Molecular cloning, sequencing, expression, and characterization of an immunogenic 43-kilodalton lipoprotein of Bartonella bacilliformis that has homology to NlpD/LppB.

A recombinant clone expressing an immunoreactive antigen of Bartonella bacilliformis was isolated by screening a genomic DNA library with serum from a patient with the chronic verruga phase of bartonellosis. The clone, pBIPIM-17, contained a partial open reading frame that expressed an immunoreactive fusion protein. Subsequent rescreening of the library by plaque hybridization resulted in the isolation of recombinant clones that contain the entire open reading frame. The open reading frame (ORF-401) is capable of encoding a protein of 401 amino acids with a predicted molecular mass of 43 kDa. The deduced amino acid sequence of the encoded protein was found to be highly homologous to a recently identified bacterial lipoprotein (LppB/NlpD) which has been associated with virulence. Evidence has been provided to show that the 43-kDa antigen of B. bacilliformis is a lipoprotein and that it is likely to use the same biosynthetic pathway as other bacterial lipoproteins. This is the first report to date that characterizes a lipoprotein of B. bacilliformis. The immunogenicity of the B. bacilliformis LppB homologue was demonstrated by Western blot analysis using sera from patients with clinical bartonellosis. Sera from patients who had a high titer for Bartonella henselae, the causative agent of bacillary angiomatosis and cat scratch disease, also recognized the recombinant 43-kDa antigen, suggesting that a homologue of this antigen is present in B. henselae. Using a cocktail of synthetic peptides corresponding to predicted major antigenic sites, polyclonal antiserum specific for the LppB homologue of B. bacilliformis was generated. This antiserum did not recognize the NlpD homologue of Escherichia coli or the 43-kDa antigen of B. henselae.

The gene encoding the 17-kDa antigen of Bartonella henselae is located within a cluster of genes homologous to the virB virulence operon.

A Bartonella henselae genomic A library was screened with antiserum generated in mice against live B. henselae. One of the immunoreactive clones expressed a 17-kDa antigen that was characterized previously as an immunodominant protein of B. henselae. Sequence analysis of the recombinant clone, pBHIM-2, revealed that the open reading frame (ORF) encoding the 17-kDa antigen was situated between homologs of virB4 and virB6, two genes that belong to the virB operon. The virB operon has been associated with the transfer of oncogenic T-DNA in Agrobacterium tumefaciens and with secretion of the pertussis toxin in Bordetella pertussis. Downstream of the virB6 gene within pBHIM-2 was a partial open reading frame that was homologous to the virB8 gene. Rescreening of the library by plaque hybridization using probes specific to the 5' and 3' ends of the pBHIM-2 insert resulted in the isolation of recombinant clones containing additional virB genes. Assembly of the sequences obtained from the recombinant clones revealed that eight of the open reading frames encode homologs of the VirB proteins. The homology and colinearity with the virB genes suggest that the gene encoding the 17-kDa antigen is expressed within the virB locus of B. henselae.

Use of the cell division protein FtsZ as a means of differentiating among Bartonella species.

Genes coding for homologs of the highly conserved cell division protein FtsZ were isolated from Bartonella henselae and Bartonella quintana, the causative agents of cat scratch disease and trench fever, respectively. DNA fragments coding for the ftsZ open reading frames (ORFs) were cloned into Escherichia coli following PCR amplification with primers based on the ftsZ sequence of the closely related species Bartonella bacilliformis. The amino acid sequences predicted from the cloned B. henselae and B. quintana ftsZ ORFs are 81 to 83% identical to the corresponding protein in B. bacilliformis. Like the FtsZ protein of B. bacilliformis, the B. henselae and B. quintana homologs are about twice as large as the FtsZ proteins reported in most other organisms. Localized sequence differences within the C-terminal coding regions of the Bartonella ftsZ genes were used as the basis for species-specific identification of these organisms at both the DNA and protein levels. Oligonucleotide primers which permit the amplification of an ftsZ fragment from each of the Bartonella species without amplifying DNA from the other two species were designed. Anti-FtsZ antisera raised in rabbits against synthetic peptides corresponding to the relatively divergent C-terminal regions were shown via Western blot analysis to react only with the FtsZ protein from the cognate Bartonella species. These observations raise the possibility that the differences in ftsZ sequences can be used as the basis for diagnostic tests to differentiate among these closely related pathogens.

Analysis of 36-kilodalton protein (PapA) associated with the bacteriophage particle of Bartonella henselae.

