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Showing papers in "Journal of Bacteriology in 2018"


Journal ArticleDOI
TL;DR: The fascinating history of CRISPR-Cas systems is discussed, from the original observation of an enigmatic sequence in E. coli to genome editing in humans and providing insights into the origin and evolution of this system from mobile genetic elements denoted casposons.
Abstract: Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are well-known acquired immunity systems that are widespread in archaea and bacteria. The RNA-guided nucleases from CRISPR-Cas systems are currently regarded as the most reliable tools for genome editing and engineering. The first hint of their existence came in 1987, when an unusual repetitive DNA sequence, which subsequently was defined as a CRISPR, was discovered in the Escherichia coli genome during an analysis of genes involved in phosphate metabolism. Similar sequence patterns were then reported in a range of other bacteria as well as in halophilic archaea, suggesting an important role for such evolutionarily conserved clusters of repeated sequences. A critical step toward functional characterization of the CRISPR-Cas systems was the recognition of a link between CRISPRs and the associated Cas proteins, which were initially hypothesized to be involved in DNA repair in hyperthermophilic archaea. Comparative genomics, structural biology, and advanced biochemistry could then work hand in hand, not only culminating in the explosion of genome editing tools based on CRISPR-Cas9 and other class II CRISPR-Cas systems but also providing insights into the origin and evolution of this system from mobile genetic elements denoted casposons. To celebrate the 30th anniversary of the discovery of CRISPR, this minireview briefly discusses the fascinating history of CRISPR-Cas systems, from the original observation of an enigmatic sequence in E. coli to genome editing in humans.

236 citations


Journal ArticleDOI
TL;DR: It is believed these survival modes represent a continuum between actively growing and dead cells, with VBNC cells being in a deeper state of dormancy than persister cells.
Abstract: Bacteria have evolved numerous means of survival in adverse environments with dormancy, as represented by "persistence" and the "viable but nonculturable" (VBNC) state, now recognized to be common modes for such survival. VBNC cells have been defined as cells which, induced by some stress, become nonculturable on media that would normally support their growth but which can be demonstrated by various methods to be alive and capable of returning to a metabolically active and culturable state. Persister cells have been described as a population of cells which, while not being antibiotic resistant, are antibiotic tolerant. This drug-tolerant phenotype is thought to be a result of stress-induced and stochastic physiological changes as opposed to mutational events leading to true resistance. In this review, we describe these two dormancy strategies, characterize the molecular underpinnings of each state, and highlight the similarities and differences between them. We believe these survival modes represent a continuum between actively growing and dead cells, with VBNC cells being in a deeper state of dormancy than persister cells.

144 citations


Journal ArticleDOI
TL;DR: It is shown that secreted outer membrane vesicles (OMVs) act as a defense mechanism that confers protection to V. cholerae against phage predation and that this OMV-mediated inhibition is phage receptor dependent.
Abstract: Novel preventatives could help in efforts to limit Vibrio cholerae infection and the spread of cholera. Bacteriophage (phage) treatment has been proposed as an alternative intervention, given the rapid replication of virulent phages, prey specificity, and relative ease of finding new virulent phages. Phage tropism is dictated in part by the presence of phage receptors on the bacterial surface. While many phages that can kill V. cholerae have been isolated, whether this pathogen is able to defend itself by neutralizing phage binding is unknown. Here, we show that secreted outer membrane vesicles (OMVs) act as a defense mechanism that confers protection to V. cholerae against phage predation and that this OMV-mediated inhibition is phage receptor dependent. Our results suggest that phage therapy or prophylaxis should take into consideration the production of OMVs as a bacterial decoy mechanism that could influence the outcome of phage treatment.IMPORTANCE Phages have been increasingly recognized for the significance of their interactions with bacterial cells in multiple environments. Bacteria use myriad strategies to defend against phage infection, including restriction modification, abortive infection, phase variation of cell surface receptors, phage-inducible chromosomal islands, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems. The data presented here suggest that the apparently passive process of OMV release can also contribute to phage defense. By considering the effect of OMVs on V. cholerae infection by three unique virulent phages, ICP1, ICP2, and ICP3, we show that, in vitro, a reproducible reduction in bacterial killing is both dose and phage receptor dependent. This work supports a role for OMVs as natural decoys to defend bacteria from phage predation.

115 citations


Journal ArticleDOI
TL;DR: Using informatics approaches applied to genomic data, it is shown that pathways of electron transfer to nitrogenase in metabolically diverse diazotrophic taxa have diversified primarily in response to host cells' acquired ability to integrate O2 or light into their energy metabolism.
Abstract: Nitrogenase catalyzes the reduction of dinitrogen (N2) using low-potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP-dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O2)-sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds, including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd-/Fld-reducing enzymes in 359 genomes of putative N2 fixers (diazotrophs). Six distinct lineages of nitrogenase were identified, and their distributions largely corresponded to differences in the host cells' ability to integrate O2 or light into energy metabolism. The predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the levels of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. The energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N2 reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation.IMPORTANCE Nitrogen fixation supplies fixed nitrogen to cells from a variety of genomic and metabolic backgrounds, including those of aerobes, facultative anaerobes, chemotrophs, and phototrophs. Here, using informatics approaches applied to genomic data, we show that pathways of electron transfer to nitrogenase in metabolically diverse diazotrophic taxa have diversified primarily in response to host cells' acquired ability to integrate O2 or light into their energy metabolism. The acquisition of two key enzyme complexes enabled aerobic and facultatively anaerobic phototrophic taxa to generate electrons of sufficiently low potential to reduce nitrogenase: the bifurcation of electrons via the Fix complex or the coupling of Fd reduction to reverse ion translocation via the Rhodobacter nitrogen fixation (Rnf) complex.

