Showing papers in "Journal of Bacteriology in 2012"
TL;DR: This minireview summarizes the recent discoveries in bacterial phylogeny, uncultured clades that do not grow on standard media and discusses the potential future of the field.
Abstract: The bacteria that can be grown in the laboratory are only a small fraction of the total diversity that exists in nature. At all levels of bacterial phylogeny, uncultured clades that do not grow on standard media are playing critical roles in cycling carbon, nitrogen, and other elements, synthesizing novel natural products, and impacting the surrounding organisms and environment. While molecular techniques, such as metagenomic sequencing, can provide some information independent of our ability to culture these organisms, it is essentially impossible to learn new gene and pathway functions from pure sequence data. A true understanding of the physiology of these bacteria and their roles in ecology, host health, and natural product production requires their cultivation in the laboratory. Recent advances in growing these species include coculture with other bacteria, recreating the environment in the laboratory, and combining these approaches with microcultivation technology to increase throughput and access rare species. These studies are unraveling the molecular mechanisms of unculturability and are identifying growth factors that promote the growth of previously unculturable organisms. This minireview summarizes the recent discoveries in this area and discusses the potential future of the field.
915 citations
TL;DR: It is used inductively coupled plasma mass spectrometry (ICP-MS) to discover that iron, calcium, and manganese are accumulated by S. Typhimurium during lag phase, while levels of cobalt, nickel, and sodium showed distinct growth-phase-specific patterns.
Abstract: Lag phase represents the earliest and most poorly understood stage of the bacterial growth cycle. We developed a reproducible experimental system and conducted functional genomic and physiological analyses of a 2-h lag phase in Salmonella enterica serovar Typhimurium. Adaptation began within 4 min of inoculation into fresh LB medium with the transient expression of genes involved in phosphate uptake. The main lag-phase transcriptional program initiated at 20 min with the upregulation of 945 genes encoding processes such as transcription, translation, iron-sulfur protein assembly, nucleotide metabolism, LPS biosynthesis, and aerobic respiration. ChIP-chip revealed that RNA polymerase was not "poised" upstream of the bacterial genes that are rapidly induced at the beginning of lag phase, suggesting a mechanism that involves de novo partitioning of RNA polymerase to transcribe 522 bacterial genes within 4 min of leaving stationary phase. We used inductively coupled plasma mass spectrometry (ICP-MS) to discover that iron, calcium, and manganese are accumulated by S. Typhimurium during lag phase, while levels of cobalt, nickel, and sodium showed distinct growth-phase-specific patterns. The high concentration of iron during lag phase was associated with transient sensitivity to oxidative stress. The study of lag phase promises to identify the physiological and regulatory processes responsible for adaptation to new environments.
523 citations
TL;DR: This review is aimed at highlighting the common themes that have emerged in recent research regarding the key components, signals, and cues that aid in the transition and those involved in establishing a more permanent surface association during initial attachment.
Abstract: The formation of bacterial biofilms is initiated by cells transitioning from the free-swimming mode of growth to a surface. This review is aimed at highlighting the common themes that have emerged in recent research regarding the key components, signals, and cues that aid in the transition and those involved in establishing a more permanent surface association during initial attachment.
319 citations
TL;DR: The results support a mechanism for daptomycin with a primary effect on cell membranes that in turn redirects the localization of proteins involved in cell division and cell wall synthesis, causing dramatic cell wall and membrane defects, which may ultimately lead to a breach in the cell membrane and cell death.
Abstract: Daptomycin is a lipopeptide antibiotic used clinically for the treatment of certain types of Gram-positive infections, including those caused by methicillin-resistant Staphylococcus aureus (MRSA). Details of the mechanism of action of daptomycin continue to be elucidated, particularly the question of whether daptomycin acts on the cell membrane, the cell wall, or both. Here, we use fluorescence microscopy to directly visualize the interaction of daptomycin with the model Gram-positive bacterium Bacillus subtilis. We show that the first observable cellular effects are the formation of membrane distortions (patches of membrane) that precede cell death by more than 30 min. Membrane patches are able to recruit the essential cell division protein DivIVA. Recruitment of DivIVA correlates with membrane defects and changes in cell morphology, suggesting a localized alteration in the activity of enzymes involved in cell wall synthesis that could account for previously described effects of daptomycin on cell wall morphology and septation. Membrane defects colocalize with fluorescently labeled daptomycin, DivIVA, and fluorescent reporters of peptidoglycan biogenesis (Bocillin FL and BODIPY FL-vancomycin), suggesting that daptomycin plays a direct role in these events. Our results support a mechanism for daptomycin with a primary effect on cell membranes that in turn redirects the localization of proteins involved in cell division and cell wall synthesis, causing dramatic cell wall and membrane defects, which may ultimately lead to a breach in the cell membrane and cell death. These results help resolve the longstanding questions regarding the mechanism of action of this important class of antibiotics.