A library of Bartonella henselae DNA was screened with antibody raised to the bacteriophage particle associated with this organism. A clone was isolated that expresses a 36-kD protein (termed PapA for particle-associated protein) when examined by immunoblot analysis using antibody raised to the particle. Southern blot hybridization indicates that the gene is present on the bacterial chromosome and packaged into the 14-kb particle-associated DNA. A papA-specific probe hybridized to multiple bands of B. henselae genomic DNA digested with several different restriction endonucleases. Thus, the gene is present in multiple copies on the genome or in different arrangements within a given population of B. henselae cells. The gene coding for PapA has been sequenced and codes for a 326-amino-acid protein with a deduced molecular weight of 36,161 daltons. The deduced protein shows 33.3% identity over a 108-amino-acid sequence with the P-min gene product of Escherichia coli. P-min is partially located within the invertible P region of the excisable element e14, found on the E. coli chromosome. Taken together, these results suggest that papA is present on a mobile genetic element of the B. henselae genome and is also packaged into the bacteriophage particle.

The 75-kilodalton antigen of Bartonella bacilliformis is a structural homolog of the cell division protein FtsZ.

A genomic library of Bartonella bacilliformis was constructed and screened with human anti-Bartonella serum from a patient with the chronic, verruga peruana phase of bartonellosis. An immunoreactive clone isolated from this library was found to code for a 591-amino-acid protein with a high degree of sequence similarity to the FtsZ family of proteins. The degree of amino acid identity between the B. bacilliformis protein (FtsZ[Bb]) and the other FtsZ proteins is especially pronounced over the N-terminal 321 amino acids (N-terminal domain) of the sequence, with values ranging from 45% identity for the homolog from Micrococcus luteus (FtsZ[Ml]) to 91% identity for the homolog from Rhizobium melliloti, (FtsZ[Rm1]). All of the functional domains required for FtsZ activity are conserved in FtsZ(Bb) and are located within the N-terminal domain of the protein. FtsZ(Bb) is approximately twice as large as most of the other FtsZ proteins previously reported, a property it shares with FtsZ(Rm1). Like the Rhizobium homolog, FtsZ(Bb) has a C-terminal region of approximately 256 amino acids that is absent in the other FtsZ proteins. Evidence is presented that implicates this region in the protein's antigenicity and suggests that, unlike most other FtsZ homologs, FtsZ(Bb) is at least partly exposed at the cell surface. PCR analysis revealed that an ftsZ gene similar in size to the B. bacilliformis gene is present in Bartonella henselae, a bacterium that is closely related to B. bacilliformis.

Identification and differentiation of Mycobacterium avium and M. intracellulare by PCR.

Known DNA sequences coding for the 16S rRNAs of 14 slowly growing Mycobacterium species were analyzed. Three sets of primers were synthesized: MAV and MIN, for M. avium and M. intracellulare, respectively, and MYCOB, for the slowly growing mycobacteria. Whole-cell DNAs of 14 reference species were extracted and amplified by PCR with the MYCOB, MAV, and MIN primers. The MYCOB primer amplified a 0.9-kb segment from the DNAs of all 14 species. The MAV and MIN primers each amplified one highly specific 1.3-kb segment from the homologous DNA, respectively. DNAs from each of 10 clinical isolates of M. avium and M. intracellulare identified by conventional methods were amplified with the MYCOB as well as the MAV and MIN primers; 9 of 10 isolates of each species were identified with their respective primers. One isolate of M. intracellulare was subsequently found to have been mislabeled. One isolate designated M. avium reacted only with the MYCOB primer. The hypervariable region of this strain was shown by DNA sequence analysis to be distinct from all known 16S rRNA sequences of Mycobacterium spp. Our data indicate that the currently identified M. avium-M. intracellulare complex includes strains genetically diverse from M. avium and M. intracellulare.

Detection of Clostridium botulinum type F using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify a portion of the Clostridium botulinum type F toxin gene. An 1137-bp fragment was amplified from 11 different strains of type F C. botulinum with primers derived from the published sequence of type F strain no. 202. This fragment was not amplified from the DNA of C. botulinum types A, B and E, or from other clostridial organisms examined. When used as a hybridization probe, the 1137-bp PCR-generated fragment generated from one of the type F strains (the proteolytic strain type F Langeland) hybridized to the PCR products from all other type F toxin-producing strains tested. Portions of fragments amplified from the type F Langeland strain were sequenced. The sequence of this strain was found to exhibit approximately 3% variation from the published sequence of the non-proteolytic type F strain no. 202. Primers designed to pair with the regions of maximum sequence variation between strain 202 and the Langeland strain gave amplification products only with DNA from type F strains that exhibited the same proteolytic properties as the strain from which the primer sequences were derived. These findings underscore the need to consider variations in sequence when designing oligonucleotide probes and PCR primers in order to avoid false negative results.

