Bruce C. Kline

Recombinant Mycobacterium tuberculosis KatG(S315T) Is a Competent Catalase-Peroxidase with Reduced Activity toward Isoniazid

The presence of KatG(S315T), a mutation frequently detected in clinical isolates of Mycobacterium tuberculosis, has been associated with loss of catalase-peroxidase activity and resistance to isoniazid therapy. Wild-type KatG and KatG(S315T) were expressed in a heterologous host (Escherichia coli) and purified to homogeneity, and enzymatic activity was measured. The catalase activity for KatG(S315T) was reduced 6-fold, and its peroxidase activity was decreased <2-fold, compared with the activities for wild-type KatG. Pyridine hemochrome analysis demonstrated 1.1 +/- 0.1 hemes/subunit for wild-type KatG and 0.9 +/- 0.1 hemes/subunit for KatG(S315T), indicating that the difference in enzymatic activity is not the result of incomplete heme cofactor incorporation in KatG(S315T). High-performance liquid chromatography analysis showed that wild-type KatG was more efficient than KatG(S315T) at converting isoniazid to isonicotinic acid. These results demonstrate that KatG(S315T), as expressed in E. coli, is a competent catalase-peroxidase that exhibits a reduced ability to metabolize isoniazid.

The F plasmid ccd autorepressor is a complex of CcdA and CcdB proteins

SummaryThe ccd operon of plasmid F produces three proteins, CcdA, CcdB, and RepD. Prior research has established that the operon is autorepressed and that at least CcdB, but not RepD, is required for autorepression. A role for CcdA in autorepression was suggested but not clearly shown. We now present a series of biochemical experiments which show that both CcdA and CcdB proteins are required for maximal formation of protein-ccd operator complexes. We also show that CcdA and CcdB are present in a complex whether or not ccd operator is present. The clear implication is that autorepressor is a complex of CcdA and CcdB. We also map the start site of the ccd transcript thus providing the first experimental evidence for the location of the ccd promoter.

A two-stage molecular model for control of mini-F replication.

It is known that mini-F replication requires production of a 29,000-Da protein, protein E, and origin of replication sequences mapping around 45. kb. Further, control of replication is determined by two genes, copA and copB. In the present work a description is given of the cloning of an F restriction fragment containing the amino terminal portion of the protein E gene, repE, and associate promoter activity. It is shown that expression of this promoter is negatively regulated in trans by sequences taken from the F replication region of copA+ plasmids. However, the same sequences taken from six different copA- plasmids failed to repress expression of the promoter. Since prior studies have shown that copA+ determines a repressor of replication, it is now suggested that the above results are an accounting of where this repressor works. A hypothesis is also proposed to explain control of F replication by the copA and copB regulatory genes.

A comparison of 16S ribosomal DNA sequences from five isolates of Helicobacter pylori

Other workers have found that clinical isolates of Helicobacter pylori exhibit very extensive DNA sequence polymorphisms when they are examined by ribotyping or some other genomic sequence characterization technique. In fact, it is rare to find similar clones, much less identical ones, among isolates. We found that the levels of divergence between the 16S ribosomal DNA sequences of individual organisms and the consensus sequence of the five isolates which we examined ranged from 0.2 to 0.5%. In contrast, other workers have shown that levels of divergence between the 16S ribosomal DNA sequence of H. pylori and the 16S ribosomal DNA sequences of four other Helicobacter species range from 2.7 to 8.0%. Our results show that the H. pylori 16S ribosomal DNA is not very polymorphic and support the conclusion that H. pylori is a unique species.

Genetic studies of F plasmid maintenance genes involved in copy number control, incompatability, and partitioning

Abstract We have identified a 2.1-kilobase (kb) region (44.1 to 46.19 kb) in F that is necessary and sufficient to form low copy number minireplicons. Within this region we have mapped (i) mutations ( cop ) inducing 4.4- to 28-fold increases in copy number and (ii) two separate regions that determine incompatability ( inc B and inc C). The 2.1-kilobase region has also been shown by others to contain (i) an origin of replication, ori (ii) a locus ( aos ) necessary for sensitivity to the plasmid replication inhibitor, acridine orange, and (iii) nine, 19- to 22-base-pair direct repeat sequences organized in two clusters. In the present work we more accurately locate the aos locus and show that it, as well as ori , inc B, inc C, and some cop mutations, map within or overlap the direct repeat regions. Analysis of other cop mutations indicates that they reduce or destroy the incompatability reaction associated with the 2.1-kb region; however, these cop mutations do not map within the inc B or inc C determinants. A 2-fold copy number elevation and unstable plasmid maintenance also results from deletion of the 46.19- to 49.2-kb region. Results described here and elsewhere suggest that the instability of the deletion mutant reflects the loss of partitioning gene, a gene that is probably identical to an inc locus, inc D, that had been identified in this region in prior work. Whether or not the inc D locus has anything to do with the slight copy number elevation is unknown.

Nonintegrated plasmid-folded chromosome complexes: genetic studies on formation and possible relationship to plasmid replication.

