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Marta Perego

Publications and source records attributed to Marta Perego.

7 recordsLinked to original sources
PubMed2003

TPR-mediated interaction of RapC with ComA inhibits response regulator-DNA binding for competence development in Bacillus subtilis.

The Bacillus subtilis Rap family of proteins are characterized by protein-protein interaction modules containing the so-called tetratricopeptide repeats (TPRs). The six TPR motifs of RapC mediate its interaction with the pentapeptide inhibitor PhrC (ERGMT) or with its target protein ComA, a phosphorylation-dependent response regulator transcription factor for genetic competence. Our results show that RapC interaction with ComA inhibits the response regulator's ability to bind its target DNA promoter but does not affect its phosphorylation state. RapC binds equally well to ComA or to ComA approximately P. The PhrC pentapeptide binds to RapC and inhibits its interaction with ComA. The D195 residue in TPR3 and the P263 residue in TPR5 of RapC are critical for the interaction with PhrC as their mutation to asparagine or leucine, respectively, prevents peptide inhibitory activity. The RapC mechanism of regulating ComA activity is a new example of how TPR motifs and their structural organization have been adapted for different specific functions within the B. subtilis Rap family.

Amino Acid Motifs↗
PubMed2003

Molecular analysis of Phr peptide processing in Bacillus subtilis.

In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins. Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development. The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F approximately P intermediate response regulator of the sporulation phosphorelay. The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation. The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form. The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme. In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide. This processing event is most likely independent of type I signal peptidase activity. In vivo and in vitro analyses indicate that none of the five signal peptidases of B. subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing. However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth.

Bacillus subtilis↗
PubMed2002

Biochemical characterization of aspartyl phosphate phosphatase interaction with a phosphorylated response regulator and its inhibition by a pentapeptide.

The RapA and RapB proteins are aspartyl phosphate phosphatases that specifically dephosphorylate the Spo0F approximately P intermediate response regulator of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis. The approximately 48-kDa His-tag derivative proteins were purified by metal affinity chromatography, and their molecular and biochemical characteristics were studied. RapA and RapB were found to be dimers in solution. Enzymatic activity was strongly dependent upon maintaining reducing conditions during purification and storage. RapA phosphatase activity on Spo0F approximately P is inhibited in vivo by a pentapeptide generated from the phrA gene. Native gel assays demonstrated that the RapA dimer forms a stable complex with two molecules of Spo0F approximately P or with its PhrA pentapeptide inhibitor. The pentapeptide was shown to displace Spo0F approximately P from a preformed complex with RapA. The structural organization of Rap phosphatases in tetratricopeptide repeats provides insights on the mechanisms of RapA interaction with its substrate and its inhibitor.

Base Sequence↗
PubMed2002

Interaction surface of the Spo0A response regulator with the Spo0E phosphatase.

Spo0A~P is the essential response regulator and transcription factor for sporulation initiation in Bacillus subtilis. The phosphorylation level of Spo0A in the cell is determined by the sensor kinase activity of the phosphorelay, donating phosphoryl groups, and the antagonistic effects of dephosphorylation mediated by the Rap and Spo0E families of phosphatases. In this study, spo0A mutations were generated that encoded proteins less sensitive to the activity of Spo0E than the wild-type protein. The Spo0A substitutions N12K, P60S, L62P and F88L are surface exposed and localize to the same face of the molecule as the active site and in its close proximity on the beta1-alpha1, beta3-alpha3 and beta4-alpha4 loops. The corresponding surface in the Spo0F response regulator was shown previously to be involved in the interaction with the RapB phosphatase, as well as the KinA histidine kinase and the Spo0B phosphotransferase. Thus, residues occupying the same position (N12:Q12, F88:Y84) and the same loops in Spo0A or Spo0F are involved in the interaction with the structurally unrelated Spo0E and RapB phosphatases, respectively, in addition to kinases and phosphotransferase. The specificity in phosphatase target recognition must be the result of side-chain variability within the response regulators and the interactions they promote. The residues involved in Spo0E interaction are identical in all Spo0A orthologues from spore-forming Bacilli encoding Spo0E phosphatases.

Amino Acid Sequence↗
PubMed2002

Overexpression of the PepF oligopeptidase inhibits sporulation initiation in Bacillus subtilis.

The yjbG gene encoding the homologue of the PepF1 and PepF2 oligoendopeptidases of Lactococcus lactis (Monnet et al., J. Biol. Chem. 269:32070-32076, 1994; Nardi et al., J. Bacteriol. 179:4164-4171, 1997) has been identified in Bacillus subtilis as an inhibitor of sporulation initiation when present in the cells on a multicopy plasmid. Genetic analysis suggested that the inhibitory effect is due to hydrolysis of the PhrA peptide in a form as small as the pentapeptide (ARNQT). Inactivation of PhrA results in deregulation of the RapA phosphatase and thus dephosphorylation of the Spo0F approximately P response regulator component of the phosphorelay for sporulation initiation. When overexpressed, the B. subtilis PepF is most likely hydrolyzing additional peptides of the Phr family, as is the case for PhrC involved in control of competence development. Chromosomal inactivation of the yjbG/pepF gene did not give rise to any detectable phenotype. The function of PepF in B. subtilis remains unknown. Limited experiments with a yjbG paralogue called yusX indicated that a frameshift is present, making the corresponding gene product inactive.

ATP-Binding Cassette Transporters↗
PubMed2002

Characterization of the parB-like yyaA gene of Bacillus subtilis.

We have characterized the yyaA gene of Bacillus subtilis, located near the origin of chromosome replication (oriC). Its protein product is similar to the Spo0J protein, which belongs to the ParB family of chromosome- and plasmid-partitioning proteins. Insertional inactivation of the yyaA gene had no apparent effect on chromosome organization and partitioning during vegetative growth or sporulation. Subcellular localization of YyaA by immunofluorescence microscopy indicated that it colocalizes with the nucleoid, and gel retardation studies confirmed that YyaA binds relatively nonspecifically to DNA. Overexpression of yyaA caused a sporulation defect characterized by the formation of multiple septa within the cell. This phenotype indicates that YyaA may have a regulatory role at the onset of sporulation.

Bacillus subtilis↗