Molecular characterization of the essential response regulator protein YycF in Bacillus subtilis

The response regulator YycF is essential for cell growth in gram-positive bacteria including Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumoniae. To study the function of YycF in the essential process, we characterized a YycF (H215P) mutation that caused temperature-sensitive growth in B. subtilis. The response regulators YycF and YycF (H215P) were analyzed using circular dichroism spectroscopy, whose T(m) values were 56.0 and 45.9 degrees C, respectively, suggesting that YycF (H215P) significantly affects the protein structure with an increase in temperature. Furthermore, using the gel mobility shift assay and DNase I footprinting, we investigated the effect of YycF (H215P) on binding to the YycF box of ftsAZ operon of B. subtilis. The replacement of the histidine 215 with proline resulted in a decrease of the DNA-binding ability of YycF in vitro. In vivo, using Escherichia coli two-hybrid and homodimerization assays, we clarified that His 215 of YycF plays a crucial role in the homodimerization of the protein. Thus the essential genes involved in growth of B. subtilis appear to be regulated by the homodimer of YycF. These results suggest that the YycF dimerization is an excellent target for the discovery of novel antibiotics.     

Biochemical characterization of the first essential two-component signal transduction system from Staphylococcus aureus and Streptococcus pneumoniae

The YYCFG two-component signal transduction system (TCSTS) has been shown to be essential to the viability of several gram-positive bacteria. However, the function of the gene pair remains unknown. Interestingly, while both components are essential to Staphylococcus aureus and Bacillus subtilis, only the response regulator (YYCF) is essential to Streptococcus pneumoniae. To study this essential TCSTS further, the S. pneumoniae and S. aureus truncated YycG histidine kinase and full-length YycF response regulator proteins were characterized at a biochemical level. The recombinant proteins from both organisms were expressed in Escherichia coli and purified. The YycG autophosphorylation activities were activated by ammonium. The apparent K(m )(ATP) of S. aureus YycG autophosphorylation was 130 microM and S. pneumoniae was 3.0 microM. Each had similar K(cat )values of 0.036 and 0.024 min(-1), respectively. Cognate phosphotransfer was also investigated indicating different levels of the phosphorylated YycG intermediates during the reaction. The S. pneumoniae YycG phosphorylated intermediate was not detectable in the presence of its cognate YycF, while phosphorylated S. aureus YycG and YycF were detected concurrently. In addition, noncognate phosphotransfer was demonstrated between the two species. These studies thoroughly compare the essential YycFG TCSTS from the two species at the biochemical level and also establish methods for assaying the activities of these antibacterial targets.     

Interactions between the YycFG and PhoPR two-component systems in Bacillus subtilis: the PhoR kinase phosphorylates the non-cognate YycF response regulator upon phosphate limitation

Two-component signal transduction systems (TCS) are an important mechanism by which bacteria sense and respond to their environment. Although each two-component system appears to detect and respond to a specific signal(s), it is now evident that they do not always act independently of each other. In this paper we present data indicating regulatory links between the PhoPR two-component system that participates in the cellular response to phosphate limitation, and the essential YycFG two-component system in Bacillus subtilis. We show that the PhoR sensor kinase can activate the YycF response regulator during a phosphate limitation-induced stationary phase, and that this reaction occurs in the presence of the cognate YycG sensor kinase. Phosphorylation of YycF by PhoR also occurs in vitro, albeit at a reduced level. However, the reciprocal cross-phosphorylation does not occur. A second level of interaction between PhoPR and YycFG is indicated by the fact that cells depleted for YycFG have a severely deficient PhoPR-dependent phosphate limitation response and that YycF can bind directly to the promoter of the phoPR operon. YycFG-depleted cells neither activate expression of phoA and phoPR nor repress expression of the essential tagAB and tagDEF operons upon phosphate limitation. This effect is specific to the PhoPR-dependent phosphate limitation response because PhoPR-independent phosphate limitation responses can be initiated in YycFG-depleted cells.     

