Isolation and characterization of Caulobacter mutants impaired in adaptation to stationary phase

The entry into stationary phase causes a change in the pattern of gene expression of bacteria, when the cells must express a whole set of genes involved mainly with resistance to starvation and to environmental stresses. As an attempt to identify genes important for the survival of Caulobacter crescentus in stationary phase, we have screened a library of 5,000 clones generated by random transposon mutagenesis for mutants that showed reduced viability after prolonged growth. Four clones were selected, which displayed either lower viability or a longer time of recovery from stationary phase. The genes disrupted were identified, and the gene products were found to be mainly involved with amino acid metabolism (glutamate N-acetyltransferase, 4-hydroxyphenylpyruvate dioxygenase and L-aspartate oxidase) or with recombination (exonuclease RecJ). Each mutant was tested for resistance to stresses, such as oxidative, saline, acidic, heat and UV exposure, showing different responses. Although the mutations obtained were not in genes involved specifically in stationary phase, our results suggest that amino acids metabolism may play an important role in keeping viability during this growth phase.

Cloning and characterization of the gene encoding the PepF endopeptidase from the aquatic bacterium Caulobacter crescentus

The metallopeptidases have a very important role in bacteria, being involved in several processes that rely on protein turnover, such as nutrition, degradation of signal peptides, protein localization and virulence. We have cloned and characterized the gene of the metalloendopeptidase PepF from the aquatic bacterium Caulobacter crescentus. The gene upstream of pepF (orf1) encodes a conserved hypothetical protein found in Mycobacterium and Streptomyces. pepF is co-transcribed with the gene downstream (orf3), which encodes a protein that belongs to the ABC1 protein kinase family, suggesting that these two proteins may share a common function in the cell. The C. crescentus PepF protein possesses the conserved HEXGH motif present in zinc binding domains of PepF homologs. Disruption of the pepF gene by insertion of a vector sequence did not produced any growth defect, but the mutant strain possesses only 30% of the specific activity of endopeptidases present in the wild type strain. Deletions and point mutations in the regulatory region showed that there are two putative promoter regions, and the operon expression is independent of the transcription regulator CtrA. The results indicate that PepF is not essential for either growth or development of this bacterium using peptides as the sole carbon source, suggesting that other peptidases can be sharing this function.

As metalopeptidases têm papel importante em bactérias, estando envolvidas em vários processos que dependem de reciclagem de proteínas, como nutrição, degradação de peptídios-sinal, localização de proteínas e virulência. Nós clonamos e caracterizamos o gene da metaloendopeptidase PepF da bactéria aquática Caulobacter crescentus. O gene a montante de pepF (orf1) codifica uma proteína hipotética conservada encontrada em Mycobacterium e Streptomyces. pepF é co-transcrito com o gene a juzante (orf3), que codifica uma proteína pertencente à família ABC1 de proteínas quinases, sugerindo que estas duas proteínas podem compartilhar uma função comum na célula. A proteína PepF de C. crescentus possui o motivo HEXGH presente nos domínios de ligação de zinco de homólogos de PepF. A interrupção do gene pepF por inserção de sequência de DNA de um vetor não gerou nenhum defeito de crescimento, mas a linhagem mutante possui apenas 30% da atividade especifica de endopeptidases presentes na linhagem selvagem. Deleções e mutações pontuais na região regulatória mostraram que há duas possíveis regiões promotoras, e a expressão do operon é independente do regulador de transcrição CtrA. Os resultados indicam que PepF não é essencial para o crescimento desta bactéria utilizando peptídios como fonte de carbono, sugerindo que outras peptidases possam estar realizando esta função.

Cloning and characterization of the gene encoding the PepF endopeptidase from the aquatic bacterium Caulobacter crescentus

The metallopeptidases have a very important role in bacteria, being involved in several processes that rely on protein turnover, such as nutrition, degradation of signal peptides, protein localization and virulence. We have cloned and characterized the gene of the metalloendopeptidase PepF from the aquatic bacterium Caulobacter crescentus. The gene upstream of pepF (orf1) encodes a conserved hypothetical protein found in Mycobacterium and Streptomyces. pepF is co-transcribed with the gene downstream (orf3), which encodes a protein that belongs to the ABC1 protein kinase family, suggesting that these two proteins may share a common function in the cell. The C. crescentus PepF protein possesses the conserved HEXGH motif present in zinc binding domains of PepF homologs. Disruption of the pepF gene by insertion of a vector sequence did not produced any growth defect, but the mutant strain possesses only 30(per cente) of the specific activity of endopeptidases present in the wild type strain. Deletions and point mutations in the regulatory region showed that there are two putative promoter regions, and the operon expression is independent of the transcription regulator CtrA. The results indicate that PepF is not essential for either growth or development of this bacterium using peptides as the sole carbon source, suggesting that other peptidases can be sharing this function. (au)