Characterization of a restriction endonuclease, PhaI, from Pasteurella haemolytica serotype A1 and protection of heterologous DNA by a cloned PhaI methyltransferase gene.

Pasteurella haemolytica is the leading cause of economic loss to the beef cattle industry in the United States and an important etiologic agent worldwide. Study of P. haemolytica is hindered by researchers' inability to genetically manipulate the organism. A new restriction endonuclease, PhaI, an isoschizomer of SfaNI (R. J. Roberts, Methods Enzymol. 65:19-36, 1980), was isolated from P. haemolytica serotype 1, strain NADC-D60, obtained from pneumonic bovine lung. PhaI recognizes the 5-base nonpalindromic sequences 5'-GCATC-3' and 5'-GATGC-3'. Cleavage occurs 5 bases 3' from the former recognition site and 9 bases 5' from the latter recognition site. A gene encoding a methyltransferase which protects against PhaI cleavage was cloned from P. haemolytica NADC-D60 into Escherichia coli. Whereas unmethylated plasmid DNA containing a P. haemolytica origin of replication was unable to transform P. haemolytica when introduced by electroporation, the same plasmid DNA obtained from E. coli which contained a cloned PhaI methyltransferase gene could do so. The data indicate that PhaI is an effective barrier to the introduction and establishment of exogenous DNA in P. haemolytica.     

Cloning, nucleotide sequence, and expression of the Pasteurella haemolytica A1 glycoprotease gene.

Pasteurella haemolytica serotype A1 secretes a glycoprotease which is specific for O-sialoglycoproteins such as glycophorin A. The gene encoding the glycoprotease enzyme has been cloned in the recombinant plasmid pH1, and its nucleotide sequence has been determined. The gene (designated gcp) codes for a protein of 35.2 kDa, and an active enzyme protein of this molecular mass can be observed in Escherichia coli clones carrying pPH1. In vivo labeling of plasmid-encoded proteins in E. coli maxicells demonstrated the expression of a 35-kDa protein from pPH1. The amino-terminal sequence of the heterologously expressed protein corresponds to that predicted from the nucleotide sequence. The glycoprotease is a neutral metalloprotease, and the predicted amino acid sequence of the glycoprotease contains a putative zinc-binding site. The gene shows no significant homology with the genes for other proteases of procaryotic or eucaryotic origin. However, there is substantial homology between gcp and an E. coli gene, orfX, whose product is believed to function in the regulation of macromolecule biosynthesis.     

Secretion of the Pasteurella leukotoxin by Escherichia coli

Nucleic acid sequence analysis has indicated that the leukotoxin determinant from Pasteurella haemolytica is related to the hemolysin determinant from E. coli. The cloning and expression in E. coli of the lktCA genes has been previously reported, but the existence of leukotoxin secretory genes equivalent to hlyBD has not been documented. In this report we demonstrate that a 4.0 kb segment of P. haemolytica genomic DNA distal to the lktA gene, when expressed in trans to the previous cloned lktCA genes, allow the synthesis and secretion of active leukotoxin from E. coli. Complementation analysis using the cloned hlyB and hlyD genes indicates that this secretory locus derived from P. haemolytica contains two genes which we designate, by analogy, lktB and lktD.     

Secretion of the Pasteurella leukotoxin by Escherichia coli

Abstract Nucleic acid sequence analysis has indicated that the leukotoxin determinant from Pasteurella haemolytica is related to the hemolysin determinant from E. coli . The cloning and expression in E. coli of the lktCA genes has been previously reported, but the existence of leukotoxin secretory genes equivalent to hlyBD has not been documented. In this report we demonstrate that a 4.0 kb segment of P. haemolytica genomic DNA distal to the lktA gene, when expressed in trans to the previous cloned lktCA genes, allow the synthesis and secretion of active leukotoxin from E. coli . Complementation analysis using the cloned hlyB and hlyD genes indicates that this secretory locus derived from P. haemolytica contains two genes which we designate, by analogy, lktB and lktD .     

Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene

The leukotoxin produced by the oral bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis. In order to elucidate the structure of the leukotoxin, molecular cloning of the leukotoxin gene was carried out. A DNA library of A. actinomycetemcomitans, strain JP2, was constructed by partial digestion of genomic DNA with Sau3AI and ligation of 0.5 to 5.0 kilobase pair fragments into the Bam HI site of the plasmid vector pENN-vrf. After transformation into E. coli RR1 (lambda cI857), the clones were screened for the production of A. actinomycetemcomitans leukotoxin with polyclonal antibody. Six immunoreactive clones were identified. The clones expressed proteins which ranged from 21-80 kilodaltons, and the clone designated pII-2, producing the largest protein was selected for further study. Antibodies eluted from immobilized pII-2 protein also recognized the native A. actinomycetemcomitans leukotoxin molecule indicating that both molecules shared at least one epitope. DNA sequence analysis demonstrated that there are regions of significant amino acid sequence homology between the cloned A. actinomycetemcomitans leukotoxin and two other cytolysins, Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin, suggesting that a family of cytolysins may exist which share a common mechanism of killing but vary in their target cell specificity.     

