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1.
Mol Microbiol ; 98(2): 329-42, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26179020

RESUMO

Bacteriophages are present in virtually all ecosystems, and bacteria have developed multiple antiphage strategies to counter their attacks. Clostridium difficile is an important pathogen causing severe intestinal infections in humans and animals. Here we show that the conserved cell-surface protein CwpV provides antiphage protection in C. difficile. This protein, for which the expression is phase-variable, is classified into five types, each differing in their repeat-containing C-terminal domain. When expressed constitutively from a plasmid or the chromosome of locked 'ON' cells of C. difficile R20291, CwpV conferred antiphage protection. Differences in the level of phage protection were observed depending on the phage morphological group, siphophages being the most sensitive with efficiency of plaquing (EOP) values of < 5 × 10(-7) for phages ϕCD38-2, ϕCD111 and ϕCD146. Protection against the myophages ϕMMP01 and ϕCD52 was weaker, with EOP values between 9.0 × 10(-3) and 1.1 × 10(-1). The C-terminal domain of CwpV carries the antiphage activity and its deletion, or part of it, significantly reduced the antiphage protection. CwpV does not affect phage adsorption, but phage DNA replication is prevented, suggesting a mechanism reminiscent of superinfection exclusion systems normally encoded on prophages. CwpV thus represents a novel ubiquitous host-encoded and phase-variable antiphage system in C. difficile.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/crescimento & desenvolvimento , Parede Celular/química , Clostridioides difficile/metabolismo , Clostridioides difficile/virologia , Animais , Proteínas de Bactérias/genética , Bacteriófagos/patogenicidade , Bacteriófagos/fisiologia , Parede Celular/metabolismo , Clostridioides difficile/química , Clostridioides difficile/genética , DNA Viral/genética , Humanos , Análise de Sequência de DNA
2.
Mol Microbiol ; 90(1): 195-207, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23927792

RESUMO

In Mycobacterium tuberculosis, the genes Rv1954A-Rv1957 form an operon that includes Rv1955 and Rv1956 which encode the HigB toxin and the HigA antitoxin respectively. We are interested in the role and regulation of this operon, since toxin-antitoxin systems have been suggested to play a part in the formation of persister cells in mycobacteria. To investigate the function of the higBA locus, effects of toxin expression on mycobacterial growth and transcript levels were assessed in M. tuberculosis H37Rv wild type and in an operon deletion background. We show that expression of HigB toxin in the absence of HigA antitoxin arrests growth and causes cell death in M. tuberculosis. We demonstrate HigB expression to reduce the abundance of IdeR and Zur regulated mRNAs and to cleave tmRNA in M. tuberculosis, Escherichia coli and Mycobacterium smegmatis. This study provides the first identification of possible target transcripts of HigB in M. tuberculosis.


Assuntos
Toxinas Bacterianas/biossíntese , Mycobacterium tuberculosis/crescimento & desenvolvimento , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Toxinas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica , Viabilidade Microbiana , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium tuberculosis/genética , Estabilidade de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
3.
Microbiology (Reading) ; 160(Pt 2): 373-384, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24327425

RESUMO

Burkholderia cepacia complex (Bcc) bacteria possess biotechnologically useful properties that contrast with their opportunistic pathogenicity. The rhizosphere fitness of Bcc bacteria is central to their biocontrol and bioremediation activities. However, it is not known whether this differs between species or between environmental and clinical strains. We investigated the ability of 26 Bcc strains representing nine different species to colonize the roots of Arabidopsis thaliana and Pisum sativum (pea). Viable counts, scanning electron microscopy and bioluminescence imaging were used to assess root colonization, with Bcc bacteria achieving mean (±sem) levels of 2.49±0.23×10(6) and 5.16±1.87×10(6) c.f.u. per centimetre of root on the A. thaliana and P. sativum models, respectively. The A. thaliana rhizocompetence model was able to reveal loss of colonization phenotypes in Burkholderia vietnamiensis G4 transposon mutants that had only previously been observed in competition experiments on the P. sativum model. Different Bcc species colonized each plant model at different rates, and no statistical difference in root colonization was observed between isolates of clinical or environmental origin. Loss of the virulence-associated third chromosomal replicon (>1 Mb DNA) did not alter Bcc root colonization on A. thaliana. In summary, Bcc bacteria possess intrinsic root colonization abilities irrespective of their species or source. As Bcc rhizocompetence does not require their third chromosomal replicon, the possibility of using synthetic biology approaches to engineer virulence-attenuated biotechnological strains is tractable.


