Journal of Bacteriology最新文献

Bacteria find suitable locations for colonization by sensing and responding to surfaces. Complex signaling repertoires control surface colonization, and surface contact sensing by the flagellum plays a central role in activating colonization programs. Caulobacter crescentus adheres to surfaces using a polysaccharide adhesin called the holdfast. In C. crescentus, disruption of the flagellum through interactions with a surface or mutation of flagellar genes increases holdfast production. Our group previously identified several C. crescentus genes involved in flagellar surface sensing. One of these, fssF, codes for a protein with homology to the flagellar C-ring protein FliN. We show here that a fluorescently tagged FssF protein localizes to the flagellated pole of the cell and requires all components of the flagellar C-ring for proper localization, supporting the model that FssF associates with the C-ring. Deleting fssF results in a severe motility defect, which we show is due to a disruption of chemotaxis. Epistasis experiments demonstrate that fssF promotes adhesion through a stator-dependent pathway when late-stage flagellar mutants are disrupted. Separately, we find that disruption of chemotaxis through deletion of fssF or other chemotaxis genes results in a hyperadhesion phenotype. Key genes in the surface sensing network (pleD, motB, and dgcB) contribute to both ∆flgH-dependent and ∆fssF-dependent hyperadhesion, but these genes affect adhesion differently in the two hyperadhesive backgrounds. Our results support a model in which the stator subunits of the flagella incorporate both mechanical and chemical signals to regulate adhesion.IMPORTANCEBacterial biofilms pose a threat in clinical and industrial settings. Surface sensing is one of the first steps in biofilm formation. Studying surface sensing can improve our understanding of biofilm formation and develop preventative strategies. In this study, we use the freshwater bacterium Caulobacter crescentus to study surface sensing and the regulation of surface attachment. We characterize a previously unstudied gene, fssF, and find that it localizes to the cell pole in the presence of three proteins that make up a component of the flagellum called the C-ring. Additionally, we find that fssF is required for chemotaxis behavior but dispensable for swimming motility. Lastly, our results indicate that deletion of fssF and other genes required for chemotaxis results in a hyperadhesive phenotype. These results support that surface sensing requires chemotaxis for a robust response to a surface.

Bacteria and phages are locked in a co-evolutionary arms race where each entity evolves mechanisms to restrict the proliferation of the other. Phage-encoded defense inhibitors have proven powerful tools to interrogate how defense systems function. A relatively common defense system is BREX (bacteriophage exclusion); however, how BREX functions to restrict phage infection remains poorly understood. A BREX system encoded by the sulfamethoxazole and trimethoprim (SXT) integrative and conjugative element, VchInd5, was recently identified in Vibrio cholerae, the causative agent of the diarrheal disease cholera. The lytic phage ICP1 (International Centre for Diarrhoeal Disease Research, Bangladesh cholera phage 1) that co-circulates with V. cholerae encodes the BREX-inhibitor OrbA, but how OrbA inhibits BREX is unclear. Here, we determine that OrbA inhibits BREX using a unique mechanism from known BREX inhibitors by directly binding to the BREX component BrxC. BrxC has a functional ATPase domain that, when mutated, not only disrupts BrxC function but also alters how BrxC multimerizes. Furthermore, we find that OrbA binding disrupts BrxC-BrxC interactions. We determine that OrbA cannot bind BrxC encoded by the distantly related BREX system encoded by the aSXT VchBan9, and thus fails to inhibit this BREX system that also circulates in epidemic V. cholerae. Lastly, we find that homologs of the VchInd5 BrxC are more diverse than the homologs of the VchBan9 BrxC. These data provide new insight into the function of the BrxC ATPase and highlight how phage-encoded inhibitors can disrupt phage defense systems using different mechanisms.IMPORTANCEWith renewed interest in phage therapy to combat antibiotic-resistant pathogens, understanding the mechanisms bacteria use to defend themselves against phages and the counter-strategies phages evolve to inhibit defenses is paramount. Bacteriophage exclusion (BREX) is a common defense system with few known inhibitors. Here, we probe how the vibriophage-encoded inhibitor OrbA inhibits the BREX system of Vibrio cholerae, the causative agent of the diarrheal disease cholera. By interrogating OrbA function, we have begun to understand the importance and function of a BREX component. Our results demonstrate the importance of identifying inhibitors against defense systems, as they are powerful tools for dissecting defense activity and can inform strategies to increase the efficacy of some phage therapies.

