Salivaricin D, a Novel Intrinsically Trypsin-Resistant Lantibiotic from Streptococcus salivarius 5M6c Isolated from a Healthy Infant

ABSTRACTIn this work, we purified and characterized a newly identified lantibiotic (salivaricin D) fromStreptococcus salivarius5M6c. Salivaricin D is a 34-amino-acid-residue peptide (3,467.55 Da); the locus of the gene encoding this peptide is a 16.5-kb DNA segment which contains genes encoding the precursor of two lantibiotics, two modification enzymes (dehydratase and cyclase), an ABC transporter, a serine-like protease, immunity proteins (lipoprotein and ABC transporters), a response regulator, and a sensor histidine kinase. The immunity gene (salI) was heterologously expressed in a sensitive indicator and provided significant protection against salivaricin D, confirming its immunity function. Salivaricin D is a naturally trypsin-resistant lantibiotic that is similar to nisin-like lantibiotics. It is a relatively broad-spectrum bacteriocin that inhibits members of many genera of Gram-positive bacteria, including the important human pathogensStreptococcus pyogenesandStreptococcus pneumoniae. Thus,Streptococcus salivarius5M6c may be a potential biological agent for the control of oronasopharynx-colonizing streptococcal pathogens or may be used as a probiotic bacterium.

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Regulation of Virulence by the RevR Response Regulator in Clostridium perfringens

Infection and Immunity ◽

10.1128/iai.00060-11 ◽

2011 ◽

Vol 79 (6) ◽

pp. 2145-2153 ◽

Cited By ~ 24

Author(s):

Thomas J. Hiscox ◽

Anjana Chakravorty ◽

Jocelyn M. Choo ◽

Kaori Ohtani ◽

Tohru Shimizu ◽

...

Keyword(s):

Clostridium Perfringens ◽

Response Regulator ◽

Hydrolytic Enzymes ◽

Differentially Expressed ◽

Wild Type ◽

Content Type ◽

Gram Positive Bacteria ◽

First Response ◽

Wild Type Phenotype ◽

Genes Encoding

ABSTRACTClostridium perfringenscauses clostridial myonecrosis or gas gangrene and produces several extracellular hydrolytic enzymes and toxins, many of which are regulated by the VirSR signal transduction system. TherevRgene encodes a putative orphan response regulator that has similarity to the YycF (WalR), VicR, PhoB, and PhoP proteins from other Gram-positive bacteria. RevR appears to be a classical response regulator, with an N-terminal receiver domain and a C-terminal domain with a putative winged helix-turn-helix DNA binding region. To determine its functional role, arevRmutant was constructed by allelic exchange and compared to the wild type by microarray analysis. The results showed that more than 100 genes were differentially expressed in the mutant, including several genes involved in cell wall metabolism. TherevRmutant had an altered cellular morphology; unlike the short rods observed with the wild type, the mutant cells formed long filaments. These changes were reversed upon complementation with a plasmid that carried the wild-typerevRgene. Several genes encoding extracellular hydrolytic enzymes (sialidase, hyaluronidase, and α-clostripain) were differentially expressed in therevRmutant. Quantitative enzyme assays confirmed that these changes led to altered enzyme activity and that complementation restored the wild-type phenotype. Most importantly, therevRmutant was attenuated for virulence in the mouse myonecrosis model compared to the wild type and the complemented strains. These results provide evidence that RevR regulates virulence inC. perfringens; it is the first response regulator other than VirR to be shown to regulate virulence in this important pathogen.

