Journal of Bacteriology最新文献

Fusobacterium nucleatum is a Gram-negative anaerobe associated with periodontitis and colorectal cancer. It secretes putrescine, a polyamine that promotes biofilm formation by oral co-colonizers and enhances the proliferation of cancer cells. However, the physiological importance of putrescine for F. nucleatum itself remains unexplored. Here, we show that putrescine biosynthesis, mediated by the ornithine decarboxylase gene oda, is essential for F. nucleatum viability. Deletion of oda was only possible when a functional copy was provided in trans, and CRISPR interference of oda expression resulted in complete growth defects. The essentiality of oda was conserved across multiple subspecies. Supplementation with exogenous putrescine enabled the isolation of a conditional oda mutant whose growth was strictly putrescine-dependent. Putrescine depletion caused filamentation, membrane disruption, detergent hypersensitivity, and lysis in hypoosmotic conditions, indicating a critical role in maintaining cell envelope integrity. RNA sequencing revealed broad transcriptional remodeling under putrescine-limited conditions, including upregulation of genes involved in lipid metabolism, osmoprotection, and cell wall remodeling. Notably, oda transcript levels increased when putrescine was depleted, suggesting a negative feedback mechanism. These findings demonstrate that putrescine is not only an extracellular communal metabolite but is also vital for the cellular integrity and survival of F. nucleatum under anaerobic conditions.

Importance: Fusobacterium nucleatum is a prominent member of the oral microbiota and has been linked to various human diseases, including periodontitis, preterm birth, and colorectal cancer. Despite its clinical significance, the metabolic requirements that support its growth and viability remain poorly understood. In this study, we identify the oda gene, which encodes ornithine decarboxylase, as essential for F. nucleatum survival due to its role in putrescine biosynthesis. We demonstrate that depletion of putrescine leads to severe growth and morphological defects, accompanied by widespread transcriptional changes. These findings reveal an underappreciated metabolic vulnerability and highlight the critical role of polyamine homeostasis in maintaining cellular integrity in this notorious anaerobe.

The alarmone (p)ppGpp (ppGpp) accumulates in response to starvation and other stress, leading to inhibition of multiple biosynthetic pathways and, at high concentrations, suppression of bacterial growth. Growth suppression by ppGpp is implicated in the formation of persister cells, which survive antibiotic challenge only to regrow once the drug is removed. However, there is also evidence that low levels of ppGpp contribute to resistance to certain cell wall-active antibiotics in actively growing cells. To characterize ppGpp's contribution to antibiotic resistance, we measured MICs of a panel of β-lactams in actively growing Escherichia coli cells overexpressing a ppGpp synthase (relA*). Cells engineered to modestly overproduce ppGpp exhibited up to 64-fold increases in resistance to PBP2-targeting β-lactams only, with mecillinam the most dramatically affected. Resistance required the transcription factor DksA and the class A penicillin-binding protein (PBP) PBP1B. PBP1B variants defective for transpeptidase activity, glycosyltransferase activity, or both were incapable of supporting resistance, suggesting the full enzymatic activity of PBP1B is required for resistance. Transcriptomics revealed that ppGpp overproduction leads to increased expression of lpoB, which encodes an activator of PBP1B. LpoB was required for mecillinam resistance, with an lpoB deletion mutant exhibiting a loss of ppGpp-dependent resistance. An lpoB deletion strain expressing an LpoB-bypass variant of PBP1B (mrcB*) exhibited an intermediate level of resistance. Together, these results suggest that ppGpp overproduction and the LpoB-dependent enzymatic activity of PBP1B function synergistically to promote survival in the presence of PBP2 inhibitors.

Importance: Antimicrobial resistance is an increasing global health threat, but its underlying molecular mechanisms remain incompletely understood. This work clarifies ppGpp's role in mediating antibiotic resistance in Escherichia coli. Elevated levels of ppGpp caused resistance to β-lactam antibiotics targeting the cell wall synthesis enzyme PBP2. Resistance required transcriptional regulation by ppGpp and enzymatic activity of the cell wall enzyme PBP1B. ppGpp overproduction was found to increase expression of the PBP1B activator lpoB. Because ppGpp levels are controlled by nutritional conditions, this work suggests that nutritional availability may impact antibiotic efficacy.

High-temperature requirement A (HtrA) aids in protein homeostasis by playing a key dual role as a chaperone and protease. HtrA ensures protein folding quality control during secretion and protects cells against protein aggregation by degrading misfolded proteins. HtrA proteins are typically composed of a protease domain and at least one PDZ domain, proposed to help regulate their activity and interactions with substrates. In gram-positive bacteria, HtrA contributes to critical cellular functions and has been linked to processes such as maintaining envelope integrity, stress resistance, and virulence. In addition, HtrA has been shown to contribute to the modulation of competence and biofilm dynamics as well as the degradation of host proteins in infection models. In some gram-positive bacteria, HtrA expression is regulated by two-component systems, but many HtrA upstream signals and downstream targets remain unclear. As antibiotic resistance continues to rise, HtrA is gaining attention as a promising target of inhibition for new antibacterial strategies. However, a lack of structural information, unclear regulatory mechanisms, and unknown substrates make designing effective HtrA inhibitors challenging. This review highlights these knowledge gaps and aims to spark more focused research on HtrA in gram-positive species.

