Molecular cloning and analysis of a region of the Bartonella bacilliformis genome encoding NlpD,L-isoaspartyl methyltransferase and YajC homologs

The NlpD/LppB homolog of the human pathogen, Bartonella bacilliformis, is an immunogenic 43-kDa protein that is encoded by a 1206-bp open reading frame (ORF-401). The regions flanking the nlpD/lppB gene of B. bacilliformis were sequenced to determine if it is located within the rpoS operon, as it is in most bacteria. We report that the B. bacilliformis nlpD/lppB gene is located immediately downstream of pcm, a gene encoding a 25-kDa protein, L-isoaspartyl protein carboxyl methyltransferase, that is a component of the rpoS operon in other bacteria. However, the genomic organization downstream of the B. bacilliformis nlpD/lppB gene appears to be distinct. In other bacteria, the third gene in the operon is rpoS, a gene that codes for an alternative sigma factor of RNA polymerase. In B. bacilliformis, an open reading frame encoding a protein homologous to the immunodominant YajC protein is located directly downstream of the nlpD/lppB gene. We show that Bartonella henselae, a close relative of B. bacilliformis, also shares this unusual organizational feature. Thus, the genomic organization of the nlpD/lppB genes of B. bacilliformis, and B. henselae appears to be unique among all bacteria for which the sequence of this region has been reported.     

The 65-kilodalton antigen of Mycobacterium tuberculosis.

The immune response of the host to the antigens of Mycobacterium tuberculosis plays the key role in determining immunity from infection with as well as the pathogenicity of this organism. A 65-kilodalton (kDa) protein has been identified as one of the medically important antigens of M. tuberculosis. The gene encoding this antigen was isolated from a lambda gt11-M. tuberculosis recombinant DNA library using monoclonal antibodies directed against the 65-kDa antigen as the specific probes. The nucleotide sequence of this gene was determined, and a 540-amino-acid sequence was deduced. This sequence was shown to correspond to that of the 65-kDa antigen by constructing a plasmid in which this open reading frame was fused to the lacZ gene. The resulting fusion protein reacted specifically with the anti-65-kDa protein antibodies. A second long open reading frame was found downstream of the 65-kDa antigen gene which could encode a protein of 517 amino acids. This putative protein contained 29 tandemly arranged partial or complete matches to a pentapeptide sequence.     

Isolation and characterization of the 54-kDa and 22-kDa chitinase genes of Serratia marcescens KCTC2172

A DNA fragment (pCHI5422) containing two genes encoding a 54-kDa and a 22-kDa chitinase was isolated from a cosmid DNA library of Serratia marcescens KCTC2172. The complete nucleotide sequence of pCHI5422 consisting of 4581 bp was determined. The nucleotide sequence of the 22-kDa chitinase consists of 681 bp of open reading frame encoding 227 amino acids and is located 1422 bp downstream of the translation termination codon of the 54-kDa chitinase sequence. The 54-kDa chitinase gene consisted of 1497 bp in a single open reading frame encoding 499 amino acids. The genes encoding the 54-kDa and 22-kDa chitinase were separately subcloned in Escherichia coli and the individual chitinases were expressed and purified from the culture broth using chitin affinity chromatography. When chitohexaose was used as substrate, the major product of the enzymatic reaction of both the 54-kDa and 22-kDa chitinases was a (GlcNAc)₂ dimer with a minor amount of monomer. The specific activity of the 54-kDa and 22-kDa chitinases were 300 μM (min)⁻¹ mg⁻¹ and 17 μM (min)⁻¹ mg⁻¹ on the natural swollen chitin, respectively.     

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197.     

Cloning and expression of the bovine cardiac sodium-calcium exchanger.