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Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

Applied and Environmental Microbiology ◽

10.1128/aem.01741-13 ◽

2013 ◽

Vol 79 (18) ◽

pp. 5566-5575 ◽

Cited By ~ 64

Author(s):

Jens Buchholz ◽

Andreas Schwentner ◽

Britta Brunnenkan ◽

Christina Gabris ◽

Simon Grimm ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Pyruvate Dehydrogenase ◽

Pyruvate Dehydrogenase Complex ◽

Fed Batch ◽

Complex Activity ◽

Gene Encoding ◽

Content Type ◽

Genes Encoding ◽

Cell Densities ◽

Dehydrogenase Complex

ABSTRACTExchange of the nativeCorynebacterium glutamicumpromoter of theaceEgene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutateddapApromoter variants led to a series ofC. glutamicumstrains with gradually reduced growth rates and PDHC activities. Upon overexpression of thel-valine biosynthetic genesilvBNCE, all strains producedl-valine. Among these strains,C. glutamicum aceEA16 (pJC4ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of thepqoandppcgenes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities,C. glutamicum aceEA16 Δpqo Δppc(pJC4ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter)l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression ofilvBNCDinstead ofilvBNCEtransformed thel-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with aYP/Sof 0.24 mol per mol of glucose and aQPof 6.9 mM per h [0.8 g/(liter × h)]. The replacement of theaceEpromoter by thedapA-A16 promoter in the twoC. glutamicuml-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate thatC. glutamicumstrains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

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Mapping of the Denitrification Pathway in Burkholderia thailandensis by Genome-Wide Mutant Profiling

Journal of Bacteriology ◽

10.1128/jb.00304-20 ◽

2020 ◽

Vol 202 (23) ◽

Author(s):

Alessandra Vitale ◽

Sarah Paszti ◽

Kohei Takahashi ◽

Masanori Toyofuku ◽

Gabriella Pessi ◽

...

Keyword(s):

Biofilm Formation ◽

Burkholderia Pseudomallei ◽

Anaerobic Growth ◽

Gene Encoding ◽

Terminal Electron Acceptor ◽

Burkholderia Thailandensis ◽

Content Type ◽

Sufficient Energy ◽

Genes Encoding ◽

Human Infections

ABSTRACT Burkholderia thailandensis is a soil saprophyte that is closely related to the pathogen Burkholderia pseudomallei, the etiological agent of melioidosis in humans. The environmental niches and infection sites occupied by these bacteria are thought to contain only limited concentrations of oxygen, where they can generate energy via denitrification. However, knowledge of the underlying molecular basis of the denitrification pathway in these bacteria is scarce. In this study, we employed a transposon sequencing (Tn-Seq) approach to identify genes conferring a fitness benefit for anaerobic growth of B. thailandensis. Of the 180 determinants identified, several genes were shown to be required for growth under denitrifying conditions: the nitrate reductase operon narIJHGK2K1, the aniA gene encoding a previously unknown nitrite reductase, and the petABC genes encoding a cytochrome bc1, as well as three novel regulators that control denitrification. Our Tn-Seq data allowed us to reconstruct the entire denitrification pathway of B. thailandensis and shed light on its regulation. Analyses of growth behaviors combined with measurements of denitrification metabolites of various mutants revealed that nitrate reduction provides sufficient energy for anaerobic growth, an important finding in light of the fact that some pathogenic Burkholderia species can use nitrate as a terminal electron acceptor but are unable to complete denitrification. Finally, we demonstrated that a nitrous oxide reductase mutant is not affected for anaerobic growth but is defective in biofilm formation and accumulates N2O, which may play a role in the dispersal of B. thailandensis biofilms. IMPORTANCE Burkholderia thailandensis is a soil-dwelling saprophyte that is often used as surrogate of the closely related pathogen Burkholderia pseudomallei, the causative agent of melioidosis and a classified biowarfare agent. Both organisms are adapted to grow under oxygen-limited conditions in rice fields by generating energy through denitrification. Microoxic growth of B. pseudomallei is also considered essential for human infections. Here, we have used a Tn-Seq approach to identify the genes encoding the enzymes and regulators required for growth under denitrifying conditions. We show that a mutant that is defective in the conversion of N2O to N2, the last step in the denitrification process, is unaffected in microoxic growth but is severely impaired in biofilm formation, suggesting that N2O may play a role in biofilm dispersal. Our study identified novel targets for the development of therapeutic agents to treat meliodiosis.