Species of the Vibrio genus occupy diverse aquatic environments ranging from brackish water to warm equatorial seas to salty coastal regions. More than 80 species of Vibrio have been identified, many of them as pathogens of marine organisms, including fish, shellfish, and corals, causing disease and wreaking havoc on aquacultures and coral reefs. Moreover, many Vibrio species associate with and thrive on chitinous organisms abundant in the ocean. Among the many diverse Vibrio species, the most well-known and studied is Vibrio cholerae, discovered in the 19th century to cause cholera in humans when ingested. The V. cholerae field blossomed in the late 20th century, with studies broadly examining V. cholerae evolution as a human pathogen, natural competence, biofilm formation, and virulence mechanisms, including toxin biology and virulence gene regulation. This review discusses some of the historic discoveries of V. cholerae biology and ecology as one of the fundamental model systems of bacterial genetics and pathogenesis.

Type IVa pili (T4aP) are widespread and enable bacteria to translocate across surfaces. T4aP engage in cycles of extension, surface adhesion, and retraction, thereby pulling cells forward. Accordingly, the number and localization of T4aP are critical to efficient translocation. Here, we address how T4aP formation is regulated in Myxococcus xanthus, which translocates with a well-defined leading and lagging cell pole using T4aP at the leading pole. This localization is orchestrated by the small GTPase MglA and its downstream effector SgmX that both localize at the leading pole and recruit the PilB extension ATPase to the T4aP machinery at this pole. Here, we identify the previously uncharacterized protein SopA and show that it interacts directly with SgmX, localizes at the leading pole, stimulates polar localization of PilB, and is important for T4aP formation. We corroborate that MglA also recruits FrzS to the leading pole, and FrzS stimulates SgmX recruitment. In addition, FrzS and SgmX separately recruit SopA. Precise quantification of T4aP-formation and T4aP-dependent motility in various mutants supports a model whereby the main pathway for stimulating T4aP formation is the MglA/SgmX pathway. FrzS stimulates this pathway by recruiting SgmX and SopA. SopA stimulates the MglA/SgmX pathway by stimulating the function of SgmX, likely by promoting the SgmX-dependent recruitment of PilB to the T4aP machinery. The architecture of the MglA/SgmX/FrzS/SopA protein interaction network for orchestrating T4aP formation allows for combinatorial regulation of T4aP levels at the leading cell pole resulting in discrete levels of T4aP-dependent motility.

Importance: Type IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in translocation across surfaces, surface adhesion, biofilm formation, and virulence. T4aP-dependent translocation crucially depends on the number of pili. To address how the number of T4aP is regulated, we focused on M. xanthus, which assembles T4aP at the leading cell pole and is a model organism for T4aP biology. Our results support a model whereby the four proteins MglA, SgmX, FrzS, and the newly identified SopA protein establish a highly intricate interaction network for orchestrating T4aP formation at the leading cell pole. This network allows for combinatorial regulation of the number of T4aP resulting in discrete levels of T4aP-dependent motility.

Cobamides (Cbas) are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life as co-catalyst of diverse reactions. There are several structural features that distinguish Cbas from one another. The most relevant of those features discussed in this review is the lower ligand, which is the nucleobase of a ribotide located in the lower face of the cyclic tetrapyrrole ring. The above-mentioned ribotide is known as the nucleotide loop, which is attached to the ring by a short linker. In Cbas, the nucleobase of the ribotide can be benzimidazole or derivatives of it, purine or derivatives of it, or phenolic compounds. Given the importance of Cbas in prokaryotic metabolism, it is not surprising that prokaryotes have evolved enzymes that cleave part or the entire nucleotide loop. This function is advantageous when Cbas contain nucleobases that somehow interfere with the function of Cba-dependent enzymes in the organism. After cleavage, Cbas are rebuilt via the nucleotide loop assembly (NLA) pathway, which includes enzymes that activate the nucleobase and the ring intermediate, followed by condensation of activated intermediates and a final dephosphorylation reaction. This exchange of nucleobases is known as Cba remodeling. The NLA pathway is used to salvage Cba precursors from the environment.

