Crystal structure of the "PhoU-like" phosphate uptake regulator from Aquifex aeolicus

The phoU gene of Aquifex aeolicus encodes a protein called PHOU_AQUAE with sequence similarity to the PhoU protein of Escherichia coli. Despite the fact that there is a large number of family members (more than 300) attributed to almost all known bacteria and despite PHOU_AQUAE's association with the regulation of genes for phosphate metabolism, the nature of its regulatory function is not well understood. Nearly one-half of these PhoU-like proteins, including both PHOU_AQUAE and the one from E. coli, form a subfamily with an apparent dimer structure of two PhoU domains on the basis of their amino acid sequence. The crystal structure of PHOU_AQUAE (a 221-amino-acid protein) reveals two similar coiled-coil PhoU domains, each forming a three-helix bundle. The structures of PHOU_AQUAE proteins from both a soluble fraction and refolded inclusion bodies (at resolutions of 2.8 and 3.2A, respectively) showed no significant differences. The folds of the PhoU domain and Bag domains (for a class of cofactors of the eukaryotic chaperone Hsp70 family) are similar. Accordingly, we propose that gene regulation by PhoU may occur by association of PHOU_AQUAE with the ATPase domain of the histidine kinase PhoR, promoting release of its substrate PhoB. Other proteins that share the PhoU domain fold include the coiled-coil domains of the STAT protein, the ribosome-recycling factor, and structural proteins like spectrin.     

Role of PhoU in phosphate transport and alkaline phosphatase regulation.

The negative regulatory function of PhoU in alkaline phosphatase (AP) was suggested by the behavior of K10 phoU35 carrying a missense mutation whose product was detected by immunoblotting. To define more clearly the regulatory function of this protein for the synthesis of AP, we constructed a null mutation. The constitutive synthesis of AP in this phoU deletion strain confirmed the negative role of PhoU. However, the expression of the PhoU protein from an isopropyl-beta-D-thiogalactopyranoside-inducible promoter had no effect on the repression of AP synthesis. Furthermore, the involvement of PhoU in free-Pi uptake was demonstrated. These results provide evidence that PhoU participates in Pi transport and in the regulatory role of the phosphate-specific transport system.     

Use of the rep technique for allele replacement to construct mutants with deletions of the pstSCAB-phoU operon: evidence of a new role for the PhoU protein in the phosphate regulon.

The phosphate regulon is negatively regulated by the PstSCAB transporter and PhoU protein by a mechanism that may involve protein-protein interaction(s) between them and the Pi sensor protein, PhoR. In order to study such presumed interaction(s), mutants with defined deletions of the pstSCAB-phoU operon were made. This was done by construction of M13 recombinant phage carrying these mutations and by recombination of them onto the chromosome by using a rep host (which cannot replicate M13) for allele replacement. These mutants were used to show that delta (pstSCAB-phoU) and delta (pstB-phoU) mutations abolished Pi uptake by the PstSCAB transporter, as expected, and that delta phoU mutations had no effect on uptake. Unexpectedly, delta phoU mutations had a severe growth defect, and this growth defect was (largely) alleviated by a compensatory mutation in the pstSCAB genes or in the phoBR operon, whose gene products positively regulate expression of the pstSCAB-phoU operon. Because delta phoU mutants that synthesize a functional PstSCAB transporter constitutively grew extremely poorly, the PhoU protein must have a new role, in addition to its role as a negative regulator. A role for the PhoU protein in intracellular Pi metabolism is proposed. Further, our results contradict those of M. Muda, N. N. Rao, and A. Torriani (J. Bacteriol. 174:8057-8064, 1992), who reported that the PhoU protein was required for Pi uptake.     

Regulation of the phosphate regulon in Escherichia coli K-12: regulation of the negative regulatory gene phoU and identification of the gene product.

