Gene structure in the histidine operon of Escherichia coli. Identification and nucleotide sequence of the hisB gene

Summary The bifunctional enzyme imidazoleglycerolphosphate dehydratase and histidinolphosphate phosphatase is encoded by the hisB gene. The fourth gene of the histidine operon, hisB, was cloned and mapped on a 2,300 base pair DNA fragment. In the present study we report the complete nucleotide sequence of the hisB gene of Escherichia coli. The gene is 1,068 nucleotides long and codes for a protein of 355 amino acids with an apparent molecular weight of 39,998 daltons. The protein product(s) of the hisB region of both Salmonella typhimurium and E. coli were identified by subcloning and expression in an in vitro translation system. In both organisms the hisB gene directed the synthesis of a single protein with an apparent molecular weight of 40,500 daltons, consistent with the data derived from the nucleotide sequence analysis.     

The evolution of the histidine biosynthetic genes in prokaryotes: A common ancestor for the hisA and hisF genes

The hisA and hisF genes belong to the histidine operon that has been extensively studied in the enterobacteria Escherichia coli and Salmonella typhimurium where the hisA gene codes for the phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxamide isomerase (EC 5.3.1.16) catalyzing the fourth step of the histidine biosynthetic pathway, and the hisF gene codes for a cyclase catalyzing the sixth reaction. Comparative analysis of nucleotide and predicted amino acid sequence of hisA and hisF genes in different microorganisms showed extensive sequence homology (43% considering similar amino acids), suggesting that the two genes arose from an ancestral gene by duplication and subsequent evolutionary divergence. A more detailed analysis, including mutual information, revealed an internal duplication both in hisA and hisF genes in each of the considered microorganisms. We propose that the hisA and hisF have originated from the duplication of a smaller ancestral gene corresponding to half the size of the actual genes followed by rapid evolutionary divergence. The involvement of gene elongation, gene duplication, and gene fusion in the evolution of the histidine biosynthetic genes is also discussed. Correspondence to: M. Bazzicalupo     

Primary and secondary structural homologies between the HIS4 gene product of Saccharomyces cerevisiae and the hisIE and hisD gene products of Escherichia coli and Salmonella typhimurium

Summary A detailed comparative analysis of the Escherichia coli and Salmonella typhimurium hisIE and hisD gene products and the functionally equivalent, single, HIS4 gene product of Saccharomyces cerevisiae permitted several insights concerning the relationship between these genes. Our analysis supports the idea that HIS4 results from the fusion of hisIE and hisD. The comparison permitted a more precise definition of the functional domains of hisI/HIS4A and hisE/HIS4B as well as the two functional domains of hisD/HIS4C. The homologies between the bacterial and yeast sequences suggest a region of the hisD/HIS4C protein that may constitute one of the active centres. A large fragment at the amino terminal region of the yeast protein is missing from the bacterial hisIE gene product and is probably not needed for catalytic activity. Another region of non-homology in the yeast protein is probably a peptide bridge connecting the HIS4AB domain to HIS4C. Although the overall homology at the level of amino acid sequence is modest (about 38%) there is a striking similarity when the hydropathic patterns and predicted secondary structural configurations of these proteins are compared.     

Nucleotide sequence of the trpB gene in Escherichia coli and Salmonella typhimurium

We have obtained the entire nucleotide sequence of the penultimate gene of the tryptophan operon, trpB, in Escherichia coli and Salmonella typhimurium. The amino acid sequence deduced for the E. coli gene product is in agreement with earlier, fragmentary protein sequence results. The trpB nucleotide sequences for the two bacterial species are perfectly colinear and show 85% identity. Most of the nucleotide differences found are without consequence for the amino acid sequence, which shows greater than 96% identity. The degree of conservation of both the nucleotide and amino acid sequences is significantly greater than for trpA, the adjacent gene encoding the other subunit of the same enzyme. When synonymous third codon position nucleotide differences are examined, they seem to be distributed at random throughout trpB and trpA, except for one completely conserved 66 basepair long region within trpB.     