276 citations
TL;DR: These phages are only the second identified group of naturally occurring phages demonstrated to be blocked for replication by a nonengineered CRISPR/Cas system, and the results provide the first evidence that the P. aeruginosa type I-F CRIS PR-Cas system can function in phage resistance.
Abstract: Here we report the isolation of 6 temperate bacteriophages (phages) that are prevented from replicating within the laboratory strain Pseudomonas aeruginosa PA14 by the endogenous CRISPR/Cas system of this microbe. These phages are only the second identified group of naturally occurring phages demonstrated to be blocked for replication by a nonengineered CRISPR/Cas system, and our results provide the first evidence that the P. aeruginosa type I-F CRISPR/Cas system can function in phage resistance. Previous studies have highlighted the importance of the protospacer adjacent motif (PAM) and a proximal 8-nucleotide seed sequence in mediating CRISPR/Cas-based immunity. Through engineering of a protospacer region of phage DMS3 to make it a target of resistance by the CRISPR/Cas system and screening for mutants that escape CRISPR/Cas-mediated resistance, we show that nucleotides within the PAM and seed sequence and across the non-seed-sequence regions are critical for the functioning of this CRISPR/Cas system. We also demonstrate that P. aeruginosa can acquire spacer content in response to lytic phage challenge, illustrating the adaptive nature of this CRISPR/Cas system. Finally, we demonstrate that the P. aeruginosa CRISPR/Cas system mediates a gradient of resistance to a phage based on the level of complementarity between CRISPR spacer RNA and phage protospacer target. This work introduces a new in vivo system to study CRISPR/Cas-mediated resistance and an additional set of tools for the elucidation of CRISPR/Cas function.
263 citations
TL;DR: The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state.
Abstract: Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptome responses of Pseudomonas aeruginosa growing in biofilms by using a microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNA levels for these housekeeping genes were low in cells at the bottom of the biofilms. Selective green fluorescent protein (GFP) labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNA levels for genes regulated by the hypoxia-induced regulator Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-term anoxia. Transcripts for ribosomal proteins were associated primarily with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (the rmf and PA4463 genes) at the bottom of the biofilms. The dormant biofilm cells of a P. aeruginosa Δrmf strain had decreased membrane integrity, as shown by propidium iodide staining. Using selective GFP labeling and cell sorting, we show that the dividing cells are more susceptible to killing by tobramycin and ciprofloxacin. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state.
223 citations
TL;DR: In this article, electron cryotomography was used to show that actin homolog MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native actin B expressed to the same levels does not.
Abstract: Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not. In contrast, mCherry fused to an internal loop (MreB-RFPSW) does not induce helices. The helices are therefore an artifact of the placement of the fluorescent protein tag. YFP-MreB helices were also clearly distinguishable from the punctate, “patchy” localization patterns of MreB-RFPSW, even by standard light microscopy. The many interpretations in the literature of such punctate patterns as helices should therefore be reconsidered.
198 citations
TL;DR: It is shown that outpatient sputum samples have significantly higher bacterial diversity than inpatients, but maintenance treatment with tobramycin did not impact overall diversity.
Abstract: Diverse microbial communities chronically colonize the lungs of cystic fibrosis patients. Pyrosequencing of amplicons for hypervariable regions in the 16S rRNA gene generated taxonomic profiles of bacterial communities for sputum genomic DNA samples from 22 patients during a state of clinical stability (outpatients) and 13 patients during acute exacerbation (inpatients). We employed quantitative PCR (qPCR) to confirm the detection of Pseudomonas aeruginosa and Streptococcus by the pyrosequencing data and human oral microbe identification microarray (HOMIM) analysis to determine the species of the streptococci identified by pyrosequencing. We show that outpatient sputum samples have significantly higher bacterial diversity than inpatients, but maintenance treatment with tobramycin did not impact overall diversity. Contrary to the current dogma in the field that Pseudomonas aeruginosa is the dominant organism in the majority of cystic fibrosis patients, Pseudomonas constituted the predominant genera in only half the patient samples analyzed and reported here. The increased fractional representation of Streptococcus in the outpatient cohort relative to the inpatient cohort was the strongest predictor of clinically stable lung disease. The most prevalent streptococci included species typically associated with the oral cavity (Streptococcus salivarius and Streptococcus parasanguis) and the Streptococcus milleri group species. These species of Streptococcus may play an important role in increasing the diversity of the cystic fibrosis lung environment and promoting patient stability.
173 citations
TL;DR: These two mechanosensitive channel families are compared and contrast, their potential physiological roles are discussed, and some of the most relevant data that underlie the current models for their structure and function are reviewed.
