Molecular cloning and characterization of the ftsEX genes of Neisseria gonorrhoeae CH811 encoding a putative ABC transporter and identification of their flanking genes.
|Title:||Molecular cloning and characterization of the ftsEX genes of Neisseria gonorrhoeae CH811 encoding a putative ABC transporter and identification of their flanking genes.|
|Abstract:||Cell division is an essential process in any living cell. In Escherichia coli, the majority of cell division genes are clustered at two loci on the chromosome. The ftsY, ftsE and ftsX genes constitute one of these clusters. The hypothesis of this Ph.D. research project was that the ftsY, ftsE and ftsX genes are present and clustered in N. gonorrhoeae, and that their gene products are involved in an uncharacterized aspect of cell division. The plasmid pSB19 was isolated while screening a genomic library of N. gonorrhoeae strain CH811. The analysis of the DNA sequence of the insert of pSB19 showed a complete open-reading frame (ORF) showing sequence similarity with the ftsX gene of E. coli. Two other partial ORFs respectively encoding a protein similar to 3-phosphoglycerate kinase and a protein similar E. coli FtsE were identified downstream and upstream of ftsX. The partial ftsE homologue overlapped ftsX by 4 base pairs (bp). To fully characterize the gonococcal ftsE and ftsX genes, the complete ftsE gene was required. A DNA fragment containing the 5$\sp\prime$-section of ftsE with 2.3 kilobases of sequence upstream of ftsE was amplified by inverse PCR, cloned and sequenced. The gonococcal ftsE gene comprised 651 bp and encoded a polypeptide of 216 amino acid (aa) residues. The gonococcal FtsE protein shared 60 to 71% similarity and 32 to 49% identity with other known bacterial FtsE homologues, and shared significant aa sequence similarities with numerous other ATP-binding domains of ABC transporters. The gonococcal ftsX gene included 918 bp encoding a protein of 305 aa residues that shared 47 to 55% similarity and 19 to 29% identity with its bacterial homologues, and did not share significant similarity to other protein sequences included in the public databases. Sequence analyses performed on N. gonorrhoeae FtsE and FtsX and on the five other known bacterial homologues of FtsE and FtsX predicted that FtsE did not contain transmembrane segments while FtsX was predicted to contain four of them. The size of FtsX varied between bacterial species and this variation appeared attributable to the amino-terminal section of the protein. N. gonorrhoeae FtsX was predicted to adopt a membrane topology that would locate both its amino- and carboxy-terminal ends in the cytoplasm. Identical topologies were predicted for the other known FtsX homologues. The presence of ftsE and ftsX was verified in ten other Neisseria species by Southern hybridization. ftsE and ftsX probes hybridized restriction fragments in each species, suggesting that ftsE and ftsX were present in each species tested. The 4 bp overlap ovserved between ftsE and ftsX suggested that these genes were co-transcribed. The co-transcription of ftsE and ftsX was confirmed by in vitro transcription/translation experiments. The results obtained suggest that FtsE and FtsX respectively constitute the ATP-binding and membrane domains of a putative ABC transporter that transports an unidentified substrate from the cell. It cannot be excluded that this transporter participates in the cell division process, but it appears not to be essential since a mutant in which ftsX had been disrupted was viable while other cell division genes were shown to be essential. (Abstract shortened by UMI.)|
|Collection||Thèses, 1910 - 2010 // Theses, 1910 - 2010|