Journal of Bacteriology最新文献

Chromosome segregation in bacteria is a critical process ensuring that each daughter cell receives an accurate copy of the genetic material during cell division. Active segregation factors, such as the ParABS system or SMC complexes, are usually essential for this process, but they are surprisingly dispensable in Streptococcus pneumoniae. Rather, chromosome segregation in S. pneumoniae relies on the protein Regulator of Chromosome Segregation (RocS), although the molecular mechanisms involved remain elusive. By combining genetics, in vivo imaging, and biochemical approaches, we dissected the molecular features of RocS involved in chromosome segregation. We investigated the respective functions of the three RocS domains, specifically the C-terminal amphipathic helix (AH), the N-terminal DNA-binding domain (DBD), and the coiled-coil domain (CCD) separating the AH and the DBD. Notably, we found that a single AH is not sufficient for membrane binding and that RocS requires prior oligomerization to interact with the membrane. We further demonstrated that this self-interaction was driven by the N-terminal part of the CCD. On the other hand, we revealed that the C-terminal part of the CCD corresponds to a domain of unknown function (DUF 536) and is defined by three conserved glutamines, which play a crucial role in RocS-mediated chromosome segregation. Finally, we showed that the DBD is phosphorylated by the unique serine-threonine kinase of S. pneumoniae StkP and that mimicking this phosphorylation abrogated RocS binding to DNA. Overall, this study offers new insights into chromosome segregation in Streptococci and paves the way for a deeper understanding of RocS-like proteins in other bacteria.IMPORTANCEBacteria have evolved a variety of mechanisms to properly segregate their genetic material during cell division. In this study, we performed a molecular dissection of the chromosome partitioning protein Regulator of Chromosome Segregation (RocS), a pillar element of chromosome segregation in S. pneumoniae that is also generally conserved in the Streptococcaceae family. Our systematic investigation sheds light on the molecular features required for successful pneumococcal chromosome segregation and the regulation of RocS by phosphorylation. In addition, our study also revealed that RocS shares functional domains with the Par protein, involved in an atypical plasmid segregation system. Therefore, we expect that our findings may serve to extend our understanding of RocS and RocS-like proteins while broadening the repertoire of partitioning systems used in bacteria.

In this issue (J Bacteriol. 206: e0014024, https://doi.org/10.1128/jb.00140-24), Ridone and Baker describe hybrids between two 5:2 heteroheptameric ion-powered motors. Chimeras were constructed between stator units of a bacterial flagellum and ExbBD of the Ton outer-membrane transport system. Only one of the 14 hybrids supported swimming in Escherichia coli. Three additional residue changes at sites distant from the hybrid region enhanced motility. This work suggests that flagellar stator units and ExbBD share an ancestor that diverged during evolution to perform different tasks.

Rickettsia rickettsii is an obligate intracellular, tick-borne bacterium that causes Rocky Mountain spotted fever. The demanding nature of cultivating these bacteria within host cells and the labor involved in obtaining clonal isolates have severely limited progress regarding the development of compatible genetic tools to study this pathogen. Conditional expression of genes that might be toxic or have an otherwise undesirable effect is the next logical goal to expand upon the constitutive expression plasmids generated thus far. We describe the construction of an inducible promoter system based on the tet-On system, leveraging design elements from the anhydrotetracycline-inducible promoter system used for Borrelia burgdorferi and one of the few characterized rickettsial promoters for the outer membrane gene, rompB (sca5). The functionality of this promoter is demonstrated via fluorescence of induced mScarlet production and was then used to construct a generalized inducible expression vector for R. rickettsii. The development of a functional inducible promoter was then applied to the construction of a CRISPR interference plasmid as a means to reduce or essentially silence the transcription of targeted genes. We demonstrate the viability of a simplified, single vector CRISPRi system to disrupt gene expression in R. rickettsii targeting the type IV secreted effector rarP2 and autotransporter peptidase rapL as examples.

Importance: This work expands upon the genetic toolbox available for R. rickettsii. We describe both an inducible promoter and CRISPRi system compatible with Rickettsia, which may provide key instruments for the development of further tools. The development of an inducible promoter system allows for the overexpression of genes, which might be toxic when expressed constitutively. The CRISPRi system enables the ability to knock down genes with specificity, and critically, genes that may be essential and could not otherwise be knocked out. These developments may provide the foundation for unlocking genetic tools for other pathogens of the order Rickettsiales, such as the Anaplasma, Orientia, and Ehrlichia for which there are currently no inducible promoters or CRISPRi platforms.

