RESUMO
In Caulobacter crescentus, the alternative sigma factor sigma54 plays an important role in the expression of late flagellar genes. Sigma54-dependent genes are temporally and spatially controlled, being expressed only in the swarmer pole of the predivisional cell. The only sigma54 activator described so far is the FlbD protein, which is involved in activation of the class III and IV flagellar genes and repression of the fliF promoter. To identify new roles for sigma54 in the metabolism and differentiation of C. crescentus, we cloned and characterized a gene encoding a putative sigma54 activator, named tacA. The deduced amino acid sequence from tacA has high similarity to the proteins from the NtrC family of transcriptional activators, including the aspartate residues that are phosphorylated by histidine kinases in other activators. The promoter region of the tacA gene contains a conserved sequence element present in the promoters of class II flagellar genes, and tacA shows a temporal pattern of expression similar to the patterns of these genes. We constructed an insertional mutant that is disrupted in tacA (strain SP2016), and an analysis of this strain showed that it has all polar structures, such as pili, stalk, and flagellum, and displays a motile phenotype, indicating that tacA is not involved in the flagellar biogenesis pathway. However, this strain has a high percentage of filamentous cells and shows a clear-plaque phenotype when infected with phage phiCb5. These results suggest that the TacA protein could mediate the effect of sigma54 on a different pathway in C. crescentus.
Assuntos
Proteínas de Bactérias , Caulobacter crescentus/genética , Proteínas de Ligação a DNA , RNA Polimerases Dirigidas por DNA/genética , Regulação Bacteriana da Expressão Gênica/genética , Fator sigma/genética , Transativadores/genética , Sequência de Aminoácidos , Bacteriófagos/fisiologia , Sequência de Bases , Caulobacter crescentus/virologia , Clonagem Molecular , Flagelos/genética , Genes Bacterianos/genética , Dados de Sequência Molecular , Fenótipo , Regiões Promotoras Genéticas/genética , RNA Polimerase Sigma 54 , Proteínas Recombinantes de Fusão , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição Gênica/genéticaRESUMO
The bacterial heat shock proteins DnaK and DnaJ are members of a class of molecular chaperones that are required for a wide variety of cellular functions at normal growth temperatures. In Caulobacter crescentus, the expression of the dnaKJ operon is regulated both temporally during the normal cell cycle and by heat shock. Analysis of deletions and base substitutions in the 5' region of the operon established the presence of two functional promoters: a heat shock-inducible promoter, P1, with characteristics of a sigma 32 promoter, and an adjacent sigma 70-like promoter, P2. Transcription initiating at the sigma 70-like promoter is under strict temporal control, whereas transcription initiating at the heat shock promoter at 30 degrees C is not. Transcription of dnaKJ occurs during a short period in the cell cycle, concomitant with the onset of DNA replication. Deletions in the 5' region have also revealed that all cis-acting sites required for temporal control of transcription reside within 50 bases of the P2 start site. Transcripts initiating from either the P1 or the P2 promoter have an RNA leader sequence with a high probability of forming an extensive secondary structure. Deletion of this leader sequence resulted in an increased rate of expression in both transcriptional and translational fusions. Although the temporal control of expression at physiological temperatures is not affected by the presence or absence of the leader sequence, changes in mRNA secondary structure may contribute to the modulation of DnaK and DnaJ levels at normal temperatures and during heat shock.
Assuntos
Proteínas de Bactérias/genética , Caulobacter crescentus/genética , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/genética , Óperon/genética , Sequência de Bases , Clonagem Molecular , Proteínas de Choque Térmico HSP40 , Dados de Sequência Molecular , Regiões Promotoras Genéticas/fisiologia , Deleção de Sequência , Transcrição Gênica/fisiologiaRESUMO
Caulobacter crescentus has a single dnaK gene that is highly homologous to the hsp70 family of heat shock genes. Analysis of the cloned and sequenced dnaK gene has shown that the deduced amino acid sequence could encode a protein of 67.6 kilodaltons that is 68% identical to the DnaK protein of Escherichia coli and 49% identical to the Drosophila and human hsp70 protein family. A partial open reading frame 165 base pairs 3' to the end of dnaK encodes a peptide of 190 amino acids that is 59% identical to DnaJ of E. coli. Northern blot analysis revealed a single 4.0-kilobase mRNA homologous to the cloned fragment. Since the dnaK coding region is 1.89 kilobases, dnaK and dnaJ may be transcribed as a polycistronic message. S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence. A single start site of transcription was identified during heat shock at 42 degrees C, and the predicted promoter sequence conformed to the consensus heat shock promoters of E. coli. At normal growth temperature (30 degrees C), a different start site was identified 3' to the heat shock start site that conformed to the E. coli sigma 70 promoter consensus sequence. S1 protection assays and analysis of expression of the dnaK gene fused to the lux transcription reporter gene showed that expression of dnaK is temporally controlled under normal physiological conditions and that transcription occurs just before the initiation of DNA replication. Thus, in both human cells (I. K. L. Milarski and R. I. Morimoto, Proc. Natl. Acad. Sci. USA 83:9517-9521, 1986) and in a simple bacterium, the transcription of a hsp70 gene is temporally controlled as a function of the cell cycle under normal growth conditions.