88 citations


Journal ArticleDOI
TL;DR: It is described by mutant studies in Acetobacterium woodii that the main function of Rnf is to energetically link cellular pools of ferredoxin and NAD+ when ferredox is greater than NADH, exergonic electron flow from ferred toxin to NAD+ generates a chemiosmotic potential, essential for energy conservation during autotrophic growth.
Abstract: The Rnf complex is a respiratory enzyme that catalyzes the oxidation of reduced ferredoxin to the reduction of NAD+, and the negative free energy change of this reaction is used to generate a transmembrane ion gradient. In one class of anaerobic acetogenic bacteria, the Rnf complex is believed to be essential for energy conservation and autotrophic growth. We describe here a methodology for markerless mutagenesis in the model bacterium of this class, Acetobacterium woodii, which enabled us to delete the rnf genes and to test their in vivo role. The rnf mutant did not grow on H2 plus CO2, nor did it produce acetate or ATP from H2 plus CO2, and ferredoxin:NAD+ oxidoreductase activity and Na+ translocation were also completely lost, supporting the hypothesis that the Rnf complex is the only respiratory enzyme in this metabolism. Unexpectedly, the mutant also did not grow on low-energy substrates, such as ethanol or lactate. Oxidation of these substrates is not coupled to the reduction of ferredoxin but only of NAD+, and we speculated that the growth phenotype is caused by a loss of reduced ferredoxin, indispensable for biosynthesis and CO2 reduction. The electron-bifurcating hydrogenase of A. woodii reduces ferredoxin, and indeed, the addition of H2 to the cultures restored growth on ethanol and lactate. This is consistent with the hypothesis that endergonic reduction of ferredoxin with NADH is driven by reverse electron transport catalyzed by the Rnf complex, which renders the Rnf complex essential also for growth on low-energy substrates.IMPORTANCE Ferredoxin and NAD+ are key electron carriers in anaerobic bacteria, but energetically, they are not equivalent, since the redox potential of ferredoxin is lower than that of the NADH/NAD+ couple. We describe by mutant studies in Acetobacterium woodii that the main function of Rnf is to energetically link cellular pools of ferredoxin and NAD+ When ferredoxin is greater than NADH, exergonic electron flow from ferredoxin to NAD+ generates a chemiosmotic potential. This is essential for energy conservation during autotrophic growth. When NADH is greater than ferredoxin, Rnf works in reverse. This reaction is essential for growth on low-energy substrates to provide reduced ferredoxin, indispensable for biosynthesis and CO2 reduction. Our studies put a new perspective on the cellular function of the membrane-bound ion-translocating Rnf complex widespread in bacteria.

84 citations


Journal ArticleDOI
TL;DR: The key inhibitory mechanism of pyoverdine was chelation of iron and denial of iron to A. fumigatus, and it was identified that iron deprivation could be a therapeutic approach to the control of pathogen growth.
Abstract: Pseudomonas aeruginosa and Aspergillus fumigatus are common opportunistic bacterial and fungal pathogens, respectively. They often coexist in airways of immunocompromised patients and individuals with cystic fibrosis, where they form biofilms and cause acute and chronic illnesses. Hence, the interactions between them have long been of interest and it is known that P. aeruginosa can inhibit A. fumigatusin vitro We have approached the definition of the inhibitory P. aeruginosa molecules by studying 24 P. aeruginosa mutants with various virulence genes deleted for the ability to inhibit A. fumigatus biofilms. The ability of P. aeruginosa cells or their extracellular products produced during planktonic or biofilm growth to affect A. fumigatus biofilm metabolism or planktonic A. fumigatus growth was studied in agar and liquid assays using conidia or hyphae. Four mutants, the pvdD pchE, pvdD, lasR rhlR, and lasR mutants, were shown to be defective in various assays. This suggested the P. aeruginosa siderophore pyoverdine as the key inhibitory molecule, although additional quorum sensing-regulated factors likely contribute to the deficiency of the latter two mutants. Studies of pure pyoverdine substantiated these conclusions and included the restoration of inhibition by the pyoverdine deletion mutants. A correlation between the concentration of pyoverdine produced and antifungal activity was also observed in clinical P. aeruginosa isolates derived from lungs of cystic fibrosis patients. The key inhibitory mechanism of pyoverdine was chelation of iron and denial of iron to A. fumigatusIMPORTANCE Interactions between human pathogens found in the same body locale are of vast interest. These interactions could result in exacerbation or amelioration of diseases. The bacterium Pseudomonas aeruginosa affects the growth of the fungus Aspergillus fumigatus Both pathogens form biofilms that are resistant to therapeutic drugs and host immunity. P. aeruginosa and A. fumigatus biofilms are found in vivo, e.g., in the lungs of cystic fibrosis patients. Studying 24 P. aeruginosa mutants, we identified pyoverdine as the major anti-A. fumigatus compound produced by P. aeruginosa Pyoverdine captures iron from the environment, thus depriving A. fumigatus of a nutrient essential for its growth and metabolism. We show how microbes of different kingdoms compete for essential resources. Iron deprivation could be a therapeutic approach to the control of pathogen growth.

83 citations


Journal ArticleDOI
TL;DR: This work presents the first combinatorial genetic analysis of five different electron conduits via creation of new markerless deletion strains and complementation vectors in Gram-negative metal-reducing bacteria.
Abstract: At least five gene clusters in the Geobacter sulfurreducens genome encode putative "electron conduits" implicated in electron transfer across the outer membrane, each containing a periplasmic multiheme c-type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single-gene-cluster deletions and all possible multiple-deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III) and Mn(IV) oxides, and graphite electrodes poised at +0.24 V and -0.1 V versus the standard hydrogen electrode (SHE). Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously described omcBC cluster caused defects, but deletion of additional components in an ΔomcBC background, such as extEFG, were needed to produce defects greater than 50% compared to findings with the wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the ΔextABCD mutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III) oxide, Mn(IV) oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than the wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth conditions showed all of these ext clusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing only extABCD detected no significant changes in expression of genes encoding known redox proteins or pilus components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth of G. sulfurreducens, depending on the available extracellular electron acceptor.IMPORTANCE Gram-negative metal-reducing bacteria utilize electron conduits, chains of redox proteins spanning the outer membrane, to transfer electrons to the extracellular surface. Only one pathway for electron transfer across the outer membrane of Geobacter sulfurreducens has been linked to Fe(III) reduction. However, G. sulfurreducens is able to respire a wide array of extracellular substrates. Here we present the first combinatorial genetic analysis of five different electron conduits via creation of new markerless deletion strains and complementation vectors. Multiple conduit gene clusters appear to have overlapping roles, including two that have never been linked to metal reduction. Another recently described cluster (ExtABCD) was the only electron conduit essential during electrode reduction, a substrate of special importance to biotechnological applications of this organism.

