RESUMO
The E. coli chromosome replication arms are polarized by motifs such as RRNAGGGS oligomers, found preferentially on leading strands. Their skew increases regularly from the origin to dif (the site in the center of the terminus where chromosome dimer resolution occurs), to reach a value of 90% near dif. Convergent information indicates that polarization in opposite directions from the dif region controls tightly the activity of dif, probably by orienting mobilization of the terminus at cell division. Another example of polarization is the presence, in the region peripheral to the terminus, of small non-divisible zones whose inversion interferes with spatial separation of sister nucleoids. The two phenomena may contribute to the organization of the Ter macrodomain.
Assuntos
Cromossomos Bacterianos , Replicação do DNA , DNA Bacteriano/metabolismo , DNA Circular/metabolismo , Escherichia coli/genética , Motivos de Aminoácidos/fisiologia , Sítios de Ligação , Inversão Cromossômica , Proteínas de Membrana/metabolismo , Modelos Genéticos , Ligação ProteicaRESUMO
Chromosome dimers form in bacteria by recombination between circular chromosomes. Resolution of dimers is a highly integrated process involving recombination between dif sites catalysed by the XerCD recombinase, cell division and the integrity of the division septum-associated FtsK protein and the presence of dif inside a restricted region of the chromosome terminus, the dif activity zone (DAZ). We analyse here how these phenomena collaborate. We show that (i) both inter- and intrachromosomal recombination between dif sites are activated by their presence inside the DAZ; (ii) the DAZ-specific activation only occurs in conditions supporting the formation of chromosome dimers; (iii) overexpression of FtsK leads to a general increase in dif recombination irrespective of dif location; (iv) overexpression of FtsK does not improve the ability of dif sites inserted outside the DAZ to resolve chromosome dimers. Our results suggest that the formation of an active XerCD-FtsK-dif complex is restricted to when a dimer is present, the features of chromosome organization that determine the DAZ playing a central role in this control.
Assuntos
Cromossomos Bacterianos , Escherichia coli/genética , Recombinação Genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Divisão Celular , Dimerização , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Recombinases Rec A/genética , Recombinases Rec A/metabolismoRESUMO
Enterococcus faecalis induces the synthesis of at least 42 proteins during 24 h of glucose starvation. Because of its induction during carbohydrate and complete starvation (incubation in tap water) and CdCl(2) and bile salts stresses, one of these proteins (Gls24) was qualified as a "general stress protein" and was analyzed at the molecular level. Its corresponding gene, gls24, seems to be the penultimate gene of an operon composed, altogether, of six open reading frames (ORFs). The ORF preceding gls24 (orf4) showed very strong identity with gls24. The deduced polypeptides of these two genes showed similarity with a 20-kDa hypothetical protein from Lactococcus lactis and an alkaline stress protein from Staphylococcus aureus with no previously known biological significance. Data from the operon sequence and Northern analysis led to the conclusions that (i) gls24 possesses its own promoter which is especially induced at the onset of starvation and (ii) the operon promoter is stress inducible in exponential-phase cells. A mutation in the gls24 gene led to a severe reduction of growth rate and reduction of survival against 0.3% bile salts in the 24-h-starved cells compared to the wild-type strain. Moreover, the chain length of the mutant is significantly reduced during growth. These results argue strongly for a role of the protein Gls24 and/or GlsB in morphological changes and in stress tolerance in E. faecalis. Comparison of two-dimensional protein gels from wild-type cells with those from gls24 mutant cells revealed a pleiotropic effect of the mutation on gene expression. At least nine proteins were present in larger amounts in the mutant. For six of them, the corresponding N-terminal microsequence has been obtained. Three of these sequences map in genes coding for L-lactate dehydrogenase, lipoamide dehydrogenase, and pyruvate decarboxylase, all involved in pyruvate metabolism.
Assuntos
Proteínas de Bactérias , Enterococcus faecalis/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Óperon , Sequência de Aminoácidos , Sequência de Bases , Cloreto de Cádmio/farmacologia , Meios de Cultura , Enterococcus faecalis/genética , Enterococcus faecalis/ultraestrutura , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/química , Dados de Sequência Molecular , Mutagênese Insercional , Fases de Leitura Aberta , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Transcrição GênicaRESUMO
The genome sequence of Enterococcus faecalis, led us to discover that gls24, encoding a general stress protein, seems to be in the second last position of a putative six-gene operon structure. Interestingly, another gene named orf4 located just upstream from gls24 shows strong identity (72%) with this last. To determine the role of the orf4 gene in E. faecalis, we have constructed a mutant strain by homologous recombination. Phenotypic analysis of these cells, reveals that Orf4 is probably involved in cell structure in stationary phase.