肺炎链球菌pcsB组成型表达的yycF缺陷菌株的构建及其致病能力

【背景】YycFG双组分系统是肺炎链球菌(Streptococcus pneumoniae,S. pn)应对外界环境的重要信息传递系统,其中表达反应调节子YycF的编码基因是肺炎链球菌生长的必需基因,但其是否调控细菌毒力尚不清楚。【目的】构建肺炎链球菌pcsB组成型表达及yycF缺陷菌,分析YycF对肺炎链球菌生物学特征和毒力的影响。【方法】采用Janus cassette (JC)反选的方法构建pcsB组成型表达菌株(Pc-PcsB~+),从该菌株出发用替代失活的方法构建yycF缺陷菌株(Pc-PcsB~+DyycF),比较野生株D39rpsl41、pcsB组成型表达株及yycF缺陷株的生长特性、荚膜多糖(capsular polysaccharide,CPS)含量、粘附侵袭能力和致病性的差异。【结果】成功构建pcsB组成型表达的yycF缺陷菌株(Pc-PcsB+DyycF);yycF缺陷导致细菌生长缓慢、分裂异常、胞内荚膜多糖和小分子荚膜多糖增多;体外实验结果显示,yycF缺陷菌株粘附能力较Pc-PcsB~+菌株减弱(P=0.006)。体内毒力实验显示,感染野生菌的小鼠全部死亡,感染Pc-PcsB+和Pc-PcsB~+DyycF菌株的小鼠死亡率分别为91.7%、75%,二者没有统计学差异(P=0.183),但Pc-PcsB~+DyycF菌株感染组有降低趋势;定殖结果显示,yycF缺陷菌株感染组的肺匀浆菌载量显著低于对照组(P=0.033)。【结论】成功构建yycF缺陷菌株,并初步证明yycF基因会影响肺炎链球菌的生物性状和致病能力,为后续探讨YycFG双组分系统对肺炎链球菌致病能力调控机制的研究奠定了基础。     

YycH regulates the activity of the essential YycFG two-component system in Bacillus subtilis

Of the numerous two-component signal transduction systems found in bacteria, only a very few have proven to be essential for cell viability. Among these is the YycF (response regulator)-YycG (histidine kinase) system, which is highly conserved in and specific to the low-G+C content gram-positive bacteria. Given the pathogenic nature of several members of this class of bacteria, the YycF-YycG system has been suggested as a prime antimicrobial target. In an attempt to identify genes involved in regulation of this two-component system, a transposon mutagenesis study was designed to identify suppressors of a temperature-sensitive YycF mutant in Bacillus subtilis. Suppressors could be identified, and the prime target was the yycH gene located adjacent to yycG and within the same operon. A lacZ reporter assay revealed that YycF-regulated gene expression was elevated in a yycH strain, whereas disruption of any of the three downstream genes within the operon, yycI, yycJ, and yycK, showed no such effect. The concentrations of both YycG and YycF, assayed immunologically, remained unchanged between the wild-type and the yycH strain as determined by immunoassay. Alkaline phosphatase fusion studies showed that YycH is located external to the cell membrane, suggesting that it acts in the regulation of the sensor domain of the YycG sensor histidine kinase. The yycH strain showed a characteristic cell wall defect consistent with the previously suggested notion that the YycF-YycG system is involved in regulating cell wall homeostasis and indicating that either up- or down-regulation of YycF activity affects this homeostatic mechanism.     

Identification of genes controlled by the essential YycG/YycF two-component system of Staphylococcus aureus

The YycG/YycF essential two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low-G+C gram-positive bacteria, including several pathogens such as Staphylococcus aureus. By studying growth of S. aureus cells where the yyc operon is controlled by an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter, we have shown that this system is essential in S. aureus during growth at 37 degrees C and that starvation for the YycG/YycF regulatory system leads to cell death. During a previous study of the YycG/YycF TCS of B. subtilis, we defined a potential YycF consensus recognition sequence, consisting of two hexanucleotide direct repeats, separated by five nucleotides [5'-TGT(A/T)A(A/T/C)-N(5)-TGT(A/T)A(A/T/C)-3']. A detailed DNA motif analysis of the S. aureus genome indicates that there are potentially 12 genes preceded by this sequence, 5 of which are involved in virulence. An in vitro approach was undertaken to determine which of these genes are controlled by YycF. The YycG and YycF proteins of S. aureus were overproduced in Escherichia coli and purified. Autophosphorylation of the YycG kinase and phosphotransfer to YycF were shown in vitro. Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified YycF to the promoter region of the ssaA gene, encoding a major antigen and previously suggested to be controlled by YycF. YycF was also shown to bind specifically to the promoter regions of two genes, encoding the IsaA antigen and the LytM peptidoglycan hydrolase, in agreement with the proposed role of this system in controlling virulence and cell wall metabolism.     

