Journal of Bacteriology最新文献

The transposon Tn10 is a prevalent composite element often detected in enteric bacteria, including those obtained from clinical samples. The Tn10 is flanked by two IS10 elements that work together in mediating transposition. IS10-right (IS10R) promotes transposition, while IS10-left lacks a functional transposase and cannot transpose independently. IS10R contains a weak promoter crucial for transposase transcription (pIN), along with two outward-oriented promoters, pOUT and OUTIIp, which may influence the expression of adjacent genes flanking the transposition site. Here, we report the identification of a novel outward-facing promoter, pOUT70, and a functional translation initiation region (TIR) within the last 70 nucleotides of IS10R. Furthermore, we show that pOUT70 is negatively regulated by ArgR and positively controlled by IHF, and we demonstrate that pOUT70 enables growth phase-dependent expression of a truncated yet constitutively active version of the histidine kinase BarA. These findings underscore the significance of IS elements in enhancing downstream gene expression, and highlights the role of outward-facing promoters in derepressing virulence factors or acquiring antibiotic resistance.

Importance: Mobile genetic elements are small DNA fragments that can relocate within the genome, causing either gene inactivation or enhanced gene expression. Our research identified a new functional promoter and mRNA translation region within the IS10R element, which is part of the widely distributed Tn10 transposon. We found that the global regulators ArgR and IHF control the activity of this promoter. Additionally, insertion of this mini-Tn10 derivative into the barA gene resulted in the expression of a truncated but constitutive active form of the BarA sensor kinase. Overall, our work sheds light on how mobile genetic elements could impact the physiology and virulence of opportunistic pathogenic bacteria.

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.

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.

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.

ATP, most widely known as the primary energy source for numerous cellular processes, also exhibits the characteristics of a biological hydrotrope. The viable but nonculturable (VBNC) and persister states are two prevalent dormant phenotypes employed by bacteria to survive challenging environments, both of which are associated with low metabolic activity. Here, we investigate the intracellular ATP concentration of individual VBNC and persister cells using a sensitive ATP biosensor QUEEN-7μ and reveal that both types of cells possess a lower intracellular ATP concentration than culturable and sensitive cells, although there is a certain overlap in the intracellular ATP concentrations between antibiotic-sensitive cells and persisters. Moreover, we successfully separated VBNC cells from culturable cells using fluorescence-activated cell sorting based on the intracellular ATP concentration threshold of 12.5 µM. Using an enriched VBNC cell population, we confirm that the precipitation of proteins involved in key biological processes promotes VBNC cell formation. Notably, using green light-illuminated proteorhodopsin (PR), we demonstrate that VBNC cells can be effectively resuscitated by elevating their intracellular ATP concentration. These findings highlight the crucial role of intracellular ATP concentration in the regulation of bacterial cell fate and provide new insights into the formation of VBNC and persister cells.IMPORTANCEThe viable but nonculturable (VBNC) and persister states are two dormant phenotypes employed by bacteria to counter stressful conditions and play a crucial role in chronic and recurrent bacterial infections. However, the lack of precise detection methods poses significant threats to public health. Our study reveals lower intracellular ATP concentrations in these states and establishes an ATP threshold for distinguishing VBNC from culturable cells. Remarkably, we revive VBNC cells by elevating their intracellular ATP levels. This echoes recent eukaryotic studies where modulating metabolism impacts outcomes like osteoarthritis treatment and lifespan extension in Caenorhabditis elegans. Our findings underscore the crucial role of intracellular ATP levels in governing bacterial fate, emphasizing ATP manipulation as a potential strategy to steer bacterial behavior.

In response to starvation, endospore-forming bacteria differentiate into stress-resistant spores that can remain dormant for years yet rapidly germinate and resume growth when nutrients become available. To identify uncharacterized factors involved in the exit from dormancy, we performed a transposon-sequencing screen taking advantage of the loss of spore heat resistance that accompanies germination. We reasoned that transposon insertions that impair but do not block germination will lose resistance more slowly than wild type after exposure to nutrients and will therefore survive heat treatment. Using this approach, we identified most of the known germination genes and several new ones. We report an initial characterization of 15 of these genes and a more detailed analysis of one (ymaF). Spores lacking ymaF (renamed gerY) are impaired in germination in response to both L-alanine and L-asparagine, D-glucose, D-fructose, and K⁺. GerY is a soluble protein synthesized under σ^E control in the mother cell. A YFP-GerY fusion localizes around the developing and mature spore in a manner that depends on CotE and SafA, indicating that it is a component of the spore coat. Coat proteins encoded by the gerP operon and gerT are also required for efficient germination, and we show that spores lacking two or all three of these loci have more severe defects in the exit from dormancy. Our data are consistent with a model in which GerY, GerT, and the GerP proteins are required for efficient transit of nutrients through the coat to access the germination receptors, but each acts independently in this process.

Importance: Pathogens in the orders Bacillales and Clostridiales resist sterilization by differentiating into stress-resistant spores. Spores are metabolically inactive and can remain dormant for decades, yet upon exposure to nutrients, they rapidly resume growth, causing food spoilage, food-borne illness, or life-threatening disease. The exit from dormancy, called germination, is a key target in combating these important pathogens. Here, we report a high-throughput genetic screen using transposon sequencing to identify novel germination factors that ensure the efficient exit from dormancy. We identify several new factors and characterize one in greater detail. This factor, renamed GerY, is part of the proteinaceous coat that encapsulates the dormant spore. Our data suggest that GerY enables efficient transit of nutrients through the coat to trigger germination.