77 citations


Journal ArticleDOI
TL;DR: The CRISPR interference system for gene repression in Pseudomonas spp.
Abstract: Pseudomonas spp. are widely used model organisms in different areas of research. Despite the relevance of Pseudomonas in many applications, the use of protein depletion tools in this host remains limited. Here, we developed the CRISPR interference system for gene repression in Pseudomonas spp. using a nuclease-null Streptococcus pasteurianus Cas9 variant (dead Cas9, or dCas9). We demonstrate a robust and titratable gene depletion system with up to 100-fold repression in β-galactosidase activity in P. aeruginosa and 300-fold repression in pyoverdine production in Pseudomonas putida. This inducible system enables the study of essential genes, as shown by ftsZ depletions in P. aeruginosa, P. putida, and Pseudomonas fluorescens that led to phenotypic changes consistent with depletion of the targeted gene. Additionally, we performed the first in vivo characterization of protospacer adjacent motif (PAM) site preferences of S. pasteurianus dCas9 and identified NNGCGA as a functional PAM site that resulted in repression efficiencies comparable to the consensus NNGTGA sequence. This discovery significantly expands the potential genomic targets of S. pasteurianus dCas9, especially in GC-rich organisms. IMPORTANCEPseudomonas spp. are prevalent in a variety of environments, such as the soil, on the surface of plants, and in the human body. Although Pseudomonas spp. are widely used as model organisms in different areas of research, existing tools to deplete a protein of interest in these organisms remain limited. We have developed a robust and inducible gene repression tool in P. aeruginosa, P. putida, and P. fluorescens using the Streptococcus pasteurianus dCas9. This method of protein depletion is superior to existing methods, such as promoter replacements and addition of degradation tags, because it does not involve genomic modifications of the target protein, is titratable, and is capable of repressing multiple genes simultaneously. This gene repression system now enables easy depletion of specific proteins in Pseudomonas, accelerating the study and engineering of this widely used model organism.

73 citations


Journal ArticleDOI
TL;DR: This study is the first survey of the lysogenic phage population within the urinary microbiota and finds that the abundance of prophage exceeds that of the bacteria, and the observed variation between the phage populations of women with and without overactive bladder symptoms suggests that phages may contribute to urinary health.
Abstract: Bacterial viruses (bacteriophages) play a significant role in microbial community dynamics Within the human gastrointestinal tract, for instance, associations among bacteriophages (phages), microbiota stability, and human health have been discovered In contrast to the gastrointestinal tract, the phages associated with the urinary microbiota are largely unknown Preliminary metagenomic surveys of the urinary virome indicate a rich diversity of novel lytic phage sequences at an abundance far outnumbering that of eukaryotic viruses These surveys, however, exclude the lysogenic phages residing within the bacteria of the bladder To characterize this phage population, we examined 181 genomes representative of the phylogenetic diversity of bacterial species within the female urinary microbiota and found 457 phage sequences, 226 of which were predicted with high confidence Phages were prevalent within the bladder bacteria: 86% of the genomes examined contained at least one phage sequence Most of these phages are novel, exhibiting no discernible sequence homology to sequences in public data repositories The presence of phages with substantial sequence similarity within the microbiota of different women supports the existence of a core community of phages within the bladder Furthermore, the observed variation between the phage populations of women with and without overactive bladder symptoms suggests that phages may contribute to urinary health To complement our bioinformatic analyses, viable phages were cultivated from the bacterial isolates for characterization; a novel coliphage was isolated, which is obligately lytic in the laboratory strain Escherichia coli C Sequencing of bacterial genomes facilitates a comprehensive cataloguing of the urinary virome and reveals phage-host interactionsIMPORTANCE Bacteriophages are abundant within the human body However, while some niches have been well surveyed, the phage population within the urinary microbiome is largely unknown Our study is the first survey of the lysogenic phage population within the urinary microbiota Most notably, the abundance of prophage exceeds that of the bacteria Furthermore, many of the prophage sequences identified exhibited no recognizable sequence homology to sequences in data repositories This suggests a rich diversity of uncharacterized phage species present in the bladder Additionally, we observed a variation in the abundances of phages between bacteria isolated from asymptomatic "healthy" individuals and those with urinary symptoms, thus suggesting that, like phages within the gut, phages within the bladder may contribute to urinary health

70 citations


Journal ArticleDOI
TL;DR: Approximate levels of antibiotic resistance in A. baumannii isolates are unlikely to be reached solely due to the overproduction of RND efflux pumps, and the impact of efflux pump overproduction on antibiotic susceptibility is significantly lower than the previously reported for clinical isolates.
Abstract: Antibiotic-resistant Acinetobacter baumannii causes infections that are extremely difficult to treat. A significant role in these resistance profiles is attributed to multidrug efflux pumps, especially those belonging to the resistance-nodulation-cell division (RND) superfamily of transporters. In this study, we analyzed functions and properties of RND efflux pumps in A. baumannii ATCC 17978. This strain is susceptible to antibiotics and does not contain mutations that are commonly selected upon exposure to high concentrations of antibiotics. We constructed derivatives of ATCC 17978 lacking chromosomally encoded RND pumps and complemented these strains by the plasmid-borne genes. We analyzed the substrate selectivities and efficiencies of the individual pumps in the context of native outer membranes and their hyperporinated variants. Our results show that inactivation of AdeIJK provides the strongest potentiation of antibiotic activities, whereas inactivation of AdeFGH triggers the overexpression of AdeAB. The plasmid-borne overproduction complements the hypersusceptible phenotypes of the efflux deletion mutants to the levels of the parental ATCC 17978. Only a few antibiotics strongly benefitted from the overproduction of efflux pumps and antibacterial activities of some of those depended on the synergistic interaction with the low permeability barrier of the outer membrane. Either overproduction or inactivation of efflux pumps change dramatically the lipidome of ATCC 17978. We conclude that efflux pumps of A. baumannii are tightly integrated into physiology of this bacterium and that clinical levels of antibiotic resistance in A. baumannii isolates are unlikely to be reached solely due to the overproduction of RND efflux pumps. IMPORTANCE RND-type efflux pumps are important contributors in development of clinical antibiotic resistance in A. baumannii. However, their specific roles and the extent of contribution to antibiotic resistance remain unclear. We analyzed antibacterial activities of antibiotics in strains with different permeability barriers and found that the role of active efflux in antibiotic resistance of A. baumannii is limited to a few select antibiotics. Our results further show that the impact of efflux pump overproduction on antibiotic susceptibility is significantly lower than the previously reported for clinical isolates. Additional mechanisms of resistance, in particular those that improve the permeability barriers of bacterial cells and act synergistically with active efflux pumps are likely involved in antibiotic resistance of clinical A. baumannii isolates.

64 citations


Journal ArticleDOI
TL;DR: The recognition of different morphotypes of NTM has been established and characterized since the 1950s, but the mechanisms that underlie colony phenotype change and subsequent differences in pathogenicity are just beginning to be explored.
Abstract: Nontuberculous mycobacteria (NTM) include species that colonize human epithelia, as well as species that are ubiquitous in soil and aquatic environments. NTM that primarily inhabit soil and aquatic environments include the Mycobacterium avium complex (MAC) (M. avium and Mycobacterium intracellulare) and the Mycobacterium abscessus complex (MABSC) (M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii) and can be free living, biofilm associated, or amoeba associated. Although NTM are rarely pathogenic in immunocompetent individuals, individuals who are immunocompromised, due to either an inherited or acquired immunodeficiency, are highly susceptible to NTM infection (NTMI). Several characteristics, such as biofilm formation and the ability of select NTM species to form distinct colony morphotypes, all may play a role in pathogenesis that is not observed in the related, well-characterized pathogen Mycobacterium tuberculosis. Different morphotypes of NTM have been recognized and characterized since the 1950s, but the mechanisms that underlie colony phenotype change and subsequent differences in pathogenicity are just beginning to be explored. Advances in genomic analysis have led to progress in identifying genes important to the pathogenesis and persistence of MAC disease as well as in illuminating genetic aspects of different colony morphotypes. Here we review recent literature regarding NTM ecology and transmission, as well as the factors which regulate colony morphotype and pathogenicity.

