Molecular cloning and nucleotide sequence of the HU-1 gene of Escherichia coli

Summary The Escherichia coli HU-1 was cloned by use of mixed synthetic oligonucleotides (17-mer) predicted from a portion of its amino acid sequence. The amino acid sequence of the HU-1 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream from the translational initiation codon (GUG) of the HU-1 gene.     

Cloning and sequencing of the HU-2 gene of Escherichia coli

Summary The Escherichia coli HU-2 gene was cloned using a DNA fragment from the HU-1 gene as a probe. The amino acid sequence of the HU-2 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream of the translation initiation codon (AUG) and a possible rhoindependent terminater site downstream of the termination codon (UAA) of the gene.     

Amino acid substitution in the C-terminal arm domain of HU-2 results in an enhanced affinity for DNA.

Three mutants of the Escherichia coli hupA gene, encoding the HU-2 protein, were constructed by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The resulting HupAN10, HupAN11 and HupAN12 proteins contained Thr59-->Lys, Gln64-->Lys and Asn53-->Arg substitutions, respectively. These amino acid (aa) changes increased the positive charge of the N-terminal half of the two-strand, antiparallel beta-ribbon of the arm structure, which is believed to be a domain for DNA binding. The three mutant proteins bound to DNA more tightly than wild-type HU-2, and their affinities for DNA increased in the order of HupAN10, HupAN11, HupAN12. The mutant proteins showed a slightly increased HU activity for supporting Mu phage development. A mutant HU-2 protein with increased basicity, but with an altered aa sequence in the arm region due to a frameshift mutation, was also constructed. This mutant protein showed a reduced affinity to DNA and was unable to support Mu growth, suggesting that a unique aa sequence of the arm domain, rather than mere basicity of this domain, is required for efficient binding to DNA.     

An archaebacterial gene from Methanococcus vannielii encoding a protein homologous to the ribosomal protein L10 family

An open reading frame upstream of the Methanococcus vannielii L12 gene has been detected. The beginning of this open reading frame agrees with the N-terminal region of a protein (MvaL10) which has been isolated from the 50 S ribosomal subunit of M. vannielii and sequenced. The length of this gene is 1008 nucleotides, coding for 336 amino acids. Excellent sequence similarities were found to the L10-like ribosomal proteins from Halobacterium halobium and man. The N-terminal part of the MvaL10 protein shows significant sequence similarities to the E. coli L10 protein. MvaL10 is more than twice as long as E. coli L10 but is of length similar to those of the homologous halobacterial and human proteins. Interestingly, the C-terminal region of MvaL10 shows exceptionally high similarity to the C-terminal sequence of the MvaL12 protein. This is not the case for the E. coli proteins but was also observed for the human, Halobacterium and Sulfolobus proteins.     

Characterization of a bordetella pertussis diaminopimelate (DAP) biosynthesis locus identifies dapC, a novel gene coding for an N-succinyl-L,L-DAP aminotransferase

The functional complementation of two Escherichia coli strains defective in the succinylase pathway of meso-diaminopimelate (meso-DAP) biosynthesis with a Bordetella pertussis gene library resulted in the isolation of a putative dap operon containing three open reading frames (ORFs). In line with the successful complementation of the E. coli dapD and dapE mutants, the deduced amino acid sequences of two ORFs revealed significant sequence similarities with the DapD and DapE proteins of E. coli and many other bacteria which exhibit tetrahydrodipicolinate succinylase and N-succinyl-L,L-DAP desuccinylase activity, respectively. The first ORF within the operon showed significant sequence similarities with transaminases and contains the characteristic pyridoxal-5'-phosphate binding motif. Enzymatic studies revealed that this ORF encodes a protein with N-succinyl-L,L-DAP aminotransferase activity converting N-succinyl-2-amino-6-ketopimelate, the product of the succinylase DapD, to N-succinyl-L,L-DAP, the substrate of the desuccinylase DapE. Therefore, this gene appears to encode the DapC protein of B. pertussis. Apart from the pyridoxal-5'-phosphate binding motif, the DapC protein does not show further amino acid sequence similarities with the only other known enzyme with N-succinyl-L,L-DAP aminotransferase activity, ArgD of E. coli.     

