Growth phase-associated changes in protein expression in Mycobacterium smegmatis identify a new low molecular weight heat shock protein.

De novo protein synthesis and the heat-shock response during different stages of bacterial culture of Mycobacterium smegmatis LR222 were investigated. A discontinuance in the increase in number of colony forming units occurred at mid-exponential phase of growth. This coincided with a plateau in the ATP content of the culture, a reduction in the synthesis of exponential phase proteins (58, 30.5, and 20 kDa), a transitory synthesis of a 32 kDa protein and the induction of stationary-phase proteins (48, 46, 31, 25, and 20 kDa). The response to heat shock showed a growth-phase dependency, with the highest fold-induction of the 75 kDa (DnaK) protein occurring during the transitory cessation in the increase in CFU, while the greatest increase of the 95 kDa, 66 kDa (GroEL), and approximately 17 kDa (a doublet) proteins occurred during stationary phase. The approximately 17 kDa doublet was resolved into four polypeptides by two-dimensional electrophoresis. Mass spectrometric analysis of the sequence of one polypeptide (named Hsp17-2, 16.8 kDa) revealed significant homology to a conserved, 16.2 kDa, hypothetical protein of unknown function in Mycobacterium tuberculosis H37Rv. The increased synthesis of Hsp17-2 in response to heat shock suggests that it may represent a new low molecular weight heat shock protein.

The Mycobacterium tuberculosis mysB gene product is a functional equivalent of the Escherichia coli sigma factor, KatF.

Mycobacterium tuberculosis, the causative agent of tuberculosis, may remain dormant within its host for many years. The nature of this dormant or latent state is not known, but it may be a specialized form of the stationary growth phase. In Escherichia coli, KatF (or RpoS) is the major stationary phase sigma factor regulating an array of genes expressed in this phase of growth. A potential M. tuberculosis katF homologue was cloned using a fragment of the E. coli katF gene as a probe. DNA sequence analysis of a resultant clone showed 100% identity to a fragment of DNA encoding the M. tuberculosis mysA and mysB genes. Overexpression of mysB in M. bovis BCG resulted in an increase in katG mRNA and catalase and peroxidase activity, and an increase in sensitivity of the cells to isoniazid. An increase in katG promoter activity from a reporter vector was demonstrated when mysB was overexpressed from the same plasmid, indicating a direct relationship between MysB and katG expression.

Characterization of a Mycobacterium tuberculosis homologue of the Streptomyces coelicolor whiB gene.

SETTING: Molecular Research Laboratory, Department of Medical Microbiology, University of Cape Town and Groote Schuur Hospital. OBJECTIVE: Characterize Mycobacterium tuberculosis homologue of the Streptomyces coelicolor, sporulation specific, whiB regulatory gene. DESIGN: The M. tuberculosis whiB3 gene was isolated by enriched cloning of a 2.8 kb BamHl fragment to which the S. coelicolor whiB gene hybridized. Expression of the gene was analysed by S1 nuclease analysis and promoter studies. RESULTS: An open reading frame within the 2.8 kb BamHl fragment was identified as the M. tuberculosis whiB3 gene, one of four whiB homologues in the M. tuberculosis genome. The deduced amino acid sequence has a 92% identity with a M. leprae protein, and 32% identity with the S. coelicolor WhiB protein. S1 nuclease analysis showed that the M. tuberculosis whiB3 gene is constitutively expressed by the cells in liquid culture. Primer extension analysis revealed three transcriptional start sites. Expression from the three potential promoters is growth phase-dependent. CONCLUSION: The M. tuberculosis whiB3 gene is expressed throughout growth, but expression from the individual promoters is growth phase dependent.

Novel method for rapid measurement of growth of mycobacteria in detergent-free media.

We describe a novel, rapid, and inexpensive method for the measurement of growth of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium smegmatis in the presence or absence of detergent. The method, which employs hot NaOH treatment of mycobacterial cells to release total cellular protein, compares favorably with other methods for monitoring mycobacterial growth but is particularly useful for heavily clumped cultures grown in defined minimal medium.

Characterisation of a lipoprotein in Mycobacterium bovis (BCG) with sequence similarity to the secreted protein MPB70.