The phoU gene is one of the negative regulatory genes of the pho regulon of Escherichia coli. The DNA fragment carrying phoU has been cloned on pBR322 (Amemura et al., J. Bacteriol. 152:692-701, 1982). Further subcloning, Tn1000 insertion inactivation, and complementation tests localized the phoU gene within a 1.1-kilobase region on the cloned DNA fragment. The gene product of phoU was identified by the maxicell method as a protein with an approximate molecular weight of 27,000. A hybrid plasmid that contains a phoU'-lac'Z fused gene was constructed in vitro. This plasmid enabled us to study phoU gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the pho regulon, and phoU gene expression in these strains was studied under limited and excess phosphate conditions. It was found that phoU is expressed at a higher level when the cells are cultured under the excess phosphate condition. The higher phoU expression was observed in a phoB mutant and a phoR-phoM double mutant. The implications of these findings for the regulation of pho genes are discussed.     

From cell membrane to nucleotides: the phosphate regulon in Escherichia coli

Most of the essential cellular components, like nucleic acids, lipids and sugars, are phosphorylated. The phosphate equilibrium in Escherichia coli is regulated by the phosphate (Pi) input from the surrounding medium. Some 90 proteins are synthesized at an increased rate during Pi starvation and the global control of the cellular metabolism requires cross-talk with other regulatory mechanisms. Since the Pi concentration is normally low in E. coli's natural habitat, these cells have devised a mechanism for synthesis of about 15 proteins to accomplish two specific functions: transport of Pi and its intracellular regulation. The synthesis of these proteins is controlled by two genes (the phoB-phoR operon), involving both negative and positive functions. PhoR protein is a histidine protein kinase, induced in Pi starvation and is a transmembrane protein. It phosphorylates the regulator protein PhoB which is also Pi starvation-induced. The PhoB phosphorylated form binds specifically to a DNA sequence of 18 nucleotides (the pho Box), which is part of the promoters of the Pho genes. The genes controlled by phoB constitute the Pho regulon. The repression of phoA (the gene encoding alkaline phosphatase) by high Pi concentrations in the medium requires the presence of an intact Pst operon (pstS, pstC, pstA, pstB and phoU) and phoR. The products of pstA and pstC are membrane bound, whereas the product of pstS is periplasmic and PstB and PhoU proteins are cytoplasmic. The function of the PhoU protein may be regulated by cofactor nucleotides and may be involved in signaling the activation of the regulon via PhoR.     

鸟分枝杆菌PhoP功能分析及其基因突变株的构建

【目的】对鸟分枝杆菌PhoP的功能进行分析及构建PhoP基因突变株,为深入研究PhoP的调控机制打下基础。【方法】利用PCR扩增出鸟分枝杆菌PhoP DNA结合区(PhoPC)编码序列,与表达载体p GEX-4T-3连接后,转化入大肠杆菌BL21(DE3)中表达GST-PhoPC融合蛋白。用凝血酶去除GST标签,制备PhoPC蛋白;利用PCR扩增出鸟分枝杆菌PhoP基因及其下游基因MAV0127、PhoU和Amt的启动子片段,采用凝胶迁移率移动试验(EMSA)分别检测PhoPC与PhoP、MAV0127、PhoU和Amt的启动子结合的情况。通过PCR扩增PhoP基因上、下游片段,构建PhoP基因缺失性同源核苷酸片段,与自杀质粒p GMB151连接后,通过电转化导入鸟分枝杆菌进行同源交换,利用PCR筛选出PhoP基因缺失突变株。【结果】EMSA结果显示,鸟分枝杆菌PhoP能与PhoP、MAV0127及Amt基因启动子结合,不能与PhoU结合。通过PCR和序列分析证实基因突变株的PhoP基因缺失了309个碱基。【结论】PhoP不仅可调控其下游基因MAV0127和Amt的转录水平,还可调控其自身基因的转录,但不参与调节PhoU二元调控系统。构建了PhoP基因缺失突变株,为进一步研究其在鸟分枝杆菌的调控功能奠定了基础。     

Accumulation of inorganic polyphosphate in phoU mutants of Escherichia coli and Synechocystis sp. strain PCC6803

The biological process for phosphate (P(i)) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the P(i) regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more P(i) from the medium than the wild-type strain removed.     