An analysis of the codon usage of Pasteurella haemolytica A1

Analysis of approximately 17 kbp of nucleotide sequences from three different regions of the genome of Pasteurella haemolytica A1 showed that the mol% G+C of P. haemolytica A1 DNA is 38.5%. When only the coding sequences (approx. 10 kbp) were analysed, a similar value of 38.8% was obtained. A comparison of the relative synonymous codon usage values of the cloned genes showed that P. haemolytica A1 has a very different codon usage pattern from that of Escherichia coli.     

Molecular gene cloning and nucleotide sequencing and construction of an aroA mutant of Pasteurella haemolytica serotype A1.

The aroA gene of Pasteurella haemolytica serotype A1 was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829. The nucleotide sequence of a 2.2-kb fragment encoding aroA predicted an open reading frame product 434 amino acids long that shows homology to other bacterial AroA proteins. Several strategies to inactivate aroA were unsuccessful. Gene replacement was finally achieved by constructing a replacement plasmid with aroA inactivated by insertion of a P. haemolytica ampicillin resistance fragment into a unique NdeI site in aroA. A hybrid plasmid was constructed by joining the aroA replacement plasmid with a 4.2-kb P. haemolytica plasmid which encodes streptomycin resistance. Following PhaI methylation, the replacement plasmid was introduced by electroporation into P. haemolytica NADC-D60, a plasmidless strain of serotype 1A. Allelic exchange between the replacement plasmid and the chromosome of P. haemolytica gave rise to an ampicillin-resistant mutant which grew on chemically defined P. haemolytica medium supplemented with aromatic amino acids but failed to grow on the same medium lacking tryptophan. Southern blot analysis confirmed that aroA of the mutant was inactivated and that the mutant was without a plasmid.     

Neisseria meningitidis transferrin-binding protein 1 expressed in Escherichia coli is surface exposed and binds human transferrin

Abstract A gene library of Neisseria meningitidis B15 P1.16 DNA was established in λ Zap II and clones containing DNA encoding transferrin binding protein 1 (TBP-1) identified following hybridisation with a 63-bp DNA probe based on the codon assignment for the first 21 N-terminal amino acids of TBP-1. Sequencing of the cloned DNA demonstrated that all of the intergenic DNA (i.e. upstream of bp-1 running through to the 3' end of the transferrin-binding protein 2 gene) and approx. 15% of tbp-1 had been cloned. The complete gene was generated using a polymerase chain reaction, with the primer for the 3' end being based on tbp-A of N. gonorrhoeae , and the approx. 2.9-kb DNA product cloned into pGem-3Z. The expressed protein (approx. 100 kDa) reacted with antiserum to an N-terminal peptide of TBP-1. In addition, the native product was surface-expressed by Escherichia coli and bound human transferrin.     

Acid phosphatases of Escherichia coli: molecular cloning and analysis of agp, the structural gene for a periplasmic acid glucose phosphatase.

Several unknown Escherichia coli genes for different species of acid phosphatase were cloned in vivo with the plasmid Mu dII4042. When present in a multicopy state, each gene promoted hydrolysis of p-nitrophenyl-phosphate at acidic pH. Among seven recombinant clones that encoded periplasmic acid phosphatase activities, five different genes could be distinguished by the pH optimum and substrate preference for the enzyme and by the restriction enzyme pattern. A 1.7-kilobase recombinant DNA fragment, common to two clones, was inserted into plasmid pBR322 and shown to contain a new gene, agp, which leads to the overexpression of the periplasmic acid glucose-1-phosphatase, a dimer of a 44-kilodalton polypeptide. Fusions of agp to gene phoA deprived of its own signal sequence conferred an alkaline phosphatase-positive phenotype to bacteria, showing the presence of an export signal on agp. The resulting hybrid proteins were characterized by immunoprecipitation with an antiserum directed against purified acid phosphatase or against alkaline phosphatase, showing that agp is the structural gene of the acid phosphatase. The beginning, the orientation, and the end of gene agp on the cloned DNA fragment were determined by the characteristics of such hybrid proteins.     

Cloning, nucleotide sequence, and expression of the Escherichia coli fabD gene, encoding malonyl coenzyme A-acyl carrier protein transacylase.

