Isolation and characterization of nocardia-like variants of Mycobacterium smegmatis

Orange-red-pigmented (OR) colonies were isolated from cream-yellow-pigmented Mycobacterium smegmatis after exposure to either mycobacteriophage MC4 or ultraviolet light; these variant strains were designated OR4 and ORuv, respectively. Early subculture of OR-colonies did not show any segregation of parental-type cells. However, colonies resembling the parental strains, possibly representing a back mutant (REV-OR4), were occasionally found during subculture of established OR-colonies or upon treatment with N-nitrose-N'-nitro-N-methylguanidine. The OR-variants were characterized by their lytic response to nocardiophage, but not to mycobacteriophages, presence of alpha-branched, beta-hydroxylated fatty acids of the Nocardia-type, and a guanine plus cytosine value of deoxyribonucleic acid (DNA) between 62 and 64 mol%. They were more resistant to the lethal action of both ultraviolet light and mitomycin C treatment than the parental and back mutant strains. Although the OR-variants in this study possess characteristics common to the genus Nocardia or some of the 'rhodochrous' mycobacteria, evidence is presented that they form a new class of mycobacterial variants.     

Phosphatidylinositol synthesis in mycobacteria

The metabolism and synthesis of an important mycobacterial lipid component, phosphatidylinositol (PI), and its metabolites, was studied in Mycobacterium smegmatis and M. smegmatis subcellular fractions. Little is known about the synthesis of PI in prokaryotic cells. Only a cell wall fraction (P60) in M. smegmatis was shown to possess PI synthase activity. Product was identified as PI by migration on TLC, treatment with phospholipase C and ion exchange chromatography. PI was the only major product (92.3%) when both cells and P60 fraction were labeled with [3H]inositol. Also, a neutral lipid inositol-containing product (4.1% of the total label) was identified in the P60 preparations. Strangely, PI synthase substrates, CDP-dipalmitoyl-DAG and CDP-NBD-DAG, added to the assay did not stimulate [3H]PI and NBD-PI yield by M. smegmatis. At the same time, addition of both substrates to rat liver and Saccharomyces cerevisiae PI synthase assays resulted in an increase in the product yield. Upon addition of CHAPS to the mycobacterial PI synthase assay, both substrates were utilized in a dose-dependent manner for the synthesis of NBD-PI and [3H]PI. These results demonstrate a strict substrate specificity of mycobacterial PI synthase toward endogenous substrates. K(m) of the enzyme toward inositol was shown to be 25 microM; Mg2+ stimulated the enzyme to a greater degree than Mn2+. Structural analogs of myo-inositol, epi-inositol and scyllo-inositol and Zn2+ were shown to be more potent inhibitors of mycobacterial PI synthase than of mammalian analogs. Lack of sequence homology with mammalian PI synthases, different kinetic characteristics, existence of selective inhibitors and an important physiological role in mycobacteria, suggest that PI synthase may be a good potential target for antituberculosis therapy.     

Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries

The Tn611 transposon was inserted into pCG63, a temperature-sensitive plasmid isolated from an Escherichia coli-mycobacterial shuttle vector which contains the pAL5000 and pUC18 replicons. The resulting plasmid, pCG79, was used to generate a large number of insertional mutations in Mycobacterium smegmatis. These are the first mycobacterial insertional mutant libraries to be constructed by transposition directly into a mycobacterium. No highly preferential insertion sites were detected by Southern blot analysis of the chromosomal DNAs isolated from the insertion mutants. Auxotrophic mutants with various phenotypes were isolated at a frequency ranging from 0.1 to 0.4%, suggesting that the libraries are representative. The pCG79 system thus seems to be a useful tool for the study of M. smegmatis genetics and may be applicable to other mycobacteria, such as the M. tuberculosis complex.     

Cloning and characterization of arylamine N-acetyltransferase genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: increased expression results in isoniazid resistance

Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it. nat genes were cloned from Mycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli and M. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT from M. smegmatis and cross-reacts with recombinant NAT from M. tuberculosis. Overexpression of mycobacterial nat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatis as the host using the vector pACE-1, NAT proteins from M. tuberculosis and M. smegmatis are soluble. M. smegmatis transformants induced to express the M. tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.     