Leaderless bacteriocins are a unique class of bacteriocins that possess antimicrobial activity after translation and have few cases of documented resistance. Aureocin A53 and lacticin Q are considered two of the most well-studied leaderless bacteriocins. Here, we used in silico genome mining to search for novel aureocin A53-like leaderless bacteriocins in GenBank and MGnify. We identified 757 core peptides across 430 genomes with 75 species found currently without characterized leaderless bacteriocin production. These include putative novel species containing bacteriocin gene clusters (BGCs) from the genera Streptomyces (sp. NBC_00237) and Agrococcus (sp. SL85). To date, all characterized leaderless bacteriocins have been found within the phylum Bacillota, but this study identified 97 core peptides within the phylum Actinomycetota. Members of this phylum are traditionally associated with the production of antibiotics, such is the case with the genus Streptomyces. Actinomycetota is an underexplored phylum in terms of bacteriocin production with no characterized leaderless bacteriocin production to date. The two novel leaderless bacteriocins arcanocin and arachnicin from Actinomycetota members Arcanobacterium sp. and Arachnia sp., respectively, were chemically synthesized and antimicrobial activity was verified. These peptides were encoded in human gut (PRJNA485056) and oral (PRJEB43277) microbiomes, respectively. This research highlights the biosynthetic potential of Actinomycetota in terms of leaderless bacteriocin production and describes the first antimicrobial peptides encoded in the genera Arcanobacterium and Arachnia.IMPORTANCEBacteriocins are gathering attention as alternatives to current antibiotics given the increasing incidence of antimicrobial resistance. Leaderless bacteriocins are considered a commercially attractive subclass of bacteriocins due to the ability to synthesize active peptide and low levels of documented resistance. Therefore, in this work, we mined publicly available data to determine how widespread and diverse leaderless bacteriocins are within the domain of bacteria. Actinomycetota, known for its antibiotic producers but lacking described and characterized bacteriocins, proved to be a rich source of leaderless bacteriocins-97 in total. Two such peptides, arcanocin and arachnicin, were chemically synthesized and have antimicrobial activity. These bacteriocins may provide a novel source of novel antimicrobials that could aid in the development of future alternative antimicrobials and highlight that the Actinomycetota are an underexplored resource of bacteriocin peptides.

NADPH is a redox cofactor that drives the anabolic reactions. Although major NADPH generation reactions have been identified in Escherichia coli, some minor reactions have not been identified. In the present study, we explored novel NADPH generation reactions by monitoring the fluorescence dynamics after the addition of carbon sources to starved cells, using a metagenome-derived blue fluorescent protein (mBFP) as an intracellular NADPH reporter. Perturbation analyses were performed on a glucose-6-phosphate isomerase (PGI) deletion strain and its parental strain. Interestingly, mBFP fluorescence increased not only in the parental strain but also in the ΔPGI strain after the addition of xylose. Because the ΔPGI strain cannot metabolize xylose through the oxidative pentose phosphate pathway, this suggests that an unexpected NADPH generation reaction contributes to an increase in fluorescence. To unravel this mystery, we deleted the NADPH generation enzymes including transhydrogenase, isocitrate dehydrogenase, NADP⁺-dependent malic enzyme, glucose-6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH) in the ΔPGI strain, and revealed that G6PDH and 6PGDH contribute to an increase in fluorescence under xylose conditions. In vitro assays using purified enzymes showed that G6PDH can produce NADPH using erythrose-4-phosphate (E4P) as a substitute for glucose-6-phosphate. Because the Km (0.65 mM) for E4P was much higher than the reported intracellular E4P concentrations in E. coli, little E4P must be metabolized through this bypass in the parental strain. However, the flux would increase when E4P accumulates in the cells owing to genetic modifications. This finding provides a metabolic engineering strategy for generating NADPH to produce useful compounds using xylose as a carbon source.IMPORTANCEBecause NADPH is consumed during the synthesis of various useful compounds, enhancing NADPH regeneration is highly desirable in metabolic engineering. In this study, we explored novel NADPH generation reactions in Escherichia coli using a fluorescent NADPH reporter and found that glucose-6-phosphate dehydrogenase can produce NADPH using erythrose-4-phosphate as a substrate under xylose conditions. Xylose is an abundant sugar in nature and is an attractive carbon source for bioproduction. Therefore, this finding contributes to novel pathway engineering strategies using a xylose carbon source in E. coli to produce useful compounds that consume NADPH for their synthesis.