The Escherichia coli fabD gene encoding malonyl coenzyme A-acyl carrier protein transacylase (MCT) was cloned by complementation of a thermosensitive E. coli fabD mutant (fabD89). Expression of the fabD gene in an appropriate E. coli expression vector resulted in an accumulation of the MCT protein of up to 10% of total soluble protein, which was accompanied by an approximately 1,000-fold increase in the MCT activity. DNA sequence analysis and expression studies revealed that the fabD gene is part of an operon consisting of at least three genes involved in fatty acid biosynthesis. Comparison with available DNA and protein data bases suggest that a 3-ketoacyl-acyl carrier protein synthase and a ketoacyl-acyl carrier protein reductase gene are located immediately upstream and downstream, respectively, of fabD within this fab operon. Western immunoblot analysis with antiserum raised against wild-type E. coli MCT showed that the fabD89 allele encodes a polypeptide with an apparent molecular weight of 27,000 in addition to the normal MCT protein of 32,000. The nature of the temperature-sensitive fabD89 gene product is discussed.     

Cloning of the gene for delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni

We have cloned an approx. 5-kb fragment of Pseudomonas testosteroni DNA containing the structural gene of delta 5-3-ketosteroid isomerase into the EcoRI site of the lambda gt11 genome. Escherichia coli infected with these recombinant phages produce a polypeptide which is recognized by antiserum raised against the purified isomerase. Four of the recombinant lambda gt11 clones contain significant levels of isomerase activity and produce an immunopositive polypeptide of the same apparent Mr as the native isomerase obtained from P. testosteroni. The approx. 5-kb fragment hybridizes to synthetic 21-mer and 17-mer oligodeoxynucleotide mixtures corresponding to the 5' and 3' regions, respectively, of the expected nucleotide sequence of the gene.     

envM genes of Salmonella typhimurium and Escherichia coli.

Conjugation and bacteriophage P1 transduction experiments in Escherichia coli showed that resistance to the antibacterial compound diazaborine is caused by an allelic form of the envM gene. The envM gene from Salmonella typhimurium was cloned and sequenced. It codes for a 27,765-dalton protein. The plasmids carrying this DNA complemented a conditionally lethal envM mutant of E. coli. Recombinant plasmids containing gene envM from a diazaborine-resistant S. typhimurium strain conferred the drug resistance phenotype to susceptible E. coli cells. A guanine-to-adenine exchange in the envM gene changing a Gly codon to a Ser codon was shown to be responsible for the resistance character. Upstream of envM a small gene coding for a 10,445-dalton protein was identified. Incubating a temperature-sensitive E. coli envM mutant at the nonpermissive temperature caused effects on the cells similar to those caused by treatment with diazaborine, i.e., inhibition of fatty acid, phospholipid, and lipopolysaccharide biosynthesis, induction of a 28,000-dalton inner membrane protein, and change in the ratio of the porins OmpC and OmpF.     

Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2.

Western blot (immunoblot) analysis of Bacillus subtilis cell extracts detected two proteins that cross-reacted with monospecific polyclonal antibody raised against Escherichia coli initiation factor 2 alpha (IF2 alpha). Subsequent Southern blot analysis of B. subtilis genomic DNA identified a 1.3-kilobase (kb) HindIII fragment which cross-hybridized with both E. coli and Bacillus stearothermophilus IF2 gene probes. This DNA was cloned from a size-selected B. subtilis plasmid library. The cloned HindIII fragment, which was shown by DNA sequence analysis to encode the N-terminal half of the B. subtilis IF2 protein and 0.2 kb of upstream flanking sequence, was utilized as a homologous probe to clone an overlapping 2.76-kb ClaI chromosomal fragment containing the entire IF2 structural gene. The HindIII fragment was also used as a probe to obtain overlapping clones from a lambda gt11 library which contained additional upstream and downstream flanking sequences. Sequence comparisons between the B. subtilis IF2 gene and the other bacterial homologs from E. coli, B. stearothermophilus, and Streptococcus faecium displayed extensive nucleic acid and protein sequence homologies. The B. subtilis infB gene encodes two proteins, IF2 alpha (78.6 kilodaltons) and IF2 beta (68.2 kilodaltons); both were expressed in B. subtilis and E. coli. These two proteins cross-reacted with antiserum to E. coli IF2 alpha and were able to complement in vivo an E. coli infB gene disruption. Four-factor recombination analysis positioned the infB gene at 145 degrees on the B. subtilis chromosome, between the polC and spcB loci. This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis.     

Gonococcal transferrin-binding protein 1 is required for transferrin utilization and is homologous to TonB-dependent outer membrane receptors.