Abstract: Single-celled organisms must survive exposure to environmental extremes. Perhaps one of the most variable and potentially life-threatening changes that can occur is that of a rapid and acute decrease in external osmolarity. This easily translates into several atmospheres of additional pressure that can build up within the cell. Without a protective mechanism against such pressures, the cell will lyse. Hence, most microbes appear to possess members of one or both families of bacterial mechanosensitive channels, MscS and MscL, which can act as biological emergency release valves that allow cytoplasmic solutes to be jettisoned rapidly from the cell. While this is undoubtedly a function of these proteins, the discovery of the presence of MscS homologues in plant organelles and MscL in fungus and mycoplasma genomes may complicate this simplistic interpretation of the physiology underlying these proteins. Here we compare and contrast these two mechanosensitive channel families, discuss their potential physiological roles, and review some of the most relevant data that underlie the current models for their structure and function.
171 citations
TL;DR: The effect of c-di-GMP on the motility of a gram-positive bacterium and on aggregation of C. difficile cells is demonstrated, which may be relevant to the function of this signaling molecule during infection.
Abstract: Clostridium difficile-associated disease is increasing in incidence and is costly to treat. Our understanding of how this organism senses its entry into the host and adapts for growth in the large bowel is limited. The small-molecule second messenger cyclic diguanylate (c-di-GMP) has been extensively studied in Gram-negative bacteria and has been shown to modulate motility, biofilm formation, and other processes in response to environmental signals, yet little is known about the functions of this signaling molecule in Gram-positive bacteria or in C. difficile specifically. In the current study, we investigated the function of the second messenger c-di-GMP in C. difficile. To determine the role of c-di-GMP in C. difficile, we ectopically expressed genes encoding a diguanylate cyclase enzyme, which synthesizes c-di-GMP, or a phosphodiesterase enzyme, which degrades c-di-GMP. This strategy allowed us to artificially elevate or deplete intracellular c-di-GMP, respectively, and determine that c-di-GMP represses motility in C. difficile, consistent with previous studies in Gram-negative bacteria, in which c-di-GMP has a negative effect on myriad modes of bacterial motility. Elevated c-di-GMP levels also induced clumping of C. difficile cells, which may signify that C. difficile is capable of forming biofilms in the host. In addition, we directly quantified, for the first time, c-di-GMP production in a Gram-positive bacterium. This work demonstrates the effect of c-di-GMP on the motility of a Gram-positive bacterium and on aggregation of C. difficile, which may be relevant to the function of this signaling molecule during infection.
166 citations
TL;DR: The genome sequence of a strain of Klebsiella pneumoniae isolated from human sputum in 2011 in Shanghai, China is reported, which contains one chromosome, three multidrug resistance plasmids, including a carbapenemase producer, and three small Plasmids.
Abstract: Klebsiella pneumoniae is an important pathogen commonly associated with opportunistic infections. Here we report the genome sequence of a strain, HS11286, isolated from human sputum in 2011 in Shanghai, China. It contains one chromosome (5.3 Mb), three multidrug resistance plasmids (∼110 kb), including a carbapenemase producer, and three small plasmids (∼3 kb).
TL;DR: It is demonstrated that dispersion by Pseudomonas aeruginosa in response to sudden changes in nutrient concentrations resulted in increased phosphodiesterase activity and reduction of c-di-GMP levels compared to biofilm and planktonic cells, and proposed a role of DipA in enabling dispersion in P. aerug inosa biofilms.
Abstract: Although little is known regarding the mechanism of biofilm dispersion, it is becoming clear that this process coincides with alteration of cyclic di-GMP (c-di-GMP) levels. Here, we demonstrate that dispersion by Pseudomonas aeruginosa in response to sudden changes in nutrient concentrations resulted in increased phosphodiesterase activity and reduction of c-di-GMP levels compared to biofilm and planktonic cells. By screening mutants inactivated in genes encoding EAL domains for nutrient-induced dispersion, we identified in addition to the previously reported ΔrbdA mutant a second mutant, the ΔdipA strain (PA5017 [dispersion-induced phosphodiesterase A]), to be dispersion deficient in response to glutamate, nitric oxide, ammonium chloride, and mercury chloride. Using biochemical and in vivo studies, we show that DipA associates with the membrane and exhibits phosphodiesterase activity but no detectable diguanylate cyclase activity. Consistent with these data, a ΔdipA mutant exhibited reduced swarming motility, increased initial attachment, and polysaccharide production but only somewhat increased biofilm formation and c-di-GMP levels. DipA harbors an N-terminal GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain and two EAL motifs within or near the C-terminal EAL domain. Mutational analyses of the two EAL motifs of DipA suggest that both are important for the observed phosphodiesterase activity and dispersion, while the GAF domain modulated DipA function both in vivo and in vitro without being required for phosphodiesterase activity. Dispersion was found to require protein synthesis and resulted in increased dipA expression and reduction of c-di-GMP levels. We propose a role of DipA in enabling dispersion in P. aeruginosa biofilms.