Aeromonas caviae, Gram-negative bacteria ubiquitous in the environment, are an emerging human pathogen associated with various infectious diseases, particularly gastroenteritis. Despite recent studies demonstrating A. caviae is the most predominant Aeromonas species underlying human infection, A. caviae remains understudied, and no A. caviae-specific virulence factors associated with human disease have been identified. To identify A. caviae-specific putative virulence factors, we conducted comparative genomic analyses among clinical Aeromonas isolates (n = 431), which identified a variant of flgB, predicted to encode a polar flagellum machinery protein, as over-represented in A. caviae isolates. To examine the role of flgB in virulence and host-pathogen interactions, we generated an A. caviae flgB deletion mutant and genetic complementation constructs. Swimming motility and polar flagella assembly were abolished in the mutant and functionally rescued with genetic complementation. As it remains unknown where A. caviae infects the human gastrointestinal tract, we assessed host-pathogen interactions in HT-29 and Caco2 human intestinal cell lines, representative of the large and small intestine, respectively. Deletion of flgB significantly decreased bacterial adherence in only HT-29 cells and also decreased production of proinflammatory cytokines, IL-8, IL-13, IL-1β, and IL-6, by both cell types. Given the lack of relevant mammalian models for studying most enteric pathogens in vivo, we characterized in vivo virulence in a Galleria mellonella larval survival model, where the flgB deletion modestly attenuated virulence. Deletion of flgB altered aspects of virulence and host-pathogen interactions, and this study provides a framework for identification and characterization of A. caviae-specific putative virulence factors.IMPORTANCEAeromonas caviae is an emerging human bacterial pathogen associated with gastroenteritis, wound infections, and numerous other infectious diseases. Recent studies demonstrate that A. caviae accounts for the greatest burden of human Aeromonas infections. Despite this, A. caviae is understudied as a human pathogen. To address this gap in knowledge, this study characterizes A. caviae-specific virulence genes. We examined 431 clinical Aeromonas isolates using comparative genomics and identified and functionally characterized a putative A. caviae-specific virulence factor, flgB. Genetic deletion of flgB in A. caviae resulted in deficiencies in bacterial motility, adherence, host-cell proinflammatory cytokine production, and in vivo virulence in an invertebrate model. This work establishes the foundation for further study of additional A. caviae-specific virulence factors.

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.

In Gammaproteobacteria, the biogenesis of the flagellar filaments is regulated by a flagellum-specific sigma factor (FliA). In Alphaproteobacteria, however, a flagellum-specific sigma factor is not known. In our search for a FliA-like sigma factor in Azospirillum brasilense Sp7, we found an extracytoplasmic function sigma factor (RpoE6) located in a gene cluster consisting of FlgK, FlgL, FliD, FliS, and FliC2. A strain lacking RpoE6 (ΔrpoE6) was compromised in expressing a small flagellin, FliC2, which contributes to the polar flagellum structure and function in swimming motility, though there was no detectable defect in swimming motility in swim plates. A non-polar inactivation of this sigma factor also led to a growth defect that depended on aeration. FliC2, like the major flagellin of the polar flagellum, is also likely glycosylated. A hypothetical protein, possibly behaving as an anti-sigma factor (Asf6), encoded downstream of rpoE6, interacts with RpoE6. A mutant lacking Asf6 (Δasf6) overproduces FliC2 and possesses thinner flagellar filaments compared to the wild type, suggesting that the abundance of FliC2 is oppositely regulated by RpoE6 and Asf6. Overexpression of rpoE6 in A. brasilense upregulates many proteins, including those involved in flagellar filament (FliD, FliS, and FliC2), required for growth on fructose and glycerol, as well as in the upregulation of another sigma factor, RpoE8. Our data support a role for RpoE6 in coupling FliC2 expression with genes involved in glycerol and fructose metabolism. Conservation of the genomic organization of rpoE6 in related Alphaproteobacteria suggests a similar function in other species.IMPORTANCEAzospirillum brasilense is a common plant growth-promoting rhizobacterium that colonizes crop rhizospheres using chemotaxis via its polar flagellum. However, our understanding of the structure, composition, and biogenesis of the polar flagellum in A. brasilense is limited. Unlike Gammaproteobacteria, where FliA and FlgM regulate flagellum biogenesis, no sigma or anti-sigma factors are known to regulate chemotactic motility in Alphaproteobacteria, including A. brasilense. This study identifies an extracytoplasmic function sigma factor (RpoE6) and its anti-sigma factor (Asf6) which modulate the expression of the minor flagellin FliC2, thereby altering the polar flagellum structure. Unlike FliA and FlgM, this regulatory pair also controls other functions such as glycerol and fructose utilization. To our knowledge, this is the first report of an alternative sigma factor regulating a flagellin in Alphaproteobacteria.