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Studies on Bd0934 and Bd3507, Two Secreted Nucleases from Bdellovibrio bacteriovorus, Reveal Sequential Release of Nucleases during the Predatory Cycle

Journal of Bacteriology ◽

10.1128/jb.00150-20 ◽

2020 ◽

Vol 202 (18) ◽

Author(s):

Ewa Bukowska-Faniband ◽

Tilde Andersson ◽

Rolf Lood

Keyword(s):

Antibiotic Resistance ◽

Life Cycle ◽

Antibiotic Resistance Genes ◽

Human Pathogens ◽

Temporal Expression ◽

Bdellovibrio Bacteriovorus ◽

Exogenous Dna ◽

Content Type ◽

Genes Encoding ◽

Predatory Bacteria

ABSTRACT Bdellovibrio bacteriovorus is an obligate predatory bacterium that invades and kills a broad range of Gram-negative prey cells, including human pathogens. Its potential therapeutic application has been the subject of increased research interest in recent years. However, an improved understanding of the fundamental molecular aspects of the predatory life cycle is crucial for developing this bacterium as a “living antibiotic.” During intracellular growth, B. bacteriovorus secretes an arsenal of hydrolases, which digest the content of the host cell to provide growth nutrients for the predator, e.g., prey DNA is completely degraded by the nucleases. Here, we have, on a genetic and molecular level, characterized two secreted DNases from B. bacteriovorus, Bd0934 and Bd3507, and determined the temporal expression profile of other putative secreted nucleases. We conclude that Bd0934 and Bd3507 are likely a part of the predatosome but are not essential for the predation, host-independent growth, prey biofilm degradation, and self-biofilm formation. The detailed temporal expression analysis of genes encoding secreted nucleases revealed that these enzymes are produced in a sequential orchestrated manner. This work contributes to our understanding of the sequential breakdown of the prey nucleic acid by the nucleases secreted during the predatory life cycle of B. bacteriovorus. IMPORTANCE Antibiotic resistance is a major global concern with few available new means to combat it. From a therapeutic perspective, predatory bacteria constitute an interesting tool. They not only eliminate the pathogen but also reduce the overall pool of antibiotic resistance genes through secretion of nucleases and complete degradation of exogenous DNA. Molecular knowledge of how these secreted DNases act will give us further insight into how antibiotic resistance, and the spread thereof, can be limited through the action of predatory bacteria.

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An intramembrane sensory circuit monitors sortase A–mediated processing of streptococcal adhesins

Science Signaling ◽

10.1126/scisignal.aas9941 ◽

2019 ◽

Vol 12 (580) ◽

pp. eaas9941 ◽

Cited By ~ 2

Author(s):

Jeffrey W. Hall ◽

Bruno P. Lima ◽

Gaetan G. Herbomel ◽

Tata Gopinath ◽

LeAnna McDonald ◽

...

Keyword(s):

Cell Membrane ◽

Biofilm Formation ◽

Response Regulator ◽

Sortase A ◽

Gram Positive Bacteria ◽

Regulatory Circuit ◽

Bacterial Adhesins ◽

Genes Encoding ◽

Quality Control Mechanism ◽

Cognate Response Regulator

Bacterial adhesins mediate adhesion to substrates and biofilm formation. Adhesins of the LPXTG family are posttranslationally processed by the cell membrane–localized peptidase sortase A, which cleaves the LPXTG motif. This generates a short C-terminal peptide (C-pep) that remains in the cell membrane, whereas the mature adhesin is incorporated into the cell wall. Genes encoding adhesins of the oral bacteriumStreptococcus gordoniiwere differentially expressed depending on whether the bacteria were isolated from saliva or dental plaque and appeared to be coordinately regulated. Deletion ofsspAandsspB (sspAB), both of which encode LPXTG-containing adhesins, unexpectedly enhanced adhesion and biofilm formation. C-peps produced from a model LPXTG-containing adhesin localized to the cell membrane and bound to and inhibited the intramembrane sensor histidine kinase SGO_1180, thus preventing activation of the cognate response regulator SGO_1181. The absence of SspAB C-peps induced the expression of thescaCBAoperon encoding the lipoprotein adhesin ScaA, which was sufficient to preserve and even enhance biofilm formation. This C-pep–driven regulatory circuit also exists in pathogenic streptococci and is likely conserved among Gram-positive bacteria. This quality control mechanism ensures that the bacteria can form biofilms under diverse environmental conditions and may play a role in optimizing adhesion and biofilm formation.