Bacteriophage-like particle of Rochalimaea henselae.

An extracellular particle approximately 40 nM in diameter was detected in culture supernatant from the fastidious bacterium Rochalimaea henselae. This particle has at least three associated proteins and contains 14 kbp linear DNA segments that are heterogeneous in sequence. The 14 kbp DNA was also present in R. henselae cells as an extrachromosomal element for all 14 strains tested. Despite attempts to induce lysis of R. henselae, plaque formation was not observed. A similar particle, also containing 14 kbp DNA, was observed in Bartonella bacilliformis, and may be analogous to a bacteriophage that has been described elsewhere for B. bacilliformis.

Syrian hamster monomorphic N-acetyltransferase (NAT1) alleles: amplification, cloning, sequencing, and expression in E. coli.

N-acetyltransferases have an important role in the metabolism of arylamine and hydrazine drugs and carcinogens. Human N-acetylation phenotype may predispose individuals toward a variety of drug and xenobiotic-induced toxicities and carcinogenesis. Syrian hamsters express two N-acetyltransferase isozymes; one varies with acetylator genotype (polymorphic) and has been termed NAT2; the other does not (monomorphic) and has been termed NAT1. The intronless NAT1 coding region was cloned via the polymerase chain reaction from homozygous rapid acetylator and homozygous slow acetylator congenic and inbred hamster genomic DNA templates and sequenced. The NAT1 alleles from the homozygous rapid (NAT1) and homozygous slow (NAT1s) acetylator hamsters differed in one nucleotide, but the mutation is silent with no change in deduced amino acid sequence. To characterize the enzyme products of the NAT1 alleles, we developed a prokaryotic-expression system. The NAT1r and NAT1s alleles were amplified by expression-cassette polymerase chain reaction and subcloned into the tac promoter-based plasmid vector pKK223-3 for over-production of recombinant NAT1 in E. coli strain JM105. Induced cultures from selected NAT1-inserted transformants yielded high levels of soluble protein capable of N-acetylation, O-acetylation, and N,O-acetylation. The recombinant NAT1r and NAT1s proteins did not differ in substrate specificity, specific activity, Michaelis-Menten kinetic properties, intrinsic stability, and electrophoretic mobility. Also, the over-expressed NAT1 proteins displayed substrate-specificity and electrophoretic mobilities characteristic of NAT1 isolated from Syrian hamster liver and colon cytosols.

Polymorphic arylamine N-acetyltransferase encoding gene (NAT2) from homozygous rapid and slow acetylator congenic Syrian hamsters.

The nucleotide (nt) and deduced amino acid (aa) sequences were determined for polymorphic arylamine N-acetyl-transferase (NAT2) and its gene, NAT2, from homozygous rapid and slow acetylator congenic Syrian hamsters. The slow acetylator (NAT2s) allele contained three point mutations which differed from the rapid acetylator allele (NAT2r); two mutations were silent, and the third mutation resulted in a premature stop codon. The NAT2s allele contained a truncated open reading frame of 726 nt encoding a 242-aa protein, which is 48-aa shorter than NAT2r.

The ImmC region of phage P1 codes for a gene whose product promotes lytic growth.

The ImmC region of the temperate bacteriophage P1 contains c1, a gene that codes for a repressor of lytic growth. Located in the region upstream of c1 are four open reading frames capable of coding for low-molecular-weight proteins. The efficiency of lysogeny by P1+Cm was found to be reduced by almost 10(5)-fold when the host cells carry this region of ImmC on a multicopy plasmid. The sequences responsible for interfering with lysogen formation were localized to one of the small open reading frames (orf4) within ImmC. Insertions and deletions within orf4 suppress the virulent phenotype of P1virC mutants when introduced into the phage by recombination. These virC-suppressed mutant phage were found to be incapable of lytic growth unless the product of orf4 is provided in trans. The presence of orf4 was observed to interfere with repression by the c1 protein of ImmC-encoded promoters fused to lacZ. For this reason, we suggest that orf4 corresponds to coi, a gene previously proposed to code for an inactivator of c1-mediated repression.

