RESUMO
In Neisseria gonorrhoeae, cytokinesis involves Escherichia coli homologues of minC, minD and minE which are encoded as part of a min operon. MinD, a 30 kD protein component of the MinC-MinD septum inhibitory complex, together with MinE, mediates cell division site selection. Gonococci mutated in minD display aberrant cytokinesis, abnormal morphology, defective microcolony formation and virulence. minD is 274 bp upstream of oxyR, another min operon gene in N. gonorrhoeae, which encodes a redox-responsive transcriptional regulator implicated in responses to oxidative stress. In this study, we aimed to examine the oxyR-mediated regulation of minD. We observed the cotranscription of oxyR with the minCDE gene cluster. The mutation of oxyR resulted in non-midline formation of the division septum, anomalous DNA segregation, and increased aggregation of bacterial cells. qRT-PCR and Western Blot analysis revealed upregulation of minD in an oxyR mutant as compared to its isogenic wild-type N. gonorrhoeae strain in stationary phase. Furthermore, the exposure to oxidative stress in the form of H2O2 increased MinD expression levels in wild-type N. gonorrhoeae. Using ß-galactosidase activity-based promoter assays, we found that oxyR negatively regulates the promoter region (PminD) upstream of minD. Our results demonstrate the involvement of oxyR in cell division and minD expression in N. gonorrhoeae.
Assuntos
Adenosina Trifosfatases/biossíntese , Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica , Neisseria gonorrhoeae/genética , Proteínas Repressoras/metabolismo , Western Blotting , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Peróxido de Hidrogênio/toxicidade , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/metabolismo , Neisseria gonorrhoeae/fisiologia , Estresse Oxidativo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genéticaRESUMO
The initiation of bacteriophage λ replication depends upon interactions between the oriλ DNA site, phage proteins O and P, and E. coli host replication proteins. P exhibits a high affinity for DnaB, the major replicative helicase for unwinding double stranded DNA. The concept of P-lethality relates to the hypothesis that P can sequester DnaB and in turn prevent cellular replication initiation from oriC. Alternatively, it was suggested that P-lethality does not involve an interaction between P and DnaB, but is targeted to DnaA. P-lethality is assessed by examining host cells for transformation by ColE1-type plasmids that can express P, and the absence of transformants is attributed to a lethal effect of P expression. The plasmid we employed enabled conditional expression of P, where under permissive conditions, cells were efficiently transformed. We observed that ColE1 replication and plasmid establishment upon transformation is extremely sensitive to P, and distinguish this effect from P-lethality directed to cells. We show that alleles of dnaB protect the variant cells from P expression. P-dependent cellular filamentation arose in ΔrecA or lexA[Ind-] cells, defective for SOS induction. Replication propagation and restart could represent additional targets for P interference of E. coli replication, beyond the oriC-dependent initiation step.
Assuntos
Bacteriófago lambda/metabolismo , Proteínas Virais/metabolismo , Alelos , Bacteriófago lambda/genética , Bacteriófago lambda/crescimento & desenvolvimento , Replicação do DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Ordem dos Genes , Genes Letais , Teste de Complementação Genética , Mutação , Fenótipo , Plasmídeos/genética , Resposta SOS em Genética , Transativadores/genética , Transativadores/imunologia , Transativadores/metabolismo , Transformação Bacteriana , Proteínas Virais/genética , Proteínas Virais/imunologia , Replicação ViralRESUMO
Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, p(O) is required for IP, as are iterons ITN3-4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop(+)oriλ(+) sequence.