Comparative analysis of phenotypic and genotypic characteristics of two desulfurizing bacterial strains, Mycobacterium phlei SM120-1 and Mycobacterium phlei GTIS10

AIM: To compare few phenotypic and genotypic characteristics of two desulfurizing bacterial strains, Mycobacterium phlei SM120-1 and Mycobacterium phlei GTIS10. METHODS AND RESULTS: In the present study, dibenzothiophene (DBT) desulfurizing activity, composition of fatty acids of cell membranes, DBT sulfone monoxygenase gene (bdsA) and the selection pressure applied during the growth and enrichment of the bacterial strains M. phlei SM120-1 and M. phlei GTIS10 were compared in our laboratory. The DBT desulfurization activity of M. phlei SM120-1 was found to be 0.17 +/- 0.02 micromol 2-HBP min(-1) (gram dry cell weight)(-1) and that of the bacterial strain M. phlei GTIS10 was 1.09 +/- 0.05 micromol 2-HBP min(-1) (gram dry cell weight)(-1). Fatty acid methyl ester analysis of cell membranes of these two bacterial strains in the presence of light gas oil showed that both the strains had different fatty acid profiles in their cell membranes. Comparison of the full gene sequences of the desulfurization gene bdsA in the two bacterial strains showed significant difference in the bdsA gene sequences. There was a significant difference observed in the selection pressure applied during the growth and enrichment of the two bacterial strains. CONCLUSIONS: The results of the comparative study of the bacterial strains, M. phlei SM120-1 and M. phlei GTIS10 showed that there were considerable differences in the phenotypic and genotypic characteristics of these two strains. SIGNIFICANCE AND IMPACT OF STUDY: The present study would broaden the understanding of biodesulfurization trait at intra-species level.     

Complete genome sequence of Mycobacterium phlei type strain RIVM601174

Mycobacterium phlei is a rapidly growing nontuberculous Mycobacterium species that is typically nonpathogenic, with few reported cases of human disease. Here we report the whole genome sequence of M. phlei type strain RIVM601174.     

Isolation and characterization of a moderate thermophile,Mycobacterium phlei GTIS10, capable of dibenzothiophene desulfurization

An organism, identified as Mycobacterium phlei GTIS10, was isolated based on its ability to use dibenzothiophene (DBT) as a sole source of sulfur for growth at 30-52 degrees C. Similar to other biodesulfurization-competent organisms, M. phlei GTIS10 converts DBT to 2-hydroxybiphenyl (2-HBP), as detected by HPLC. The specific desulfurization activity of the 50 degrees C M. phlei GTIS10 culture was determined to be 1.1+/-0.07 micromol 2-HBP min(-1) (g dry cell)(-1). M. phlei GTIS10 can also utilize benzothiophene and thiophene as sulfur sources for growth. The dszABC operon of M. phlei GTIS10 was cloned and sequenced and was found to be identical to that of Rhodococcus erythropolis IGTS8. The presence of the R. erythropolis IGTS8 120-kb plasmid pSOX, which encodes the dszABC operon, has been demonstrated in M. phlei GTIS10. Even though identical dsz genes are contained in both cultures, the temperature at which resting cells of R. erythropolisIGTS8 reach the highest rate of DBT metabolism is near 30 degrees C whereas the temperature that shows the highest activity in resting cell cultures of M. phlei GTIS10 is near 50 degrees C, and activity is detectable at temperatures as high as 57 degrees C. In M. phlei GTIS10, the rate-limiting step in vivo appears to be the conversion of DBT to dibenzothiophene sulfone catalyzed by the product of the dszC gene, DBT monooxygenase. The thermostability of individual desulfurization enzymes was determined and 2-hydroxybiphenyl-2-sulfinate sulfinolyase, encoded by dszB, was found to be the most thermolabile. These results demonstrate that the thermostability of individual enzymes determined in vitro is not necessarily a good predictor of the functional temperature range of enzymes in vivo.     

Effect of lysozyme on mycobacteria

The effect of lysozyme on the growth of several strains of mycobacteria was examined at pH 5.0-7.0 in Dubos medium containing various concentrations of lysozyme (100-2,000 microgram/ml). Mycobacterium smegmatis and M. phlei were susceptible to lysozyme at pH 5.0-7.0. The effect of lysozyme was marked between pH 6.0 and 7.0 and the colony counts were reduced to approximately 0.1-10% after incubation with 100 micrograms of lysozyme per ml for 48 hr. At pH 5.0, 10-40% of the organisms survived treatment with 1,000 micrograms of lysozyme per ml for 48 hr. M. bovis strain BCG, M. tuberculosis, and M. fortuitum appeared to be more resistant to lysozyme than M. smegmatis and M. phlei. M. smegmatis and M. phlei did not contain detectable amounts of poly-L-glutamic acid, although the susceptibility of the mycobacteria to lysozyme did not correlate with the amounts of the polymer in the cell walls. The role of lysozyme in animal infections with so-called saprophytic mycobacteria is discussed.     