Primary structure and mapping of the hupA gene of Salmonella typhimurium.

In bacteria, the complex nucleoid structure is folded and maintained by negative superhelical tension and a set of type II DNA-binding proteins, also called histonelike proteins. The most abundant type II DNA-binding protein is HU. Southern blot analysis showed that Salmonella typhimurium contained two HU genes that corresponded to Escherichia coli genes hupA (encoding HU-2 protein) and hupB (encoding HU-1). Salmonella hupA was cloned, and the nucleotide sequence of the gene was determined. Comparison of hupA of E. coli and S. typhimurium revealed that the HU-2 proteins were identical and that there was high conservation of nucleotide sequences outside the coding frames of the genes. A 300-member genomic library of S. typhimurium was constructed by using random transposition of MudP, a specialized chimeric P22-Mu phage that packages chromosomal DNA unidirectionally from its insertion point. Oligonucleotide hybridization against the library identified one MudP insertion that lies within 28 kilobases of hupA; the MudP was 12% linked to purH at 90.5 min on the standard map. Plasmids expressing HU-2 had a surprising phenotype; they caused growth arrest when they were introduced into E. coli strains bearing a himA or hip mutation. These results suggest that IHF and HU have interactive roles in bacteria.     

Widespread protein sequence similarities: origins of Escherichia coli genes.

To learn more about the evolutionary origins of Escherichia coli genes, we surveyed systematically for extended sequence similarities among the 1,264 amino acid sequences encoded by chromosomal genes of E. coli K-12 in SwissProt release 26 by using the FASTA program and imposing the following criteria: (i) alignment of segments at least 100 amino acids long and (ii) at least 20% amino acid identity. Altogether, 624 extended alignments meeting the two criteria were identified, corresponding to 577 protein sequences (45.6% of the 1,264 E. coli protein sequences) that had an extended alignment with at least one other E. coli protein sequence. To exclude alignments of questionable biological significance, we imposed a high threshold on the number of gaps allowed in each of the 624 extended alignments, giving us a subset of 464 proteins. The population of 464 alignments has the following characteristics expressed as median values of the group: 254 amino acids in the alignment, representing 86% of the length of the protein, 33% of the amino acids in the alignment being identical, and 1.1 gaps introduced per 100 amino acids of alignment. Where functions are known, nearly all pairs consist of functionally related proteins. This implies that the sequence similarity we detected has biological meaning and did not arise by chance. That a major fraction of E. coli proteins form extended alignments strongly suggests the predominance of duplication and divergence of ancestral genes in the evolution of E. coli genes. The range of degrees of similarity shows that some genes originated more recently than others. There is no evidence of genome doubling in the past, since map distances between genes of sequence-related proteins show no coherent pattern of favored separations.     

Structural homologies in alanine-rich acidic ribosomal proteins from procaryotes and eucaryotes.

The amino acid composition and amino-terminal sequence have been determined for the alanine-rich, acidic ribosomal 'A' protein (equivalent to Escherichia coli L7/L12) from three procaryotic cell types that live under extreme environmental conditions (Arthrobacter glacialis, Clostridium pasteurianum, and Bacillus stearothermophilus) as well as from wheat germ, a eucaryote source. These data are compared with previously published 'A' protein sequences from other procaryotes and eucaryotes. All the procaryotic 'A' proteins, with the exception of the very acidic 'A' protein from Halobacterium cutirubrum, show similar charge, size, and amino acid composition, as well as an extensive sequence homology in the N-terminal region. Some differences are observed between gram-negative and gram-positive bacteria. The 'A' proteins from eucaryotes contain two tyrosine molecules, an amino acid absent in procaryotic 'A' proteins, as well as a reduced number of valine residues and an increased amount of aspartic acid. The N-terminal sequence of wheat germ 'A' protein shows considerable homology with other eucaryotic 'A' proteins and also with H. cutirubrum. It also shows some sequence homology with E. coli 'A' proteins.     