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Coordinated Zinc Homeostasis Is Essential for the Wild-Type Virulence of Brucella abortus

Journal of Bacteriology ◽

10.1128/jb.02543-14 ◽

2015 ◽

Vol 197 (9) ◽

pp. 1582-1591 ◽

Cited By ~ 15

Author(s):

Lauren M. Sheehan ◽

James A. Budnick ◽

R. Martin Roop ◽

Clayton C. Caswell

Keyword(s):

Mouse Model ◽

Brucella Abortus ◽

Zinc Uptake ◽

Zinc Homeostasis ◽

Zinc Toxicity ◽

Uptake System ◽

Bacterial Cells ◽

Gene Encoding ◽

Content Type ◽

Genes Encoding

ABSTRACTMetal homeostasis in bacterial cells is a highly regulated process requiring intricately coordinated import and export, as well as precise sensing of intracellular metal concentrations. The uptake of zinc (Zn) has been linked to the virulence ofBrucella abortus; however, the capacity ofBrucellastrains to sense Zn levels and subsequently coordinate Zn homeostasis has not been described. Here, we show that expression of the genes encoding the zinc uptake system ZnuABC is negatively regulated by the Zn-sensing Fur family transcriptional regulator, Zur, by direct interactions between Zur and the promoter region ofznuABC. Moreover, the MerR-type regulator, ZntR, controls the expression of the gene encoding the Zn exporter ZntA by binding directly to its promoter. Deletion ofzurorzntRalone did not result in increased zinc toxicity in the corresponding mutants; however, deletion ofzntAled to increased sensitivity to Zn but not to other metals, such as Cu and Ni, suggesting that ZntA is a Zn-specific exporter. Strikingly, deletion ofzntRresulted in significant attenuation ofB. abortusin a mouse model of chronic infection, and subsequent experiments revealed that overexpression ofzntAin thezntRmutant is the molecular basis for its decreased virulence.IMPORTANCEThe importance of zinc uptake forBrucellapathogenesis has been demonstrated previously, but to date, there has been no description of how overall zinc homeostasis is maintained and genetically controlled in the brucellae. The present work defines the predominant zinc export system, as well as the key genetic regulators of both zinc uptake and export inBrucella abortus. Moreover, the data show the importance of precise coordination of the zinc homeostasis systems as disregulation of some elements of these systems leads to the attenuation ofBrucellavirulence in a mouse model. Overall, this study advances our understanding of the essential role of zinc in the pathogenesis of intracellular bacteria.

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Genetic and Functional Investigation of Zn 2 Cys 6 Transcription Factors RSE2 and RSE3 in Podospora anserina

Eukaryotic Cell ◽

10.1128/ec.00172-13 ◽

2013 ◽

Vol 13 (1) ◽

pp. 53-65 ◽

Cited By ~ 10

Author(s):

Elodie Bovier ◽

Carole H. Sellem ◽

Adeline Humbert ◽

Annie Sainsard-Chanet

Keyword(s):

Transcription Factors ◽

Phosphoenolpyruvate Carboxykinase ◽

Constitutive Expression ◽

Podospora Anserina ◽

Loss Of Function ◽

Gene Encoding ◽

Content Type ◽

Zinc Cluster ◽

Wide Range ◽

Genes Encoding

ABSTRACT In Podospora anserina , the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase ( aox ) when the mitochondrial electron transport chain is impaired. In parallel, they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase ( pck ) and fructose-1,6-biphosphatase ( fbp ). Orthologues of these transcription factors are present in a wide range of filamentous fungi, and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organization of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of the aox gene. We identified 10 independent mutations in 9 positions in rse2 and 11 mutations in 5 positions in rse3 . Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-terminal activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations in rse2 or rse3 . It showed that in addition to aox , fbp , and pck , RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn 2 Cys 6 transcription factor, a flavohemoglobin, and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both an rse2 gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids, and α-ketoglutarate pools.