TL;DR: OxyR, PerR, and OhrR as mentioned in this paper sense oxidants by reversible oxidation of specific cysteine residues, while PerR senses oxidants via the Fe-catalyzed oxidation of histidine residues.
Abstract: The ability to maintain intracellular concentrations of toxic reactive oxygen species (ROS) within safe limits is essential for all aerobic life forms. In bacteria, as well as other organisms, ROS are produced during the normal course of aerobic metabolism, necessitating the constitutive expression of ROS scavenging systems. However, bacteria can also experience transient high-level exposure to ROS derived either from external sources, such as the host defense response, or as a secondary effect of other seemingly unrelated environmental stresses. Consequently, transcriptional regulators have evolved to sense the levels of ROS and coordinate the appropriate oxidative stress response. Three well-studied examples of these are the peroxide responsive regulators OxyR, PerR, and OhrR. OxyR and PerR are sensors of primarily H(2)O(2), while OhrR senses organic peroxide (ROOH) and sodium hypochlorite (NaOCl). OxyR and OhrR sense oxidants by means of the reversible oxidation of specific cysteine residues. In contrast, PerR senses H(2)O(2) via the Fe-catalyzed oxidation of histidine residues. These transcription regulators also influence complex biological phenomena, such as biofilm formation, the evasion of host immune responses, and antibiotic resistance via the direct regulation of specific proteins.
TL;DR: In vivo assays demonstrated that all of the three GlnR binding boxes were required forglnR-mediated activation of amtB transcription under the nitrogen-limited condition, attributable to the PhoP/GlnR counter-regulatory function subjected to further experimental proof.
Abstract: The transcription of amtB in Streptomyces coelicolor has been proposed to be counter-regulated by GlnR (a global regulator for nitrogen metabolism) and PhoP (a global regulator for phosphate metabolism). However, the GlnR-protected region, which was deduced to be two 22-bp GlnR binding boxes (gTnAc-n6-GaAAc-n6-GtnAC-n6-GAAAc-n6, abbreviated as a1-b1 and a2-b2), was separated from the PhoP-protected region in the promoter of amtB, leaving the mechanism for this regulation undefined. In this study, another 22-bp GlnR binding box, which consisted of a3-site-n6-b3-site (a3-b3) overlapping with the PhoP-binding sequences, was identified in the promoter region of amtB by a DNase I footprinting assay. An electrophoretic mobility shift assay (EMSA) using purified recombinant GlnR and the synthetic amtB promoter fragments with the three GlnR binding boxes individually mutated demonstrated that every box was involved in GlnR binding in vitro. Further in vivo assays using the egfp reporter gene fused to various kinds of mutated promoter regions of amtB demonstrated that all of the three GlnR binding boxes were required for GlnR-mediated activation of amtB transcription under the nitrogen-limited condition. The results of EMSA using the amtB promoter with mixtures of recombinant His-tagged GlnR and Trx-His-S-tagged PhoP inferred that PhoP might compete against GlnR from binding at the a3-b3 site, attributable to the PhoP/GlnR counter-regulatory function subjected to further experimental proof.
TL;DR: It is proposed that polyamines are produced under Mg(2+)-limiting conditions as an organic polycation to bind lipopolysaccharide (LPS) and to stabilize and protect the outer membrane against antibiotic and oxidative damage.
Abstract: Extracellular DNA acts as a cation chelator and induces the expression of antibiotic resistance genes regulated by Mg 2+ levels. Here we report the characterization of novel DNA-induced genes in Pseudomonas aeruginosa that are annotated as homologs of the spermidine synthesis genes speD ( PA4773 ) and speE ( PA4774 ). The addition of sublethal concentrations of DNA and membrane-damaging antibiotics induced expression of the genes PA4773 to PA4775 , as shown using transcriptional lux fusions and quantitative RT-PCR. Exogenous polyamine addition prevented DNA- and peptide-mediated gene induction. Mutation of PA4774 resulted in an increased outer membrane (OM) susceptibility phenotype upon polymyxin B, CP10A, and gentamicin treatment. When the membrane-localized fluorescent probe C 11 -BODIPY 581/591 was used as an indicator of peroxidation of membrane lipids, the PA4774 :: lux mutant demonstrated an increased susceptibility to oxidative membrane damage from H 2 O 2 treatment. Addition of exogenous polyamines protected the membranes of the PA4774 :: lux mutant from polymyxin B and H 2 O 2 treatment. Polyamines from the outer surface were isolated and shown to contain putrescine and spermidine by using high-performance liquid chromatography and mass spectrometry. The PA4774 :: lux mutant did not produce spermidine on the cell surface, but genetic complementation restored surface spermidine production as well as the antibiotic and oxidative stress resistance phenotypes of the membrane. We have identified new functions for spermidine on the cell surface and propose that polyamines are produced under Mg 2+ -limiting conditions as an organic polycation to bind lipopolysaccharide (LPS) and to stabilize and protect the outer membrane against antibiotic and oxidative damage.