Cloning, expression, and nucleotide sequence of genes involved in production of pediocin PA-1, and bacteriocin from Pediococcus acidilactici PAC1.0.

The production of pediocin PA-1, a small heat-stable bacteriocin, is associated with the presence of the 9.4-kbp plasmid pSRQ11 in Pediococcus acidilactici PAC1.0. It was shown by subcloning of pSRQ11 in Escherichia coli cloning vectors that pediocin PA-1 is produced and, most probably, secreted by E. coli cells. Deletion analysis showed that a 5.6-kbp SalI-EcoRI fragment derived from pSRQ11 is required for pediocin PA-1 production. Nucleotide sequence analysis of this 5.6-kbp fragment indicated the presence of four clustered open reading frames (pedA, pedB, pedC, and pedD). The pedA gene encodes a 62-amino-acid precursor of pediocin PA-1, as the predicted amino acid residues 19 to 62 correspond entirely to the amino acid sequence of the purified pediocin PA-1. Introduction of a mutation in pedA resulted in a complete loss of pediocin production. The pedB and pedC genes, encoding proteins of 112 and 174 amino acid residues, respectively, are located directly downstream of the pediocin structural gene. Functions could not be assigned to their gene products; mutation analysis showed that the PedB protein is not involved in pediocin PA-1 production. The mutation analysis further revealed that the fourth gene, pedD, specifying a relatively large protein of 724 amino acids, is required for pediocin PA-1 production in E. coli. The predicted pedD protein shows strong similarities to several ATP-dependent transport proteins, including the E. coli hemolysin secretion protein HlyB and the ComA protein, which is required for competence induction for genetic transformation in Streptococcus pneumoniae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mammalian mitochondrial ribosomal proteins (2). Amino acid sequencing, characterization, and identification of corresponding gene sequences

Four different classes of mammalian mitochondrial ribosomal proteins were identified and characterized. Mature proteins were purified from bovine liver and subjected to N-terminal or matrix-assisted laser-desorption mass spectroscopic amino acid sequencing after tryptic in-gel digestion and high pressure liquid chromatography separation of the resulting peptides. Peptide sequences obtained were used to virtually screen expressed sequence tag data bases from human, mouse, and rat. Consensus cDNAs were assembled in silico from various expressed sequence tag sequences identified. Deduced mammalian protein sequences were characterized and compared with ribosomal protein sequences of Escherichia coli and yeast mitochondria. Significant sequence similarities to ribosomal proteins of other sources were detected for three out of four different mammalian protein classes determined. However, the sequence conservation between mitochondrial ribosomal proteins of mammalian and yeast origin is much less than the sequence conservation between cytoplasmic ribosomal proteins of the same species. In particular, this is shown for the mammalian counterparts of the E. coli EcoL2 ribosomal protein (MRP-L14), that do not conserve the specific and functional highly important His(229) residue of E. coli and the corresponding yeast mitochondrial Rml2p.     

Cloning, expression, and nucleotide sequence of genes involved in production of pediocin PA-1, and bacteriocin from Pediococcus acidilactici PAC1.0.