Journal ArticleDOI
TL;DR: This work shows that the marine pathogen Vibrio harveyi packages one of its QS molecules, the long-chain ketone CAI-1, into outer membrane vesicles (OMVs), which helps facilitate their distribution between bacteria that live in aqueous environments.
Abstract: Many bacteria use extracellular signaling molecules to coordinate group behavior, a process referred to as quorum sensing (QS). However, some QS molecules are hydrophobic in character and are probably unable to diffuse across the bacterial cell envelope. How these molecules are disseminated between bacterial cells within a population is not yet fully understood. Here, we show that the marine pathogen Vibrio harveyi packages the hydrophobic QS molecule CAI-1, a long-chain amino ketone, into outer membrane vesicles. Electron micrographs indicate that outer membrane vesicles of variable size are predominantly produced and released into the surroundings during the stationary phase of V. harveyi, which correlates with the timing of CAI-1-dependent signaling. The large vesicles (diameter, <55 nm) can trigger a QS phenotype in CAI-1-nonproducing V. harveyi and Vibrio cholerae cells. Packaging of CAI-1 into outer membrane vesicles might stabilize the molecule in aqueous environments and facilitate its distribution over distances. IMPORTANCE Formation of membrane vesicles is ubiquitous among bacteria. These vesicles are involved in protein and DNA transfer and offer new approaches for vaccination. Gram-negative bacteria use hydrophobic signaling molecules, among others, for cell-cell communication;however, due to their hydrophobic character, it is unclear how these molecules are disseminated between bacterial cells. Here, we show that the marine pathogen Vibrio harveyi packages one of its QS molecules, the long-chain ketone CAI-1, into outer membrane vesicles (OMVs). Isolated CAI-1-containing vesicles trigger a QS phenotype in CAI-1 nonproducing V. harveyi and also in Vibrio cholerae cells. Packaging of CAI-1 into OMVs not only solubilizes, stabilizes, and concentrates this class of molecules, but facilitate their distribution between bacteria that live in aqueous environments.

Journal ArticleDOI
TL;DR: The identification of carB, which codes for the large subunit of carbamoyl-phosphate synthetase, as a persister gene that contributes to multidrug tolerance in P. aeruginosa supports the hypothesis that a drop in intracellular ATP is a general mechanism of persister formation in bacteria.
Abstract: Persisters represent a small subpopulation of cells within a bacterial culture that are tolerant to killing by antibiotics. Persisters have been linked to recalcitrant infections caused by numerous bacterial pathogens, including Pseudomonas aeruginosa A classic example is the incurable infection of the airways for patients with cystic fibrosis. The genetic mediators of persister formation for P. aeruginosa are poorly understood. We generated a high-density transposon insertion library of P. aeruginosa PAO1 and determined the relative frequency of each insertion following fluoroquinolone treatment using transposon sequencing (Tn-seq). Of the 4,411 disrupted genes included in the screen, 137 had a ≥10-fold impact on survival. The gene disruption that resulted in the lowest survival rate was disruption of carB, which codes for the large subunit of carbamoyl phosphate synthetase (CPSase). CPSase is a metabolic enzyme that is involved in pyrimidine and arginine synthesis. Disruption of carB resulted in survival rates that were reduced by up to 2,500-fold following antibiotic treatment, and this phenotype was abolished by the addition of uracil, highlighting the importance of de novo pyrimidine biosynthesis for persister formation. Disruption of carB resulted in intracellular ATP accumulation, and lowering ATP levels using arsenate restored the antibiotic tolerance profile of the mutant to levels similar to those seen with the wild type. A decrease in ATP would lead to reduced antibiotic target activity and increased survival.IMPORTANCE Antibiotic treatment of P. aeruginosa residing in the lung of cystic fibrosis patients is ineffective. Treatment failure is attributed in part to antibiotic-tolerant phenotypic variants known as persister cells. Understanding how these cells emerge will likely inform future therapeutic strategies. In the current study, we identified carB, which codes for the large subunit of carbamoyl-phosphate synthetase, as a persister gene that contributes to multidrug tolerance in P. aeruginosa Disruption of carB resulted in a metabolic perturbation that increased cellular ATP and reduced persister formation. Conversely, lowering ATP in the mutant restored antibiotic tolerance. Our data support the hypothesis that a drop in intracellular ATP is a general mechanism of persister formation in bacteria.

Journal ArticleDOI
TL;DR: The abundance of oxidants and reductants must be balanced for an organism to thrive and bacteria have evolved methods to mitigate their deleterious consequences through the expression of detoxification enzymes, antioxidants, and systems to repair or degrade damaged proteins and DNA.
Abstract: The abundance of oxidants and reductants must be balanced for an organism to thrive. Bacteria have evolved methods to prevent redox imbalances and to mitigate their deleterious consequences through the expression of detoxification enzymes, antioxidants, and systems to repair or degrade damaged proteins and DNA. Regulating these processes in response to redox changes requires sophisticated surveillance strategies ranging from metal chelation to direct sensing of toxic reactive oxygen species. In the case of bacterial pathogens, stress that threatens to disrupt redox homeostasis can derive from endogenous sources (produced by the bacteria) or exogenous sources (produced by the host). This minireview summarizes the sources of redox stress encountered during infection, the mechanisms by which bacterial pathogens diminish the damaging effects of redox stress, and the clever ways some organisms have evolved to thrive in the face of redox challenges during infection.