Two clones (p17 and p13), each containing the complete coding sequence for the bovine cardiac Na+/Ca2+ exchanger, were obtained from a lambda gt10 cDNA library by screening with cDNA probes from the canine exchanger. The coding sequence of clone p17 was 92 and 98% identical to the canine cDNA at the nucleotide and amino acid levels, respectively. Nine of the 21 amino acid differences between the two exchangers were found within the 32-amino acid signal sequence. The sequenced portions of the 3' untranslated regions of the cow and dog clones were 88% identical. Na+/Ca2+ exchange activity was expressed in Xenopus laevis oocytes injected with cRNA from clone p17, and in COS cells transfected with expression vectors containing p17. Immunoprecipitation of 35S-labeled proteins from transfected cells with an antibody against the N-terminal portion of the bovine exchanger showed the presence of a 120-kDa protein corresponding to the intact cardiac exchanger. The second bovine clone (p13) did not express exchange activity in either of the above expression systems, presumably because it contained a 300-bp insert with multiple stop codons which interrupted the coding sequence. Comparison of the 5' untranslated regions of p13 and p17 revealed a 156-bp segment in p17 that was apparently spliced out of p13. This segment contained a short open reading frame. A chimera encoding the 5' untranslated region of p13 and the coding sequence of p17 exhibited only a modest (74%) increase in expressed exchange activity in transfected cells compared to p17, suggesting that the presence of the upstream open reading frame in p17 did not greatly reduce translation efficiency. The results suggest that alternate splicing mechanisms may be involved in processing mRNA for the bovine cardiac exchanger.     

Antigenic proteins of Mycobacterium leprae. Complete sequence of the gene for the 18-kDa protein

Recombinant clones expressing antigenic determinants of the 18-kDa protein antigen from Mycobacterium leprae recognized by the L5 monoclonal antibody were isolated from a lambda gt11 expression library and their nucleotide sequences determined. All clones expressed the M. leprae-specific determinant as part of a large fusion protein with Escherichia coli beta-galactosidase. The deduced amino acid sequence of the coding region indicated that all the lambda gt11 recombinant clones contained an incomplete M. leprae gene sequence representing the carboxy-terminal two-thirds (111 amino acids) of the 18-kDa gene and coding for a peptide of m.w. 12,432. Subsequent isolation and sequencing of a 3.2kb BamHI-PstI DNA fragment from a genomic M. leprae cosmid library permitted the deduction of the complete 148 amino acid sequence with a predicted m.w. of 16,607. A second open reading frame 560 bases downstream from the 18-kDa coding sequence was found to code for a putative protein of 137 amino acids (m.w. = 15,196). Neither this nor the 18-kDa amino acid sequence displayed any significant homologies with any proteins in the GENBANK, EMBL, or NBRF data bases. Crude lysates from recombinant lambda gt11 clones expressing part of the 18-kDa protein have been reported to stimulate the proliferation of some M. leprae-specific helper T cell clones. Thus, it is significant that the complete 18-kDa sequence contains five short peptides predicted to be possible helper T cell antigenic epitopes based on their propensity to form amphipathic helices. Although three of these occur within the 111 amino acid carboxy-terminal peptide expressed by lambda gt11 clones, the most highly amphipathic peptide is found in the amino-terminal region not present in the lambda gt11 recombinants.     

Cloning of a novel pilin-like gene from Bordetella pertussis: homology to the fim2 gene

A search for pilin genes in a Bordetella pertussis (Bp) genomic library has led to the identification of several clones which hybridize to synthetic oligonucleotides with sequences derived from amino acid sequences of Bp fimbrial subunits. One of these clones (corresponding to a gene we have named fimX) contains an open reading frame encoding a protein with a molecular weight of about 20 kD and a sequence similar but not identical to the fimbrial subunit fim2 and to other fimbrial protein sequences. In this communication we present the cloning and nucleotide sequence of the fimX gene and its homology to the fim2 gene. A genomic analysis on the positional relationship between the two genes is also presented.     

Biosynthesis of riboflavin: cloning, sequencing, mapping, and expression of the gene coding for GTP cyclohydrolase II in Escherichia coli.

GTP cyclohydrolase II catalyzes the first committed step in the biosynthesis of riboflavin. The gene coding for this enzyme in Escherichia coli has been cloned by marker rescue. Sequencing indicated an open reading frame of 588 bp coding for a 21.8-kDa peptide of 196 amino acids. The gene was mapped to a position at 28.2 min on the E. coli chromosome and is identical with ribA. GTP cyclohydrolase II was overexpressed in a recombinant strain carrying a plasmid with the cloned gene. The enzyme was purified to homogeneity from the recombinant strain. The N-terminal sequence determined by Edman degradation was identical to the predicted sequence. The sequence is homologous to the 3' part of the central open reading frame in the riboflavin operon of Bacillus subtilis.     

