Journal of Bacteriology最新文献

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.

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.

Glycolytic activity is required for Staphylococcus aureus (S. aureus) to establish an infection. These data indicate that carbon catabolite repression, mediated by CcpA, is active during the initial stages of infection. CcpA represses both the biosynthesis and catabolism of arginine; therefore, it is hypothesized that arginine must be transported by S. aureus from host tissue to facilitate growth during the establishment of an infection. Within S. aureus USA300, two known arginine/ornithine antiporters, ArcD1 and ArcD2, are encoded on the two copies of the arginine deiminase operon (native and arginine catabolite mobile genetic element-derived). However, when both antiporters are inactivated via allelic replacement, no growth defect is observed in defined medium where arginine is required for growth, indicating that S. aureus contains additional arginine transporters. Using the toxic arginine analog, canavanine, we identified a novel S. aureus arginine transporter, SAUSA300_2383 (Arginine Transporter; ArgT). Transcriptional analysis found that argT was regulated by both the canonical arginine biosynthesis repressor AhrC and CcpA; thus, its transcription is repressed during growth in medium containing glucose and is therefore not the primary arginine transporter during growth in medium containing glucose. However, we found that growth is dependent upon ArgT during growth in medium lacking proline, which suggests that S. aureus has evolved a specific response to accommodate proline-depleted growth conditions.IMPORTANCEStaphylococcus aureus is a leading cause of both community and hospital-acquired infection worldwide. In addition, S. aureus is resistant to many commonly used antibiotics, which make the treatment of bacteremia, infective endocarditis, and other invasive diseases more challenging. It is essential to obtain a basic understanding of how S. aureus survives in a variety of host niches, including those niches where S. aureus is dependent upon amino acid catabolism. We hypothesize that arginine acquisition is critical for S. aureus pathogenesis; therefore, identifying these transporters is essential for the development of novel therapeutic strategies.

This study aimed to determine reasons that longer sporulation times in liquid or on plates have large effects on Bacillus subtilis spore germination and resistance. B. subtilis spores were prepared for 3/30/60 days in liquid or on plates and their germination, resistance, levels of core water and Ca²⁺-dipicolinic acid (CaDPA), and inner membrane (IM) fluidity and permeability were measured. Liquid spores of 3/30/60 days had no differences in wet heat resistance, while plate spores of 30/60 days had higher wet heat resistance than 3-day plate spores. There were minimal increases in 3- to 60-day liquid spores' resistance to UV radiation and chemicals, while plate spores of 30/60 days had increased resistance to chemicals. Germinant receptor (GR) germination with L-valine was identical with 3- to 60-day liquid or plate spores, while plate spores of 30/60 days germinated faster than 3-day spores with the L-asparagine, D-glucose, D-fructose, and KCl mixture. Three-day liquid or plate spores germinated faster than 30- and 60-day spores with the GR-independent germinant, dodecylamine, and 30-day plate spores had lower IM permeability and higher IM rigidity than 3-day plate spores or 30-day liquid spores. However, levels of two spore core components' modulating spore wet heat resistance, water, and CaDPA were identical in spores of 3/30/60 days. Thus, increased IM rigidity and decreased IM permeability appear to be major factors in increased resistance of spores prepared on plates for long periods. However, precisely how these longer times cause increased IM rigidity and lower permeability, but not with spores prepared in liquid, is not clear.IMPORTANCECells of some Bacillota cause food spoilage and human diseases. These organisms' ability to do this is exacerbated by forming hard-to-kill dormant spores because of their resistance and ability to come "back to life" in germination. We examined two Bacillus subtilis sporulation parameters, liquid versus solid media and sporulation time, measuring effects on spore resistance and germination. We found (i) an effector of spore heat resistance, core water content, is not changed by different sporulation media or times; and (ii) spores' IM becomes more rigid and less permeable in spores made on solid media and for longer times. This knowledge may influence how spores are prepared as probiotics or as standards for analyses of autoclave function.