TL;DR: Evidence is presented that conserved rickettsial genes associated with an intracellular lifestyle were acquired via MGEs, especially the RAGE, through a continuum of genomic invasions, making REIS one of the most plastic and repetitive bacterial genomes sequenced to date.
Abstract: We present the draft genome for the Rickettsia endosymbiont of Ixodes scapularis (REIS), a symbiont of the deer tick vector of Lyme disease in North America. Among Rickettsia species (Alphaproteobacteria: Rickettsiales), REIS has the largest genome sequenced to date (>2 Mb) and contains 2,309 genes across the chromosome and four plasmids (pREIS1 to pREIS4). The most remarkable finding within the REIS genome is the extraordinary proliferation of mobile genetic elements (MGEs), which contributes to a limited synteny with other Rickettsia genomes. In particular, an integrative conjugative element named RAGE (for Rickettsiales amplified genetic element), previously identified in scrub typhus rickettsiae (Orientia tsutsugamushi) genomes, is present on both the REIS chromosome and plasmids. Unlike the pseudogene-laden RAGEs of O. tsutsugamushi, REIS encodes nine conserved RAGEs that include F-like type IV secretion systems similar to that of the tra genes encoded in the Rickettsia bellii and R. massiliae genomes. An unparalleled abundance of encoded transposases (>650) relative to genome size, together with the RAGEs and other MGEs, comprise ∼35% of the total genome, making REIS one of the most plastic and repetitive bacterial genomes sequenced to date. We present evidence that conserved rickettsial genes associated with an intracellular lifestyle were acquired via MGEs, especially the RAGE, through a continuum of genomic invasions. Robust phylogeny estimation suggests REIS is ancestral to the virulent spotted fever group of rickettsiae. As REIS is not known to invade vertebrate cells and has no known pathogenic effects on I. scapularis, its genome sequence provides insight on the origin of mechanisms of rickettsial pathogenicity.
TL;DR: Gardnerella vaginalis appears to include four nonrecombining groups/clades of organisms with distinct gene pools and genomic properties, which may confer distinct ecological properties, and it may be appropriate to treat these four groups as separate species.
Abstract: Gardnerella vaginalis is associated with a spectrum of clinical conditions, suggesting high degrees of genetic heterogeneity among stains. Seventeen G. vaginalis isolates were subjected to a battery of comparative genomic analyses to determine their level of relatedness. For each measure, the degree of difference among the G. vaginalis strains was the highest observed among 23 pathogenic bacterial species for which at least eight genomes are available. Genome sizes ranged from 1.491 to 1.716 Mb; GC contents ranged from 41.18% to 43.40%; and the core genome, consisting of only 746 genes, makes up only 51.6% of each strain's genome on average and accounts for only 27% of the species supragenome. Neighbor-grouping analyses, using both distributed gene possession data and core gene allelic data, each identified two major sets of strains, each of which is composed of two groups. Each of the four groups has its own characteristic genome size, GC ratio, and greatly expanded core gene content, making the genomic diversity of each group within the range for other bacterial species. To test whether these 4 groups corresponded to genetically isolated clades, we inferred the phylogeny of each distributed gene that was present in at least two strains and absent in at least two strains; this analysis identified frequent homologous recombination within groups but not between groups or sets. G. vaginalis appears to include four nonrecombining groups/clades of organisms with distinct gene pools and genomic properties, which may confer distinct ecological properties. Consequently, it may be appropriate to treat these four groups as separate species.
TL;DR: It is shown here that the cariogenic organism Streptococcus mutans forms persister cells showing noninherited multidrug tolerance, and this is the first study reporting the induction of bacterial persistence using a quorum-sensing regulatory system.