The pathogenic Neisseria species are capable of utilizing transferrin as their sole source of iron. A neisserial transferrin receptor has been identified and its characteristics defined; however, the biochemical identities of proteins which are required for transferrin receptor function have not yet been determined. We identified two iron-repressible transferrin-binding proteins in Neisseria gonorrhoeae, TBP1 and TBP2. Two approaches were taken to clone genes required for gonococcal transferrin receptor function. First, polyclonal antiserum raised against TBP1 was used to identify clones expressing TBP1 epitopes. Second, a wild-type gene copy was cloned that repaired the defect in a transferrin receptor function (trf) mutant. The clones obtained by these two approaches were shown to overlap by DNA sequencing. Transposon mutagenesis of both clones and recombination of mutagenized fragments into the gonococcal chromosome generated mutants that showed reduced binding of transferrin to whole cells and that were incapable of growth on transferrin. No TBP1 was produced in these mutants, but TBP2 expression was normal. The DNA sequence of the gene encoding gonococcal TBP1 (tbpA) predicted a protein sequence homologous to the Escherichia coli and Pseudomonas putida TonB-dependent outer membrane receptors. Thus, both the function and the predicted protein sequence of TBP1 were consistent with this protein serving as a transferrin receptor.     

Cloning of a chromosomal fragment from Lactococcus lactis subsp. lactis partially complementing Escherichia coli recA functions

A recA-like gene was isolated from a gene library of Lactococcus lactis subsp. lactis by intergeneric complementation of an E. coli recA mutant. A plasmid was obtained which fully complemented the RecA response to DNA damaging agents and UV inducibility of prophage, but not P1 plating efficiency in an E. coli recA mutant. The cloned DNA fragment also partially complemented the rec mutation in Lc. lactis MMS36. Hybridization studies showed that there was no detectable sequence homology between the recA gene of E. coli and Lc. lactis subsp. lactis chromosomal DNA.     

Characterization of a CACAG pentanucleotide repeat in Pasteurella haemolytica and its possible role in modulation of a novel type III restriction-modification system

In a previous study, a recombinant plasmid that contains a CACAG pentanucleotide repeat was isolated from a Pasteurella haemolytica A1 library. Southern hybridization analysis using a (CACAG)5probe indicated the presence of two loci that contain the pentanucleotide repeats on the genome of P.haemolytica A1. Additional hybridization analyses against genomic DNA from related microorganisms indicated that the repeats are only present in P.haemolytica and Pasteurella trehalosi T3. The various serotypes of P.haemolytica werefound to have either one or two of the CACAG repeat-containing loci. Examination of the locus designated Rpt2 by PCR and sequence analysis indicated that the number of CACAG repeats could change upon serial subculture which most likely occurs as a result of DNA slipped-strand mispairing. A plasmid carrying the Rpt2 locus was isolated and characterized. Sequenceanalysis indicated that the CACAG repeats are contained within the 5'-end of a gene that showed homology to mod genes of type III restriction-modification systems. A second open reading frame downstream was identified which showed homology to res genes of type III restriction-modification systems. Both the modification and restriction proteins could be expressed and polypeptides of the expected sizes were detected by SDS-PAGE. Restriction activity could also be detected in crude cytoplasmic extracts of Escherichia coli strains carrying the mod and res genes on recombinant plasmids.     

Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology.

The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes.     

A mutation in the Neisseria gonorrhoeae rfaD homolog results in altered lipooligosaccharide expression.

The gonococcal lsi-6 locus was cloned and shown by DNA sequence analysis to have homology with the E. coli rfaD gene, which encodes ADP-L-glycero-D-mannoheptose epimerase. This enzyme is involved in the biosynthesis of the lipopolysaccharide precursor ADP-L-glycero-D-mannoheptose. A site-directed frameshift mutation in lsi-6 was constructed by PCR amplification and introduced into the chromosome of Neisseria gonorrhoeae MS11 P+ by transformation. The lipooligosaccharides (LOS) of mutant and parental strains were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lsi-6 mutant produced LOS components with apparent molecular masses of 2.6 and 3.6 kDa as compared with a 3.6-kDa band of the MS11 P+ strain. The parental LOS phenotype was expressed when a revertant was constructed by transformation of the cloned wild-type gene into the lsi-6 mutant. The immunoreactivity of LOS from parental and constructed strains was examined by SDS-PAGE and Western blotting. Only the parental and reconstructed wild-type strains produced a 3.6-kDa LOS component that reacted with monoclonal antibody 2-1-L8. These results suggest that the lsi-6 locus is involved in gonococcal LOS biosynthesis and that the nonreactive mutant 3.6-kDa LOS component contains a conformational change or altered saccharide composition that interferes with immunoreactivity.