Crystal structure of a PhoU protein homologue: a new class of metalloprotein containing multinuclear iron clusters

PhoU proteins are known to play a role in the regulation of phosphate uptake. In Thermotoga maritima, two PhoU homologues have been identified bioinformatically. Here we report the crystal structure of one of the PhoU homologues at 2.0 A resolution. The structure of the PhoU protein homologue contains a highly symmetric new structural fold composed of two repeats of a three-helix bundle. The structure unexpectedly revealed a trinuclear and a tetranuclear iron cluster that were found to be bound on the surface. Each of the two multinuclear iron clusters is coordinated by a conserved E(D)XXXD motif pair. Our structure reveals a new class of metalloprotein containing multinuclear iron clusters. The possible functional implication based on the structure are discussed.     

Phosphate-specific transport system of Escherichia coli: nucleotide sequence and gene-polypeptide relationships.

The DNA nucleotide sequence of four genes for the phosphate-specific transport system of Escherichia coli is reported. Along with the DNA sequence for the phoS gene reported previously (Surin et al., J. Bacteriol. 157:772-778, 1984; Magota et al., J. Bacteriol. 157:909-917, 1984), this study completes the nucleotide sequence of the phosphate-specific transport region. The complete sequence (including phoS) contains five open reading frames oriented in the same direction, each preceded by a putative ribosome-binding site near the presumed translation initiation codon ATG. The complete sequence is transcribed counterclockwise, in the order phoS pstC pstA pstB phoU. Genetic complementation shows that of the four open reading frames in the new sequence, three correspond to known mutant alleles; the fourth, which was designated pstC, has not been described before and could not be related to any known mutant allele. We have confirmed that pstA was allelic to phoT32. The pstC, pstB, and phoU gene products were identified as peripheral membrane proteins. The pstA gene product appears to be an integral membrane protein.     

Nucleotide sequence of the tcml gene (ribosomal protein L3) of Saccharomyces cerevisiae.

The yeast tcml gene, which codes for ribosomal protein L3, has been isolated by using recombinant DNA and genetic complementation. The DNA fragment carrying this gene has been subcloned and we have determined its DNA sequence. The 20 amino acid residues at the amino terminus as inferred from the nucleotide sequence agreed exactly with the amino acid sequence data. The amino acid composition of the encoded protein agreed with that determined for purified ribosomal protein L3. Codon usage in the tcml gene was strongly biased in the direction found for several other abundant Saccharomyces cerevisiae proteins. The tcml gene has no introns, which appears to be atypical of ribosomal protein structural genes.     

Analysis of the Escherichia coli K-12 cysB gene and its product using the method of gene fusion

The amino-terminal structure and the essential functional region of the cysB gene product of Escherichia coli K-12 were analyzed by the method of gene fusion. The translational start codon of the cysB gene was located by determining the amino-terminal sequence of a hybrid protein containing the first 31 amino acid residues of the CysB protein at the amino terminus of beta-galactosidase(LacZ protein). The fact that two other CysB'-'LacZ hybrid polypeptides expressed a normal CysB activity indicated that the functional region of the CysB protein was located within the first 215 amino acid residues of the total 324 amino acids deduced from the nucleotide sequence.     

Determinations of the DNA sequence of the mreB gene and of the gene products of the mre region that function in formation of the rod shape of Escherichia coli cells.

The 6.5-kilobase mre region at 71 min in the Escherichia coli chromosome map, where genes involved in formation of a rod-shaped cell form a gene cluster, was analyzed by in vivo protein synthesis in a maxicell system and by base sequencing of DNA. An open reading frame that may code for a protein with an Mr of about 37,000 on sodium dodecyl sulfate-polyacrylamide gels was found and was correlated with the mreB gene. N-terminal amino acid sequencing of the hybrid mreB-lacZ protein confirmed the production by mreB of a protein of 347 amino acid residues with a molecular weight of 36,958. The amino acid sequence of this protein deduced from the DNA sequence showed close similarity with that of a protein of the ftsA gene which is involved in cell division of E. coli. Three other contiguous genes that formed three proteins with Mrs of about 40,000, 22,000, and 51,000, respectively, were detected downstream of the mreB gene by in vivo protein synthesis. The mreB protein and some of these three proteins may function together in determination of cell shape.