Abstract: Within a given microbial population, a small subpopulation known as dormant persister cells exists. This persistence property ensures the survival of the population as a whole in the presence of lethal factors. Although persisters are highly important in antibiotic therapy, the mechanism for persistence is still not thoroughly understood. We show here that the cariogenic organism Streptococcus mutans forms persister cells showing noninherited multidrug tolerance. We demonstrated that the ectopic expression of the type II toxin-antitoxin systems, MazEF and RelBE, caused an increase in the number of persisters. In a search for additional persistence genes, an expression library was constructed, and several clones exhibiting a significant difference in persister formation after prolonged antibiotic treatment were selected. The candidate persister genes include genes involved in transcription/replication, sugar metabolism, cell wall synthesis, and energy metabolism, clearly pointing to redundant pathways for persister formation. We have previously reported that the S. mutans quorum-sensing peptide, CSP pheromone, was a stress-inducible alarmone capable of conveying sophisticated messages in the bacterial population. In this study, we demonstrate the involvement of the intraspecies quorum-sensing system during the formation of stress-induced multidrug-tolerant persisters. To the best of our knowledge, this is the first study reporting the induction of bacterial persistence using a quorum-sensing regulatory system.
TL;DR: In this paper, a screen of fatty acids as growth inhibitors of Staphylococcus aureus revealed structure-specific antibacterial activity, including oleate (18:1Δ9) and palmitoleate (16:1 Δ9).
Abstract: The skin represents an important barrier for pathogens and is known to produce fatty acids that are toxic toward gram-positive bacteria. A screen of fatty acids as growth inhibitors of Staphylococcus aureus revealed structure-specific antibacterial activity. Fatty acids like oleate (18:1Δ9) were nontoxic, whereas palmitoleate (16:1Δ9) was a potent growth inhibitor. Cells treated with 16:1Δ9 exhibited rapid membrane depolarization, the disruption of all major branches of macromolecular synthesis, and the release of solutes and low-molecular-weight proteins into the medium. Other cytotoxic lipids, such as glycerol ethers, sphingosine, and acyl-amines blocked growth by the same mechanisms. Nontoxic 18:1Δ9 was used for phospholipid synthesis, whereas toxic 16:1Δ9 was not and required elongation to 18:1Δ11 prior to incorporation. However, blocking fatty acid metabolism using inhibitors to prevent acyl-acyl carrier protein formation or glycerol-phosphate acyltransferase activity did not increase the toxicity of 18:1Δ9, indicating that inefficient metabolism did not play a determinant role in fatty acid toxicity. Nontoxic 18:1Δ9 was as toxic as 16:1Δ9 in a strain lacking wall teichoic acids and led to growth arrest and enhanced release of intracellular contents. Thus, wall teichoic acids contribute to the structure-specific antimicrobial effects of unsaturated fatty acids. The ability of poorly metabolized 16:1 isomers to penetrate the cell wall defenses is a weakness that has been exploited by the innate immune system to combat S. aureus.
TL;DR: It is reported here that the 2 NADPH molecules required for CO(2) reduction to acetic acid are generated by the reduction of 2 NADP(+) molecules with 1 NADH and 1 Fd(red)(2-) catalyzed by the electron-bifurcating NADH-dependent reduced ferredoxin:NADP(+) oxidoreductase (NfnAB).
Abstract: Moorella thermoacetica ferments glucose to three acetic acids. In the oxidative part of the fermentation, the hexose is converted to 2 acetic acids and 2 CO2 molecules with the formation of 2 NADH and 2 reduced ferredoxin (Fdred2−) molecules. In the reductive part, 2 CO2 molecules are reduced to acetic acid, consuming the 8 reducing equivalents generated in the oxidative part. An open question is how the two parts are electronically connected, since two of the four oxidoreductases involved in acetogenesis from CO2 are NADP specific rather than NAD specific. We report here that the 2 NADPH molecules required for CO2 reduction to acetic acid are generated by the reduction of 2 NADP+ molecules with 1 NADH and 1 Fdred2− catalyzed by the electron-bifurcating NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (NfnAB). The cytoplasmic iron-sulfur flavoprotein was heterologously produced in Escherichia coli, purified, and characterized. The purified enzyme was composed of 30-kDa (NfnA) and 50-kDa (NfnB) subunits in a 1-to-1 stoichiometry. NfnA harbors a [2Fe2S] cluster and flavin adenine dinucleotide (FAD), and NfnB harbors two [4Fe4S] clusters and FAD. M. thermoacetica contains a second electron-bifurcating enzyme. Cell extracts catalyzed the coupled reduction of NAD+ and Fd with 2 H2 molecules. The specific activity of this cytoplasmic enzyme was 3-fold higher in H2-CO2-grown cells than in glucose-grown cells. The function of this electron-bifurcating hydrogenase is not yet clear, since H2-CO2-grown cells additionally contain high specific activities of an NADP+-dependent hydrogenase that catalyzes the reduction of NADP+ with H2. This activity is hardly detectable in glucose-grown cells.
TL;DR: This work created a sequence-verified collection of 9,437 transposon mutants that provides genome coverage and includes two mutants for most genes and should help minimize missed or erroneous genotype-phenotype assignments in studies using the new library.