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GH51 Arabinofuranosidase and Its Role in the Methylglucuronoarabinoxylan Utilization System in Paenibacillus sp. Strain JDR-2

Applied and Environmental Microbiology ◽

10.1128/aem.01684-14 ◽

2014 ◽

Vol 80 (19) ◽

pp. 6114-6125 ◽

Cited By ~ 5

Author(s):

Neha Sawhney ◽

James F. Preston

Keyword(s):

Abc Transporters ◽

Transcriptional Regulators ◽

Fermentable Sugars ◽

Efficient Utilization ◽

Content Type ◽

Thermochemical Pretreatment ◽

Reverse Transcription Pcr ◽

Paenibacillus Sp ◽

Intracellular Enzymes ◽

Genes Encoding

ABSTRACTMethylglucuronoarabinoxylan (MeGAXn) from agricultural residues and energy crops is a significant yet underutilized biomass resource for production of biofuels and chemicals. Mild thermochemical pretreatment of bagasse yields MeGAXnrequiring saccharifying enzymes for conversion to fermentable sugars. A xylanolytic bacterium,Paenibacillussp. strain JDR-2, produces an extracellular cell-associated GH10 endoxylanse (XynA1) which efficiently depolymerizes methylglucuronoxylan (MeGXn) from hardwoods coupled with assimilation of oligosaccharides for further processing by intracellular GH67 α-glucuronidase, GH10 endoxylanase, and GH43 β-xylosidase. This process has been ascribed to genes that comprise a xylan utilization regulon that encodes XynA1and includes a gene cluster encoding transcriptional regulators, ABC transporters, and intracellular enzymes that convert assimilated oligosaccharides to fermentable sugars. Here we show thatPaenibacillussp. JDR-2 utilized MeGAXnwithout accumulation of oligosaccharides in the medium. ThePaenibacillussp. JDR-2 growth rate on MeGAXnwas 3.1-fold greater than that on oligosaccharides generated from MeGAXnby XynA1. Candidate genes encoding GH51 arabinofuranosidases with potential roles were identified. Following growth on MeGAXn, quantitative reverse transcription-PCR identified a cluster of genes encoding a GH51 arabinofuranosidase (AbfB) and transcriptional regulators which were coordinately expressed along with the genes comprising the xylan utilization regulon. The action of XynA1on MeGAXngenerated arabinoxylobiose, arabinoxylotriose, xylobiose, xylotriose, and methylglucuronoxylotriose. Recombinant AbfB processed arabinoxylooligosaccharides to xylooligosaccharides and arabinose. MeGAXnprocessing byPaenibacillussp. JDR-2 may be achieved by extracellular depolymerization by XynA1coupled to assimilation of oligosaccharides and further processing by intracellular enzymes, including AbfB.Paenibacillussp. JDR-2 provides a GH10/GH67 system complemented with genes encoding intracellular GH51 arabinofuranosidases for efficient utilization of MeGAXn.

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Structural basis of trehalose recycling by the ABC transporter LpqY-SugABC

Science Advances ◽

10.1126/sciadv.abb9833 ◽

2020 ◽

Vol 6 (44) ◽

pp. eabb9833

Author(s):

Fengjiang Liu ◽

Jingxi Liang ◽

Bing Zhang ◽

Yan Gao ◽

Xiuna Yang ◽

...

