ptsH Gene Research Articles

Amino acid sequences of several Bacillus subtilis proteins modified by apparent guanylylation.

Bacillus subtilis cell extracts, prepared at different times during growth, contained several proteins that were apparently guanylylated in vitro with [alpha-32P]-GTP. Four of the proteins were partially purified and the N-terminal amino acid sequences (13 to 20 residues) were determined. One sequence had 84% identity to Bacillus stearothermophilus triosephosphate isomerase, two were 100% identical to the predicted sequences of the B. subtilis ptsI and ptsH genes while no identity was found for the fourth sequence. This apparent guanylylation occurred with proteins involved in glucose metabolism, although the significance is unknown.

Identification of proteins phosphorylated by ATP during sporulation of Bacillus subtilis.

Protein phosphorylation in Bacillus subtilis was assayed in vitro by using extracts prepared from cells at various times during growth and sporulation. At least six proteins were labeled in vitro by using [gamma-32P]ATP and extracts of vegetative cells. In extracts prepared at the end of exponential growth and during stationary phase, 12 to 13 proteins were labeled. Seven of the phosphoproteins were purified by fast-performance liquid chromatography and polyacrylamide gel electrophoresis, blotted to Immobilon membranes, and subjected to partial protein sequencing. One of the sequences had sequence homology (greater than 45%) to elongation factor G from several bacterial species, and four sequences matched the predicted amino-terminal sequences of the outB, orfY-tsr, orfU, and ptsH genes.

Staphylococcal phosphoenolpyruvate‐dependent phosphotransferase system

The histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Staphylococcus carnosus and purified to homogeneity. The protein sequence was determined by Edman degradation of peptides obtained by proteolytic digestion with proteases V8, trypsin and chemical cleavage with BrCN. Furthermore, immunological screening of a chromosomal S. carnosus DNA gene library in pUC19 vector enabled us to isolate S. carnosus HPr-expressing colonies. The nucleotide sequence of this ptsH gene and its flanking regions was determined by the dideoxy-chain-termination technique. Upstream, the 264-bp open reading frame of the ptsH gene is flanked by a putative S. carnosus promoter structure and a putative ptsI gene downstream suggesting that ptsH gene is the first gene in the PTS operon of S. carnosus. Comparison of the amino acid sequence of S. carnosus HPr with the HPr sequence of Staphylococcus aureus (derived from peptide sequencing) showed a high degree of similarity.

Antisense expression at theptsH-ptsIlocus ofEscherichia coli

A Mud(Ap, lac) prophage has been shown to be inserted into the ptsH gene of E. coli. The insertion is likely to have generated an operon fusion revealing antisense transcription at this locus. This suggests that the ORF previously identified, overlapping with ptsH ORF in the opposite orientation, might be functional. A model of regulation for the ptsH-ptsI-crr operon transcription is presented which accounts for antisense transcription.

Partial nucleotide sequence of the pts operon in Salmonella typhimurium: comparative analyses in five bacterial genera

The nucleotide sequence of a Salmonella typhimurium DNA segment of 549 base pairs which encompasses the operator-promoter of the pts operon, the entirety of the ptsH gene, encoding HPr of the phosphotransferase system (PTS), the first 29 nucleotides of the ptsI gene, encoding Enzyme I of the PTS, and the intercistronic region between the ptsH and ptsI genes was determined and compared with the corresponding sequence from Escherichia coli (De Reuse et al., 1985). The two sequences showed 91% overall identity, with some regions showing sequence conservation and others exhibiting relative divergence. Two open reading frames were identified in both species: one encoded HPr on the 'sense' strand (255 nucleotides; 12 nucleotide differences, no amino acid differences); the other, on the anti-sense strand, consisted of 291 nucleotides (13 nucleotide differences, 13 amino acid differences). While HPr bears a net negative charge, the putative protein encoded by the open reading frame on the anti-sense strand is strongly basic. Computer analyses of HPr proteins from five different bacterial genera revealed four regions which show strong sequence identity and therefore are presumed to be critical for maintenance of biological activity. Two of these regions were specific to Gram-positive bacteria. Proposed functions for each of these regions are discussed. Relative evolutionary distances between the HPr proteins were also computed.