The monoclonal antibody, mAb3C4, raised against sonicated Mycobacterium bovis (Mb) BCG (Tokyo strain 172) cells recognises a 23-kDa protein in the cell wall. The gene encoding this protein was cloned and sequenced and found to be 100% homologous to mpb83 and mpt83 and the putative protein to have a 76% sequence similarity to the secreted, Mb-specific protein, MPB70. MPB83 contains the amino acid (aa) sequence LAGC, which corresponds to the consensus sequence for bacterial lipoprotein modification and processing. MPB83 associated with the detergent phase when separated with Triton X-114 confirming that it is a lipoprotein. When the putative site of acylation, the Cys in the sequence LAGC, was substituted with Ser, the mutated MPB83 associated with the aqueous phase. The cloned gene was used to determine the distribution of mpb83 in various Mycobacterium species. The gene was present in the M. tuberculosis (Mt) complex organisms, as well as in M. kansasii. In addition, Southern blot analysis of Mb and Mt DNA indicated that the mpb83 and mpb70 genes are located close to each other on the genome. Western blot analysis of cell lysates of various Mycobacterium species indicated that only Mt H37Rv and H37Ra produced proteins which reacted with mAb3C4. Furthermore, only two out of six of the Mb field isolates produced detectable antigen, indicating that expression of the mpb83 gene is variable within the Mt complex organisms.

Nonopsonic binding of Mycobacterium tuberculosis to human complement receptor type 3 expressed in Chinese hamster ovary cells.

Nonopsonic invasion of mononuclear phagocytes by Mycobacterium tuberculosis is likely important in the establishment of a primary infection in the lung. M. tuberculosis binds to a variety of phagocyte receptors, of which the mannose receptor and complement receptor type 3 (CR3) may support nonopsonic binding. CR3, a beta2 integrin, is a target for diverse intracellular pathogens, but its role in nonopsonic binding remains uncertain. We have examined the binding of M. tuberculosis H37Rv to human CR3 heterologously expressed in Chinese hamster ovary (CHO) cells, thereby circumventing the problems of competing receptors and endogenously synthesized complement, which are inherent in studies with mononuclear phagocytes. The surface expression of CD11b and CD18 was assessed by immunofluorescence, immunobead binding, flow cytometry, and immunoprecipitation with anti-CD11b and anti-CD18 monoclonal antibodies (MAbs). The functional activity of the surface-expressed CD11b/CD18 (CR3) heterodimer was confirmed by rosetting with C3bi-coated microspheres. We found that M. tuberculosis bound four- to fivefold more avidly to CR3-expressing CHO cells than to wild-type cells and, importantly, that this binding was at similar levels in the presence of fresh or heat-inactivated human or bovine serum or no serum. In contrast, Mycobacterium smegmatis bound poorly to CR3-expressing CHO cells in the absence of serum, but after opsonization in serum, binding was comparable to that of M. tuberculosis. The binding of M. tuberculosis to the transfected CHO cells was CR3 specific, as it was inhibited by anti-CR3 MAbs, particularly the anti-CD11b MAbs LM2/1 (I domain epitope) and OKM1 (C-terminal epitope), neither of which inhibit C3bi binding. MAb 2LPM19c, which recognizes the C3bi-binding site on CD11b, had little or no effect on M. tuberculosis binding. The converse was found for the binding of opsonized M. smegmatis, which was strongly inhibited by 2LPM19c but unaffected by LM2/1 or OKM1. CR3-specific binding was also evidenced by the failure of M. tuberculosis to bind to CHO cells transfected with an irrelevant surface protein (angiotensin-converting enzyme) in the presence or absence of serum. We conclude that the binding of M. tuberculosis H37Rv to CR3 expressed in CHO cells is predominantly nonopsonic and that the organism likely expresses a ligand that binds directly to CR3.

16S rRNA sequence analysis of an isolate of Mycobacterium haemophilum from a heart transplant patient.

Biopsy samples from a heart transplant patient with cellulitis and bursitis yielded an isolate of Mycobacterium haemophilum. The isolate was identified on the basis of a growth requirement for haemin or ferric ammonium citrate, growth at 30 degrees C but not at 37 degrees C, negative catalase test, intracellular growth in McCoy fibroblasts and sequence identify with a portion of the 16S rRNA sequence of the type strain. In comparisons with known 16S rRNA sequences, M. haemophilum grouped with other pathogenic, slow-growing mycobacteria, showing close sequence similarity to M. marinum (98.8%) and lower similarity to M. ulcerans and M. tuberculosis complex organisms. M. haemophilum and M. marium share other features including optimal growth at 30 degrees C and the ability to cause superficial skin lesions in man.