The c1 genes of P1 and P7.

The c1 genes of the heteroimmune phages P1 and P7 were sequenced and their products were compared. P7c1 expression was correlated with the translation in vitro of a protein whose predicted molecular weight (33,000 daltons) is indistinguishable from that of the P1c1 repressor. The c1 regions from both P1 and P7 were found to contain open reading frames capable of coding for a 283-amino acid protein whose predicted secondary structure lacks the helix-turn-helix motif commonly associated with repressor proteins. Two P1c1 amber mutations were localized to the 283-amino acid open reading frame. The P1c1 and P7c1 sequences were found to differ at only 18 positions, all but two of which alter the third position of the affected codon and do not alter the amino acid sequence of the protein. Plasmids expressing the c1 gene from either phage cause the repression of transcription from a cloned promoter situated upstream of P1c1.

The c1 repressor of bacteriophage P1 operator-repressor interaction of wild-type and mutant repressor proteins.

The c1 repressor gene of bacteriophage P1 and the temperature-sensitive mutants P1c1.100 and P1c1.162 was cloned into an expression vector and the repressor proteins were overproduced. A rapid purification procedure was required for the isolation of the thermolabile repressor proteins. Identification of the highly purified protein of an apparent molecular weight of 33,000 as the product of the c1 gene was verified by (i) the coincidence of partial amino acid sequences determined experimentally to that deduced from the c1 DNA sequence, and (ii) the temperature-sensitive binding to the operator DNA of the thermolabile repressor proteins. Analysis of the products of c1-c1.100 recombinant DNAs relates the thermolability to an unknown alteration in the C-terminal half of the c1.100 repressor. Binding to the operator DNA of c1 repressor is sensitive to N-ethylmaleimide. Since the only three cysteine residues are located in the C-terminal half of the repressor it is suggested that this part of the molecule is important for the binding to the operator DNA. This assumption is supported by the findings that a 14-kDa C-terminal repressor fragment obtained by cyanogen bromide cleavage retains DNA binding properties.

The structure of tyrosine aminotransferase. Evidence for domains involved in catalysis and enzyme turnover.

The primary structure of tyrosine aminotransferase, as deduced from the nucleotide sequence of complementary DNA, was confirmed by fast atom bombardment mass spectrometry of tryptic peptides derived from the purified protein. Limited digestion of the native enzyme with trypsin released an acetylated, amino-terminal peptide; the new amino terminus in the modified enzyme was Val65. Endogenous proteases generated a chromatographically separable form of tyrosine aminotransferase that began at Lys35. Neither trypsin nor the other proteases altered the catalytic activity of tyrosine aminotransferase. Reduction of the holoenzyme with sodium borohydride yielded a major tryptic peptide containing phosphopyridoxamine bound to lysine 280, which probably functions in transamination. The carboxyl terminus of tyrosine aminotransferase contains features that typify proteins with short half-lives; it includes two negatively charged, hydrophilic segments that are enriched for glutamyl residues and are similar to a PEST region in ornithine decarboxylase (Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368). Tyrosine aminotransferase belongs to a superfamily of enzymes which includes aspartate aminotransferase and can be aligned so that many invariant, functional residues coincide. Like the isoenzymes of aspartate aminotransferase, tyrosine aminotransferase may contain two domains, with a central, catalytic core, and a small domain made up of both amino- and carboxyl-terminal components. We speculate that the exposed small domain may confer the unusually rapid degradative rate that characterizes this enzyme.

Expression of the gene encoding the 17-kilodalton antigen from Rickettsia rickettsii: transcription and posttranslational modification.

Recently, we reported the molecular cloning and nucleotide sequence analysis of a gene from Rickettsia rickettsii that codes for a 17-kilodalton antigen (17K antigen) and is preceded by sequences closely resembling the -10 and -35 consensus sequences for recognition by Escherichia coli RNA polymerase (Anderson et al., J. Bacteriol. 169:2385-2390, 1987). Experiments described in this report indicate that the start sites for initiating transcription of the 17K antigen gene are identical in the E. coli clone and in intact R. rickettsii. In each case, initiation was shown to begin 9 bases downstream of the presumed Pribnow box sequence (TATACT). A 169-base-pair fragment containing the promoter sequence initiated transcription in both directions when cloned into an E. coli promoter probe vector. The rickettsial fragment was found to contain sequences identical to the -10 region (but not the -35 region) of the E. coli promoter consensus sequence directed away from the 17K antigen gene. The amino-terminal portion (residues 17 to 20) of the deduced amino acid sequence for the 17K antigen contained the tetrapeptide Leu-Gln-Ala-Cys, a sequence that conforms favorably to those described for lipid modification and cleavage by lipoprotein signal peptidase II. The 17K antigen produced by the E. coli clone was shown to be labeled with [3H]palmitate and [3H]glycerol, indicative of lipid modification. In vitro mutagenesis designed to alter the cysteine at residue 20 to a glycine abolished incorporation of [3H]palmitate, suggesting that posttranslational modification occurs via a mechanism similar to that described for other gram-negative bacterial lipoproteins.