Magnetotactic bacteria (MTB) synthesize magnetic bacterial organelles called magnetosomes, which enable them to navigate along the geomagnetic field in aquatic environments. The actin-like cytoskeletal protein MamK forms filaments that associate with magnetosomes and mediate their positioning. Interestingly, in seven phyla, including Desulfobacterota, MTB encodes a second actin-like protein, Mad28, alongside MamK, within the magnetosome island-a genetic region responsible for magnetosome synthesis. In this study, we characterized the structure and function of this alternative magnetosome-associated cytoskeletal protein, Mad28. Magnetosome-specific localization of Mad28 in Solidesulfovibrio magneticus RS-1 was confirmed using immunoblotting, immunofluorescence microscopy, and correlative light and electron microscopy. To examine whether Mad28 and MamK have distinct or overlapping roles in magnetosome positioning, we tested the ability of Mad28^RS-1 or MamK^RS-1 to rescue the MamK-dependent static magnetosome positioning phenotype in Magnetospirillum magneticum AMB-1. Live-cell imaging revealed that MamK^RS-1 expression restored static magnetosome positioning, whereas Mad28^RS-1 expression had no effect, suggesting functional divergence between the two proteins. We further examined the potential role of Mad28 in sensing cellular geometry by comparing the localization of a Mad28-Dendra2 fusion protein in wild-type rod-shaped Escherichia coli and vibrio-shaped E. coli cells expressing Crescentin. Remarkably, Mad28 exhibited a curvature-dependent localization pattern in E. coli. These findings provide direct evidence that the actin-like protein Mad28 presents an affinity with membrane curvature in bacterial cells. In conclusion, the dual cytoskeletal systems-MamK and Mad28-contribute to magnetosome positioning through distinct mechanisms in deep-branching MTB.

Importance: Bacteria are capable of precisely positioning nanosized, membrane-enclosed organelles within their limited cellular spaces. This study shows that two distinct actin-like proteins contribute to magnetosome positioning through separate mechanisms in deep-branching magnetotactic bacteria. This contrasts with the evolutionary strategy observed in eukaryotic cells, where a single actin protein performs multiple functions. Furthermore, the findings suggest that the protein Mad28 is involved in sensing membrane curvature, introducing a novel functional property for bacterial actin-like proteins. These findings offer new insights into the role of the cytoskeleton in organelle positioning within micron-scale bacterial cells.

Thermococcales are among the most widely studied hyperthermophilic Archaea and have become key models for understanding life at extreme temperatures. Early work in the 1980s culminated in the isolation of novel Thermococcales species from hydrothermal vents that grew rapidly, tolerated extreme heat, and metabolized diverse substrates, making them uniquely amenable for laboratory studies. Their thermostable enzymes and emerging genetic tools facilitated detailed investigations of core processes such as DNA replication, repair, and transcription under conditions that challenge most life forms. These practical advantages, together with the accumulation of tools and protocols, cemented the role of Thermococcales as a model system. Here, we recount how chance discoveries, environmental adaptations, and experimental practicality intersected to establish Thermococcales as a central model for studying archaeal biology and extremophile physiology.

Cobamides play a paradoxical but critical role in the biology of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Although Mtb retains nearly all cobalamin (Cbl) biosynthetic genes and encodes multiple cobamide-requiring enzymes, experimental evidence indicates that Mtb is incapable of de novo Cbl synthesis under any tested conditions to date. Instead, an evolutionary shift appears to have occurred toward host dependency for biologically relevant cobamides or their precursors. This review highlights recent advances in our understanding of cobamide-related metabolism in Mtb, including: (i) the progressive erosion of de novo cobamide biosynthetic capacity across Mtb lineages; (ii) the role of host-derived cobamides in sustaining key mycobacterial metabolic pathways, including methionine synthesis and propionate catabolism; (iii) the impact of host immune pressures, including itaconate-mediated inhibition of methylmalonyl-CoA mutase; (iv) strategies employed by Mtb for cobamide and precursor acquisition; and (v) unique adaptations of Cbl-sensing riboswitches that regulate methionine synthesis, virulence-associated gene expression, and dormancy resuscitation. We also highlight unresolved questions, including possible niche-specific synthesis, utilization of alternate cobamide species, and the therapeutic potential of targeting cobamide-related metabolism. We review recent evidence of the centrality of cobamides in the metabolic flexibility of Mtb, virulence, and survival in the host environment, despite apparent loss of de novo biosynthetic capacity. Further mechanistic studies are required which may reveal vulnerabilities for the exploitation of cobamide acquisition, cobamide-related regulation, and the role of cobamides at the Mtb-host interface for innovative therapeutic interventions.