We identified and characterized genomic regions of Streptococcus agalactiae that are involved in the Leloir and the tagatose-6-phosphate pathways for D-galactose catabolism. The accumulation of mutations in genes coding the Leloir pathway and the absence of these genes in a significant proportion of the strains suggest that this pathway may no longer be necessary for S. agalactiae and is heading toward extinction. In contrast, a genomic region containing genes coding for intermediates of the tagatose-6-phosphate pathway, a Gat family PTS transporter, and a DeoR/GlpR family regulator is present in the vast majority of strains. By deleting genes that code for intermediates of each of these two pathways in three selected strains, we demonstrated that the tagatose-6-phosphate pathway is their sole route for galactose catabolism. Furthermore, we showed that the Gat family PTS transporter acts as the primary importer of galactose in S. agalactiae. Finally, we proved that the DeoR/GlpR family regulator is a repressor of the tagatose-6-phosphate pathway and that galactose triggers the induction of this biochemical mechanism.IMPORTANCES. agalactiae, a significant pathogen for both humans and animals, encounters galactose and galactosylated components within its various ecological niches. We highlighted the capability of this bacterium to metabolize D-galactose and showed the role of the tagatose-6-phosphate pathway and of a PTS importer in this biochemical process. Since S. agalactiae relies on carbohydrate fermentation for energy production, its ability to uptake and metabolize D-galactose could enhance its persistence and its competitiveness within the microbiome.

In most natural environments, bacteria live in polymicrobial communities where secreted molecules from neighboring species alter bacterial behaviors, including motility, but such interactions are understudied. Pseudomonas aeruginosa is a motile opportunistic pathogen that exists in diverse multispecies environments, such as the soil, and is frequently found in human wound and respiratory tract co-infections with other bacteria, including Staphylococcus aureus. Here, we show that P. aeruginosa can co-opt secreted surfactants from other species for flagellar-based surface motility. We found that exogenous surfactants from S. aureus, other bacteria, and interkingdom species enabled P. aeruginosa to switch from swarming to an alternative surface spreading motility on semi-solid surfaces and allowed for the emergence of surface motility on hard agar where P. aeruginosa was otherwise unable to move. Although active flagellar function was required for surface spreading, known motility regulators were not essential, indicating that surface spreading may be regulated by an as yet unknown mechanism. This motility was distinct from the response of most other motile bacterial species in the presence of exogenous surfactants. Mutant analysis indicated that this P. aeruginosa motility was similar to a previously described mucin-based motility, "surfing," albeit with divergent regulation. Thus, our study demonstrates that secreted surfactants from the host as well as neighboring bacterial and interkingdom species act as public goods facilitating P. aeruginosa flagella-mediated surfing-like surface motility, thereby allowing it to access different environmental niches.

Importance: Bacterial motility is an important determinant of bacterial fitness and pathogenesis, allowing expansion and invasion to access nutrients and adapt to new environments. Here, we demonstrate that secreted surfactants from a variety of foreign species, including other bacterial species, infection hosts, fungi, and plants, facilitate surface spreading motility in the opportunistic pathogen Pseudomonas aeruginosa that is distinct from established motility phenotypes. This response to foreign surfactants also occurs in Pseudomonas putida, but not in more distantly related bacterial species. Our systematic characterization of surfactant-based surface spreading shows that these interspecies surfactants serve as public goods to enable P. aeruginosa to move and explore environmental conditions when it would be otherwise immotile.

Motility allows microbes to explore and maximize success in their environment; however, many laboratory bacterial strains have a reduced or altered capacity for motility. Swimming motility in Bacillus subtilis depends on peritrichous flagella and is carried out individually as cells move by biased random walks toward attractants. Previously, we adapted Bacillus subtilis strain 3610 to the laboratory for 300 generations in lysogeny broth (LB) batch culture and isolated lab-adapted strains. Strain SH2 is motility-defective and in broth culture forms large, frequently spherical aggregates of cells. A single point mutation in the flagellin gene hag that causes amino acid 259 to switch from A to T is necessary and sufficient to cause these social cell aggregates, and aggregation occurs between flagellated cells bearing this point mutation regardless of the strain background. Cells associate when bearing this mutation, but flagellar rotation is needed to pull associating cells into spherical aggregates. Using electron microscopy, we are able to show that the SH2 flagellar filament has limited polymorphism when compared to other flagellar structures. This limited polymorphism hinders the flagellum's ability to function as a motility apparatus but appears to alter its function to that of cell aggregation/adhesion. We speculate that the genotype-specific aggregation of cells producing Hag^A259T flagella could have increased representation in a batch-culture experiment by allowing similar cells to go through a transfer together and also that this mutation could serve as an early step to evolve sociality in the natural world.IMPORTANCEThe first life forms on this planet were prokaryotic, and the earliest environments were aquatic, and from these relatively simple starting conditions, complex communities of microbes and ultimately multicellular organisms were able to evolve. Usually, motile cells in aqueous environments swim as individuals but become social by giving up motility and secreting extracellular substances to become a biofilm. Here, we identify a single point mutation in the flagellum that is sufficient to allow cells containing this mutation to specifically form large, suspended groups of cells. The specific change in the flagellar filament protein subunits causes a unique change in the flagellar structure. This could represent a distinct way for closely related cells to associate as an early precursor to sociality.