The c4 gene of phage P1.

The c4 gene of phage P1 has been localized to 335 bp of the P1EcoRI-9 fragment, within 50 bp of the EcoRI-9/14 junction. DNA sequence analysis of this fragment reveals a single open reading frame of 66 amino acids. The location of two c4 mutations, both of which produce changes in the predicted amino acid sequence in this reading frame, suggests that the reading frame codes for the c4 repressor. A region with high homology to the E. coli promoter consensus sequence is located approximately 50 bp upstream from the reading frame. Deletion of this potential promoter region abolishes expression of c4, as indicated by the loss of complementation of c4 mutants for lysogeny. Complementation is restored by the introduction of a heterologous promoter (the T7 phi 10 promoter), indicating that c4 expression is absolutely dependent on transcription of the 66-amino acid reading frame.

Interaction of the P1c1 repressor with P1 DNA: localization of repressor binding sites near the c1 gene.

The c1 repressor of phage P1 was previously shown (B.R. Baumstark and J.R. Scott, 1980, J. Mol. Biol. 140, 471-480) to bind specifically to P1BamHI-9, a 1.4-kb fragment that is closely linked to the c1 structural gene and spans the ends of the P1 genetic map. The position of the repressor binding site(s) relative to the ends of the genetic map and the c1 gene was investigated by testing cloned fragments of EcoRI-7 and BamHI-9 for c1 expression and repressor binding. Although sequences in both BamHI-9 and the adjacent 2.7-kb EcoRI/BamHI fragment were found to be required for the production of the c1 protein, c1 expression could be restored to the 2.7-kb fragment by the addition of a heterologous promoter (ptac). These observations are consistent with the localization of the c1 reading frame to the 2.7-kb fragment and at least part of the c1 promoter region to BamHI-9. The c1 repressor was shown to bind in vitro to two distinct cloned fragments of BamHI-9 derived from the far right side of the P1 map, indicating the presence of at least two recognition sites in this region. DNA sequence analysis revealed that these two fragments share a 23-bp region of homology. A synthetic DNA containing an 11-bp sequence from this region acts as an effective competitor for repressor binding in vitro, suggesting that at least part of the sequence shared by the fragments is involved in repressor-DNA recognition.

Sequence dependent electrophoretic mobilities and melting temperatures for A-T containing oligodeoxyribonucleotides.

The electrophoretic mobilities and thermal melting properties of self complementary A-T containing dodecamer oligodeoxyribonucleotides have been investigated as a function of solution conditions. The oligomers contained tracts of nonalternating A-T base pairs of 2 (d(A2T2)3), 3 (d(A3T3)2), and 6 (d(A6T6] as well as the fully alternating (d(A-T)6) sequence. The melting temperature increased with the length of the nonalternating sequence and was approximately 12 degrees C higher in the d(A6T6) sequence than in the alternating oligomer. Under denaturing conditions all oligomers had the same electrophoretic mobility on acrylamide gels. Under conditions which favor duplex formation, the oligomers exhibited significant sequence dependent mobility differences. The mobilities of two oligomers, d(A-T)6 and d(A6-T6), were approximately equal and were less than those of the other oligonucleotides. The greatest mobility was observed for d(A2T2)3. These results are best explained by a model which requires bending at a junction of two or more continuous A or T bases with another sequence.

Location by DNA sequence analysis of cop mutations affecting the number of plasmid copies of prophage P1.

The DNA sequence of six P1 cop mutants, which are altered in the control of copy number of the plasmid prophage, was compared to that of P1 wild type. Each cop mutant differs from the wild type by a single base substitution. All of these substitutions are located within a 400 base pair region of P1 DNA that also encodes rep, a gene whose product is required for P1 replication.