Intracellular accumulation of norfloxacin in Mycobacterium smegmatis

To evaluate the intracellular accumulation of norfloxacin in mycobacteria, two methods were used with Mycobacterium smegmatis. A radiometric method (K. V. Cundy, C. E. Fasching, K. E. Willard, and L. R. Peterson, J. Antimicrob. Chemother. 28:491-497, 1991) was used without great modification, but the fluorometric method (P. G. S. Mortimer and L. J. V. Piddock, J. Antimicrob. Chemother. 28:639-653, 1991) was changed considerably. Indeed, adsorption of the quinolone to the bacterial surface was characterized by measuring the level of accumulation of 0 degree C. Taking into account the adsorption, the pH of the washing buffer was increased from 7.0 to 9.0 to improve the desorption of norfloxacin from the cell surface. Both the fluorometric method, with the technical improvement, and the radiometric method could be used to estimate the intracellular accumulation of norfloxacin, which resulted from the difference between the whole uptake measured at 37 degrees C and the adsorption measured at 0 degrees C. A total of 35 ng of norfloxacin per mg of cells (dry weight) penetrated into the M. smegmatis cell, and the steady state was achieved in 5 min. Use of inhibitors of the proton motive force revealed that transport of norfloxacin was energy independent. Thus, the same mechanisms of quinolone accumulation that occur in eubacteria seem to occur in mycobacteria, at least in M. smegmatis.     

The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein

Mycobacterium marinum, like Mycobacterium tuberculosis, is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200-1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2-20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis. These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.     

Oxidative stress response and characterization of the oxyR-ahpC and furA-katG loci in Mycobacterium marinum

Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate gene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to the oxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H2O2, in keeping with the presence of a functional OxyR and its binding site within the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC and katG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.     

Structural elucidation by field desorption and electron-impact mass spectrometry of the C-mycosides isolated from Mycobacterium smegmatis

C-mycosides are superficial type-specific glycopeptidolipids of mycobacterial origin. In the present work, we have shown that field desorption mass spectrometry using the cationization method is a useful method for the molecular weight determination of such compounds. Complementary structural information has been obtained by electron-impact mass spectrometry. Combination of both methods has permitted the elucidation of the structure of the C-mycosides of Mycobacterium smegmatis, ATCC 607. This structure is similar to those described elsewhere, but some minor differences are observed in the lipid portion, mainly in the double bond location.     

Suppression of the growth of six potentially-pathogenic mycobacteria by beta-lactam/beta-lactamase-inhibitors

Drug-resistant tuberculosis and opportunistic infections by mycobacteria in immunocompromised subjects are not readily controlled with the antimycobacterial drugs now available. beta-Lactam antibiotics, the most widely used antibacterial agents, are ineffective against mycobacteria since they synthesize beta-lactamases. The beta-lactam/beta-lactamase-inhibitor combinations are used at present to treat infections caused by other beta-lactamase-positive organisms. Six potentially-pathogenic mycobacteria: Mycobacterium avium, M. chelonei, M. haemophilum, M. microti, M. scrofulaceum and M. simiae, were cultured in 7H9 medium (containing Tween 80 and albumin, dextrose, catalase) at 37 degrees C for 10-14 days, with or without various concentrations (2-100 micrograms/ml) of ampicillin/sulbactam, amoxicillin/clavulanate and piperacillin/tazobactam. More than 50-80% inhibition of the mycobacterial growth was observed at drug levels of 40-100 micrograms/ml in the medium; the drugs were active even when the detergent (Tween 80) was omitted. Against four of the mycobacteria, ampicillin/sulbactam proved to be the most active. The beta-lactam/beta-lactamase-inhibitor combinations may be of use as rational therapeutic agents against mycobacterial infections.     

PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples

A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks species-specific sequences within the genes coding for 16S rRNA. The PCR product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3), M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples.     

Molecular and immunological analyses of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kilodalton protein

The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the Mycobacterium avium homolog of the highly immunogenic M. leprae 35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host M. smegmatis, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from M. leprae-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from Streptomyces griseus and the cyanobacterium Synechoccocus sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the M. avium 35-kDa protein and SrpI of Synechoccocus was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of S. griseus were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the M. avium 35-kDa protein was present in all 30 M. avium clinical isolates tested but absent from M. intracellulare, M. tuberculosis, or M. bovis BCG. Mice infected with M. avium, but not M. bovis BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with M. avium.     

Ultrastructural studies in epidermolysis bullosa hereditaria. III. Recessive dystrophic types with dermolytic blistering (Hallopeau-Siemens types and inverse type)

In this paper the authors describe and analyse results that they obtained by infection of the guinea pig organism carried out by subcutaneous or intratracheal application with five mycobacterial strains, namely Myco bovis BCG-Praha, Myco the H37Ra, Myco Kansasii, Myco fortuitum and Myco smegmatis. At predetermined time intervals following subcutaneously or intratracheally performed infection (on 7th, 16th, 28th and 56th day after infection) transplantation of a Deals' guinea pig sarcoma cell suspension was carried out in guinea pigs by the intraltracheal route. As it appears from the results gained the applied mycobacteria exhibit a partial inhibition of growth od Deals' guinea pig sarcoma cells of different character. From among the utilized strains the Myco bovis BCP-Praha and the Myco tbc H37Ra exhibited the highest, Myco fortuitum and Myco smegmatis the lowest inhibitory activity. Intratracheally performed infections yielded in general better results on the growth inhibition than infections carried out with the same strain but by the subcutaneous route. Furthermore, in the experiments reported on in the present paper the authors could verify their earlier experience, namely that inhibition of growth of sarcoma cells is most pronounced at the time of maximal biological activity (logarithmic phase of multiplication) of the applied mycobacterium.     

