RESUMO
The Vibrio cholerae (Vc) gene (tagA) coding for the TagA lipoprotein has been isolated. Sequencing of tagA revealed the presence of an open reading frame (ORF) of 568 amino acids with a characteristic signal peptidase II cleavage site at the N terminus. Electrophoretic analysis of proteins synthesized by Escherichia coli (Ec) cells following T7 promoter/RNA polymerase-directed expression of tagA, revealed a closely migrating doublet of proteins corresponding to two species of TagA. Computer-generated alignment algorithms predict that a homology exists between Vc TagA and Ec K99 fimbriae biogenesis determinant FanD.
Assuntos
Proteínas de Bactérias , Proteínas de Escherichia coli , Genes Bacterianos , Lipoproteínas/genética , Vibrio cholerae/genética , Algoritmos , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Bacteriófago T7/genética , Sequência de Bases , Clonagem Molecular , Proteínas de Ligação a DNA/fisiologia , Escherichia coli/genética , Fímbrias Bacterianas/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/fisiologiaRESUMO
The toxin-coregulated pilus (TCP)-encoding gene cluster (tcp) specifies a type-IV pilus that is a major colonization determinant of Vibrio cholerae. We have identified a gene 200 bp upstream from the tcp cluster that requires ToxR for expression. We have designated this gene tagD (ToxR-activated gene) and have shown that tagD is encoded on a 600-nt transcript. The deduced tagD product is a 164-amino-acid polypeptide (20 kDa). Interestingly, TagD shares a high degree of similarity to a protein of Streptococcus sanguis 12 that is thought to play a role in fimbriae synthesis or assembly. The high degree of similarity between tagD and the Ss 12 protein provides preliminary evidence that tagD represents an additional member of the tcp cluster.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/fisiologia , Genes Bacterianos/genética , Peroxidases , Fatores de Transcrição/fisiologia , Vibrio cholerae/genética , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Sequência de Bases , Escherichia coli/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica/genética , Dados de Sequência Molecular , Família Multigênica/genética , RNA Bacteriano/biossíntese , RNA Mensageiro/biossíntese , Regulon/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Virulence gene activation in Vibrio cholerae is under the control of the ToxR-ToxT regulatory cascade. The ToxR regulon consists of genes required for toxin-coregulated-pilus (TCP) biogenesis, accessory colonization factor genes, cholera toxin genes, and ToxR-activated genes (tag) of unknown function. The tagB gene was isolated by using a tagB::TnphoA fusion junction to probe a V. cholerae )395 bacteriophage lambda library. Nucleotide sequence analysis revealed that tagB is identical to tcpI, a gene which encodes a protein that negatively regulates the synthesis of the major pilin subunit of TCP (TcpA). Our results show that the tcpI gene encodes a 620-amino-acid protein that shares extensive sequence similarity with the highly conserved signaling domain in methyl-accepting chemotaxis proteins. Expression of tcpI in Escherichia coli results in the synthesis of a 71-kDa polypeptide that becomes localized to the inner membrane. Similarly, TcpI-PhoA alkaline phosphatase activity is enriched in V. cholerae inner membrane preparations. Colonies of V. cholerae tcpI::TnphoA mutant cells display increased swarming on solid media when compared with those of the parental V. cholerae O395. Taken together, these observations suggest that TcpI may play a dual role in promoting vibrio colonization of the small bowel. In response to the appropriate environmental signal(s), TcpI permits maximum expression of tcpA while simultaneously reducing vibrio chemotaxis-directed motility. We believe coordinate regulation of colonization and motility determinants, in such a fashion, facilitates efficient V. cholerae microcolony formation.