High level kanamycin resistance associated with the hyperproduction of AAC(3)II and a generalised reduction in the accumulation of aminoglycosides in Acinetobacter spp.

The clinical isolate of Acinetobacter baumannii strain SAK contains an actively transcribed aacC2 gene and a latent aadB gene that encode the aminoglycoside-modifying enzymes, AAC(3)II and AAD(2"), respectively. In an attempt to activate the aadB gene, the strain was cultured in the presence of kanamycin which is a substrate for AAD(2"). Although it was possible to isolate kanamycin resistant derivatives these were not associated with detectable AAD(2") activity. Instead, there was a marked increase in the level of AAC(3)II activity which was associated with amplification of the aacC2 gene.

Characterization of a replicon of the moderately promiscuous plasmid, pGSH5000, with features of both the mini-replicon of pCU1 and the ori-2 of F.

The dominant, polA1-independent replicon of pGSH500, rep beta (1.8 kb), consists of a cis-acting oriV region of 245 bp; a repB gene that is essential for autonomous replication and 18, 30 to 36 bp iterons which constitute the inc/cop region. The molecular organization of rep beta resembles that of mini-pCU1 (IncN). Furthermore, there is a 58% identity between the Rep proteins of these replicons. RepB also shows a 31% identity with RepE of mini-F. In addition, an 80% identity over 200 bp was identified between the cis-acting beta oriV region and the equivalent region of ori-2 (mini-F). Replicons with deletions of repB could be complemented by Rep (pCU1) and RepE (mini-F) in trans, supporting the hypothesis that rep beta is a natural hybrid between a pCU1-like and F-like replicon.

Plasmid profiles of "Campylobacter upsaliensis" isolated from blood cultures and stools of paediatric patients.

Seventy-three clinical isolates of "Campylobacter upsaliensis" were screened for the presence of plasmids. Plasmid bands were found in 68 (93%) isolates, from which 14 plasmid types were identified. Type 5 was found only in blood-culture isolates, whereas types 7-14 were found only in faecal isolates. Plasmid-free isolates and the other plasmid profiles were present in both faecal and blood isolates. The reproducibility of these profiles was largely dependent on the method of plasmid isolation. The chloroform-phenol lysis method was the most efficient and reliable method of preparing plasmid DNA for plasmid profile analysis. The success of this method may be largely attributable to two factors: (1) the efficacy of cell lysis was independent of either an alkaline agent or of heat; (2) the inactivation of nucleases by the utilisation of chloroform-phenol to lyse the cells. Furthermore, this method of plasmid DNA preparation is ideally suited for use on clinical isolates, especially when rapid plasmid profile analysis may be required.

Cloning and expression in Escherichia coli of a recA homologue from Mycobacterium tuberculosis.

A 3.8 kb PstI fragment of Mycobacterium tuberculosis was cloned in a recA-deleted Escherichia coli by selecting transformants with increased EMS resistance. The cloned fragment restored homologous recombination in Hfr crosses and conferred resistance to long wave (302 nm) but not short wave (254 nm) UV light. E. coli containing the 3.8 kb PstI fragment produced a 38-40 kDa protein which cross-reacted with antibodies raised against the E. coli RecA protein. The cloned DNA thus probably encodes a RecA homologue.

Identification of an Acinetobacter baumannii gene region with sequence and organizational similarity to Tn2670.

A chloramphenicol resistance gene was cloned from chromosomal DNA prepared from a clinical Acinetobacter baumannii isolate. Sequence analysis of this gene (cat) and the flanking DNA regions shows that this gene is linked to Tn21 and to IS1 in a manner similar to that found in Tn2670.

Aminoglycoside-O-phosphotransferase (APH (3'')) activity in a clinical isolate of Acinetobacter calcoaceticus.

An isolate of Acinetobacter calcoaceticus var. anitratus containing an aminoglycoside-aminocyclitol antibiotic-modifying enzyme, which phosphorylates streptomycin at the 3" position, is described. The enzyme, APH(3"), was identified on the basis of its substrate specificity; in particular, its ability to phosphorylate streptomycin but not streptidine.