Abstract: Mutant hunts using comprehensive sequence-defined libraries make it possible to identify virtually all of the nonessential functions required for different bacterial processes. However, the success of such screening depends on the accuracy of mutant identification in the mutant library used. To provide a high-quality library for Pseudomonas aeruginosa PAO1, we created a sequence-verified collection of 9,437 transposon mutants that provides genome coverage and includes two mutants for most genes. Mutants were cherry-picked from a larger library, colony-purified, and resequenced both individually using Sanger sequencing and in a pool using Tn-seq. About 8% of the insertion assignments were corrected, and in the final library nearly 93% of the transposon locations were confirmed by at least one of the resequencing procedures. The extensive sequence verification and inclusion of more than one mutant for most genes should help minimize missed or erroneous genotype-phenotype assignments in studies using the new library.
TL;DR: A comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae revealed characteristics of horizontal gene transfer and it appears that variation in the distributed genome is a common strategy of C.diphtheria to establish differences in host-pathogen interactions.
Abstract: Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ωtox+ phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox+ corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.
TL;DR: It is proposed that the integration of QS with c-di-GMP allows bacteria to assimilate information about the local bacterial population density with other physicochemical environmental signals within the broader c- DiGMP signaling network.
Abstract: Bacteria sense and respond to environmental cues to control important developmental processes. Two widely conserved and important strategies that bacteria employ to sense changes in population density and local environmental conditions are quorum sensing (QS) and cyclic di-GMP (c-di-GMP) signaling, respectively. The importance of these pathways in controlling a broad variety of functions, including virulence, biofilm formation, and motility, has been recognized in many species. Recent research has shown that these pathways are intricately intertwined. Here we review the regulatory connections between QS and c-di-GMP signaling. We propose that the integration of QS with c-di-GMP allows bacteria to assimilate information about the local bacterial population density with other physicochemical environmental signals within the broader c-di-GMP signaling network.
TL;DR: To aid in the investigation of the Populus deltoides microbiome, draft genome sequences for 21 Pseudomonas strains and 19 other diverse bacteria isolated from Populus Deltoides roots were generated.
Abstract: To aid in the investigation of the Populus deltoides microbiome, we generated draft genome sequences for 21 Pseudomonas strains and 19 other diverse bacteria isolated from Populus deltoides roots. Genome sequences for isolates similar to Acidovorax, Bradyrhizobium, Brevibacillus, Caulobacter, Chryseobacterium, Flavobacterium, Herbaspirillum, Novosphingobium, Pantoea, Phyllobacterium, Polaromonas, Rhizobium, Sphingobium, and Variovorax were generated.
TL;DR: Five steps of microbial IMS are presented: culturing, matrix application, dehydration of the sample, data acquisition, and data analysis/interpretation, which address the common challenges encountered during sample preparation, matrix selection and application, and sample adherence to the MALDI target plate.
Abstract: Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry (IMS) applied directly to microbes on agar-based medium captures global information about microbial molecules, allowing for direct correlation of chemotypes to phenotypes. This tool was developed to investigate metabolic exchange factors of intraspecies, interspecies, and polymicrobial interactions. Based on our experience of the thousands of images we have generated in the laboratory, we present five steps of microbial IMS: culturing, matrix application, dehydration of the sample, data acquisition, and data analysis/interpretation. We also address the common challenges encountered during sample preparation, matrix selection and application, and sample adherence to the MALDI target plate. With the practical guidelines described herein, microbial IMS use can be extended to bio-based agricultural, biofuel, diagnostic, and therapeutic discovery applications.
TL;DR: The complete 3.0-Mb genome sequence of the multilocus sequence type 17 vancomycin-resistant Enterococcus faecium strain Aus0004, isolated from the bloodstream of a patient in Melbourne, Australia, in 1998, is reported.
Abstract: Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infections in health care facilities around the globe. In particular, infections caused by vancomycin-resistant Enterococcus faecium are becoming increasingly common. Comparative and functional genomic studies of E. faecium isolates have so far been limited owing to the lack of a fully assembled E. faecium genome sequence. Here we address this issue and report the complete 3.0-Mb genome sequence of the multilocus sequence type 17 vancomycin-resistant Enterococcus faecium strain Aus0004, isolated from the bloodstream of a patient in Melbourne, Australia, in 1998. The genome comprises a 2.9-Mb circular chromosome and three circular plasmids. The chromosome harbors putative E. faecium virulence factors such as enterococcal surface protein, hemolysin, and collagen-binding adhesin. Aus0004 has a very large accessory genome (38%) that includes three prophage and two genomic islands absent among 22 other E. faecium genomes. One of the prophage was present as inverted 50-kb repeats that appear to have facilitated a 683-kb chromosomal inversion across the replication terminus, resulting in a striking replichore imbalance. Other distinctive features include 76 insertion sequence elements and a single chromosomal copy of Tn1549 containing the vanB vancomycin resistance element. A complete E. faecium genome will be a useful resource to assist our understanding of this emerging nosocomial pathogen.