An open reading frame upstream from the nifH gene of Klebsiella pneumoniae

An open reading frame upstream from nifHDK operon of Klebsiella pneumoniae had been described. The orientation of this open reading frame is opposite to that of nifHDK and sequence homology was found between the open reading frame promoter and the promoter of nifHDK operon. A recombinant plasmid carrying the promoter region of the open reading frame fused to the beta-galactosidase gene was constructed. Strains of E.coli were transformed with the plasmid containing this open reading frame promoter-lacZ fusion or co-transformed with it and a plasmid carrying the nifA gene. An appreciable activity of beta-galactosidase was found in strains which received both plasmids, indicating that the promoter of the open reading frame can be activated by the product of nifA gene. Thus, the open reading frame found between nifHDK operon and nifJ behaves just like other nif genes of K.pneumoniae in requiring the product of nifA as the positive effector for expression.     

Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae.

Colonization of the swine respiratory tract by Mycoplasma hyopneumoniae is accomplished by specific binding to the cilia of the mucosal epithelial cells. Previous studies have implicated a 97-kDa outer membrane-associated protein, P97, that appeared to mediate this interaction. In order to further define the role of P97 in adherence to porcine cilia, the structural gene was cloned and sequenced, and the recombinant products were analyzed. Monoclonal antibodies were used to identify recombinant clones in a genomic library expressed in an opal suppressor host because of alternate codon usage by mycoplasmas. The gene coding for P97 was then identified by Tn1000 mutagenesis of recombinant clones. DNA sequence analysis revealed an open reading frame coding for a 124.9-kDa protein with a hydrophobic transmembrane spanning domain. The N-terminal sequence of purified P97 mapped at amino acid position 195 of the translated sequence, indicating that a processing event had occurred in M. hyopneumoniae. Both recombinant P97 protein expressed in an Escherichia coli opal suppressor host and M. hyopneumoniae bound specifically to swine cilia, and the binding was inhibited by heparin and fucoidan, thus supporting the hypothesis that P97 was actively involved in binding to swine cilia in vivo.     

Molecular cloning of a Brassica napus thiohydroximate S-glucosyltransferase gene and its expression in Escherichia coli

A genomic clone encoding a thiohydroximate S -glucosyltransferase ( S -GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers. Its corresponding cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library screening. The genomic clone was 5 896 bp long and contained a 173-bp intron. At least two copies of the S -GT gene were present in B. napus . The full-length cDNA clone was 1.5 kb long and contained an open reading frame encoding a 51-kDa polypeptide. The deduced amino acid sequence shared a significant degree of homology with other glucosyltransferases characterized in other species, including a highly conserved motif within this family of enzymes corresponding to the glucose-binding domain. The recombinant protein was expressed in Escherichia coli , and the enzyme activity was tested by a biochemical assay based on the measure of glucose incorporation. The high thiohydroximate S -GT activity detected from the recombinant protein confirmed that this clone was indeed a S -glucosyltransferase.     

Cloning, sequencing, and expression of a gene encoding a 100-kilodalton mosquitocidal toxin from Bacillus sphaericus SSII-1.

A cosmid library was prepared from a partial BamHI digest of total DNA from Bacillus sphaericus SSII-1. Two hundred fifty Escherichia coli clones were screened for toxicity against larvae of the mosquito Culex quinquefasciatus. One toxic clone, designated pKF2, was chosen for further study. Two toxic subclones, designated pXP33 and pXP34, obtained by ligating PstI-derived fragments of pKF2 into pUC18, contained the same 3.8-kb fragment, but in opposite orientations. Sequence analysis revealed the presence of an open reading frame corresponding to a 100-kDa protein and the 3' end of a further open reading frame having significant homology to open reading frames of transposons Tn501 and Tn21. The sequence of the SSII-1 toxin was compared with those of known toxins and was found to show regional homology to those of ADP-ribosyltransferase toxins. The distribution of the toxin gene among other B. sphaericus strains was examined.