Inosine 5'-monophosphate dehydrogenase (IMPDH), known as GuaB in bacteria, catalyzes the rate-limiting step in de novo guanine biosynthesis and is conserved from humans to bacteria. We developed a series of potent inhibitors that selectively target GuaB over its human homolog. Here, we show that these GuaB inhibitors are bactericidal, generate phenotypic signatures that are distinct from other antibiotics, and elicit different time-kill kinetics and regulatory responses in two important Gram-negative pathogens: Acinetobacter baumannii and Escherichia coli. Specifically, the GuaB inhibitor G6 rapidly kills A. baumannii but only kills E. coli after 24 h. After exposure to G6, the expression of genes involved in purine biosynthesis and stress responses change in opposite directions while siderophore biosynthesis is downregulated in both species. Our results suggest that different species respond to GuaB inhibition using distinct regulatory programs and possibly explain the different bactericidal kinetics upon GuaB inhibition. The comparison highlights opportunities for developing GuaB inhibitors as novel antibiotics.IMPORTANCEA. baumannii is a priority bacterial pathogen for which development of new antibiotics is urgently needed due to the emergence of multidrug resistance. We recently developed a series of specific inhibitors against GuaB, a bacterial inosine 5'-monophosphate dehydrogenase, and achieved sub-micromolar minimum inhibitory concentrations against A. baumannii. GuaB catalyzes the rate-limiting step of de novo guanine biosynthesis and is highly conserved across bacterial pathogens. This study shows that inhibition of GuaB induced a bacterial morphological profile distinct from that of other classes of antibiotics, highlighting a novel mechanism of action. Moreover, our transcriptomic analysis showed that regulation of de novo purine biosynthesis and stress responses of A. baumannii upon GuaB inhibition differed significantly from that of E. coli.

The lipopeptide antibiotic daptomycin exhibits bactericidal activity against Gram-positive bacteria by forming a complex with phosphatidylglycerol (PG) and lipid II in the cell membrane, causing membrane perforation. With the emergence of daptomycin-resistant bacteria, understanding the mechanisms of bacterial resistance to daptomycin has gained great importance. In this study, we aimed to identify the genetic factors contributing to daptomycin resistance in Bacillus subtilis, a model Gram-positive bacterium. Our findings demonstrated that overexpression of ugtP, which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in B. subtilis. Specifically, overexpression of ugtP resulted in increased levels of diglucosyldiacylglycerol (Glc₂DAG) and decreased levels of acidic phospholipids cardiolipin and PG, as well as the basic phospholipid lysylphosphatidylglycerol. However, ugtP overexpression did not alter the cell surface charge and the susceptibility to the cationic antimicrobial peptide nisin or the cationic surfactant hexadecyltrimethylammonium bromide. Furthermore, by serial passaging in the presence of daptomycin, we obtained daptomycin-resistant mutants carrying ugtP mutations. These mutants showed increased levels of Glc₂DAG and a >4-fold increase in the minimum inhibitory concentration of daptomycin. These results suggest that increased Glc₂DAG levels, driven by ugtP overexpression, modify the phospholipid composition and confer daptomycin resistance in B. subtilis without altering the cell surface charge of the bacteria.IMPORTANCEDaptomycin is one of the last-resort drugs for the treatment of methicillin-resistant Staphylococcus aureus infections, and the emergence of daptomycin-resistant bacteria has become a major concern. Understanding the mechanism of daptomycin resistance is important for establishing clinical countermeasures against daptomycin-resistant bacteria. In the present study, we found that overexpression of ugtP, which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in B. subtilis, a model Gram-positive bacteria. The overexpression of UgtP increased diglucosyldiacylglycerol levels, resulting in altered phospholipid composition and daptomycin resistance. These findings are important for establishing clinical strategies against daptomycin-resistant bacteria, including their detection and management.