Journal ArticleDOI
TL;DR: It is found that many morons confer selective advantages for the bacterial host, some of which correlate with increased bacterial virulence.
Abstract: The viruses that infect bacteria, known as phages, play a critical role in controlling bacterial populations in many diverse environments, including the human body. This control stems not only from phages killing bacteria but also from the formation of lysogens. In this state, the phage replication cycle is suppressed, and the phage genome is maintained in the bacterial cell in a form known as a prophage. Prophages often carry genes that benefit the host bacterial cell, since increasing the survival of the host cell by extension also increases the fitness of the prophage. These highly diverse and beneficial phage genes, which are not required for the life cycle of the phage itself, have been referred to as "morons," as their presence adds "more on" the phage genome in which they are found. While individual phage morons have been shown to contribute to bacterial virulence by a number of different mechanisms, there have been no systematic investigations of their activities. Using a library of phages that infect two different clinical isolates of P. aeruginosa, PAO1 and PA14, we compared the phenotypes imparted by the expression of individual phage morons. We identified morons that inhibit twitching and swimming motilities and observed an inhibition of the production of virulence factors such as rhamnolipids and elastase. This study demonstrates the scope of phage-mediated phenotypic changes and provides a framework for future studies of phage morons.IMPORTANCE Environmental and clinical isolates of the bacterium Pseudomonas aeruginosa frequently contain viruses known as prophages. These prophages can alter the virulence of their bacterial hosts through the expression of nonessential genes known as "morons." In this study, we identified morons in a group of Pseudomonas aeruginosa phages and characterized the effects of their expression on bacterial behaviors. We found that many morons confer selective advantages for the bacterial host, some of which correlate with increased bacterial virulence. This work highlights the symbiotic relationship between bacteria and prophages and illustrates how phage morons can help bacteria adapt to different selective pressures and contribute to human diseases.

Journal ArticleDOI
TL;DR: The results of this study provide a comprehensive view of the DNA methylation pattern in B. burgdorferi, and the accompanying gene expression profiles add to the emerging body of research on RM systems and gene regulation in bacteria.
Abstract: Prokaryote restriction modification (RM) systems serve to protect bacteria from potentially detrimental foreign DNA. Recent evidence suggests that DNA methylation by the methyltransferase (MTase) components of RM systems can also have effects on transcriptome profiles. The type strain of the causative agent of Lyme disease, Borrelia burgdorferi B31, possesses two RM systems with N6-methyladenosine (m6A) MTase activity, which are encoded by the bbe02 gene located on linear plasmid lp25 and bbq67 on lp56. The specific recognition and/or methylation sequences had not been identified for either of these B. burgdorferi MTases, and it was not previously known whether these RM systems influence transcript levels. In the current study, single-molecule real-time sequencing was utilized to map genome-wide m6A sites and to identify consensus modified motifs in wild-type B. burgdorferi as well as MTase mutants lacking either the bbe02 gene alone or both bbe02 and bbq67 genes. Four novel conserved m6A motifs were identified and were fully attributable to the presence of specific MTases. Whole-genome transcriptome changes were observed in conjunction with the loss of MTase enzymes, indicating that DNA methylation by the RM systems has effects on gene expression. Genes with altered transcription in MTase mutants include those involved in vertebrate host colonization (e.g., rpoS regulon) and acquisition by/transmission from the tick vector (e.g., rrp1 and pdeB). The results of this study provide a comprehensive view of the DNA methylation pattern in B. burgdorferi, and the accompanying gene expression profiles add to the emerging body of research on RM systems and gene regulation in bacteria.IMPORTANCE Lyme disease is the most prevalent vector-borne disease in North America and is classified by the Centers for Disease Control and Prevention (CDC) as an emerging infectious disease with an expanding geographical area of occurrence. Previous studies have shown that the causative bacterium, Borrelia burgdorferi, methylates its genome using restriction modification systems that enable the distinction from foreign DNA. Although much research has focused on the regulation of gene expression in B. burgdorferi, the effect of DNA methylation on gene regulation has not been evaluated. The current study characterizes the patterns of DNA methylation by restriction modification systems in B. burgdorferi and evaluates the resulting effects on gene regulation in this important pathogen.

Journal ArticleDOI
TL;DR: Heterologous expression of the enzyme Ddl (dipeptide ligase)—an essential enzyme involved in peptidoglycan synthesis—increases sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a native d-Ala-d- Ala dipeptidase.
Abstract: The peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poorly to peptidoglycan ending in d-alanyl-d-lactate and binds with high affinity to peptidoglycan ending in d-alanyl-d-alanine (d-Ala-d-Ala), which results in vancomycin resistance and sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is dipeptide ligase (Ddl). A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a native d-Ala-d-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated that it functions as a counterselection marker (CSM) in lactobacilli; vancomycin selection allows only those cells to grow in which the suicide vector has been lost. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately half the time required by conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance and consequently indicated the broad applicability of the use of Ddl as a counterselection marker in the genus Lactobacillus Finally, our system represents the first "plug and play" counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium.IMPORTANCE The genus Lactobacillus contains more than 200 species, many of which are exploited in the food and biotechnology industries and in medicine. Prediction of intrinsic vancomycin resistance has thus far been limited to selected Lactobacillus species. Here, we show that heterologous expression of the enzyme Ddl (dipeptide ligase)-an essential enzyme involved in peptidoglycan synthesis-increases sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we showed that Ddl is a phylogenetic marker that can be used to predict vancomycin resistance in Lactobacillus; 81% of Lactobacillus species are intrinsically resistant to vancomycin, which makes our tool broadly applicable.

Journal ArticleDOI
TL;DR: This review defines unique aspects of the germination pathways of C. difficile and compares them to those of two other well-studied organisms, Bacillus anthracis and Clostridium perfringens, and discusses the major discrepancies between current models of germination and areas of ongoing investigation.
Abstract: Germination of Clostridium difficile spores is a crucial early requirement for colonization of the gastrointestinal tract. Likewise, C. difficile cannot cause disease pathologies unless its spores germinate into metabolically active, toxin-producing cells. Recent advances in our understanding of C. difficile spore germination mechanisms indicate that this process is both complex and unique. This review defines unique aspects of the germination pathways of C. difficile and compares them to those of two other well-studied organisms, Bacillus anthracis and Clostridium perfringensC. difficile germination is unique, as C. difficile does not contain any orthologs of the traditional GerA-type germinant receptor complexes and is the only known sporeformer to require bile salts in order to germinate. While recent advances describing C. difficile germination mechanisms have been made on several fronts, major gaps in our understanding of C. difficile germination signaling remain. This review provides an updated, in-depth summary of advances in understanding of C. difficile germination and potential avenues for the development of therapeutics, and discusses the major discrepancies between current models of germination and areas of ongoing investigation.