The production of pediocin PA-1, a small heat-stable bacteriocin, is associated with the presence of the 9.4-kbp plasmid pSRQ11 in Pediococcus acidilactici PAC1.0. It was shown by subcloning of pSRQ11 in Escherichia coli cloning vectors that pediocin PA-1 is produced and, most probably, secreted by E. coli cells. Deletion analysis showed that a 5.6-kbp SalI-EcoRI fragment derived from pSRQ11 is required for pediocin PA-1 production. Nucleotide sequence analysis of this 5.6-kbp fragment indicated the presence of four clustered open reading frames (pedA, pedB, pedC, and pedD). The pedA gene encodes a 62-amino-acid precursor of pediocin PA-1, as the predicted amino acid residues 19 to 62 correspond entirely to the amino acid sequence of the purified pediocin PA-1. Introduction of a mutation in pedA resulted in a complete loss of pediocin production. The pedB and pedC genes, encoding proteins of 112 and 174 amino acid residues, respectively, are located directly downstream of the pediocin structural gene. Functions could not be assigned to their gene products; mutation analysis showed that the PedB protein is not involved in pediocin PA-1 production. The mutation analysis further revealed that the fourth gene, pedD, specifying a relatively large protein of 724 amino acids, is required for pediocin PA-1 production in E. coli. The predicted pedD protein shows strong similarities to several ATP-dependent transport proteins, including the E. coli hemolysin secretion protein HlyB and the ComA protein, which is required for competence induction for genetic transformation in Streptococcus pneumoniae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes.

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.     

YmL9, a nucleus-encoded mitochondrial ribosomal protein of yeast, is homologous to L3 ribosomal proteins from all natural kingdoms and photosynthetic organelles.

The nuclear gene for mitochondrial ribosomal protein YmL9 (MRP-L9) of yeast has been cloned and sequenced. The deduced amino acid sequence characterizes YmL9 as a basic (net charge + 30) protein of 27.5 kDa with a putative signal peptide for mitochondrial import of 19 amino acid residues. The intact MRP-L9 gene is essential for mitochondrial function and is located on chromosome XV or VII. YmL9 shows significant sequence similarities to Escherichia coli ribosomal protein L3 and related proteins from various organisms of all three natural kingdoms as well as photosynthetic organelles (cyanelles). The observed structural conservation is located mostly in the C-terminal half and is independent of the intracellular location of the corresponding genes [Graack, H.-R., Grohmann, L. & Kitakawa, M. (1990) Biol. Chem. Hoppe Seyler 371, 787-788]. YmL9 shows the highest degree of sequence similarity to its eubacterial and cyanelle homologues and is less related to the archaebacterial or eukaryotic cytoplasmic ribosomal proteins. Due to their high sequence similarity to the YmL9 protein two mammalian cytoplasmic ribosomal proteins [MRL3 human and rat; Ou, J.-H., Yen, T. S. B., Wang, Y.-F., Kam, W. K. & Rutter, W. J. (1987) Nucleic Acids Res. 15, 8919-8934] are postulated to be true nucleus-encoded mitochondrial ribosomal proteins.     

Sequencing the gene for an imipenem-cefoxitin-hydrolyzing enzyme (CfiA) from Bacteroides fragilis TAL2480 reveals strong similarity between CfiA and Bacillus cereus beta-lactamase II.

Using a newly constructed Bacteroides fragilis-Escherichia coli cloning shuttle vector, pJST61, we have cloned the cefoxitin (FOX)-imipenem (IMP) resistance determinant from B. fragilis TAL2480. FOX-IMP resistance in this strain results from the production of a periplasmic, Zn2(+)-containing beta-lactamase which hydrolyzes carbapenems and cephamycins and whose activity is resistant to clavulanic acid but sensitive to Zn2(+)-binding reagents, including EDTA. The pJST61 vector permits efficient library construction in E. coli and allows for the transfer of the library to B. fragilis recipients for the screening or selection of specific phenotypes. The library clone containing the FOX-IMP resistance gene was detected after transfer to B. fragilis TM4000 (Fox-Imps) selecting for Foxr. One of the isolates carrying plasmid pJST241 is resistant to FOX and IMP and synthesizes a periplasmic protein with substrate and inhibitor properties identical to those of strain TAL2480. On the basis of deletion analysis, Tn1000 insertion mutations, and DNA sequencing, we have defined the 747-base cfiA (FOX-IMP resistance) gene within the 3.6-kilobase cloned insert in pJST241. The cfiA gene contains an open reading frame that could code for a precursor protein of 249 amino acids and with a molecular mass of 27,260 daltons. A potential signal sequence has been identified at the N terminus of this protein; cleavage within this sequence would result in a protein of 231 amino acids with a molecular mass of 25,249 daltons. The CfiA protein shows remarkable similarities to the exported, Zn2(+)-requiring, type II beta-lactamase Blm proteins from Bacillus cereus 569/H and 5/B/6. Although overall amino acid identity is only 32%, the Zn ligand-binding His and Cys residues are precisely conserved and the amino acids in the vicinity of these sites show strong similarities (greater than 80%) when the CfiA and Blm proteins are compared.     