Flagella are trans-envelope nanomachines expressed from genes organized in a complex regulatory hierarchy governed at the highest level by transcription factors called master activators. The master activator of flagellar biosynthesis in Bacillus subtilis is a hybrid of SwrA•DegU that is required to increase flagellar density to swarm over solid surfaces. Here, we show that the ClpX unfoldase subunit of the ClpP protease is required for swarming motility, and that cells mutated for ClpX fail to swarm due to restricted levels of both SwrA and DegU. Suppressor mutations were found that increased expression of the fla/che operon under SwrA•DegU control, and mutation of the LonA protease elevates the levels of SwrA protein, while mutation of the global transcriptional regulator Spx increases transcription of both the degU and swrA genes. We conclude that ClpX promotes swarming motility via degradation of Spx, which represses motility gene transcription, including the P_fla/che, P_degU, and P_swrA promoters, each activated by DegU. The ClpX-dependent regulatory proteolysis of Spx is relieved under stress conditions, and we infer that Spx may dampen DegU-mediated positive feedback to limit cell envelope stress caused by excessive flagellar biosynthesis.

Importance: Bacterial flagella are elaborate machines that are inserted through the bacterial cell envelope. The regulation of flagellar gene expression is organized in hierarchical tiers that largely correlate with the order of flagellar assembly. Here, we show that the abundance of the master activator of flagellar gene expression in Bacillus subtilis is restricted when the transcription factor Spx accumulates. Spx is an unusual transcription factor, as it binds to RNA polymerase and renders it insensitive to transcriptional activators. We show that Spx interferes with flagellar promoters activated by the response regulator DegU and may do so to limit a DegU-mediated positive feedback loop.

PrsA1 and PrsA2 are parvulin peptidyl-prolyl isomerases that function as post-translocation secretion chaperones in Listeria monocytogenes. To assess the contributions of PrsA1 and PrsA2 to overall L. monocytogenes protein secretion, we analyzed prsA1 and prsA2 deletion mutants and PrsA2 structural variants for altered secretion profiles when compared to wild-type bacteria using tandem mass-tagged mass spectrometry. We find that prsA1 and prsA2 deletion mutants have distinctly altered secretion profiles. In addition, among the subset of known secreted proteins with significantly altered secretion abundance were those with characterized functions in virulence, cell division and cell wall assembly, and stress response. To further identify common pathways and protein factors that are altered when PrsA homologs are absent, we conducted a meta-analysis comparing our data from L. monocytogenes to recently published quantitative proteomic secretome data of prsA deletion mutants from diverse gram-positive human pathogens, including Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus. We find that when PrsA homologs are absent in diverse gram-positive bacteria, several pathways are similarly affected, including those promoting bacterial virulence, cell division and cell wall assembly, and oxidative stress resistance. Moreover, we provide evidence of novel roles for L. monocytogenes PrsA1 and PrsA2 in oxidative stress resistance and cell morphology, and PrsA2 in thermo-osmotic stress resistance. Overall, this work suggests that gram-positive PrsA homologs serve in the maturation of multiple protein substrates with varied cellular functions.

Importance: Bacterial protein secretion is critical for functions ranging from cell physiology to virulence. Here, we examine the effect of deleting two Listeria monocytogenes secretion chaperones, PrsA1 and PrsA2, and find that in the absence of one or both chaperones, secretion of several proteins implicated in key biological processes was significantly disrupted. These results, coupled with phenotypic observations of chaperone deletion mutants, reveal that PrsA1 and PrsA2 have roles in bacterial physiology and stress resistance. Furthermore, our meta-analysis of prsA deletion mutants in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus suggests that the contribution of PrsA to critical bacterial processes is well conserved in gram-positive pathogens. Our work lays the foundation for future inquiry investigating the client repertoire of these chaperones.

Photoactive Yellow Protein (PYP) is a model system for functional protein dynamics and a prototype of the PAS domain superfamily. It is a bacterial photoreceptor that triggers a range of responses in different bacteria: phototaxis, biosynthesis of photo-protective pigments, and light regulation of biofilm formation. An important gap in knowledge on PYP is the signal transduction chain that guides the initial signal from the photoreceptor to various biological responses. Here, we present an expanded set of 984 PYP homologs, providing information on sequence conservation and variation. We analyze this set of PYPs using two bioinformatics approaches to identify candidate proteins that are functionally related to PYP. First, we identified 153 multi-domain proteins containing PYP and analyzed the domain composition of these proteins. Specific preferences for N- or C-terminal placement of the PYP domain were observed. Second, we identified 113 predicted multi-gene operons containing the pyp gene. These two approaches yielded multiple candidates for proteins in the signal transduction chain associated with PYP, particularly histidine kinase (implying phosphorylation), methyl accepting chemotaxis protein (implying phototaxis), and GGDEF and EAL proteins (implying a role of c-di-GMP and biofilm formation). Some of these candidates were present only in multi-domain proteins and others only in pyp operons. Overexpression of the PYP domain from the MCP-fusion protein from Nitrincola alkalilacustris yielded a protein with an absorbance maximum of 447 nm and an overall photocycle rate of 0.5 s. Our results provide a clear basis for future experimental work on identifying signal transduction partners of PYP.