Competence repression under oxygen limitation through the two-component MicAB signal-transducing system in Streptococcus pneumoniae and involvement of the PAS domain of MicB

Abstract

In Streptococcus pneumoniae, a fermentative aerotolerant and catalase-deficient human pathogen, oxidases with molecular oxygen as substrate are important for virulence and for competence. The signal-transducing two-component systems CiaRH and ComDE mediate the response to oxygen, culminating in competence. In this work we show that the two-component MicAB system, whose MicB kinase carries a PAS domain, is also involved in competence repression under oxygen limitation. Autophosphorylation of recombinant MicB and phosphotransfer to recombinant MicA have been demonstrated. Mutational analysis and in vitro assays showed that the C-terminal part of the protein and residue L100 in the N-terminal cap of its PAS domain are both crucial for autokinase activity in vitro. Although no insertion mutation in micA was obtained, expression of the mutated allele micA59DA did not change bacterial growth and overcame competence repression under microaerobiosis. This was related to a strong instability of MicA59DA-PO(4) in vitro. Thus, mutations which either reduced the stability of MicA-PO(4) or abolished kinase activity in MicB were related to competence derepression under microaerobiosis, suggesting that MicA-PO(4) is involved in competence repression when oxygen becomes limiting. The micAB genes are flanked by mutY and orfC. MutY is an adenine glycosylase involved in the repair of oxidized pyrimidines. OrfC shows the features of a metal binding protein. We did not obtain insertion mutation in orfC, suggesting its requirement for growth. It is proposed that MicAB, with its PAS motif, may belong to a set of functions important in the protection of the cell against oxidative stress, including the control of competence.

FIG. 1

Organization of the mutY-micAB-orfC locus with the conserved modules in MicA and MicB and strategy for producing the recombinant proteins. (A) Gene organization with significant conserved motifs in MicA, MicB, and OrfC. RD, receiver domain; HTH, helix turn helix; TM, transmembrane domain; HK, histidine kinase; MβL, metallo-β-lactamase. (B) Site-specific mutagenesis of MicA and MicB. (C) Cloning strategy for the nucleotide sequence determination of micAB and orfC and the expression of recombinant proteins in E. coli (the sizes of the recombinant proteins were calculated with the addition of 2.5 kDa for the His tag). (D) Sequence alignment of candidate PAS domains in MicB orthologues from B. subtilis (YYCG Bacsu, GenBank no. D78193), L. lactis (L1KINC Llact, GenBank no. AF178425), S. aureus (YYCG Staur, GenBank no. AF136709), FixL from S. meliloti (FIXL Rhime, GenBank no. J03174), and the PAS consensus sequence (http://smart.embl-heidelberg.de/). Identical residues are in bold. Residues chosen as mutagenesis targets are underlined.

FIG. 2

Effect of the micB100LR and micB::km mutations on competence control by O₂. Strains Cp1015 (A), Cp7003 (B), and Cp7002 (C) were grown under aerobiosis or microaerobiosis. Aliquots were withdrawn at 30-min intervals and analyzed for change in biomass by determinations of the optical density (OD) at 400 nm (black bars), transformability (grey bars), or by Northern blotting. For Northern blotting, a DNA probe specific for comE (see Materials and Methods) was used to detect comCDE transcripts. We used 16S rRNA as a qualitative and quantitative internal control. Quantitative densitometry of 16S rRNA gave less than 25% variability between samples taken throughout the period of growth for a given culture. The experiment was repeated with independent cultures to test reproducibility. The columns are aligned with the corresponding signals in the Northern blots.

FIG. 3

Effect of the micA59DA mutation on competence control by O₂. Strains Cp7009 and Cp1015 were grown under aerobiosis or microaerobiosis. Aliquots of cultures were withdrawn at 30-min intervals and analyzed as described in the legend to Fig. 1. For Northern blotting, probes specific for comE and micB (see Materials and Methods) were used to detect comCDE and micAB RNA, respectively. The signal for 16S rRNA differed by less than 24% between wells. The experiment was repeated with independent cultures to check reproducibility.

FIG. 4

Purification of MicA and MicB* recombinant proteins from E. coli extracts. Cultures of strain TOP10 of E. coli producing the recombinant pneumococcal proteins (see Fig. 1) were induced by IPTG and lysed, and the recombinant proteins were purified as described in Materials and Methods. The proteins were analyzed by SDS-PAGE, and the gels were stained with Coomassie blue. Lane M, molecular mass standard; lanes 1 and 6, total protein from IPTG-induced cultures of E. coli TOP10 containing pTrcHis2A; lanes 2, 4, 7, 9, and 11, total protein from IPTG-induced bacteria containing pPMAE, pPMAE59, pPMBE1, pPMBE100, and pPMBEN, respectively; lanes 3, 5, 8, 10, and 12, purified MicA, MicA59DA, MicB*, MicB*100LR, and MicB*-N, respectively.

FIG. 5

MicB* autokinase activity lies in the C-terminal part of the protein and is under the control of PAS. MicB*, MicB*-N, and MicB*100LR were autophosphosphorylated with [γ-³²P]ATP at 23°C in 48 μl of phosphorylation buffer (see Materials and Methods) containing 12 pmol of purified protein. At intervals, 16-μl aliquots were mixed with 2× loading buffer and run on SDS-12% PAGE gels for analysis as described in Materials and Methods. (C) Control in which [α-³²P]/ATP replaced [γ-³²P]/ATP in the reaction mixture containing MicB*.

FIG. 6

Phosphotransfer from MicB-PO₄ to MicA and MicA59DA and properties of the phosphorylated proteins. (A) The MicA and MicA59DA proteins (9 μg corresponding to 300 pmol) were added to the MicB* phosphorylation mixture at 240 s, and aliquots were analyzed at intervals. Ct, control sample containing MicA in the absence of MicB*. (B) MicA-PO₄ and MicA59DA-PO₄ were incubated with 0.1 N HCl or 0.1 N NaOH for 20 min at 23°C, and proteins were analyzed and compared to the control incubated with H₂O. (C) MicA-PO₄ and MicA59DA-PO₄ were incubated at 23°C in phosphorylation buffer supplemented with 10 mM ATP. Aliquots were analyzed at intervals. Proteins were subjected to electrophoresis in 12% polyacrylamide gels. Signals on autoradiographs were quantified by densitometry (see Materials and Methods) and are expressed in arbitrary units. The bars are aligned with the corresponding signals.