Bovine heart mitochondrial F6: HPLC purification, NH2-terminal sequence and the possible structural relatedness to other components of ATPase complexes

The mitochondrial factor F6 has been purified by reverse-phase HPLC and the molecular weight (8500), amino acid composition and about 25% of the amino acid sequence determined. In the NH2-terminal sequence of the first 18 amino acids (NKELDPVQKLFVDKIREY), six identities with the NH2-terminal sequence of the oligomycin-sensitivity conferring protein (OSCP) are apparent, as well as less striking similarities with the OSCP related subunit delta of E. coli F1. The possibility that F6, OSCP and subunit delta of E. coli F1 could have evolved from a common ancestral gene is supported by apparent gene duplication within the OSCP and subunit delta sequences.     

Sequence of a cDNA clone encoding a rat Reg-2 protein.

We report here the nucleotide sequence of a rat cDNA clone encoding a protein homologous to the Reg (regenerating) protein. The encoded protein, designated Reg-2, shows 60%, 78% and 61% similarities with the reported amino acid sequences of the rat, bovine and human proteins, respectively.     

An Escherichia coli protein with homology to the H-protein of the glycine cleavage enzyme complex from pea and chicken liver.

The nucleotide sequence of an Escherichia coli gene which presumably encodes the H-protein of the glycine cleavage (GCV) enzyme complex is presented. The gene, designated gcvH, encodes a polypeptide of 128 amino acids with a calculated molecular weight of 13,665 daltons. The translation start site was determined by N-terminal amino acid sequence analysis of a gcvH-lacZ encoded fusion protein. The E. coli H-protein shows extensive homology with the H-proteins from the pea (Pisum sativum) and the chicken liver GCV enzyme complexes. 85 of 128 amino acid residues are identical or chemically similar between the E. coli and the pea H-proteins, and 74 of 128 amino acid residues are identical or chemically similar between the E. coli and the chicken liver H-proteins. All three proteins have identical amino acid sequences from residues 61-65. This sequence contains the lysyl residue involved in lipoic acid attachment in the chicken liver H-protein.     

Amino acid and cDNA sequences of a methionine-rich 2S protein from sunflower seed (Helianthus annuus L.)

The amino acid sequence of a methionine-rich 2S seed protein from sunflower (Helianthus annuus L.) and the sequence of a cDNA clone which codes for the entire primary translation product have been determined. The mature protein consists of a single polypeptide chain of 103 amino acids (molecular mass 12133 Da) which contains 16 residues of methionine and 8 residues of cysteine. The cDNA sequence established that the protein is synthesized as a precursor of 141 residues with a typical hydrophobic signal sequence of 25 residues followed by a further 13-residue hydrophobic pro-sequence which is presumably removed by post-translational cleavage. The sequence of the mature protein and that deduced from the cDNA were identical with no evidence of processing at the C-terminus. Comparison of the sunflower methionine-rich protein sequence with sequences of other seed 2S proteins from dicotyledons and monocotyledons showed limited but distinct sequence similarities; in particular the arrangement of the cysteine residues was conserved. The sunflower protein shows 34% identity with the methionine-rich Brazil nut 2S protein and the prepro regions of the precursors of these two proteins show about 50% identity. This similarity indicates that these methionine-rich 2S proteins have diverged as a subclass of the 2S superfamily of proteins which contain only 2-3% methionine. While the related 2S proteins from other dicotyledons are processed to a small and large subunit, the sunflower protein is not cleaved in this way.