Journal ArticleDOI
TL;DR: A specific biofilm-inducing signal that coordinately controls two polysaccharides, identifies a new regulator, and clarifies the regulatory control over biofilm formation by V. fischeri cells is identified.
Abstract: Vibrio fischeri uses biofilm formation to promote symbiotic colonization of its squid host, Euprymna scolopes Control over biofilm formation is exerted at the level of transcription of the symbiosis polysaccharide (syp) locus by a complex set of two-component regulators. Biofilm formation can be induced by overproduction of the sensor kinase RscS, which requires the activities of the hybrid sensor kinase SypF and the response regulator SypG and is negatively regulated by the sensor kinase BinK. Here, we identify calcium as a signal that promotes biofilm formation by biofilm-competent strains under conditions in which biofilms are not typically observed (growth with shaking). This was true for RscS-overproducing cells as well as for strains in which only the negative regulator binK was deleted. The latter results provided, for the first time, an opportunity to induce and evaluate biofilm formation without regulator overexpression. Using these conditions, we determined that calcium induces both syp-dependent and bacterial cellulose synthesis (bcs)-dependent biofilms at the level of transcription of these loci. The calcium-induced biofilms were dependent on SypF, but SypF's Hpt domain was sufficient for biofilm formation. These data suggested the involvement of another sensor kinase(s) and led to the discovery that both RscS and a previously uncharacterized sensor kinase, HahK, functioned in this pathway. Together, the data presented here reveal both a new signal and biofilm phenotype produced by V. fischeri cells, the coordinate production of two polysaccharides involved in distinct biofilm behaviors, and a new regulator that contributes to control over these processes.IMPORTANCE Biofilms, or communities of surface-attached microorganisms adherent via a matrix that typically includes polysaccharides, are highly resistant to environmental stresses and are thus problematic in the clinic and important to study. Vibrio fischeri forms biofilms to colonize its symbiotic host, making this organism useful for studying biofilms. Biofilm formation depends on the syp polysaccharide locus and its regulators. Here, we identify a signal, calcium, that induces both SYP-PS and cellulose-dependent biofilms. We also identify a new syp regulator, the sensor kinase HahK, and discover a mutant phenotype for the sensor kinase RscS. This work thus reveals a specific biofilm-inducing signal that coordinately controls two polysaccharides, identifies a new regulator, and clarifies the regulatory control over biofilm formation by V. fischeri.

Journal ArticleDOI
TL;DR: This work shows that RpoN plays critical and global roles in the regulation of bacterial pathogenicity and fitness and the functional characterization of RPON-mediated regulation will improve the current understanding of the regulatory network of quorum sensing and virulence in P. aeruginosa and other bacteria.
Abstract: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen of humans, particularly those with cystic fibrosis. As a global regulator, RpoN controls a group of virulence-related factors and quorum-sensing (QS) genes in P. aeruginosa To gain further insights into the direct targets of RpoN in vivo, the present study focused on identifying the direct targets of RpoN regulation in QS and the type VI secretion system (T6SS). We performed chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) that identified 1,068 binding sites of RpoN, mostly including metabolic genes, a group of genes in QS (lasI, rhlI, and pqsR) and the T6SS (hcpA and hcpB). The direct targets of RpoN have been verified by electrophoretic mobility shifts assays (EMSA), lux reporter assay, reverse transcription-quantitative PCR, and phenotypic detection. The ΔrpoN::Tc mutant resulted in the reduced production of pyocyanin, motility, and proteolytic activity. However, the production of rhamnolipids and biofilm formation were higher in the ΔrpoN::Tc mutant than in the wild type. In summary, the results indicated that RpoN had direct and profound effects on QS and the T6SS.IMPORTANCE As a global regulator, RpoN controls a wide range of biological pathways, including virulence in P. aeruginosa PAO1. This work shows that RpoN plays critical and global roles in the regulation of bacterial pathogenicity and fitness. ChIP-seq provided a useful database to characterize additional functions and targets of RpoN in the future. The functional characterization of RpoN-mediated regulation will improve the current understanding of the regulatory network of quorum sensing and virulence in P. aeruginosa and other bacteria.

Journal ArticleDOI
TL;DR: Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RSmF binding activity in vivo and in vitro are more stringent than those for RSMA.
Abstract: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA. IMPORTANCE CsrA homologs are present in many bacteria. The opportunistic pathogen Pseudomonas aeruginosa uses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activity in vivo. Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activity in vivo and in vitro are more stringent than those for RsmA.

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TL;DR: An overview of the major findings for the Gram-negative inner membrane flippase Wzx is provided, with a particular focus on substrate preference.
Abstract: Translocation of lipid-linked oligosaccharides is a common theme across prokaryotes and eukaryotes. For bacteria, such activity is used in cell wall construction, polysaccharide synthesis, and the relatively recently discovered protein glycosylation. To the best of our knowledge, the Gram-negative inner membrane flippase Wzx was the first protein identified as being involved in oligosaccharide translocation, and yet we still have only a limited understanding of this protein after 3 decades of research. At present, Wzx is known to be a multitransmembrane protein with enormous sequence diversity that flips oligosaccharide substrates with varied degrees of preference. In this review, we provide an overview of the major findings for this protein, with a particular focus on substrate preference.

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TL;DR: This work establishes that S. aureus is capable of utilizing the fatty acids present in low-density lipoproteins to bypass both chemical and genetic inhibition of fatty acid synthesis, and suggests that human lipoprotein particles are a viable source of exogenous fatty acids for S.Aureus during infection.
Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) is a threat to global health. Consequently, much effort has focused on the development of new antimicrobials that target novel aspects of S. aureus physiology. Fatty acids are required to maintain cell viability, and bacteria synthesize fatty acids using the type II fatty acid synthesis (FASII) pathway. FASII is significantly different from human fatty acid synthesis, underscoring the therapeutic potential of inhibiting this pathway. However, many Gram-positive pathogens incorporate exogenous fatty acids, bypassing FASII inhibition and leaving the clinical potential of FASII inhibitors uncertain. Importantly, the source(s) of fatty acids available to pathogens within the host environment remains unclear. Fatty acids are transported throughout the body by lipoprotein particles in the form of triglycerides and esterified cholesterol. Thus, lipoproteins, such as low-density lipoprotein (LDL), represent a potentially rich source of exogenous fatty acids for S. aureus during infection. We sought to test the ability of LDLs to serve as a fatty acid source for S. aureus and show that cells cultured in the presence of human LDLs demonstrate increased tolerance to the FASII inhibitor triclosan. Using mass spectrometry, we observed that host-derived fatty acids present in the LDLs are incorporated into the staphylococcal membrane and that tolerance to triclosan is facilitated by the fatty acid kinase A, FakA, and Geh, a triacylglycerol lipase. Finally, we demonstrate that human LDLs support the growth of S. aureus fatty acid auxotrophs. Together, these results suggest that human lipoprotein particles are a viable source of exogenous fatty acids for S. aureus during infection.IMPORTANCE Inhibition of bacterial fatty acid synthesis is a promising approach to combating infections caused by S. aureus and other human pathogens. However, S. aureus incorporates exogenous fatty acids into its phospholipid bilayer. Therefore, the clinical utility of targeting bacterial fatty acid synthesis is debated. Moreover, the fatty acid reservoir(s) exploited by S. aureus is not well understood. Human low-density lipoprotein particles represent a particularly abundant in vivo source of fatty acids and are present in tissues that S. aureus colonizes. Herein, we establish that S. aureus is capable of utilizing the fatty acids present in low-density lipoproteins to bypass both chemical and genetic inhibition of fatty acid synthesis. These findings imply that S. aureus targets LDLs as a source of fatty acids during pathogenesis.