Evaluation of polymerase chain reaction-restriction enzyme analysis of mycobacteria cultured in BACTEC 12B bottles

Polymerase chain reaction with restriction enzyme analysis (PRA) was first tested on 15 reference strains and 50 subcultured clinical isolates of mycobacteria according to the reference algorithm by Telenti et al [1]. Next, we evaluated the application of this method to 108 isolates from liquid media (BACTEC 12B). Of them, 15 M. tuberculosis complex and 81 mycobacteria other than tuberculosis (MOTT) had comparable results with both PRA and the BACTEC 460 TB systems. However, seven M. tuberculosis complex and three potentially pathogenic MOTT were identified by PRA rather than the BACTEC TB system. PRA seems to be an efficient method for the identification of mycobacteria to the species level and a good aid to detect potentially pathogenic mycobacteria, especially in mixed mycobacterial cultures.     

Investigation of mycobacterial recA function: protein introns in the RecA of pathogenic mycobacteria do not affect competency for homologous recombination

The recA locus of pathogenic mycobacteria differs from that of non-pathogenic species in that it contains large intervening sequences termed protein introns or inteins that are excised by an unusual protein-splicing reaction. In addition, a high degree of illegitimate recombination has been observed in the pathogenic Mycobacterium tuberculosis complex. Homologous recombination is the main mechanism of integration of exogenous nucleic acids in M. smegmatis, a non-pathogenic mycobacterium species that carries an inteinless RecA and is amenable to genetic manipulations. To investigate the function of recA in mycobacteria, recA- strains of M. smegmatis were generated by allelic exchange techniques. These strains are characterized (i) by increased sensitivity towards DNA-damaging agents [ethylmethylsulphonate (EMS), mitomycin C, UV irradiation] and (ii) by the inability to integrate nucleic acids by homologous recombination. Transformation efficiencies using integrative or replicative vectors were not affected in recA- mutants, indicating that in mycobacteria RecA does not affect plasmid uptake or replication. Complementation of the recA- mutants with the recA from M. tuberculosis restored resistance towards EMS, mitomycin C and UV irradiation. Transformation of the complemented strains with suicide vectors targeting the pyrF gene resulted in numerous allelic exchange mutants. From these data, we conclude that the intein apparently does not interfere with RecA function, i.e. with respect to competency for homologous recombination, the RecAs from pathogenic and non-pathogenic mycobacteria are indistinguishable.     

Magnocellular vasopressinergic neurons in explant cultures are rescued from cell death by ciliary neurotrophic factor and leukemia inhibiting factor

The genus Mycobacterium includes the major human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. The development of rational drug treatments for the diseases caused by these and other mycobacteria requires the establishment of basic molecular techniques to determine the genetic basis of pathogenesis and drug resistance. To date, the ability to manipulate and move DNA between mycobacterial strains has relied on the processes of transformation and transduction. Here, we describe a naturally occurring conjugation system present in Mycobacterium smegmatis, which we anticipate will further facilitate the ability to manipulate the mycobacterial genome. Our data rule out transduction and transformation as possible mechanisms of gene transfer in this system and are most consistent with conjugal transfer. We show that recombinants are not the result of cell fusion and that transfer occurs from a distinct donor to a recipient. One of the donor strains is mc(2)155, a highly transformable derivative that is considered the prototype laboratory strain for mycobacterial genetics; the demonstration that it is conjugative should increase its genetic manipulability dramatically. During conjugation, extensive regions of chromosomal DNA are transferred into the recipient and then integrated into the recipient chromosome by multiple recombination events. We propose that DNA transfer is occurring by a mechanism similar to Hfr conjugation in Escherichia coli.     

