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Evidence that the Essential Response Regulator YycF in Streptococcus pneumoniae Modulates Expression of Fatty Acid Biosynthesis Genes and Alters Membrane Composition

AuthorsMohedano Bonillo, Mari Luz CSIC ORCID ; Overweg, Karin; Fuente, Alicia de la; Reuter, Mark; Altabe, Silvia; Mulholland, Francis; Mendoza, Diego de; López, Paloma CSIC ORCID ; Wells, Jerry M.
Issue Date2005
PublisherAmerican Society for Microbiology
CitationJournal of Bacteriology, 187(7):2357-2367 (2005)
AbstractThe YycFG two-component system, originally identified in Bacillus subtilis, is highly conserved among gram-positive bacteria with low G+C contents. In Streptococcus pneumoniae, the YycF response regulator has been reported to be essential for cell growth, but the signal to which it responds and the gene members of the regulon remain unclear. In order to investigate the role of YycFG in S. pneumoniae, we increased the expression of yycF by using a maltose-inducible vector and analyzed the genome-wide effects on transcription and protein expression during the course of yycF expression. The induction of yycF expression increased histidine kinase yycG transcript levels, suggesting an autoregulation of the yycFG operon. Evidence from both proteomic and microarray transcriptome studies as well as analyses of membrane fatty acid composition indicated that YycFG is involved in the regulation of fatty acid biosynthesis pathways and in determining fatty acid chain lengths in membrane lipids. In agreement with recent transcriptome data on pneumococcal cells depleted of YycFG, we also identified several other potential members of the YycFG regulon that are required for virulence and cell wall biosynthesis and metabolism. Two-component signal transduction systems (TCSs) seem to be a fundamental constituent of the regulatory organization in bacteria, and they frequently control the expression of virulence factors and adaptive responses (12; reviewed in reference 3). TCSs typically comprise a histidine kinase (HK) in the membrane that senses an environmental stimulus and a cognate response regulator (RR) in the cytoplasm that controls gene expression through binding to DNA promoter sequences. Signal transduction between the sensor protein and the response regulator is achieved by phosphoryl transfer from a histidine residue in the conserved histidine kinase catalytic site to an aspartate residue in the N-terminal receiver domain of the response regulator. Modulation of the phosphorylation state of the RR by the HK is the basis for their effects on the control of gene expression (23). In the genome of the human pathogen Streptococcus pneumoniae, 13 TCSs have been identified, and among them, only YycFG is essential for this bacterium (27, 41). YycFG homologues are found in several gram-positive pathogens (16), and in S. pneumoniae, this system has been designated MicAB (14), VicRK (42), and 492hkrr (41), but it is referred to here as YycFG.
Gene knockout studies of the 34 TCSs in B. subtilis showed that YycFG is the only essential TCS. Both the yycF and yycG genes are required for viability (16), as is also the case for Staphylococcus aureus (29). However, for S. pneumoniae, only the response regulator yycF gene was found to be essential (27, 41), suggesting that YycF functions independently of the adjacent histidine kinase YycG, possibly participating in signal transduction with other TCS histidine kinases. Although the role of YycFG remains unclear, these proteins may have relevance as novel targets for antimicrobial intervention (38). In S. aureus, a point mutation in the yycF gene conferred temperature-dependent sensitivity to several macrolide and lincosamide antibiotics and also sensitivity to unsaturated long-chain fatty acids, suggesting that this TCS plays a role in cell permeability or the regulation of cell wall and membrane composition (29). In B. subtilis, at a nonpermissive temperature, a conditional null mutant of yycFG formed chains of cells, some of them lacking cytoplasmic contents (16), whereas the overexpression of YycF led to the production of minicells and a reduction in the cell length as well as a transcriptional induction of the ftsAZ cell division operon (17). Further clues that YycF is important for cell division were provided by a hybrid regulator/transcriptome approach in B. subtilis (24). This involved the construction of chimeric response regulators by fusing the receiver domain of a regulator of known function with the DNA binding domain of a regulator of unknown function. Thus, microarray analysis under low-phosphate conditions revealed that a gene (yocH) encoding a potential autolysin was induced more than fourfold in the presence of the hybrid PhoP-YycF regulator. The identification of the consensus recognition sequence for YycF, consisting of two hexanucleotide direct repeats separated by five nucleotides, enabled 10 genes involved in cell division, cell wall metabolism, and a membrane-bound transport system to be identified as potential members of the YycFG regulon in B. subtilis. Similarly, for S. aureus, 12 genes potentially regulated by YycF were identified, including the gene for LytM (a peptidoglycan hydrolase), and binding of YycF to this motif was confirmed in vitro (13). However, none of these genes in S. aureus are known to be essential. The functions controlled by YycFG in S. pneumoniae are also not fully understood. Mutation of the putative phosphorylation site in the receiver domain of YycF substantially reduced the stability of YycF-PO4 in vitro, although a clone carrying this mutation did not show altered growth properties compared to the wild-type strain (14). As stated above, the YycG histidine kinase is not essential in S. pneumoniae, although a recent study (32) showed that YycG is conditionally required for growth when levels of YycF are reduced by means of a regulatable promoter. In that study, a microarray analysis of mutants with reduced expression of the YycFG TCS showed an altered expression of 49 genes in 10 clusters, encoding proteins of unknown function or proteins involved in transport, heat shock, the cell surface, carbohydrate metabolism, and amino acid biosynthesis. Of these, pcsB, encoding a possible extracellular cell wall hydrolase which is also found in other streptococci, may be the essential target of YycF in S. pneumoniae, as its constitutive expression suppressed the essentiality of yycF.
It was recently shown through a DNA microarray analysis of B. subtilis two-component regulatory systems that overproduction of the response regulator of a two-component system affects the regulation of genes controlled by the system (35). In order to further elucidate the role of YycFG in S. pneumoniae, we increased the expression of yycF by using a maltose-inducible vector, and here we describe the effects on transcription, protein expression, and fatty acid membrane composition, as determined by the use of DNA microarrays, proteomics, and gas chromatography-mass spectrometry
Description11 páginas, 4 figuras, 3 tablas -- PAGS NROS. 2357-2367
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