Molecular cloning and sequencing of the extracellular pectate lyase II gene from Erwinia carotovora Er.

Erwinia carotovora Er produces three extra-cellular pectate lyases (PL I, II, and III). The gene for pectate lyase II (pelII) of E. carotovora Er was cloned and expressed both in Escherichia coli and E. carotovora Er. Localization experiments in E. coli showed that PL II was exclusively in the cytoplasmic space, while PL II was excreted into the culture medium. The complete nucleotides of the pelII gene were sequenced and found to include one open reading frame of 1122 bp coding for a protein of 374 amino acid residues. From comparison of the N-terminal amino acid sequence between the purified PL II and the deduced protein from the nucleotide sequence we reached the conclusion that the mature protein is composed of 352 amino acids with a calculated molecular weight of 38,169 and is preceded by a typical signal sequence of 22 amino acid residues. PL II had 90.1% and 82.9% homologies with PL I and PL III in amino acid sequence, respectively.     

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.     

Studies on the Components of Mammalian Urine

Pectate lyase was purified approximately 29-fold to electrophoretic homogeneity from Pseudomonas marginalis N6301. A pectate lyase (PL; EC4.2.2.2) gene of the strain was cloned and expressed in Escherichiacoli. The nucleotides of the PL gene (pel) were sequenced. An open reading frame that encodes a polypeptide (molecular weight: 40,812) composed of 380 amino acids including a 29 amino acid signal peptide was assigned. The structural gene of pel consisted of 1140 base pairs. The nucleotide sequence of the 5′-flanking region of pel showed a consensus sequence of the promoter region of the pectin lyase gene (pnl) in P. marginalis N6301, a Pribnow box, and a ribosome binding site as found in E. coli.     

Erwinia pel gene homology survey in selected bacteria

A 2.1 kb Bam H1 DNA fragment encoding a pectate lyase (PL) enzyme was isolated from an Erwinia carotovora subsp. atroseptica (Eca) cosmid library. The fragment was labeled with ³²P-CTP and hybridized to total DNA digests from selected bacteria which included plant-invasive as well as plant associative organisms. The pel gene probe hybridized to E. carotovora subsp. carotovora (Ecc) DNA under all conditions tested. Hybridization to DNAs from Agrobacterium tumefaciens and Pseudomonas marginalis was observed at low stringency conditions (45°C). No hybridization was observed between the pel gene probe and six other DNA samples.     

Low Boiling Compounds in Cellulose Cigarette Smoke

The pectate lyase I (PAL I) gene was cloned from Erwinia carotovora Er. The cloned DNA, 5.1 kb, seemed to contain the region necessary for the regulation of pectin lyase formation, as PAL I was induced by pectin in E. carotovora Er 18 containing the cloned PAL I gene. PAL I had a signal peptide of 22 amino acids which seemed to be cleaved between Gly and Ala. PAL I had a M.W. of 37,587 composed of 345 amino acids. The presence of promoter and SO sequences was suggested upstream of the translational start codon of the PAL I gene. A palindromic sequence, which might be important for the regulation of PAL I gene expression, was detected downstream from the putative promoter. Another open reading frame highly homologous to the PAL I gene was detected downstream from the PAL I gene and designated the PAL X gene. This gene had 81% homology in 606 bp from the translational start codon with the PAL I gene.     

Analysis of yeast prp20 mutations and functional complementation by the human homologue RCC1, a protein involved in the control of chromosome condensation

Summary A DNA fragment that codes for the 364 amino-terminal amino acid residues of a putative Bacillus subtilis SecA homologue has been cloned using the Escherichia coli SecA gene as a probe. The deduced amino acid sequence showed 58% identity to the aminoterminus of the E. coli SecA protein. A DNA fragment which codes for 275 amino-terminal amino acid residues of the B. subtilis SecA homologue was expressed in E. coli and the corresponding gene product was shown to be recognized by anti-E. coli SecA antibodies. This polypeptide, although only about 30% the size of the E. coli SecA protein, also restored growth of E. coli MM52 (secA ^ts) at the non-permissive temperature and the translocation defect of proOmpA in this mutant was relieved to a substantial extent.     

Colorimetric Determination of α-Amino Nitrogen in Urine and Plasma with Ninhydrin Reaction

Bacillus stearothermophilus TH 6–2 has two kinds of purine nucleoside phosphorylases (Pu-NPase I and Pu-NPase II). The Pu-NPase I is a functional homolog of eukaryotic purine nucleoside phosphorylases that can catalyze the phosphorolysis of inosine and guanosine, but not adenosine, the primary substrate of Pu-NPase II. The Pu-NPase I gene of TH 6–2 has been cloned, sequenced, and expressed in E. coli. The gene corresponded to an open reading frame of 822 nucleotides that translates into a putative 274-amino acid protein with a molecular weight of 29,637. The deduced amino terminus sequence completely coincided with that found for the purified enzyme. The cloned gene was overexpressed in E. coli by using the trc promoter to produce an active enzyme in large quantities. The amino acid sequence of Pu-NPase I shared 50% similarity with those of human and mouse purine nucleoside phosphorylases.     