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TL;DR: This work identifies and characterize some of the proteins that regulate the mycobacterial cell wall and finds that some of these regulators appear to be functionally conserved with their structural homologs in evolutionarily distant species such as Escherichia coli, but other proteins have critical regulatory functions that may be unique to the actinomycetes.
Abstract: Septation in bacteria requires coordinated regulation of cell wall biosynthesis and hydrolysis enzymes so that new septal cross-wall can be appropriately constructed without compromising the integrity of the existing cell wall. Bacteria with different modes of growth and different types of cell wall require different regulators to mediate cell growth and division processes. Mycobacteria have both a cell wall structure and a mode of growth that are distinct from well-studied model organisms and use several different regulatory mechanisms. Here, using Mycobacterium smegmatis, we identify and characterize homologs of the conserved cell division regulators FtsL and FtsB, and show that they appear to function similarly to their homologs in Escherichia coli We identify a number of previously undescribed septally localized factors which could be involved in cell wall regulation. One of these, SepIVA, has a DivIVA domain, is required for mycobacterial septation, and is localized to the septum and the intracellular membrane domain. We propose that SepIVA is a regulator of cell wall precursor enzymes that contribute to construction of the septal cross-wall, similar to the putative elongation function of the other mycobacterial DivIVA homolog, Wag31.IMPORTANCE The enzymes that build bacterial cell walls are essential for cell survival but can cause cell lysis if misregulated; thus, their regulators are also essential. The number and nature of these regulators is likely to vary in bacteria that grow in different ways. The mycobacteria are a genus that have a cell wall whose composition and construction vary greatly from those of well-studied model organisms. In this work, we identify and characterize some of the proteins that regulate the mycobacterial cell wall. We find that some of these regulators appear to be functionally conserved with their structural homologs in evolutionarily distant species such as Escherichia coli, but other proteins have critical regulatory functions that may be unique to the actinomycetes.

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TL;DR: The connection between the human host and the potential ecological role of these virulent traits of V. cholerae are discussed and unraveling these connections will help to understand the emergence of this organism and other facultative bacterial pathogens.
Abstract: Vibrio cholerae is a natural inhabitant of aquatic ecosystems. Some strains of V. cholerae can colonize the human host and cause cholera, a profuse watery diarrhea. The major pathogenicity factors and virulence regulators of V. cholerae are either encoded in mobile genetic elements acquired in the environment (e.g. pathogenicity islands or lysogenic phages) or in the core genome. Several lines of evidence indicate that the emergence of numerous virulence traits of V. cholerae occurred in its natural environment due to biotic and abiotic pressures. Here, we discuss the connection between the human host and the potential ecological role of these virulent traits. Unraveling these connections will help us understand the emergence of this organism and other facultative bacterial pathogens.

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TL;DR: The Pseudomonas fluorescens genome encodes more than 50 proteins predicted to be involved in c-di-GMP signaling, and it is demonstrated that, tested across 188 nutrients, these enzymes and effectors appeared capable of impacting biofilm formation.
Abstract: The Pseudomonas fluorescens genome encodes more than 50 proteins predicted to be involved in c-di-GMP signaling. Here, we demonstrated that, tested across 188 nutrients, these enzymes and effectors appeared capable of impacting biofilm formation. Transcriptional analysis of network members across ∼50 nutrient conditions indicates that altered gene expression can explain a subset of but not all biofilm formation responses to the nutrients. Additional organization of the network is likely achieved through physical interaction, as determined via probing ∼2,000 interactions by bacterial two-hybrid assays. Our analysis revealed a multimodal regulatory strategy using combinations of ligand-mediated signals, protein-protein interaction, and/or transcriptional regulation to fine-tune c-di-GMP-mediated responses. These results create a profile of a large c-di-GMP network that is used to make important cellular decisions, opening the door to future model building and the ability to engineer this complex circuitry in other bacteria.IMPORTANCE Cyclic diguanylate (c-di-GMP) is a key signaling molecule regulating bacterial biofilm formation, and many microbes have up to dozens of proteins that make, break, or bind this dinucleotide. A major open issue in the field is how signaling specificity is conferred in the unpartitioned space of a bacterial cell. Here, we took a systems approach, using mutational analysis, transcriptional studies, and bacterial two-hybrid analysis to interrogate this network. We found that a majority of enzymes are capable of impacting biofilm formation in a context-dependent manner, and we revealed examples of two or more modes of regulation (i.e., transcriptional control with protein-protein interaction) being utilized to generate an observable impact on biofilm formation.

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TL;DR: By defining key components of the LTA three-component glycosylation pathway and uncovering stress-induced regulation by the alternative sigma factor SigB, the role of N-acetylglucosamine tailoring during adaptation to environmental stresses can now be elucidated.
Abstract: Lipoteichoic acid (LTA) in Staphylococcus aureus is a poly-glycerophosphate polymer anchored to the outer surface of the cell membrane. LTA has numerous roles in cell envelope physiology, including regulating cell autolysis, coordinating cell division, and adapting to environmental growth conditions. LTA is often further modified with substituents, including d-alanine and glycosyl groups, to alter cellular function. While the genetic determinants of d-alanylation have been largely defined, the route of LTA glycosylation and its role in cell envelope physiology have remained unknown, in part due to the low levels of basal LTA glycosylation in S. aureus We demonstrate here that S. aureus utilizes a membrane-associated three-component glycosylation system composed of an undecaprenol (Und) N-acetylglucosamine (GlcNAc) charging enzyme (CsbB; SAOUHSC_00713), a putative flippase to transport loaded substrate to the outside surface of the cell (GtcA; SAOUHSC_02722), and finally an LTA-specific glycosyltransferase that adds α-GlcNAc moieties to LTA (YfhO; SAOUHSC_01213). We demonstrate that this system is specific for LTA with no cross recognition of the structurally similar polyribitol phosphate containing wall teichoic acids. We show that while wild-type S. aureus LTA has only a trace of GlcNAcylated LTA under normal growth conditions, amounts are raised upon either overexpressing CsbB, reducing endogenous d-alanylation activity, expressing the cell envelope stress responsive alternative sigma factor SigB, or by exposure to environmental stress-inducing culture conditions, including growth media containing high levels of sodium chloride.IMPORTANCE The role of glycosylation in the structure and function of Staphylococcus aureus lipoteichoic acid (LTA) is largely unknown. By defining key components of the LTA three-component glycosylation pathway and uncovering stress-induced regulation by the alternative sigma factor SigB, the role of N-acetylglucosamine tailoring during adaptation to environmental stresses can now be elucidated. As the dlt and glycosylation pathways compete for the same sites on LTA and induction of glycosylation results in decreased d-alanylation, the interplay between the two modification systems holds implications for resistance to antibiotics and antimicrobial peptides.