As a biological byproduct from both humans and microbes, glycerol's contribution to microbial homeostasis in the oral cavity remains understudied. In this study, we examined glycerol metabolism by Streptococcus sanguinis, a commensal associated with oral health. Genetic mutants of glucose-PTS enzyme II (manL), glycerol metabolism (glp and dha pathways), and transcriptional regulators were characterized with regard to glycerol catabolism, growth, production of hydrogen peroxide (H₂O₂), transcription, and competition with Streptococcus mutans. Biochemical assays identified the glp pathway as a novel source for H₂O₂ production by S. sanguinis that is independent of pyruvate oxidase (SpxB). Genetic analysis indicated that the glp pathway requires glycerol and a transcriptional regulator, GlpR, for expression and is negatively regulated by PTS, but not the catabolite control protein, CcpA. Conversely, deletion of either manL or ccpA increased the expression of spxB and a second, H₂O₂-non-producing glycerol metabolic pathway (dha), indicative of a mode of regulation consistent with conventional carbon catabolite repression (CCR). In a plate-based antagonism assay and competition assays performed with planktonic and biofilm-grown cells, glycerol greatly benefited the competitive fitness of S. sanguinis against S. mutans. The glp pathway appears to be conserved in several commensal streptococci and actively expressed in caries-free plaque samples. Our study suggests that glycerol metabolism plays a more significant role in the ecology of the oral cavity than previously understood. Commensal streptococci, though not able to use glycerol as a sole carbohydrate source for growth, benefit from the catabolism of glycerol through production of both ATP and H₂O₂.

Importance: Glycerol is an abundant carbohydrate in the oral cavity. However, little is understood regarding the metabolism of glycerol by commensal streptococci, some of the most abundant oral bacteria. This was in part because most streptococci cannot grow on glycerol as the sole carbon source. In this study, we show that Streptococcus sanguinis, a commensal associated with dental health, can degrade glycerol for persistence and competition through two pathways, one of which generates hydrogen peroxide at levels capable of inhibiting Streptococcus mutans. Preliminary studies suggest that several additional commensal streptococci are also able to catabolize glycerol, and glycerol-related genes are actively expressed in human dental plaque samples. Our findings reveal the potential of glycerol to significantly impact microbial homeostasis, which warrants further exploration.

Streptococcus pneumoniae (pneumococcus) causes a wide range of important human infectious diseases, including pneumonia, pneumonia-derived sepsis, otitis media, and meningitis. Pneumococcus produces numerous secreted proteins that are critical for normal physiology and pathogenesis. The membrane targeting and translocation of these secreted proteins are partly mediated by the signal recognition particle (SRP) complex, which consists of 4.5S small cytoplasmic RNA (ScRNA), and the Ffh, and FtsY proteins. Here, we report that pneumococcal ∆scRNA, ∆ffh, and ∆ftsY mutants were significantly impaired in competence induction, competence pili production, exogenous DNA uptake, and genetic transformation. Also, the ∆scRNA mutant was significantly attenuated in the mouse models of bacteremia and pneumonia. Interestingly, unlike the ∆scRNA, both ∆ffh and ∆ftsY mutants had growth defects on Todd-Hewitt Agar, which were alleviated by the provision of free amino acids or serum. Differences in nutritional requirements between ∆ffh and ∆ftsY vs ∆scRNA suggest that Ffh and FtsY may be partially functional in the absence of ScRNA. Finally, the insertase YidC2, which could functionally rescue some SRP mutations in other streptococcal species, was not essential for pneumococcal genetic transformation. Collectively, these results indicate that ScRNA is crucial for the successful development of genetic competence and virulence in pneumococcus.

Importance: Streptococcus pneumoniae (pneumococcus) causes multiple important infectious diseases in humans. The signal recognition particle (SRP) complex, which comprised 4.5S small cytoplasmic RNA (ScRNA), and the Ffh and FtsY proteins, mediates membrane targeting and translocation of secreted proteins in all organisms. However, the role of SRP and ScRNA has not been characterized during the induction of the competence system for genetic transformation and virulence in pneumococcus. By using a combination of genetic, biochemical, proteomic, and imaging approaches, we demonstrated that the SRP complex plays a significant role in membrane targeting of competence system-regulated effectors important for genetic transformation, virulence during bacteremia and pneumonia infections, and nutritional acquisition.