells, and that decreased binding was not observed for other mutants with reduced flagellation. These results imply that at least two molecular pathways contribute to C. difficile mucosal adhesion. In addition to their direct roles encoding T4P and flagellar subunits, pilA1 and fliC may contribute to regulatory networks governing other proteins relevant to mucosal adhesion.IMPORTANCEIn the context of previous work on Clostridioides difficile host adhesion by our groups and others, our results suggest that (i) at least two mechanisms exist for mucosal adhesion by C. difficile, potentially direct adhesion by flagella and another lectin-like adhesion regulated by flagellar components; (ii) mucosal binding can also contribute to C. difficile adhesion in 2D cell culture and could explain previous defects from fliC mutants; and (iii) levels of flagellation are largely insensitive to fliC transcription, implying that other factors limit flagellar production and that FliC levels may be regulated independently as part of regulatory networks within C. difficile.

Coenzyme M (2-mercaptoethane sulfonate, CoM) is an essential low-molecular-weight thiol in methanogenic archaea (methanogens) that serves as a methyl carrier and as a component of CoM-S-S-CoB heterodisulfide comprising CoM and coenzyme B (7-mercaptoheptanoylthreoninephosphate), which serves as the terminal electron acceptor in methanogenesis. Increasing the amount of CoM in Methanosarcina acetivorans cells by overexpressing its proposed biosynthesis genes results in faster growth on methanol or methanol + acetate medium in the absence of sulfide. Furthermore, CoM overproduction enhances resistance to oxidative stress in the ΔhdrABC mutant genetic background in which the nonessential methylotrophic-specific CoM-S-S-CoB heterodisulfide reductase enzyme, HdrABC, has been deleted. The ΔhdrABC mutant is resistant to 5% O₂ atmosphere, and overexpression of CoM biosynthesis genes resulted in resistance to up to 2 mM hydrogen peroxide in a stress assay. Increased resistance to O₂ is correlated with 34% lower CoM-SH level in the ΔhdrABC and 16% lower CoM-SH level in the ΔhdrABC com⁺ mutant strains versus the parent strain, while resistance to H₂O₂ stress is correlated to a higher ratio of reduced CoM-SH to total CoM in the parent versus the com⁺ (8.7%), ΔhdrABC (10%), and ΔhdrABC com⁺ (16%) mutant strains. Our study suggests increased expressions of genes encoding coenzyme M biosynthesis, in conjunction with deletion of HdrABC, increase oxidative stress resistance of the strictly anaerobic methanogen Methanosarcina acetivorans.

Importance: Methanogens are key organisms in the global carbon cycle with potential to be harnessed to produce renewable energy and transportation fuel. Methanogens are strict anaerobes found in subsurface sediment, anaerobic digesters, and digestive tracts of animals such as the rumen. Our results suggest methanogens can adapt to prolonged exposure to oxidative stress under the appropriate environmental conditions, and it may be possible to engineer thiol redox homeostasis and oxidative stress resistance in methanogens. Engineering redox homeostasis and oxidative stress resistance in methanogens and other strict anaerobes has the potential to reduce technical barriers to culturing, thus accelerating research progress on a wide variety of non-model microbes, and ultimately broadening potential biotechnology applications related to sustainable food, fuel, and biomedical uses.