Molecular cloning and nucleotide sequence of the mutT mutator of Escherichia coli that causes A:T to C:G transversion

Summary The Escherichia coli mutator gene mutT, which causes A:TC:G transversion, was cloned in pBR 322. mutT ⁺ plasmids carry a 0.9 kb PvuII DNA fragment derived from the E. coli chromosome. Specific labelling of plasmid-encoded proteins by the maxicell method revealed that mutT codes for a polypeptide of about 15,000 daltons. The protein was overproduced when the mutT gene was placed under the control of the lac regulatory region on a multicopy runaway plasmid. The nucleotide sequence of the mutT gene was determined by the dideoxy method.Abbreviations Ap ampicillin - IPTG isopropyl--d-thiogalactopyranoside - kb kilobase pair(s) - kDa kilodalton(s) - SDS sodium dodecyl sulphate - Tc tetracycline     

Identification and DNA sequence of tdcR, a positive regulatory gene of the tdc operon of Escherichia coli

Summary Efficient in vivo expression of the biodegradative threonine dehydratase (tdc) operon of Escherichia coli is dependent on a regulatory gene, tdcR. The tdcR gene is located 198 base pairs upstream of the tdc operon and is transcribed divergently from this operon. The nucleotide sequence of tdcR and two unrelated reading frames has been determined. The deduced amino acid sequence of TdcR indicates that is is a polypeptide of M_r 12000 with 99 amino acid residues and contains a potential helix-turnhelix DNA binding motif. Deletion analysis and minicell expression of the tdcR gene suggest that TdcR may serve as a trans-acting positive activator for the tdc operon.     

Molecular cloning and nucleotide sequencing of oxyR, the positive regulatory gene of a regulon for an adaptive response to oxidative stress in Escherichia coli: homologies between OxyR protein and a family of bacterial activator proteins

Summary Treatment of Escherichia coli and Salmonella typhimurium cells with a low dose of hydrogen peroxide induces expression of a large number of genes, and confers resistance to oxidative stresses. The oxyR gene encodes a positive regulatory protein for a subset of these genes involved in the defense against oxidative damage. We cloned a DNA fragment that contains the E. coli oxyR region on a plasmid vector, and analyzed the nucleotide sequence of the gene. The amino acid sequence of OxyR protein, deduced from the nucleotide sequence, shows a high degree of homology to the sequences of a number of bacterial activator proteins including LysR, cysB, IlvY, MetR and NodD. The product of the oxyR gene identified by the maxicell procedure was a 34 kDa protein, which agrees with the size predicted from the nucleotide sequence of the gene.     

The role of gene fusions in the evolution of metabolic pathways: the histidine biosynthesis case

Background

Histidine biosynthesis is one of the best characterized anabolic pathways. There is a large body of genetic and biochemical information available, including operon structure, gene expression, and increasingly larger sequence databases. For over forty years this pathway has been the subject of extensive studies, mainly in Escherichia coli and Salmonella enterica, in both of which details of histidine biosynthesis appear to be identical. In these two enterobacteria the pathway is unbranched, includes a number of unusual reactions, and consists of nine intermediates; his genes are arranged in a compact operon (hisGDC [NB]HAF [IE]), with three of them (hisNB, hisD and hisIE) coding for bifunctional enzymes. We performed a detailed analysis of his gene fusions in available genomes to understand the role of gene fusions in shaping this pathway.

Results

The analysis of HisA structures revealed that several gene elongation events are at the root of this protein family: internal duplication have been identified by structural superposition of the modules composing the TIM-barrel protein.Several his gene fusions happened in distinct taxonomic lineages; hisNB originated within γ-proteobacteria and after its appearance it was transferred to Campylobacter species (ε-proteobacteria) and to some Bacteria belonging to the CFB group. The transfer involved the entire his operon. The hisIE gene fusion was found in several taxonomic lineages and our results suggest that it probably happened several times in distinct lineages.Gene fusions involving hisIE and hisD genes (HIS4) and hisH and hisF genes (HIS7) took place in the Eukarya domain; the latter has been transferred to some δ-proteobacteria.

Conclusion

Gene duplication is the most widely known mechanism responsible for the origin and evolution of metabolic pathways; however, several other mechanisms might concur in the process of pathway assembly and gene fusion appeared to be one of the most important and common.

     

Molecular cloning of the Salmonella typhimurium lep gene in Escherichia coli

Summary A system is described which enabled the selection of a heterologous ep gene, encoding signal peptidase I, in Escherichia coli. It is based on complementation of an E. coli mutant, in which the synthesis of signal peptidase I can be regulated. With this system the lep gene of Salmonella typhimurium was cloned and the nucleotide sequence was determined. The S. typhimurium lep gene encodes a protein of 324 amino acids. Expression of the gene in the E. coli mutant resulted in suppression of growth inhibition and in the restoration of processing activity under conditions where synthesis of E. coli signal peptidase I was repressed. The cloned S. typhimurium signal peptidase I had an apparent molecular weight of 36000 daltons, which is in agreement with the calculated molecular weight of 35782 daltons. The system described for selection of the S. typhimurium lep gene may permit the cloning and expression of other heterologous signal peptidase I gen/es.     

Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene

Summary The pepM gene coding for a methionine-specific aminopeptidase was cloned from Salmonella typhimurium and its nucleotide sequence determined. The gene encoded a 264 amino acid protein that was homologous to a similar protein from Escherichia coli. The sequence of an overproducer mutant allele, pepM100, contained a single base change in the likely –35 region of the pepM promoter that increased its homology to the consensus promoter sequence. A region downstream from the pepM coding sequence contained extensive inverted repeats and was homologous to sequences found elsewhere in both Salmonella and other bacterial species.     

Complete nucleotide sequence of the Escherichia coli ptr gene encoding protease III.

The nucleotide sequence of a 3120 bp region of the E. coli chromosome that includes the entire ptr gene has been determined. The proposed coding region for Protease III is 2889 nucleotides long, which would encode a protein consisting of 962 amino acids with a calculated molecular mass of 107,719 daltons. The predicted primary structure of the protein includes a 23-residue signal sequence, cleavage of which would give rise to a mature protein of molecular mass 105,124 daltons. At its 3' end, the ptr gene overlaps the start of the recB coding sequence by 8 bases, suggesting that these genes may form part of an operon.     

Identification of a chloroform-soluble membrane miniprotein in Escherichia coli and its homolog in Salmonella typhimurium

Two homologous 29 amino acid-long highly hydrophobic membrane miniproteins were identified in the Bligh–Dyer lipid extracts of Escherichia coli and Salmonella typhimurium using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The amino acid sequences of the proteins were determined by collision-induced dissociation tandem mass spectrometry, in conjunction with a translating BLAST (tBLASTn) search, i.e., comparing the MS/MS-determined protein query sequence against the six-frame translations of the nucleotide sequences of the E. coli and S. typhimurium genomes. Further MS characterization revealed that both proteins retain the N-terminal initiating formyl-methionines. The methodologies described here may be amendable for detecting and characterizing small hydrophobic proteins in other organisms that are difficult to annotate or analyze by conventional methods.     

Cloning and complete nucleotide sequence of the Escherichia coli glutamine permease operon (glnHPQ)

Summary The glutamine permease operon encoding the high-affinity transport system of glutamine in Escherichia coli could be cloned in one of the mini F plasmids, but not in pBR322 or pACYC184, by selection for restoration of the Gln⁺ phenotype, the ability to utilize glutamine as a sole carbon source. We determined the nucleotide sequence of the glutamine permease operon, which contains the structural gene of the periplasmic glutamine-binding protein (glnH), an indispensable component of the permease activity. The N-terminal amino acid sequence and the overall amino acid composition of the purified glutamine-binding protein were in good agreement with those predicted from the nucleotide sequence, if the N-terminal 22 amino acid residues were discounted. The latter comprised two Lys residues (nos. 2 and 6) followed by 16 hydrophobic amino acid residues and was assumed to be a signal peptide for transport into the periplasmic space. There were two additional reading frames (glnP and glnQ) downstream of glnH sharing a common promoter. It was concluded that the glnP and glnQ proteins as well as the glnH protein are essential for glutamine permease activity.     

Discontinuity of homology of Escherichia coli and Salmonella typhimurium DNA in the lac region

Summary Partial homology of Salmonella typhimurium DNA to Escherichia coli DNA was demonstrated by Southern hybridization blots to exist on either side of the lac operon of E. coli but no homology was detected between S. typhimurium DNA and about 12 kb of E. coli DNA including the lac genes as well as about 5 kb of E. coli DNA between lac and proC. Thus portions of DNA seem to have been either added to the E. coli genome or deleted from the S. typhimurium genome since their divergence from a common ancestor. Although an IS1 element was located near the lac operon of E. coli, the insertional element was shown not to be near any of the junctures of discontinuity of E. coli - S. typhimurium homology near lac.     