TL;DR: The draft genome sequence of "Candidatus Methanomethylophilus alvus" Mx1201, a methanogen present in the human gut, is reported, which contains genes needed for methylotrophic methanogenesis from methanol and tri-, di-, and monomethylamine.
Abstract: We report the draft genome sequence of "Candidatus Methanomethylophilus alvus" Mx1201, a methanogen present in the human gut. It was enriched from human feces under anaerobic conditions with methanol as the substrate. Its circular genome, of around 1.7 Mb, contains genes needed for methylotrophic methanogenesis from methanol and tri-, di-, and monomethylamine.
TL;DR: The genome of L. plantarum WCFS1, first sequenced in 2001, was resequenced using Solexa technology and identified 116 nucleotide corrections and improved function prediction for nearly 1,200 proteins, with a focus on metabolic functions and cell surface-associated proteins.
Abstract: There is growing interest in the beneficial effects of Lactobacillus plantarum on human health. The genome of L. plantarum WCFS1, first sequenced in 2001, was resequenced using Solexa technology. We identified 116 nucleotide corrections and improved function prediction for nearly 1,200 proteins, with a focus on metabolic functions and cell surface-associated proteins.
TL;DR: The identification of ZapD (YacF) as a member of the E. coli midcell division machinery is reported, indicating that ZapD is dispensable for division and therefore belongs to the growing group of FtsZ-associated proteins in E coli that aid in the overall fitness of the division process.
Abstract: The tubulin homolog FtsZ forms a polymeric membrane-associated ring structure (Z ring) at midcell that establishes the site of division and provides an essential framework for the localization of a multiprotein molecular machine that promotes division in Escherichia coli. A number of regulatory proteins interact with FtsZ and modulate FtsZ assembly/disassembly processes, ensuring the spatiotemporal integrity of cytokinesis. The Z-associated proteins (ZapA, ZapB, and ZapC) belong to a group of FtsZ-regulatory proteins that exhibit functionally redundant roles in stabilizing FtsZ-ring assembly by binding and bundling polymeric FtsZ at midcell. In this study, we report the identification of ZapD (YacF) as a member of the E. coli midcell division machinery. Genetics and cell biological evidence indicate that ZapD requires FtsZ but not other downstream division proteins for localizing to midcell, where it promotes FtsZ-ring assembly via molecular mechanisms that overlap with ZapA. Biochemical evidence indicates that ZapD directly interacts with FtsZ and promotes bundling of FtsZ protofilaments. Similarly to ZapA, ZapB, and ZapC, ZapD is dispensable for division and therefore belongs to the growing group of FtsZ-associated proteins in E. coli that aid in the overall fitness of the division process.
TL;DR: This study disrupted bfrA and bfrB of M. tuberculosis and demonstrated that these genes are crucial for the storage and supply of iron for the growth of bacteria and to withstand oxidative stress in vitro, and establishes them as attractive targets for the development of new inhibitors against mycobacterial infections.
Abstract: Iron is one of the crucial elements required for the growth of Mycobacterium tuberculosis. However, excess free iron becomes toxic for the cells because it catalyzes the production of reactive oxygen radicals, leading to oxidative damage. Hence, it is essential for the pathogen to have the ability to store intracellular iron in an iron-rich environment and utilize it under iron depletion. M. tuberculosis has two iron storage proteins, namely BfrA (Rv1876; a bacterioferritin) and BfrB (Rv3841; a ferritin-like protein). However, the demonstration of biological significance requires the disruption of relevant genes and the evaluation of the resulting mutant for its ability to survive in the host and cause disease. In this study, we have disrupted bfrA and bfrB of M. tuberculosis and demonstrated that these genes are crucial for the storage and supply of iron for the growth of bacteria and to withstand oxidative stress in vitro. In addition, the bfrA bfrB double mutant (H37Rv ΔbfrA ΔbfrB) exhibited a marked reduction in its ability to survive inside human macrophages. Guinea pigs infected with H37Rv ΔbfrA ΔbfrB exhibited a marked diminution in the dissemination of the bacilli to spleen compared to that of the parental strain. Moreover, guinea pigs infected with H37Rv ΔbfrA ΔbfrB exhibited significantly reduced pathological damage in spleen and lungs compared to that of animals infected with the parental strain. Our study clearly demonstrates the importance of these iron storage proteins in the survival and pathogenesis of M. tuberculosis in the host and establishes them as attractive targets for the development of new inhibitors against mycobacterial infections.