Pseudomonas aeruginosa is a common opportunistic pathogen and a model organism for studying bacterial sociality. A social behavior of P. aeruginosa that is critical for its success as a pathogen is its ability to form protective biofilms. Many of P. aeruginosa's social phenotypes are regulated by quorum sensing-a type of cell-cell communication that allows bacteria to respond to population density. Although biofilm formation is known to be affected by quorum sensing, evidence for direct regulation of biofilm production by quorum regulators has remained elusive. In this work, we show that production of the major biofilm matrix polysaccharide Psl in P. aeruginosa PAO1 is regulated by the quorum regulators LasR and RhlR in stationary-phase cultures. Secretion of Psl into the culture medium requires LasR, RhlR, and the quorum signal molecules N-3-oxo-dodecanoyl-homoserine lactone and N-butanoyl homoserine lactone. Psl production in strains unable to synthesize the homoserine lactone signals can be restored by exogenous introduction of the signal molecules. We found that LasR and RhlR perform different roles in the regulation of Psl production: LasR acts at the promoter of the psl operon and activates transcription of the Psl biosynthetic genes, while RhlR activates translation of the psl transcripts. This work contributes to our understanding of the overlapping but distinct functions of the Las and Rhl quorum-sensing systems and implicates both in the direct regulation of biofilm matrix production.IMPORTANCEPseudomonas aeruginosa biofilms are responsible for many treatment-resistant infections in humans. Many cooperative behaviors in P. aeruginosa are controlled by quorum sensing, but evidence for a direct role of quorum sensing in the regulation of biofilm matrix production has been scant. In this work, we show that the Las and Rhl quorum-sensing systems have distinct roles in regulating production of the matrix polysaccharide Psl and that this regulation happens at the level of transcription (Las) and translation (Rhl) of the psl operon. These findings deepen our understanding of overlapping functions of Las and Rhl quorum sensing and the complex regulation of biofilm development in P. aeruginosa.

Many bacteria produce type IV pili (T4P), surfaced-exposed protein filaments that enable cells to interact with their environment and transition from planktonic to surface-adapted states. T4P are dynamic, undergoing rapid cycles of filament extension and retraction facilitated by a complex protein nanomachine powered by cytoplasmic motor ATPases. Dedicated assembly motors drive the extension of the pilus fiber into the extracellular space, but like any machine, this process is tightly organized. These motors are coordinated by various ligands and binding partners, which control or optimize their functional associations with T4P machinery before cells commit to the crucial first step of building a pilus. This review focuses on the molecular mechanisms that regulate T4P extension motor function. We discuss secondary messenger-dependent transcriptional or post-translational regulation acting both directly on the motor and through protein effectors. We also discuss the recent discoveries of naturally occurring extension inhibitors as well as alternative mechanisms of pilus assembly and motor-dependent signaling pathways. Given that T4P are important virulence factors for many bacterial pathogens, studying these motor regulatory systems will provide new insights into T4P-dependent physiology and efficient strategies to disable them.

In mycobacteria, σ^A is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes. Our knowledge about this factor in mycobacteria is limited. Here, we performed an unbiased search for interacting partners of Mycobacterium smegmatis σ^A. The search revealed a number of proteins; prominent among them was MoaB2. The σ^A-MoaB2 interaction was validated and characterized by several approaches, revealing that it likely does not require RNAP and is specific, as alternative σ factors (e.g., closely related σ^B) do not interact with MoaB2. The structure of MoaB2 was solved by X-ray crystallography. By immunoprecipitation and nuclear magnetic resonance, the unique, unstructured N-terminal domain of σ^A was identified to play a role in the σ^A-MoaB2 interaction. Functional experiments then showed that MoaB2 inhibits σ^A-dependent (but not σ^B-dependent) transcription and may increase the stability of σ^A in the cell. We propose that MoaB2, by sequestering σ^A, has a potential to modulate gene expression. In summary, this study has uncovered a new binding partner of mycobacterial σ^A, paving the way for future investigation of this phenomenon.IMPORTANCEMycobacteria cause serious human diseases such as tuberculosis and leprosy. The mycobacterial transcription machinery is unique, containing transcription factors such as RbpA, CarD, and the RNA polymerase (RNAP) core-interacting small RNA Ms1. Here, we extend our knowledge of the mycobacterial transcription apparatus by identifying MoaB2 as an interacting partner of σ^A, the primary sigma factor, and characterize its effects on transcription and σ^A stability. This information expands our knowledge of interacting partners of subunits of mycobacterial RNAP, providing opportunities for future development of antimycobacterial compounds.

The ability of bacteria to interact with and respond to their environment is crucial to their lifestyle and survival. Bacterial cells routinely need to engage with extracellular target molecules, in locations spatially separated from their cell surface. Engagement with distant targets allows bacteria to adhere to abiotic surfaces and host cells, sense harmful or friendly molecules in their vicinity, as well as establish symbiotic interactions with neighboring cells in multicellular communities such as biofilms. Binding to extracellular molecules also facilitates transmission of information back to the originating cell, allowing the cell to respond appropriately to external stimuli, which is critical throughout the bacterial life cycle. This requirement of bacteria to bind to spatially separated targets is fulfilled by a myriad of specialized cell surface molecules, which often have an extended, filamentous arrangement. In this review, we compare and contrast such molecules from diverse bacteria, which fulfil a range of binding functions critical for the cell. Our comparison shows that even though these extended molecules have vastly different sequence, biochemical and functional characteristics, they share common architectural principles that underpin bacterial adhesion in a variety of contexts. In this light, we can consider different bacterial adhesins under one umbrella, specifically from the point of view of a modular molecular machine, with each part fulfilling a distinct architectural role. Such a treatise provides an opportunity to discover fundamental molecular principles governing surface sensing, bacterial adhesion, and biofilm formation.