Sequencing and characterization of gene AlkB Caulobacter crescentus

Author(s)

Débora Colombi

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 1995-09-13

Examining board members

Suely Lopes Gomes; Roberto Vicente Santelli; José Franco da Silveira Filho

• Advisor: Suely Lopes Gomes

Abstract

Caulobacter crescentus is a gram-negative bacterium that yields two different progeny cells at each division cycle, the swarmer cell and the stalked cell, which differ in structure, developmental program and ability to replicate DNA. The alkB gene in Caulobacter crescentus was identified in a clone which contains also the heat shock genes dnaK and dnaJ (Gomes et al, 1990). In Escherichia coli the alkB gene has been shown to be involved, with alkA and ada, in the pathway for repair of DNA alkylation damage (Teo et al, 1986). The E. coli alkB gene forms an operon with the ada gene, being downstream of it. The addition of the alkylating agent MMS to E. coli cultures induces the expression of both ada and alkB, at the level of transcription. The aminoacid sequence deduced from the nucleotide sequence of the C. crescentus alkB gene has shown 42,5% identity with the AlkB protein of E. coli (Kondo et al, 1986). Contrary to what happens in E. coli, the alkB gene of Caulobacter cescentus does not form an operon with the ada gene and its expression is not induced by alkylating agents. S1 nuclease protection assays have shown the presence of a single start of transcription for the alkB gene. To investigate the expression of the alkB gene during the cell cycle of the bacterium, its regulatory region was fused to a promoterless lacZ gene present in the vector placZJ290 (Gober & Shapiro, 1992), and β-galactosidase activity and synthesis, directed by the alkB gene promoter, were determined. It was observed that expression of the alkB gene is cell cycle controlled in Caulobacter. The placZJ290 transcriptional fusion with alkB, expressed in predivisional cells did not display a polar pattern of segregation in Caulobacter crescentus. β-galactosidase synthesized in predivisional cell was partitioned in equal amounts to both cell types after cell division. An adaptive response to alkylation damage has been demonstrated in C. crescentus. The response was detected by the increased resistance to cell killing of pretreated cells with low doses of methylating agents, followed by exposure to higher doses of these agents, and by cross-reactivity of an inducible protein of 39 kDa with E. coli anti-Ada monoclonal antibodies. (AU)