Systematic inactivation and phenotypic characterization of two-component signal transduction systems of Enterococcus faecalis V583

The ability of enterococci to adapt and respond to different environmental stimuli, including the host environment, led us to investigate the role of two-component signal transduction in the regulation of Enterococcus faecalis physiology. Using a bioinformatic approach, we previously identified 17 two-component systems (TCS), consisting of a sensory histidine kinase and the cognate response regulator, as well as an additional orphan response regulator (L. E. Hancock and M. Perego, J. Bacteriol. 184:5819-5825, 2002). In an effort to identify the potential function of each TCS in the biology of E. faecalis clinical isolate strain V583, we constructed insertion mutations in each of the response regulators. We were able to inactivate 17 of 18 response regulators, the exception being an ortholog of YycF, previously shown to be essential for viability in a variety of gram-positive microorganisms. The biological effects of the remaining mutations were assessed by using a number of assays, including antibiotic resistance, biofilm formation, and environmental stress. We identified TCS related to antibiotic resistance and environmental stress and found one system which controls the initiation of biofilm development by E. faecalis.     

Purification and activity testing of the full-length YycFGHI proteins of Staphylococcus aureus

Background

The YycFG two-component regulatory system (TCS) of Staphylococcus aureus represents the only essential TCS that is almost ubiquitously distributed in Gram-positive bacteria with a low G+C-content. YycG (WalK/VicK) is a sensor histidine-kinase and YycF (WalR/VicR) is the cognate response regulator. Both proteins play an important role in the biosynthesis of the cell envelope and mutations in these proteins have been involved in development of vancomycin and daptomycin resistance.

Methodology/Principal Findings

Here we present high yield expression and purification of the full-length YycG and YycF proteins as well as of the auxiliary proteins YycH and YycI of Staphylococcus aureus. Activity tests of the YycG kinase and a mutated version, that harbours an Y306N exchange in its cytoplasmic PAS domain, in a detergent-micelle-model and a phosholipid-liposome-model showed kinase activity (autophosphorylation and phosphoryl group transfer to YycF) only in the presence of elevated concentrations of alkali salts. A direct comparison of the activity of the kinases in the liposome-model indicated a higher activity of the mutated YycG kinase. Further experiments indicated that YycG responds to fluidity changes in its microenvironment.

Conclusions/Significance

The combination of high yield expression, purification and activity testing of membrane and membrane-associated proteins provides an excellent experimental basis for further protein-protein interaction studies and for identification of all signals received by the YycFGHI system.     

Aspartate 141 is the fourth ligand of the oxygen-sensing [4Fe-4S]2+ cluster of Bacillus subtilis transcriptional regulator Fnr

The Bacillus subtilis redox regulator Fnr controls genes of the anaerobic metabolism in response to low oxygen tension. An unusual structure for the oxygen-sensing [4Fe-4S](2+) cluster was detected by a combination of genetic experiments with UV-visible and M?ssbauer spectroscopy. Asp-141 was identified as the fourth iron-sulfur cluster ligand besides three Cys residues. Exchange of Asp-141 with Ala abolished functional in vivo complementation of an fnr knock-out strain by the mutagenized fnr gene and in vitro DNA binding of the recombinant regulator FnrD141A. In contrast, substitution of Asp-141 with Cys preserved [4Fe-4S](2+) structure and regulator function.     