Antisense expression at the ptsH-ptsI locus of Escherichia coli

A Mud(Ap, lac) prophage has been shown to be inserted into the ptsH gene of E. coli. The insertion is likely to have generated an operon fusion revealing antisense transcription at this locus. This suggests that the ORF previously identified, overlapping with ptsH ORF in the opposite orientation, might be functional. A model of regulation for the ptsH-ptsI-crr operon transcription is presented which accounts for antisense transcription.

Site-directed mutagenesis with the ptsH gene of Bacillus subtilis. Isolation and characterization of heat-stable proteins altered at the ATP-dependent regulatory phosphorylation site.

The codon for Ser-46 of the ptsH gene of Bacillus subtilis was modified by site-directed mutagenesis to the codons for Ala, Thr, Tyr, and Asp. The mutant genes were overexpressed, three of the corresponding proteins were purified to homogeneity with the exception for the Asp derivative, which could not be detected, although the gene had the desired nucleotide sequence. The phosphotransferase activity of the altered proteins was determined to be 20-35% of wild type activity, which correlates well with the slow phosphorylation of heat-stable protein (HPr) by enzyme I and phosphoenolpyruvate. The ATP-dependent HPr kinase, which previously was shown to be involved in the regulation of carbohydrate uptake of Gram-positive bacteria by covalent phosphorylation of Ser-46 of HPr, is entirely inactive toward the OH group of Thr-46 and Tyr-46 proteins. In addition, we constructed a strain of B. subtilis, where the altered gene coding for the Ala-46 derivative of HPr was introduced into the bacterial chromosome. The physiological properties of this mutant are described.

The ptsH, ptsI, and crr genes of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: a complex operon with several modes of transcription

The ptsH, ptsI, and crr genes, coding for three of the proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) (HPr, enzyme I, and enzyme IIIGlc, respectively) have been studied by determination of their nucleotide sequence and analysis of their expression. The three genes constitute an operon, but analysis of the ptsH, ptsI, and crr transcripts by Northern (RNA) blotting revealed the existence of three major mRNA species. One encompassed the three cistrons, a second one the ptsH gene and part of the ptsI gene, and the third one only the distal gene crr. The short crr transcripts were initiated inside the ptsI open reading frame at points which were identified by S1 mapping. Expression of the genes was studied in vivo by using operon and protein fusions between the lacZ gene and the ptsH, ptsI, or crr gene on IncW low-copy-number plasmids. The present study showed that (i) the ptsH, ptsI, and crr genes exhibited high basal expression, (ii) transcription of the ptsH and ptsI genes was stimulated threefold by the cyclic AMP-cyclic AMP receptor protein complex and also by growth on glucose, but only in the presence of an active enzyme IIGlc, (iii) crr-specific expression was not sensitive to the complex or to growth on glucose, and (iv) under the growth conditions tested, the major part of crr transcription was initiated from internal promoters.

DominantptsHmutations of the gene coding for synthesis of the HPr protein of the phosphoenolpyruvate-dependent phosphotransferase system inEscherichia coliK-12 merodiploids

Abstract The Escherichia coli ptsI and ptsH genes code for the synthesis of two proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), namely enzyme I and protein HPr. A number of ptsI + ptsH + /F′ ptsI + ptsH merodiploids was obtained. It was shown in experiments in vivo that ptsH mutations in the transposition are dominant. Bacterial extracts from these merodiploids supported [ 14 C] methyl glucoside (MG) phosphorylation at the expense of phosphoenolpyruvate only half as much as extracts from the pts + cells. ptsI + ptsH /F′ ptsI + ptsH + merodiploids appeared to be non-viable; the reason for this lack of viability is discussed.

Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: nucleotide sequence of the ptsH gene

The nucleotide sequence of an Escherichia coli DNA segment containing the ptsH gene and the first 162 nucleotides of the ptsI gene encoding, respectively, Hpr and enzyme I of the phosphoenolpyruvate-dependent glycose phosphotransferase system (PTS), was determined. The ptsH promoter was localized using the S1 mapping technique. A nucleotide sequence very similar to the consensus binding site for cAMP receptor protein was found in the -35 region of the ptsH promoter. The ptsH gene is transcribed in the same direction as the ptsI gene and the crr gene (encoding enzyme IIIG1c of the PTS). Analysis of the nucleotide sequence substantiates the notion that the ptsH-ptsI-crr genes constitute a polycistronic operon.

Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: evidence for the existence of a single transcriptional unit

A cosmid containing the ptsH, ptsI and crr genes of Escherichia coli coding for three components of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) has been isolated. The products of these genes were identified using the maxicell technique. The cloning of the PTS region as a whole allowed us to map and order ptsH,ptsI and crr genes in a 3.8-kb DNA fragment and determine thatptsI and crr are transcribed from a common promoter. They, therefore, constitute a single transcriptional unit which is likely to include also the ptsH gene. In addition, the crr gene may have a secondary promoter located inside ptsI.

The primary structure of Salmonella typhimurium HPr, a phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system. A correction.

The protein HPr is a low-molecular-weight phosphocarrier protein of the bacterial phosphoenolpyruvate:glycose phosphotransferase system. We have recently reported the complete primary amino acid sequence of HPr isolated from Salmonella typhimurium (Weigel, N., Powers, D.A., and Roseman, S. (1982) J. Biol. Chem. 257, 14499-14509). This sequence is incorrect at certain residues; the correct primary structure of the protein is presented in this report. The corrected structure generally agrees with the primary sequence predicted for HPr from Escherichia coli (based on the nucleotide sequence of the corresponding ptsH gene). The one apparent ambiguity is at the carboxyl terminus.

Location and direction of transcription of the ptsH and ptsI genes on the Escherichia coli K12 genome.

Recombinant plasmids were constructed that carried various fragments of the DNA specifying the Escherichia coli genes ptsH and (part of) ptsI, the genes for the common components of the phosphoenolpyruvate: sugar phosphotransferase. Expression of plasmid-specified functions in minicells showed that ptsH and ptsI were transcribed clockwise. Most of the transcription of ptsI was from the ptsH promoter, but some was from a second site within or after ptsH.

Isolation and investigation of the Escherichia coli mutant with the deletion in the ptsH gene

Isolation and investigation of the Escherichia coli mutant with the deletion in the ptsH gene

Effect of ptsI and ptsH genes dosage on manifestation of glucose catabolite repression of β-galactosidase synthesis in escherichia coli K12

Effect of ptsI and ptsH genes dosage on manifestation of glucose catabolite repression of β-galactosidase synthesis in escherichia coli K12

Repression of inducible enzyme synthesis in a mutant of Escherichia coli K12 deleted for the ptsH gene

The genome of lambda phage with thermosensitive repressor was inserted into the pts region of the Escherichia coli chromosome. This lysogenic culture possessed the PTS1 phenotype at 30 degrees C. A mutant strain with a deletion covering the ptsH gene was isolated after a prophage curing procedure. The deletion nature of the pts mutation was confirmed in genetical and biochemical experiments. The deletion covered a small fragment of the bacterial genome not extending in the ptsI and lig genes. The isolated deltaptsH mutant possessed all characteristics of known pts mutants: pleiotropical disturbances of transport and utilization of a number of carbohydrates, repression of the enzyme inducible synthesis, and resistance to glucose catabolite repression. From these and other data we can conclude that the phosphorylated form of the heat-stable protein HPr is involved (directly or indirectly) in activation of the DNA transcription process.