Staphylococcus aureus is a Gram-positive, opportunistic human pathogen that is a leading cause of skin and soft tissue infections and invasive disease worldwide. Virulence in this bacterium is tightly controlled by a network of regulatory factors. One such factor is the global regulatory protein CodY. CodY links branched-chain amino acid sufficiency to the production of surface-associated and secreted factors that facilitate immune evasion and subversion. Our previous work revealed that CodY regulates virulence factor gene expression indirectly in part by controlling the activity of the SaeRS two-component system (TCS). While this is correlated with an increase in membrane anteiso-15:0 and -17:0 branched-chain fatty acids (BCFAs) derived from isoleucine, the true mechanism of control has remained elusive. Herein, we report that CodY-dependent regulation of SaeS sensor kinase activity requires BCFA synthesis. During periods of nutrient sufficiency, BCFA synthesis and Sae TCS activity are kept relatively low by CodY-dependent repression of the ilv-leu operon and the isoleucine-specific permease gene brnQ2. In a codY null mutant, which simulates extreme nutrient limitation, de-repression of ilv-leu and brnQ2 directs the synthesis of enzymes in redundant de novo and import pathways to upregulate production of BCFA precursors. Overexpression of brnQ2, independent of CodY, is sufficient to increase membrane anteiso BCFAs, Sae-dependent promoter activity, and SaeR ~P levels. Our results further clarify the molecular mechanisms by which CodY controls virulence in S. aureus.IMPORTANCEExpression of bacterial virulence genes often correlates with the exhaustion of nutrients, but how the signaling of nutrient availability and the resulting physiological responses are coordinated is unclear. In S. aureus, CodY controls the activity of two major regulators of virulence-the Agr and Sae two-component systems (TCSs)-by unknown mechanisms. This work identifies a mechanism by which CodY controls the activity of the sensor kinase SaeS by modulating the levels of anteiso branched-chain amino acids that are incorporated into the membrane. Understanding the mechanism adds to our understanding of how bacterial physiology and metabolism are linked to virulence and underscores the role virulence in maintaining homeostasis. Understanding the mechanism also opens potential avenues for targeted therapeutic strategies against S. aureus infections.

Although the development of disinfection technologies with novel mechanisms has stagnated, we demonstrate the bactericidal effects and mechanisms of high-speed nanodroplet generation technology. The first development of this technology in 2017 gushes out a water droplet of 10 nm in size at 50 m/s; however, the target surface does not become completely wet. Nanodroplets were exposed to biofilm models of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Serratia marcescens. This phenomenon was verified when the nanodroplets collide with the surface of the bacteria at an impact pressure of ~75 MPa. S. aureus was exposed to nanodroplets for 30 seconds at 75 MPa, which exploded the bacterial body and completely sterilized. Eighteen MPa damaged the bacterial surface, causing peptidoglycan leakage. S. aureus was repaired and survives in this state. In contrast, in Gram-negative bacteria, nanodroplets with 18 MPa penetrated some biofilm-forming bacteria but did not hit all of them, and the viable count was not significantly reduced. Although all three bacterial species were completely sterilized at 75 MPa, the disinfectant effect was affected by the biomass of the biofilm formed. In summary, our findings prove that nanodroplets at 18 MPa on the bacterial surface were ineffective in killing bacteria, whereas at 75 MPa, all four bacterial species were completely sterilized. The disinfection mechanism involved a high-velocity collision of nanodroplets with the bacteria, physically destroying them. Our results showed that disinfection using this technology could be an innovative method that is completely different from existing disinfection techniques.

Importance: Although existing disinfection techniques demonstrate bactericidal effects through chemical reactions, concerns regarding human toxicity and environmental contamination have been raised. To the best of our knowledge, this study is the first in the world to reveal that the use of this technology, with nanodroplets of less than 100 nm, can destroy and sterilize bacterial cells by colliding with biofilm-forming bacteria at 75 MPa. Furthermore, because this technology uses only water, it can solve the problems of human toxicity and environmental contamination caused by existing disinfection techniques. Because of its minimal water usage, it can be employed for sanitation worldwide without being limited to specific regions. Our report proposes an unprecedented physical disinfection approach that utilizes a high-speed nanodroplet generation technology.

Contact-dependent inhibition (CDI) is a mechanism of interbacterial competition in Gram-negative bacteria. Bacteria that contain CDI systems produce a large, filamentous protein, CdiA, on their cell surfaces. CdiA contains a C-terminal toxin domain that is transported across the outer membranes (OMs) of neighboring bacteria. Once inside a target bacterium, the toxin is released from the CdiA protein via a proteolytic mechanism that has not been well characterized. We have developed an in vitro assay to monitor this toxin release process and have identified several conserved amino acids that play critical roles in the autocatalytic mechanism. Our results indicate that a hydrophobic, membrane-like environment is required for CdiA to fold, and the proteolysis occurs through an asparagine cyclization mechanism. Our in vitro assay thus provides a starting point for analyzing the conformational state of the CdiA protein when it is inserted into a target cell's OM and engaged in transporting the toxin across that membrane.

Importance: It is challenging to develop new antibiotics capable of killing Gram-negative bacteria because their outer membranes are impermeable to many small molecules. Some Gram-negative bacteria, however, deliver much larger protein toxins through the outer membranes of competing bacteria in their environments using contact-dependent inhibition (CDI) systems. How these toxins traverse the outer membranes of their targets is not well understood. We have therefore developed a method to study the toxin delivery process in a highly simplified system using a fragment of a CDI protein. Our results indicate that the CDI protein assembles into a structure in the target membrane that catalyzes the release of the toxin. This CDI protein fragment enables further studies of the toxin delivery mechanism.