Stable integration and expression of the Plasmodium falciparum circumsporozoite protein coding sequence in mycobacteria

The DNA coding for the circumsporozoite protein of Plasmodium falciparum (CSP; aa 1-412) has been placed under the control of the mycobacterial promoter derived from the gene encoding the 64-kDa antigen of Mycobacterium bovis-BCG. This expression cassette was cloned into pJRD184, an Escherichia coli multicloning site vector, together with the kanamycin resistance gene from Tn903 and the attachment site and integrase gene from the temperate mycobacteriophage FRAT1. One of the resulting plasmids, pNIV2173, introduced by electroporation into both Mycobacterium smegmatis and M. bovis-BCG, integrated at a specific site in the genome of each recipient. Recombinants expressed immunoreactive polypeptides, ranging in size from 62 to 43 kDa, at a level of about 1% of total soluble proteins. Part of this material was present in the culture medium indicating that mycobacterial recombinants were able to secrete the CSP. The M. smegmatis and M. bovis-BCG recombinants, transformed with pNIV2173 and grown in absence of antibiotic, were followed for more than 400 and 50 generations respectively. Over this time span, neither DNA rearrangement nor loss of expression was observed. Inoculation of the recombinant BCG to mice did not induce humoral response to CSP nor proliferative response to CSP Th2R CD4+ T lymphocyte epitope.     

Comparison of the resistance of C57BL/6 and C3H/He mice to infection with Mycobacterium paratuberculosis

Susceptibility of C57BL/6 (Bcgs) and C3H/HeN (Bcgr) mice to an intraperitoneal infection with Mycobacterium paratuberculosis strain 19698 was compared (by histopathology and the number of mycobacteria isolated from the spleen). Mycobacterial counts from the spleen of Bcgr mice progressively decreased over the course of infection but remained unchanged in Bcgs mice. Granulomatous lesions and acid-fast bacteria were consistently present in the liver and lymph nodes of Bcgs mice, whereas lesions were transient or absent in Bcgr mice. These results indicate that Bcgr mice are inherently resistant to M. paratuberculosis, whereas Bcgs mice are inherently susceptible. These differences may prove useful in elucidating the mechanisms of resistance and susceptibility to paratuberculosis and other mycobacterial infections.     

Clinical significance of the isolation of rapidly growing mycobacteria

BACKGROUND: To assess the clinical significance of the isolates of rapid-growing mycobacteria in a Universitary hospital from Madrid (Spain). PATIENTS AND METHODS: Review of medical records from patients with isolates of rapid-growing mycobacteria identified between 1979 and 1996 in the Microbiology department of the Fundación Jiménèz Díaz (Madrid, Spain). RESULTS: Rapid-growing mycobacteria were isolated from 28 patients during the study period (13 M. chelonae, 10 M. fortuitum, 2 M. mucogenicum, 1 M. marinum, 1 M. smegmatis and 1 M. flavascens). Clinical records of 26 patients were reviewed, being the isolate significative in 10 cases (5 soft tissue infections, 2 peritonitis in patients undergoing Continuous Ambulatory Peritoneal Dialysis [CAPD], 1 urinary tract infection, 1 osteomyelitis and 1 catheter-related soft-tissue infection). No patient was HIV+. All infections cured except 2 of them (the urinary tract infection and the osteomyelitis). Catheter withdrawal was needed in 3 cases (peritonitis in CAPD and catheter-related soft-tissue infection), apart from proper antimicrobial therapy. CONCLUSION: The most frequent rapid-growing mycobacteria isolated were those of the M. fortuitum complex. In our experience, isolation of rapid-growing mycobacteria from skin and soft-tissue samples was usually clinically significant, while isolates from respiratory tract, gut and blood cultures are always nonsignificant.     

Role of relaxin and estrogen in the control of eosinophilic invasion and collagen remodeling in rat cervical tissue at term

The modified fluorescence method was used to determine the accumulation of norfloxacin by Mycobacterium aurum A+ and Mycobacterium smegmatis mc(2)155. By using an exogenous norfloxacin concentration of 10 microg/ml, a steady-state concentration (SSC) of 160 to 180 ng of norfloxacin/mg of cells was obtained for M. aurum, and an SSC of 120 to 140 ng of norfloxacin/mg of cells obtained for M. smegmatis. For both species of mycobacteria, the SSC was achieved within 5 min. The silicon oil method was investigated and gave higher SSCs than the modified fluorescence method. Further studies on the mechanism of norfloxacin accumulation by M. aurum were performed. An increase in the pH of the wash buffer from 7.0 to 9.0 did not significantly affect the final SSC obtained. Accumulation was nonsaturated over a norfloxacin concentration range of 0 to 100 microg/ml, and the proton motive force inhibitor 2,4-dinitrophenol (1 and 2 mM), whether it was added before or after norfloxacin was added, had no effect on the final SSC obtained. 2,4-Dinitrophenol also had no effect on norfloxacin accumulation by M. smegmatis. Furthermore, norfloxacin accumulation by M. aurum was unaffected by the presence of either Tween 80 or subinhibitory concentrations of ethambutol in the growth medium. Therefore, it is proposed that norfloxacin accumulation by mycobacteria occurs by simple, energy-independent diffusion.