Cloning and sequencing of an Escherichia coli K12 gene which encodes a polypeptide having similarity to the human ferritin H subunit

Summary Using lambda phage clones containing segments of the Escherichia coli K12 chromosome as hybridization probes, we found one gene at 42 min on the E. coli chromosome map, the expression of which was affected by RNase III. The sequence of the DNA fragment containing this gene (gen-165) revealed the presence of an open reading frame encoding a polypeptide of 165 amino acid residues. The amino acid sequence deduced from the nucleotide sequence exhibited a remarkable similarity to that of the human ferritin H chain.     

The araC gene of Citrobacter freundii

The araC gene of Citrobacter freundii was cloned into plasmid pBR322 and expressed in Escherichia coli and Salmonella typhimurium. The nucleotide sequence and the predicted translational product were determined and compared to those of E. coli, S. typhimurium and Erwinia carotovora. The predicted translational product is 281 amino acids (aa) long, identical in size to that of S. typhimurium, and is 11 and 29 aa shorter than that of E. coli and E. carotovora, respectively. The nucleotide sequence of the araC gene of C. freundii is 83% homologous to the araC genes of both E. coli and S. typhimurium, but only 60% homologous to that of E. carotovora with respect to the regions they share. The predicted amino acid sequence is highly conserved and shows 96% and 94% homology to S. typhimurium and E. coli, respectively. E. carotovora shows only a 58% aa homology. The activator and autoregulatory activities of each plasmid encoded AraC protein in a S. typhimurium araC::lacZ protein fusion strain were examined.     

Studies on Antioxidative Substances of Microbial Origin

Aminopeptidase T (AP-T) is a metallo-dependent dimeric enzyme of Thermus aquaticus YT-1, an extremely thermophilic bacterium. We cloned the AP-T gene from T. aquaticus YT-1 into Escherichia coli using a synthetic oligonucleotide as a hybridization probe. The nucleotide sequence of the AP-T gene was found to encode 408 amino acid residues with GTG as a start codon. The molecular weight was calculated to be 44,820. The AP-T was overproduced in E. coli (about 5% of total soluble protein) when the start codon of the gene was changed from GTG to ATG, and the gene was downstream from the tac promoter. The AP-T expressed in E. coli was heat stable and easily purified by heat treatment (80°C, 30 min). The N-terminal amino acid sequence of AP-T showed similarity with that of aminopeptidase II from Bacillus stearothermophilus.     

An Efficient Approach to a Lactosamine Synthon for the Synthesis of I-Type Antigens

The gene coding for the ATCGAT specific BanIII DNA methyltransferase (M-BanIII) of Bacillus aneurinolyticus was cloned and its nucleotides sequenced. The coding region was assigned on the nucleotide sequence on the basis of the N-terminal amino acid sequence and molecular weight of the enzyme. The M-BanIII gene coded for a protein of 580 amino acid residues (MW 66,344). Comparison with other methylases indicated that the M-BanIII sequence contained a segment of tetra-amino acids, NPPY, characteristic of N6-adenine methylases. In addition, some homologous regions were found in the sequences of type II adenine methylases PaeR7I(CTCGAG), TaqI(TCGA) and PstI(CTGCAG), containing TCGA within the recognition sequences.     

Peroxyl Radical Scavenging Activity of Caffeic Acid and Its Related Phenolic Compounds in Solution

The esterase gene (est) of Pseudomonas putida MR-2068 was cloned into Escherichia coli JM109. An 8-kb inserted DNA directed synthesis of an esterase in E. coli. The esterase gene was in a 1.1-kb PstI-ClaI fragment within the insert DNA. The complete nucleotides of the DNA fragment containing the esterase gene were sequenced and found to include a single open reading frame of 828 bp coding for a protein of 276 amino acid residues. The open reading frame was confirmed by N-terminal amino acid sequence analysis of the purified esterase. A potential Shine-Dalgarno sequence is followed by the open reading frame. The esterase activity of the recombinant E. coli was more than 200 times higher than that of parental strain, P. putida MR-2068.     

Nucleotide Sequences and Organization of the Genes for Carotovoricin (Ctv) from Erwinia carotovora Indicate That Ctv Evolved from the Same Ancestor as Salmonella typhi Prophage

Carotovoricin Er (CtvEr), which is produced by a plant soft rot disease causative agent, Erwinia carotovora subsp. carotovora Er, is a high-molecular-weight bacteriocin showing Myoviridae phage-tail-like morphology with contractile sheath and plural tail fibers. We determined the complete nucleotide sequences of CtvEr genes on the E. carotovora Er chromosome and report that CtvEr genes consist of lysis cassette, major and minor structural protein gene clusters. Four promoters were identified. The lysis gene cassette, which is composed of the genes for lysis enzyme and holin, was also identified and characterized. The nucleotide sequences and organization of the genes for CtvCGE, which is produced by E. carotovora strain CGE234-M403 with the morphology similar to CtvEr, were also determined and compared to that of CtvEr, and it was found that CtvCGE is almost identical to CtvEr except for tail fibers which are involved in the killing spectra of both bacteriocins. We also explain that the gene organization and the deduced amino acid sequences of both carotovoricins are very close to those of prophage, which is lysogenized in the chromosome on Salmonella enterica serovar Typhi CT18. These findings strongly suggest that Ctv evolved as a phage tail-like bacteriocin from a common ancestor with Salmonella typhi prophage.     