Keyword(s):

Electron Microscopy ◽

Abc Transporter ◽

Abc Transporters ◽

Unique Feature ◽

Structural Basis ◽

Human Pathogens ◽

Gram Positive Bacteria ◽

Cryo Electron Microscopy ◽

Uptake Of Nutrients ◽

Initial Mode

In bacteria, adenosine 5′-triphosphate (ATP)–binding cassette (ABC) importers are essential for the uptake of nutrients including the nonreducing disaccharide trehalose, a metabolite that is crucial for the survival and virulence of several human pathogens including Mycobacterium tuberculosis. SugABC is an ABC transporter that translocates trehalose from the periplasmic lipoprotein LpqY into the cytoplasm of mycobacteria. Here, we report four high-resolution cryo–electron microscopy structures of the mycobacterial LpqY-SugABC complex to reveal how it binds and passes trehalose through the membrane to the cytoplasm. A unique feature observed in this system is the initial mode of capture of the trehalose at the LpqY interface. Uptake is achieved by a pivotal rotation of LpqY relative to SugABC, moving from an open and accessible conformation to a clamped conformation upon trehalose binding. These findings enrich our understanding as to how ABC transporters facilitate substrate transport across the membrane in Gram-positive bacteria.

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Role of the Transporter-Like Sensor Kinase CbrA in Histidine Uptake and Signal Transduction

Journal of Bacteriology ◽

10.1128/jb.00361-15 ◽

2015 ◽

Vol 197 (17) ◽

pp. 2867-2878 ◽

Cited By ~ 13

Author(s):

Xue-Xian Zhang ◽

Jonathan C. Gauntlett ◽

Darby G. Oldenburg ◽

Gregory M. Cook ◽

Paul B. Rainey

Keyword(s):

Signal Transduction ◽

Response Regulator ◽

Nutrient Acquisition ◽

Coupled Processes ◽

Sensor Kinase ◽

Unique Type ◽

Content Type ◽

Genes Encoding ◽

Interdomain Interactions ◽

Cognate Response Regulator

ABSTRACTCbrA is an atypical sensor kinase found inPseudomonas. The autokinase domain is connected to a putative transporter of the sodium/solute symporter family (SSSF). CbrA functions together with its cognate response regulator, CbrB, and plays an important role in nutrient acquisition, including regulation ofhutgenes for the utilization of histidine and its derivative, urocanate. Here we report on the findings of a genetic and biochemical analysis of CbrA with a focus on the function of the putative transporter domain. The work was initiated with mutagenesis of histidine uptake-proficient strains to identify histidine-specific transport genes located outside thehutoperon. Genes encoding transporters were not identified, but mutations were repeatedly found incbrA. This, coupled with the findings of [3H]histidine transport assays and further mutagenesis, implicated CbrA in histidine uptake. In addition, mutations in different regions of the SSSF domain abolished signal transduction. Site-specific mutations were made at four conserved residues: W55 and G172 (SSSF domain), H766 (H box), and N876 (N box). The mutations W55G, G172H, and N876G compromised histidine transport but had minimal effects on signal transduction. The H766G mutation abolished both transport and signal transduction, but the capacity to transport histidine was restored upon complementation with a transport-defective allele of CbrA, most likely due to interdomain interactions. Our combined data implicate the SSSF domain of CbrA in histidine transport and suggest that transport is coupled to signal transduction.IMPORTANCENutrient acquisition in bacteria typically involves membrane-bound sensors that, via cognate response regulators, determine the activity of specific transporters. However, nutrient perception and uptake are often coupled processes. Thus, from a physiological perspective, it would make sense for systems that couple the process of signaling and transport within a single protein and where transport is itself the stimulus that precipitates signal transduction to have evolved. The CbrA regulator inPseudomonasrepresents a unique type of sensor kinase whose autokinase domain is connected to a transporter domain. We present genetic and biochemical evidence that suggests that CbrA plays a dual role in histidine uptake and sensing and that transport is dependent on signal transduction.

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