Chlamydia trachomatis is an obligate intracellular bacterium of major clinical significance. While untreated sexually transmitted infections can result in pelvic inflammatory disease and infertility, ocular infections can cause the blinding disease trachoma. During infection of host cells, C. trachomatis transitions between the non-replicative, infectious elementary body (EB) and the replicative, non-infectious reticulate body (RB). ObgE is a GTPase that can promote morphological differentiation in some bacteria. In C. trachomatis, obgE is maximally expressed from 16 to 24hpi, a timeframe that is associated with logarithmic growth and the onset of production of infectious progeny; therefore, ObgE is predicted to have significance during Chlamydia replication and/or morphological transitions. To determine the role of ObgE during the C. trachomatis developmental cycle, we assessed the effects of ObgE ectopic overexpression and CRISPRi-based knockdown of obgE on RB replication and EB formation. When ectopic overexpression of ObgE was induced, we observed a significant decrease in infectious progeny but no changes in bacterial ultrastructure. These data suggest that during ectopic overexpression of ObgE, RBs can transition into EBs; however, EBs are diminished in their ability to establish new infections. CRISPRi-based knockdown of obgE resulted in a 2-log decrease in bacterial yield and infectious progeny. Ultrastructural analysis revealed that knockdown of obgE resulted in small, underdeveloped inclusions with few cells inside. In total, while ectopic overexpression of ObgE negatively affects production of infectious EBs, CRISPRi-based knockdown of obgE severely affects RB replication, inclusion development, and generation of EBs.IMPORTANCEThe pathogenesis of C. trachomatis is reliant on the transition between the non-replicative, infectious elementary body (EB) and the replicative, non-infectious reticulate body (RB). Therefore, understanding the molecular determinants of Chlamydia developmental transitions is of the utmost importance. ObgE has been shown to regulate morphological transitions in other bacteria and is thus predicted to have relevance during regulation of the Chlamydia developmental cycle. Using both ectopic overexpression and CRISPRi-based knockdown of ObgE/obgE, we identify the significance of balanced ObgE expression for RB replication and the formation of infectious EBs. Our findings further expand our knowledge of how developmental transitions in Chlamydia are regulated.

Peptidoglycan synthesis is an essential driver of bacterial growth and division. The final steps of this crucial process involve the polymerization of glycan strands by shape, elongation, division, and sporulation (SEDS) family glycosyltransferases and the cross-linking of peptide cross-bridges by class B penicillin-binding proteins (bPBP). While many bacteria use distinct bPBPs to perform specialized roles during a given cellular process, some bPBPs can play redundant roles, particularly in the presence of certain cell wall stresses. Our understanding of these compensatory mechanisms, however, remains incomplete. Endospore-forming bacteria typically encode multiple bPBPs to drive morphological changes required for sporulation. The sporulation-specific bPBP, SpoVD, synthesizes the polar division septum and the cortex peptidoglycan layer during sporulation in the pathogen Clostridioides difficile. Although SpoVD catalytic activity is essential for cortex synthesis, we show that it is partially dispensable for asymmetric division. The dispensability of SpoVD's catalytic activity requires the presence of its SEDS partner, SpoVE, and another sporulation-induced bPBP, PBP3. While PBP3 localizes to the polar septum and interacts with components of the polar division machinery, the ability of PBP3 to promote division during sporulation occurs independent of its catalytic activity. Notably, this latter finding differs from previously reported modes of functional redundancy in bacteria, indicating that there are diverse mechanisms by which penicillin-binding proteins can be functionally redundant in bacteria.IMPORTANCEPeptidoglycan synthesis requires the transpeptidase activity of penicillin-binding proteins (PBPs), which have specialized functions during cell growth, division, and differentiation. However, many bacteria produce PBPs with overlapping functions, and this functional redundancy can lead to increased antibiotic resistance. While the major pathogen, Clostridioides difficile, requires the SpoVD PBP to form spores, we found that its transpeptidase activity is dispensable for asymmetric division, the first morphological stage of sporulation, because a sporulation-induced PBP, PBP3, partially substitutes for SpoVD's function during this stage. Since PBP3's ability to promote asymmetric division in this context does not depend on the its catalytic activity, unlike prior studies of PBP functional redundancy, our analyses highlight the diversity in mechanisms used to enable functional redundancy between PBPs.

Brucella species are notorious pathogens of animals and humans and cause significant morbidity and economic losses globally. These hardy bacteria have evolved to survive and replicate in host cells, particularly macrophages, and have developed a specialized quorum sensing system that is essential for navigating intracellular life. Moreover, successful infection of the host is dependent upon elements of the Brucella quorum sensing system. While quorum sensing is a thoroughly well-defined process in many Gram-negative bacteria, several unique features in the quorum sensing pathway have evolved that set Brucella apart from more established model organisms. The current review is aimed at describing the paradigmatic aspects of Brucella quorum sensing, while also underscoring the nuance and distinctiveness of quorum sensing in the brucellae, and we discuss important questions that remain unanswered in the field.