Glucosaminyl phosphatidylglycerol (GlcN-PG) was first identified in bacteria in the 1960s and was recently reported in Pseudomonas aeruginosa. Despite the important implications in altering membrane charge (by the modification of anionic phosphatidylglycerol [PG] with cationic glucosamine), the biosynthesis and functions of GlcN-PG have remained uncharacterized. Using bioinformatic and lipidomic analysis, we identified a 3-gene predicted operon, renamed as gpgSDF, that is responsible for the biosynthesis and potential transport of GlcN-PG in P. aeruginosa: gpgS encodes a novel glycotransferase that is responsible for the modification of PG with N-acetylglucosamine (GlcNAc) to produce GlcNAc-PG, and gpgD encodes a novel deacetylase that removes the acetyl group from GlcNAc-PG to produce GlcN-PG. The third gene, gpgF, is predicted to encode a flippase whose activity remains to be experimentally verified. A P. aeruginosa gpgD transposon mutant accumulates GlcNAc-PG and lacks GlcN-PG, and as expected, the complementation of gpgD restores the production of GlcN-PG. Moreover, the heterologous expression of gpgSDF in Escherichia coli resulted in the production of both GlcNAc-PG and GlcN-PG. The identification of the biosynthetic genes of GlcN-PG paves the way for the investigation of its biological and pathological functions, which has significant implications in our understanding of the unique membrane physiology, pathogenesis, and antimicrobial resistance of P. aeruginosa.

Importance: The identification of the biosynthetic genes of glucosaminyl phosphatidylglycerol (GlcN-PG) paves the way for the investigation of its biological and pathological functions, which has significant implications in our understanding of the unique membrane physiology, pathogenesis, and antimicrobial resistance of Pseudomonas aeruginosa.

YcgR is a c-di-GMP effector that inhibits chemotaxis and swimming speed in Escherichia coli and Salmonella. Genetic, biochemical, and structural studies suggest that YcgR interacts with both the bidirectional flagellar rotor and the stator to bias rotation toward counterclockwise (CCW) and reduce motor speed, but the underlying mechanism remains unresolved. Recent cryo-electron microscopy structures revealing conformational changes in the rotor-stator complex during directional switching suggested to us a mechanism by which YcgR acts. We call this the Texas 2-step model, after the country dance in which partners move smoothly in a CCW arc with quick steps followed by slow ones. In this model, YcgR first binds a MotA subunit when the rotor adopts the CCW conformation, in which stators are largely displaced from the C-ring. In the next step, the rotating MotA pentamer delivers YcgR to the rotor protein FliG, thereby slowing motor speed. We provide evidence for the first step of this model, offering testable predictions for future work.IMPORTANCEThe mechanism of YcgR action has been investigated by multiple laboratories using diverse approaches, yet no consensus has emerged. Some studies implicate the rotor, others the stator. A key complication is the involvement of four interacting proteins-MotA, FliG, FliM, and YcgR-with multiple contact sites in several of them. Recent rotor-stator cryo-electron microscopy structures revealing conformational changes during directional switching suggested a mechanism that we set out to test. Our experiments show that rotor conformation is crucial for YcgR function.

Errors in protein synthesis can influence cellular fitness and, in some cases, affect genome stability. While this connection has been explored, most investigations have focused on Enterobacteriaceae such as Escherichia coli, leaving open the question of how translational fidelity shapes mutagenesis in other bacterial groups. Would a metabolically versatile, stress-tolerant soil bacterium behave differently? We focused on the small-subunit ribosomal protein uS5 (rpsE) because, despite its role in decoding accuracy, its potential as a mutator locus has not been evaluated. To explore this, we isolated several mutants of ribosomal protein uS5 (rpsE) in Pseudomonas putida, all carrying amino acid substitutions or deletions in loop 2, via spectinomycin selection, and then quantified their effects on frameshifting and stop-codon readthrough using dual-luciferase reporters. In parallel, we measured the rate and spectrum of spontaneous rifampicin resistance (rpoB) mutations. The fitness costs of these uS5 alleles were also assessed through growth and stress tolerance assays. Several mutants displayed divergent decoding phenotypes, including both error-prone and error-restrictive profiles, and the increase in translational errors was often, but not universally, coupled with elevated mutation rate. This approach probes which uS5 perturbations confer a bona fide mutator phenotype and how decoding errors bias mutation outcomes. By extending fidelity mutagenesis studies beyond enterobacteria, our work identifies uS5 as a previously unrecognized mutator locus in P. putida and illuminates the nuanced coupling of translation accuracy and evolvability in a key environmental microbe.IMPORTANCEThe accuracy of protein synthesis is essential for cellular integrity, yet its influence on genomic stability remains poorly understood, especially outside model organisms. By examining mutations in ribosomal protein uS5 of Pseudomonas putida, a bacterium adapted to environmental stress, we reveal how altered translation fidelity can modulate spontaneous mutagenesis. Some mutations increased translational errors and mutagenesis, while others decoupled these phenotypes, highlighting mechanistic complexity. These findings suggest that the link between translation fidelity and evolvability may be context-dependent, shaped by both ribosomal structure and environmental adaptation. Our work expands fidelity mutagenesis studies into non-enteric bacteria and offers insights into how translation errors may contribute to adaptive potential in fluctuating environments.

