RESUMO
The cell surface expression of group 2 capsular polysaccharides involves the translocation of the polysaccharide from its site of synthesis on the inner face of the cytoplasmic membrane onto the cell surface. The transport process is independent of the repeat structure of the polysaccharide, and translocation across the periplasm requires the cytoplasmic membrane-anchored protein KpsE and the periplasmic protein KpsD. In this paper we establish the topology of the KpsE protein and demonstrate that the C terminus interacts with the periplasmic face of the cytoplasmic membrane. By chemical cross-linking we show that KpsE is likely to exist as a dimer and that dimerization is independent of the other Kps proteins or the synthesis of capsular polysaccharide. No interaction between KpsD and KpsE could be demonstrated by chemical cross-linking, although in the presence of both KpsE and Lpp, KpsD could be cross-linked to a 7-kDa protein of unknown identity. In addition, we demonstrate that KpsD is present not only within the periplasm but is also in both the cytoplasmic and outer membrane fractions and that the correct membrane association of KpsD was dependent on KpsE, Lpp, and the secreted polysaccharide molecule. Both KpsD and KpsE showed increased proteinase K sensitivity in the different mutant backgrounds, reflecting conformational changes in the KpsD and KpsE proteins as a result of the disruption of the transport process. Collectively the data suggest that the trans-periplasmic export involves KpsD acting as the link between the cytoplasmic membrane transporter and the outer membrane with KpsE acting to facilitate this transport process.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras , Periplasma/metabolismo , Proteínas Periplásmicas , Polissacarídeos/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Transporte Biológico , Primers do DNA , Endopeptidase K/metabolismoAssuntos
Proteínas de Ciclo Celular , Ciclinas/química , Ciclinas/genética , Mitose , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Schizosaccharomyces pombe , Trypanosoma brucei brucei/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Ciclinas/metabolismo , Mutação da Fase de Leitura , Proteínas Fúngicas/metabolismo , Genes de Protozoários , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
The transcriptional organization and regulation of region 1 expression of the Escherichia coli K5 capsule gene cluster were studied. Region 1 was transcribed as an 8.0-kb polycistronic mRNA which was processed to form a separate 1.3-kb transcript encoding the 3'-most gene kpsS. Transcription of region 1 of the E. coli K5 capsule gene cluster was directed from a single promoter 225 bp upstream of a previously unidentified gene, kpsF. The promoter had -35 and -10 consensus sequences typical of an E. coli sigma 70 promoter, with no similarities to binding sites for other sigma factors. Two integration host factor (IHF) binding site consensus sequences were identified 110 bp upstream and 130 bp downstream of the transcription start site. In addition, two AT-rich regions separated by 16 bp identified upstream of the region 1 promoter were conserved upstream of the region 3 promoter. The kpsF gene was 98.8% identical with the kpsF gene identified in the E. coli K1 antigen gene cluster and confirms that the kpsF gene is conserved among group II capsule gene clusters. An intragenic Rho-dependent transcriptional terminator was discovered within the kpsF gene. No essential role for KpsF in the expression of the K5 antigen could be established. The temperature regulation of region 1 expression was at the level of transcription, with no transcription detectable in cells grown at 18 degrees C. Mutations in regulatory genes known to control temperature-dependent expression of a number of virulence genes had no effect on the temperature regulation of region 1 expression. Likewise, RfaH, which is known to regulate expression of E. coli group II capsules had no effect on the expression of region 1. Mutations in the himA and himD genes which encode the subunits of the IHF led to a fivefold reduction in the expression of KpsE at 37 degrees C, confirming a regulatory role for IHF in the expression of region 1 genes.
Assuntos
Cápsulas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Transcrição Gênica , Proteínas de Bactérias/genética , Sequência de Bases , Dados de Sequência Molecular , Família Multigênica , Polissacarídeos Bacterianos/genética , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , Mapeamento por Restrição , Temperatura , Regiões Terminadoras GenéticasRESUMO
Inherited symbionts which selectively cause the death of male hosts are found widely across the Insecta. Previous studies have shown a single, but different micro-organism to be responsible for male-killing in each taxonomic group studied. We here produce evidence that within a group of insects, the Coccinellidae, there is more than one causal agent of male lethality. We report a novel observation of a male-killing trait in the species Coleomegilla maculata. Six of 26 crosses were found to produce a female-biased sex ratio associated with a low egg hatch-rate. The trait was matrilinearly inherited and was observed to be tetracycline-sensitive. However, tests which indicate the presence of a Rickettsia, previously found to cause male-killing in another member of the Coccinellidae, Adalia bipunctata, proved negative. We therefore conclude that the phenomenon of male-killing is multicausal, within, as well as between, taxonomic groups of the Insecta.
