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.In the catalase-negative pathogen Streptococcus pneumoniae, which has essentially fermentative metabolism, oxygen limitation in a microaerobic atmosphere abolishes developmental competence. Nox, an NADH oxidase that produces water by reducing O 2 as it recycles NADH, has been shown to contribute to competence regulation by oxygen (3,8). Studies of oxygenindependent mutant strains demonstrated the involvement of the two-component systems (TCSs) CiaRH and ComDE in this regulation (7,8). To characterize in more detail the regulatory network facilitating bacterial adaptation to oxygen availability, we searched for amino acid sequences corresponding to motifs putatively involved in O 2 and redox sensing, in the publicly available pneumococcal genome sequence (http://www .tigr.org).The PAS domain may perceive cell energetic status by sensing oxygen, redox potential, ligands, proton motive force, and light (22; for review, see reference 25). PAS domains have been found in bacterial, archaeal, and eukaryotic proteins. Redox sensing and the corresponding signal transduction via twocomponent systems carrying PAS domains is one strategy used by bacteria and archaea for adaptation to variations in ambient oxygen concentration (4). PAS domains are frequently found upstream from the kinase transmitter domain. A heme-containing domain...

Defects in ex vivo and in vivo growth and sensitivity to osmotic stress of group A Streptococcus caused by interruption of response regulator gene vicR

“…Nevertheless, the essential processes regulated by VicRK in these organisms remain unknown. Defects in morphology and cell wall synthesis, decreased competence, sensitivity to antibiotics and fatty acids, attenuated virulence, and defects in biofilm formation have been associated with null vicK mutants or conditional mutants under non-functional conditions for VicR (Martin et al, 1999;Echenique & Trombe, 2001;Kadioglu et al, 2003;Ng et al, 2004;Senadheera et al, 2005). Some of these phenotypes appear to be organism-specific; however, no studies on GAS VicRK have been reported.…”

“…VicR is essential in Bacillus subtilis (Fabret & Hoch, 1998), Staphylococcus aureus (Martin et al, 1999) and Streptococcus pneumoniae (Lange et al, 1999;Throup et al, 2000), and vicR cannot be inactivated unless VicR is provided in trans to these organisms (Throup et al, 2000) or PcsB is constitutively expressed in S. pneumoniae (Ng et al, 2003). VicK is also essential in B. subtilis, but not in S. pneumoniae and Streptococcus mutans (Fabret & Hoch, 1998;Echenique & Trombe, 2001;Senadheera et al, 2005). Since an unconditional vicR mutant is not available, various strategies, including bioinformatics, construction of hybrid regulator, depletion and overexpression of VicRK, and vicK inactivation, have been used to identify putative targets of VicRK (Fukuchi et al, 2000;Howell et al, 2003;Ng et al, 2003;Durac & Msadek, 2004;Mohedano et al, 2005;Senadheera et al, 2005).…”

Identification of Genes Controlled by the Essential YycG/YycF Two-Component System ofStaphylococcus aureus

“…Previous studies on the YycG/YycF system have shown that it is involved in competence development and virulence in S. pneumoniae (10,42). In S. aureus, the YycG/YycF system is thought to play a role in membrane permeability, and a temperature-sensitive mutant of S. aureus yycF exhibited attenuated virulence in an in vivo model (27).…”

Inactivation of the ciaH Gene in Streptococcus mutans Diminishes Mutacin Production and Competence Development, Alters Sucrose-Dependent Biofilm Formation, and Reduces Stress Tolerance

“…One of the major effects of ciaRH mutations in Streptococcus pneumoniae occurs with competence development (8,10,23). Insertional inactivation of ciaR and ciaH results in derepression of competence both in aerobic and microaerobic cultures (7).…”