Assuntos
Arabidopsis/microbiologia , Complexo Burkholderia cepacia/crescimento & desenvolvimento , Pisum sativum/microbiologia , Raízes de Plantas/microbiologia , Infecções por Burkholderia/microbiologia , Complexo Burkholderia cepacia/isolamento & purificação , Contagem de Colônia Microbiana , Elementos de DNA Transponíveis , Microbiologia Ambiental , Microscopia Eletrônica de Varredura , Mutagênese Insercional , Imagem Óptica
4.
Microbiology (Reading) ; 158(Pt 2): 308-318, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22034487

RESUMO

Thymidylate synthase (TS) enzymes catalyse the biosynthesis of deoxythymidine monophosphate (dTMP or thymidylate), and so are important for DNA replication and repair. Two different types of TS proteins have been described (ThyA and ThyX), which have different enzymic mechanisms and unrelated structures. Mycobacteria are unusual as they encode both thyA and thyX, and the biological significance of this is not yet understood. Mycobacterium tuberculosis ThyX is thought to be essential and a potential drug target. We therefore analysed M. tuberculosis thyA and thyX expression levels, their essentiality and roles in pathogenesis. We show that both thyA and thyX are expressed in vitro, and that this expression significantly increased within murine macrophages. Under all conditions tested, thyA expression exceeded that of thyX. Mutational studies show that M. tuberculosis thyX is essential, confirming that the enzyme is a plausible drug target. The requirement for M. tuberculosis thyX in the presence of thyA implies that the essential function of ThyX is something other than dTM synthesis [corrected].We successfully deleted thyA from the M. tuberculosis genome, and this deletion conferred an in vitro growth defect that was not observed in vivo. Presumably ThyX performs TS activity within M. tuberculosis ΔthyA at a sufficient rate in vivo for normal growth, but the rate in vitro is less than optimal. We also demonstrate that thyA deletion confers M. tuberculosis p-aminosalicylic acid resistance, and show by complementation studies that ThyA T202A and V261G appear to be functional and non-functional, respectively.


Assuntos
Ácido Aminossalicílico/farmacologia , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Tuberculose/microbiologia , Animais , Farmacorresistência Bacteriana , Feminino , Deleção de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Timina/metabolismo
5.
J Bacteriol ; 192(17): 4348-56, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20585061

RESUMO

Bacterial chromosomally encoded type II toxin-antitoxin (TA) loci may be involved in survival upon exposure to stress and have been linked to persistence and dormancy. Therefore, understanding the role of the numerous predicted TA loci within the human pathogen Mycobacterium tuberculosis has become a topic of great interest. Antitoxin proteins are known to autoregulate TA expression under normal growth conditions, but it is unknown whether they have a more global role in transcriptional regulation. This study focuses on analyzing the regulatory role of the M. tuberculosis HigA antitoxin. We first show that the M. tuberculosis higBA locus is functional within its native organism, as higB, higA, and Rv1957 were successfully deleted from the genome together while the deletion of higA alone was not possible. The effects of higB-Rv1957 deletion on M. tuberculosis global gene expression were investigated, and a number of potential HigA-regulated genes were identified. Transcriptional fusion and protein-DNA-binding assays were utilized to confirm the direct role of HigA in Rv1954A-Rv1957 repression, and the M. tuberculosis HigA DNA-binding motif was defined as ATATAGG(N(6))CCTATAT. As HigA failed to bind to the next-most-closely related motif within the M. tuberculosis genome, HigA may not directly regulate any other genes in addition to its own operon.


Assuntos
Antitoxinas/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/metabolismo , Antitoxinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Deleção de Genes , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Análise de Sequência com Séries de Oligonucleotídeos , Óperon , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica
6.
Microbiology (Reading) ; 155(Pt 1): 186-197, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19118359

RESUMO

Correct identification of translational start sites is important for understanding protein function and transcriptional regulation. The annotated translational start sites contained in genome databases are often predicted using bioinformatics and are rarely verified experimentally, and so are not all accurate. Therefore, we devised a simple approach for determining translational start sites using a combination of epitope tagging and frameshift mutagenesis. This assay was used to determine the start sites of three Mycobacterium tuberculosis proteins: LexA, SigC and Rv1955. We were able to show that proteins may begin before or after the predicted site. We also found that a small, non-annotated open reading frame upstream of Rv1955 was expressed as a protein, which we have designated Rv1954A. This approach is readily applicable to any bacterial species for which plasmid transformation can be achieved.


Assuntos
Proteínas de Bactérias/genética , Códon de Iniciação , Mycobacterium tuberculosis/genética , Fases de Leitura Aberta/genética , Biossíntese de Proteínas , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epitopos , Mutação da Fase de Leitura , Genoma Bacteriano , Humanos , Dados de Sequência Molecular , Mycobacterium tuberculosis/metabolismo , Fases de Leitura Aberta/fisiologia , Plasmídeos/genética , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Fator sigma/química , Fator sigma/genética , Fator sigma/metabolismo
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