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TL;DR: This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in the haloarchaeon Haloferax volcanii, demonstrating that common principles for the response to a major cellular stress exist across the 3 domains of life while uncovering pathways that might be specific to the Archaea.
Abstract: Haloarchaea in their natural environment are exposed to hypersalinity, intense solar radiation, and desiccation, all of which generate high levels of oxidative stress. Previous work has shown that haloarchaea are an order of magnitude more resistant to oxidative stress than most mesophilic organisms. Despite this resistance, the pathways haloarchaea use to respond to oxidative stress damage are similar to those of nonresistant organisms, suggesting that regulatory processes might be key to their robustness. Recently, small regulatory noncoding RNAs (sRNAs) were discovered in Archaea under a variety of environmental conditions. We report here the transcriptional landscape and functional roles of sRNAs in the regulation of the oxidative stress response of the model haloarchaeon Haloferax volcanii Thousands of sRNAs, both intergenic and antisense, were discovered using strand-specific sRNA sequencing (sRNA-seq), comprising 25 to 30% of the total transcriptome under no-challenge and oxidative stress conditions, respectively. We identified hundreds of differentially expressed sRNAs in response to hydrogen peroxide-induced oxidative stress in H. volcanii The targets of a group of antisense sRNAs decreased in expression when these sRNAs were upregulated, suggesting that sRNAs are potentially playing a negative regulatory role on mRNA targets at the transcript level. Target enrichment of these antisense sRNAs included mRNAs involved in transposon mobility, chemotaxis signaling, peptidase activity, and transcription factors.IMPORTANCE While a substantial body of experimental work has been done to uncover the functions of small regulatory noncoding RNAs (sRNAs) in gene regulation in Bacteria and Eukarya, the functional roles of sRNAs in Archaea are still poorly understood. This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in the haloarchaeon Haloferax volcanii Our work demonstrates that common principles for the response to a major cellular stress exist across the 3 domains of life while uncovering pathways that might be specific to the Archaea This work also underscores the relevance of sRNAs in adaptation to extreme environmental conditions.

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TL;DR: It is shown that the PrrF1 and PrRF2 sRNAs promote AQ production by redundantly inhibiting translation of antR, which encodes a transcriptional activator of the anthranilate degradation genes.
Abstract: Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that requires iron for growth and virulence. Under low-iron conditions, P. aeruginosa transcribes two highly identical (95%) small regulatory RNAs (sRNAs), PrrF1 and PrrF2, which are required for virulence in acute murine lung infection models. The PrrF sRNAs promote the production of 2-akyl-4(1H)-quinolone metabolites (AQs) that mediate a range of biological activities, including quorum sensing and polymicrobial interactions. Here, we show that the PrrF1 and PrrF2 sRNAs promote AQ production by redundantly inhibiting translation of antR, which encodes a transcriptional activator of the anthranilate degradation genes. A combination of genetic and biophysical analyses was used to define the sequence requirements for PrrF regulation of antR, demonstrating that the PrrF sRNAs interact with the antR 5′ untranslated region (UTR) at sequences overlapping the translational start site of this mRNA. The P. aeruginosa Hfq protein interacted with UA-rich sequences in both PrrF sRNAs (Kd [dissociation constant] = 50 nM and 70 nM). Hfq bound with lower affinity to the antR mRNA (0.3 μM), and PrrF was able to bind to antR mRNA in the absence of Hfq. Nevertheless, Hfq increased the rate of PrrF annealing to the antR UTR by 10-fold. These studies provide a mechanistic description of how the PrrF1 and PrrF2 sRNAs mediate virulence traits, such as AQ production, in P. aeruginosa. IMPORTANCE The iron-responsive PrrF sRNAs play a central role in regulating P. aeruginosa iron homeostasis and pathogenesis, yet the molecular mechanisms by which PrrF regulates gene expression are largely unknown. In this study, we used genetic and biophysical analyses to define the interactions of the PrrF sRNAs with Hfq, an RNA annealer, and the antR mRNA, which has downstream effects on quorum sensing and virulence factor production. These studies provide a comprehensive mechanistic analysis of how the PrrF sRNAs regulate virulence trait production through a key mRNA target in P. aeruginosa.

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TL;DR: This model proposes a model of how staphylococcal protein A (SpA), a B cell superantigen, modifies host immune responses during colonization to support continued persistence of S. aureus in the nasopharynx and shows that this mechanism can be thwarted by vaccine-induced anti-SpA antibodies that promote IgG responses against staphlyococcal antigens and diminish colonization.
Abstract: Staphylococcus aureus persistently colonizes the nasopharynx in humans, which increases the risk for invasive diseases, such as skin infection and bacteremia. Nasal colonization triggers IgG responses against staphylococcal surface antigens; however, these antibodies cannot prevent subsequent colonization or disease. Here, we describe S. aureus WU1, a multilocus sequence type 88 (ST88) isolate that persistently colonizes the nasopharynx in mice. We report that staphylococcal protein A (SpA) is required for persistence of S. aureus WU1 in the nasopharynx. Compared to animals colonized by wild-type S. aureus, mice colonized with the Δspa variant mount increased IgG responses against staphylococcal colonization determinants. Immunization of mice with a nontoxigenic SpA variant, which cannot cross-link B cell receptors and divert antibody responses, elicits protein A-neutralizing antibodies that promote IgG responses against colonizing S. aureus and diminish pathogen persistence. IMPORTANCEStaphylococcus aureus persistently colonizes the nasopharynx in about one-third of the human population, thereby promoting community- and hospital-acquired infections. Antibiotics are currently used for decolonization of individuals at increased risk of infection. However, the efficacy of antibiotics is limited by recolonization and selection for drug-resistant strains. Here, we propose a model of how staphylococcal protein A (SpA), a B cell superantigen, modifies host immune responses during colonization to support continued persistence of S. aureus in the nasopharynx. We show that this mechanism can be thwarted by vaccine-induced anti-SpA antibodies that promote IgG responses against staphylococcal antigens and diminish colonization.