Gene structure in the tryptophan operon of Escherichia coli: Nucleotide sequence of trpC and the flanking intercistronic regions

We have determined the DNA sequence for the portion of the Escherichia coli tryptophan (trp) operon spanning trpC, which codes for the bifunctional enzyme N-(5′-phosphoribosyl)-anthranilic acid isomerase/indole-3-glycerol phosphate synthetase. The coding region consists of 1356 nucleotides, directing the synthesis of a polypeptide 452 amino acids in length. The predicted protein sequence is consistent with the amino acid composition of the pure enzyme, and with all known partial peptide sequences derived from this molecule. The enzyme is of particular functional interest, because it contains the catalytic activities for two sequential reactions in tryptophan biosynthesis in a single polypeptide chain.The nucleotide sequences of the junctions between trpC and its flanking genes, trpD and trpB, have also been determined. The trpD-trpC junction consists of six untranslated nucleotides and translation of trpC initiates at the second of two adjacent AUG codons. The trpC termination codon is separated from trpB by 11 nucleotides. The short non-translated regions flanking trpC distinguish it from trpA and trpD, whose initiation codons overlap the termination codons of the preceding genes (trpB and trpE), respectively. These differences in the intercistronic regions may reflect functional relationships between the products of adjacent genes in the operon.     

Sequence divergence of the murB and rrfB genes from Escherichia coli and Salmonella typhimurium

The murB gene of Salmonella typhimurium was cloned and found to be 75% and 82% identical to the DNA and protein sequences, respectively, of the same gene in Escherichia coli. These identities are among the lowest recorded between the two bacteria. Nevertheless, wild-type S. typhimurium murB complemented the known temperature-sensitive E. coli mutant, and wild-type E. coli murB complemented three temperature-sensitive mutants of S. typhimurium. The 5S rRNA gene, rrfB, and the region between murB and rrfB were also cloned and sequenced. The rrfB gene of S. typhimurium differs from rrfB of E. coli in only 2 of 120 nt, but the region between murB and rrfB has diverged greatly and includes a sequence that elosely resembles a repetitive extragenic palindrome of the type normally associated with E. coli. Previous comparisons of gene divergence have suggested that the chromosomal mutation rate is lower in the vicinity of the origin of replication. However, the S. typhimurium murB gene, located 6 map minutes from the origin of replication, is highly substituted at synonymous sites and the sequence between murB and rrfB is significantly modified as well. Thus, murB is an exception to the general observation that genes near the origin of replication show less divergence than do genes elsewhere in the bacterial chromosome.Abbreviations CAI codon adaptation index - REP repetitive extragenic palindrome     

Cloning of histidine genes of Azospirillum brasilense: organization of the ABFH gene cluster and nucleotide sequence of the hisB gene

Summary A cluster of four Azospirillum brasilense histidine biosynthetic genes, hisA, hisB, hisF and hisH, was identified on a 4.5 kb DNA fragment and its organization studied by complementation analysis of Escherichia coli mutations and nucleotide sequence. The nucleotide sequence of a 1.3 kb fragment that complemented the E. coli hisB mutation was determined and an ORF of 624 nucleotides which can code for a protein of 207 amino acids was identified. A significant base sequence homology with the carboxyterminal moiety of the E. coli hisB gene (0.53) and the Saccharomyces cerevisiae HIS3 gene (0.44), coding for an imidazole glycerolphosphate dehydratase activity was found. The amino acid sequence and composition, the hydropathic profile and the predicted secondary structures of the yeast, E. coli and A. brasilense proteins were compared. The significance of the data presented is discussed.Abbreviations IGP imidazole glycerolphosphate - HP histidinolphosphate     

Structure and regulation of the Salmonella typhimurium rnc-era-recO operon

The Escherichia coli rnc-era-recO operon encodes ribonuclease III (RNase III; a dsRNA endonuclease involved in rRNA and mRNA processing and decay), Era (an essential G-protein of unknown function) and RecO (involved in the RecF homologous recombination pathway). Expression of the rnc and era genes is negatively autoregulated: RNase III cleaves the rncO ‘operator’ in the untranslated leader, destabilizing the operon mRNA. As part of a larger effort to understand RNase III and Era structure and function, we characterized rnc operon structure, function and regulation in the closely related bacterium Salmonella typhimurium. Construction of a S typhimurium strain conditionally defective for RNase III and Era expression showed that Era is essential for cell growth. This mutant strain also enabled selection of recombinant clones containing the intact S typhimurium rnc-era-recO operon, whose nucleotide sequence, predicted protein sequence, and predicted rncO RNA secondary structure were all highly conserved with those of E coli. Furthermore, genetic and biochemical analysis revealed that S typhimurium rnc gene expression is negatively autoregulated by a mechanism very similar or identical to that in E coli, and that the cleavage specificities of RNase III_S.t. and RNase III_E.c. are indistinguishable with regard to rncO cleavage and S typhimurium 23S rRNA fragmentation in vivo.