Bacillus anthracis pXO1 virulence plasmid encodes a type 1 DNA topoisomerase

The virulence plasmid pXO1 is responsible for toxin production in Bacillus anthracis. A DNA fragment from pXO1 was isolated and was shown, by sequence analysis, to contain part of a type 1 DNA topoisomerase gene. Attempts to clone the entire wild-type gene, designated topX, in Escherichia coli, were unsuccessful. In order to obtain the complete gene, it was first insertionally inactivated and then cloned in the mutated form. The deduced amino acid sequence of Topo X1 shows similarities to that of the two E. coli type 1 DNA topoisomerases. The N-terminal two-thirds of the putative B. anthracis protein exhibits strongest sequence similarity to topoisomerase III, whereas the C-terminal portion contains cysteine residues that could form three zinc-binding domains, as they do in topoisomerase I. The suggested active-site tyrosine is conserved in all three proteins. The regulation of expression from the topX promoter is modified by addition of a gyrase inhibiting antibiotic. The Topo X1 protein is likely to be involved in the stability of pXO1.     

External Loops at the C Terminus of Erwinia chrysanthemi Pectate Lyase C Are Required for Species-Specific Secretion through the Out Type II Pathway

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of E. chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E. chrysanthemi, but not E. carotovora, Pels. We exploited the high sequence similarity between E. chrysanthemi PelC and E. carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pel1. The differential secretion of these hybrid proteins by E. coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning.     

Cloning and Characterization of the Gene Encoding Pyrroloquinoline Quinone-dependent Poly (vinyl alcohol) Dehydrogenase of Pseudomonas sp. Strain VM15C

A gene library of poly (vinyl alcohol) (PVA)-degrading Pseudomonas sp. strain VM15C was constructed in Escherichia coli with the vector pUC18. Screening of this library with a chromogenic PVA dehydrogenase assay resulted in the isolation of a clone that carries the gene (pdh) for the PVA dehydrogenase, and the entire nucleotide sequence of its structural gene was determined. The gene encodes a protein of 639 amino acid residues (68,045 Da) and in the deduced amino acid sequence, some putative functional sites, a signal sequence, a heme c-binding site, and a PQQ-binding site, were detected. The amino acid sequence showed low similarity to other types of quinoprotein dehydrogenases. PVA dehydrogenase expressed in E. coli clones required PQQ. Ca²⁺, and Mg²⁺ stimulated the activity. PVA-dependent heme c reduction occurred with exogenous PQQ in cell extracts of the E. coli clone. The PVA dehydrogenase in the E. coli clone was localized in the cytoplasm.     

Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida.

Summary The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 by in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 by in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84 — Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.     

Directed evolution for increased chitinase activity

Directed evolution through DNA shuffling and screening was used to enhance the catalytic ability of a fungal, Beauveria bassiana, chitinase, Bbchit1. The Bbchit gene was first linked to various prokaryotic signal sequences and expressed in Escherichia coli. The signal peptide, PelB, from Erwinia carotovora resulted in greatest chitinase secretion into broth. The nucleotide sequence expressing PelB signal peptide was then incorporated into an E. coli vector to express Bbchit1 variants generated by three rounds of DNA shuffling. A Bbchit1 library with 150,000 variants was constructed with a nucleotide point mutation frequency of 0.6% and screened for chitinolytic activity. Two Bbchit1 variants (SHU-1 and SHU-2) were selected that showed increased chitinolytic activity compared to the wild type. Sequence analysis of these variants revealed mutations in amino acid residues that would not normally be considered for rational design of improved chitinase activity. The amino acid substitutions occurred outside of the two putative substrate-binding sites and the catalytic region.     

Spontaneous and reversible high-frequency frameshifts originating a phase transition in the carotenoid biosynthesis pathway of the phototrophic bacterium Rhodobacter sphaeroides 2.4.1

Summary The nucleotide sequence of the melB gene coding for the Na⁺(Li⁺)/melibiose symporter of Salmonella typhimurium LT2 was determined, and its amino acid sequence was deduced. It consists of 1428 bp, corresponding to a protein of 476 amino acid residues (calculated molecular weight 52800). The amino acid sequence is homologous to that of the melibiose permease of Escherichia coli K12, with 85% identical residues. All, except one, of the amino acid residues that have been reported to be important for cation or substrate recognition in the melibiose permease of E. coli are conserved in the melibiose permease of S. typhimurium. In addition, part of the sequence resembles the lactose permease of Streptococcus thermophilus, the animal glucose transporter (GLUT1), the plasmid-coded raffinose permease (RafB), and the NADH-ubiquinone oxidoreductase chain 4 (Nuo4) of Aspergillus amstelodami.The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank/EMBL Data Bank with accession number X62101