Barbara R. Baumstark

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 14kbp linear DNA segments that are heterogeneous in sequence. The 14kbp 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.

Characterization of six type A strains of Clostridium botulinum that contain type B toxin gene sequences

Six Clostridium botulinum isolates exhibiting type A toxicity as measured by the mouse bioassay were found to contain both type A and type B neurotoxin DNA sequences. The six strains were divided into three groups based on the DNA sequence of the type B neurotoxin gene. Members of each group exhibited 100% sequence identity over the 3876 bp type B toxin open reading frame. The type B toxin sequence of all groups differed at more than 60 positions when compared to the BGB control strain.

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.

Polymerase chain reaction for detection of type A Clostridium botulinum in foods

A DNA fragment of the type A Clostridium botulinum neurotoxin gene was demonstrated in canned food products with the polymerase chain reaction (PCR). The fragment, 1340-bp in size, was amplified from green peas, whole kernel corn, green beans, lima beans, black-eyed peas, and turnip greens previously inoculated with type A C. botulinum . The PCR products were identified by agarose gel electrophoresis and also confirmed by dot blot DNA hybridization with a type A specific gene probe. Some inoculated foods were PCR negative but became PCR positive after subculture and overnight incubation in brain heart infusion broth. No PCR amplification products were obtained from uninoculated foods. The procedure was highly sensitive and detected as few as 100 vegetative cells per ml brain heart infusion broth culture.

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.

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.

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

ABSTRACT A recombinant clone expressing an immunoreactive antigen ofBartonella 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 forBartonella 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 ofB. henselae.

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.

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.