Curli amyloid fibers are the key protenacious component of the extracellular matrix in Escherichia coli. The regulation of curli expression is highly complex and depends on multiple environmental responses. While many genetic determinants of curli production are known, previous studies have been conducted under various conditions and using different strains of E. coli or Salmonella. Furthermore, while the expression of curli genes in an E. coli population is known to be bimodal, the origins of this bimodality are only partially understood. Here, we systematically investigated the role of various cellular factors in the expression of the curli structural genes csgBA at the single-cell level in planktonic E. coli culture. We observed that multiple factors involved in the regulation of stress response, cell motility, cell physiology and metabolism, maintenance of DNA architecture, and second messenger signaling either promote or repress the expression of csgBA genes. We further elucidated which regulators act upstream of the master transcription factor CsgD and which are crucial for the bimodality of curli gene expression. Overall, this study provides an overview of the regulation of curli gene expression in planktonic E. coli culture in the absence of any microenvironmental gradients.IMPORTANCEThe transition from a solitary planktonic lifestyle to a multicellular biofilm is a complex developmental process involving multiple changes in bacterial cell physiology. For Enterobacteriaceae, a critical step in this process is the production of curli amyloid fibers, the main component of their extracellular matrix. A commitment to express curli genes already occurs in a subpopulation of planktonically growing Escherichia coli cells. Here, we investigated how this activation depends on multiple stress response factors, global regulators of gene expression, and the second messenger cyclic-di-GMP. We demonstrated that bimodal expression of curli structural genes in planktonic cultures requires an interplay between several transcription factors and chromosome-organizing proteins but not second messenger signaling.

Bacterial chemotaxis is an important behavior to study to understand spatial segregation of species in mixed communities and the assembly of host microbiomes. This is particularly relevant in the rhizosphere, where chemoattraction toward root exudates is an important determinant of plant colonization. However, current methods to screen chemoeffectors are limited in their throughput, creating a barrier to generating comprehensive data sets describing chemotactic profiles for species of interest. Here, we describe a novel 3D-printed capillary tube holder approach, which facilitates up to 384 simultaneous capillary tube chemotaxis assays. We optimized and benchmarked our assay using Escherichia coli K12 and Bacillus subtilis 3610 with known chemoattractants: serine and aspartate. We then tested the threshold concentration of these chemoattractants using our assay and found that we could detect chemoattraction toward concentrations spanning multiple orders of magnitude. In this paper, we describe our high-throughput chemotaxis assay in detail and provide the necessary files for 3D printing the capillary tube holder.IMPORTANCEChemotaxis is an important behavior to study to understand how microbial communities assemble and respond to their environment. Identifying chemoattractants may uncover key targets for microbiome engineering. However, the generation of large data sets describing chemotactic profiles has been limited by a lack of high-throughput tools to quantitatively screen chemotaxis. We designed a 3D-printed assay allowing for up to 384 simultaneous capillary tube chemotaxis assays and validated our method with two different bacterial species. The throughput of our approach is greatly increased by the ability to use lag time as a proxy for cell count. Our approach is easy to use and low cost, effectively lowering the barrier to expanding more comprehensive data sets describing the chemotactic profiles of different bacterial species.