Tandem DNA binding of E. coli's transactivator PhoB

In this issue of Structure, Blanco et al. describe the first structure of a two-component response regulator effector domain bound to its target DNA, showing novel tandem binding to successive direct repeat sequences of pho boxes from the phoA operon promotor.     

Crystal Structure of the Pneumococcal Vancomycin-Resistance Response Regulator DNA-Binding Domain

Vancomycin response regulator (VncR) is a pneumococcal response regulator of the VncRS two-component signal transduction system (TCS) of Streptococcus pneumoniae. VncRS regulates bacterial autolysis and vancomycin resistance. VncR contains two different functional domains, the N-terminal receiver domain and C-terminal effector domain. Here, we investigated VncR C-terminal DNA binding domain (VncRc) structure using a crystallization approach. Crystallization was performed using the micro-batch method. The crystals diffracted to a 1.964 _Å resolution and belonged to space group P212121. The crystal unit-cell parameters were a = 25.71 _Å, b = 52.97 _Å, and c = 60.61 _Å. The structure of VncRc had a helix-turn-helix motif highly similar to the response regulator PhoB of Escherichia coli. In isothermal titration calorimetry and size exclusion chromatography results, VncR formed a complex with VncS, a sensor histidine kinase of pneumococcal TCS. Determination of VncR structure will provide insight into the mechanism by how VncR binds to target genes.     

Conservation of structure and function among histidine-containing phosphotransfer (HPt) domains as revealed by the crystal structure of YPD1.

In Saccharomyces cerevisiae, the SLN1-YPD1-SSK1 phosphorelay system controls a downstream mitogen-activated protein (MAP) kinase in response to hyperosmotic stress. YPD1 functions as a phospho-histidine protein intermediate which is required for phosphoryl group transfer from the sensor kinase SLN1 to the response regulator SSK1. In addition, YPD1 mediates phosphoryl transfer from SLN1 to SKN7, the only other response regulator protein in yeast which plays a role in response to oxidative stress and cell wall biosynthesis.The X-ray structure of YPD1 was solved at a resolution of 2.7 A by conventional multiple isomorphous replacement with anomalous scattering. The tertiary structure of YPD1 consists of six alpha-helices and a short 310-helix. A four-helix bundle comprises the central core of the molecule and contains the histidine residue that is phosphorylated. Structure-based comparisons of YPD1 to other proteins having a similar function, such as the Escherichia coli ArcB histidine-containing phosphotransfer (HPt) domain and the P1 domain of the CheA kinase, revealed that the helical bundle and several structural features around the active-site histidine residue are conserved between the prokaryotic and eukaryotic kingdoms.Despite limited amino acid sequence homology among HPt domains, our analysis of YPD1 as a prototypical family member, indicates that these phosphotransfer domains are likely to share a similar fold and common features with regard to response regulator binding and mechanism for histidine-aspartate phosphoryl transfer.     

Crystal structure of master biofilm regulator CsgD regulatory domain reveals an atypical receiver domain

The master regulator CsgD switches planktonic growth to biofilm formation by activating synthesis of curli fimbriae and cellulose in Enterobacteriaceae. CsgD was classified to be the LuxR response regulatory family, while its cognate sensor histidine kinase has not been identified yet. CsgD consists of a C‐terminal DNA binding domain and an N‐terminal regulatory domain that provokes the upstream signal transduction to further modulate its function. We provide the crystal structure of Salmonella Typhimurium CsgD regulatory domain, which reveals an atypical β5α5 response regulatory receiver domain folding with the α2 helix representing as a disorder loop compared to the LuxR/FixJ canonical response regulator, and the structure indicated a noteworthy α5 helix similar to the non‐canonical master regulator VpsT receiver domain α6. CsgD regulatory domain assembles with two dimerization interfaces mainly through α1 and α5, which has shown similarity to the c‐di‐GMP independent and stabilized dimerization interface of VpsT from Vibrio cholerae respectively. The potential phosphorylation site D59 is directly involved in the interaction of interfaces I and mutagenesis studies indicated that both dimerization interfaces could be crucial for CsgD activity. The structure reveals important molecular details for the dimerization assembly of CsgD and will shed new insight into its regulation mechanism.