Abstract When folded chromosomes are purified from plasmid-containing bacteria, a reproducible fraction of the hosts covalently closed, circular (CCC) plasmid DNA copurifies with the chromosomes. From this copurification, we infer the existence of nonintegrative plasmid-chromosome (NPC) complexes. Previously, we noted that plasmids dependent on DNA polymerase III and with stringent control of replication complex to a greater extent than plasmids dependent on DNA polymerase I and with relaxed control of replication. We have examined this subject in more depth and find that: (i) The composite plasmids formed by in vitro recombination of a “stringent” with a “relaxed” replicon complex to chromosomes at the frequency of the component replicon which directs replication; (ii) all of the detectable replicative intermediates, but only 25% of the CCC forms, of plasmid ColE1 complex to chromosome; and (iii) when a mini-F plasmid is deleted for the DNA sequences which include the primary origin of replication, the complexing frequency decreases 30 to 40%. We conclude from these findings that NPC complexes either indirectly or directly relate to plasmid replication. Further, we find that the Eco RI kan + fragments of pML31 and the ampicillin resistance transposon, Tn 3 , promote complexing of both ColE1 and mini-F plasmids to host chromosomes. The biological significance of this latter complexing is unknown. However, we conclude from these studies and from point (iii) that complexing is determined in part by unique plasmid sequences.

DNA Sequence Resembling vanA and vanB in the Vancomycin-Resistant Biopesticide Bacillus popilliae

The origin of high-level vancomycin resistance in enterococci is unknown. Biopesticidal powders containing spores of Bacillus popilliae, which is vancomycin-resistant, have been used for >50 years in the United States for suppression of Japanese beetle populations. Using a polymerase chain reaction assay designed to amplify the vanB gene in enterococci, an amplicon in B. popilliae was identified and sequenced. The putative ligase gene in B. popilliae had 76.8% and 68.4%-68.9% nucleotide identity to the sequences of the vanA and vanB genes, respectively. There was 75.3% and 69.3%-69.9% identity between the translation of the putative ligase gene in B. popilliae and the translation of the vanA and vanB genes, respectively. We have identified a gene resembling vanA and vanB in B. popilliae. The gene in B. popilliae may have been a precursor to or have had an ancestral gene in common with vancomycin resistance genes in enterococci.

Incompatibility between Flac, R386, and F:pSC101 recombinant plasmids: The specificity of F incompatibility genes

Abstract The specificity of F incompatibility genes ( inc + ) has been studied with the F lac and R386 plasmids, members of the IncFI incompatibility group. Recently, two inc + regions, inc A (46.4–49.3F) and inc B (43.1–46.4F) were identified by cloning these F sequences onto pSC101 and subsequently demonstrating incompatibility of the recombinants with F lac . It is shown here that the F inc A + recombinant is incompatible with both F lac and R386 while the F inc B + recombinant is incompatible only with F lac . Also, a plasmid mutant is described that has reduced incompatibility against F lac and R386. The mutation is located on the Bam HI restriction fragment that contains the F inc A region. These genetic findings are consistent with the deduction of Palchaudhuri and Maas, based on heteroduplex analysis of IncFI plasmids, that placed the IncFI determinant in the 46.4–48.6F region. The findings also indicate that the F inc B + gene product, which has been implicated in negative control of F copy number, is specific for the F replicon.

Identification and characterization of a second copy number control gene in mini-F plasmids

SummaryWe previously reported the existence of a series of chemically induced trans recessive copy-number mutations (cop) for mini-F plasmids and the existence of a similar series of cop mutations induced by insertion of the ampicillin resistance transposon Tn3. In this paper we describe the experiments showing that these two series of mutations are in different genes. Briefly, the experiments show that the one mutant series can complement the other, that the mutations map in distinct but adjacent regions, that the copy numbers of double mutants are the products of the copy numbers determined by the single mutations, and that Tn3 does not elevate copy number by a polar effect on the adjacent cop gene defined by chemical mutagenesis. We term the latter gene copA and the gene mutated by Tn3, copB. We also demonstrate here that copB mutations are recessive to the wild type allele. Further, we have characterized copB by deletion and recombinational analysis as the series of five 19- to 22-base-pair directly repeated sequences that had previously been designated incC-that is, one of the incompatibility genes. The evidence for this conclusion is that plasmids lacking two, three or five direct repeats have their copy number elevated proportionately. Possible mechanisms for copB control of replication are discussed.

Laboratory diagnosis of Pneumocystis carinii infections by PCR directed to genes encoding for mitochondrial 5S and 28S ribosomal RNA

PCR with 5S mitochondrial ribosomal RNA (5S) target is a sensitive and specific assay for the detection of Pneumocystis carinii in clinical specimens from the respiratory tract. We developed an oligonucleotide probe directed to a 200 bp amplicon generated by fungal-specific universal primers that anneals with sequences specific for P. carinii in the 28S ribosomal RNA gene (28S). Of 50 archived bronchoalveolar lavage 1(BAL) specimens, 46 of 50 samples (92% agreement) gave the same result (23 positive, 23 negative) by PCR directed to the 5S and 28S assays. Results of calcofluor white staining of BAL smears on slides indicated agreement with the molecular results in 43 of 46 (93.5%) assays. PCR detection of P. carinii by amplification of 28S ribosomal gene target by fungal-specific primers and an organism-specific probe provides an alternate genomic target for the laboratory diagnosis of this organism.