Phagocytosis of mycobacteria by U937 cells: a rapid method for monitoring uptake and separating phagocytosed and free bacteria by magnetic beads

A human-derived monocytic cell line (U937) was induced to phagocytose Mycobacterium phlei by the addition of phorbol myristate acetate (PMA) to the culture medium for 50-60 h. Cells not treated with PMA were unable to phagocytose M. phlei. Magnetic beads enabled a rapid and highly efficient separation of phagocytosed and free bacteria to be achieved, an approach which is particularly useful if colony plating is used to enumerate bacterial survival within phagocytic cells. Fluorescence-activated cell sorting (FACS) analysis showed that 98% of U937 cells contained viable bacteria after 3 h.     

Alterations in lipid constituents during growth of Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354.

Phospholipids of Mycobacterium phlei ATCC 354 and Mycobacterium smegmatis CDC 46 consist of cardiolipin, phosphatidyl ethanolamine, tri-acylated dimannophosphoinositide, tetra-acylated dimannophosphoinositide and tetra-acylated pentamannosphosphoinositide. A comparative study of lipid patterns of M. phlei ATCC 354 and of M. smegmatis CDC 46 in relation to age of culture revealed higher total lipid level and increased activity of malate-vitamin K reductase, a phospholipid requiring enzyme, during the early logarithmic growth phase of the former. No appreciable change occurred in the latter. The high total lipid content coincides with an increase in phospholipid, brought about apparently by the increase in malate-vitamin K reductase. Changes in cardiolipin and phosphatidyl ethanolamine appeared to be unique to M. phlei ATCC 354. However, in both bacterial species, a decrease in glyceride and a progressive increase in tuberculostearic acid with a concomitant decrease in oleic acid, occurred with ageing.     

Characterization of mycobacterial immunoprecipitates by selective staining of enzymes

Immune precipitation patterns of Mycobacterium intracellulare, M. phlei and M. smegmatis were analysed by selective enzyme staining procedures in order to characterize individual mycobacterial antigens. Enzyme activity was shown in eight precipitinogens of M. intracellulare, seven of M. phlei, and six of M. smegmatis. The identification of mycobacterial precipitinogens as enzymes is important since only a few mycobacterial antigens have been functionally characterized.     

Mycobacteria possess a surprisingly small number of ribosomal RNA genes in relation to the size of their genome

DNAs from Mycobacterium tuberculosis, M. intracellulare, M. phlei and M. smegmatis were digested by restriction enzymes and hybridized with three probes consisting of the 5' (16S rRNA), the middle (16S and 23S rRNA), and the 3' (23S and 5S rRNA) portions of the Escherichia coli rrnB operon. The resulting hybridization patterns indicate that slow-growing Mycobacteria species (i.e., M. tuberculosis and M. intracellulare), with genome size 3.13 - 4.29 X 10(9) daltons, appear to possess only one rRNA operon, whereas fast-growing species (i.e., M. phlei and M. smegmatis), with genome size 4.30 - 5.20 X 10(9) daltons, appear to possess two rRNA operons.     

Correlation between calmodulin-like protein, phospholipids, and growth in glucose-grown Mycobacterium phlei.

In Mycobacterium phlei TMC 1548 supplementation of growth medium containing 2% v/v glycerol with glucose (up to 5% w/v) resulted in an increase in growth (yield of cells), in amount of total phospholipids, and in each of the individual phospholipids (cardiolipin, phosphatidylethanolamine, phosphatidylinositol and its mannosides, and phosphatidylglycerol). However, when the medium was supplemented with a higher concentration (7.5% w/v) of glucose, both growth and phospholipid levels decreased to near control values (2% v/v glycerol alone). Cyclic AMP levels, which decreased at all concentrations of glucose, had no relation to phospholipid content or growth. The presence of a protein that possesses the property of stimulating c-AMP phosphodiesterase activity was recently demonstrated in Mycobacterium smegmatis (Falah et al. 1988. FEMS Microbiol. Lett. 56: 89-93). In M. phlei the level of this calmodulin-like protein (assayed by radioimmunoassay) changed with different concentrations of glucose in the growth medium in a manner identical with that of phospholipids. We suggest that in mycobacteria (i) intracellular calmodulin-like protein levels are affected by glucose concentration in the growth medium and (ii) there is a positive correlation between the levels of calmodulin-like protein, total and individual phospholipids, and growth (yield of cells) in glucose-grown M. phlei.     

Isolation of a Soluble Resistance-Enhancing Factor from Mycobacterium phlei

Fox, Alfred E. (Warner-Lambert Research Institute, Morris Plains, N.J.), Joachim Anschel, George L. Evans, Raam R. Mohan, and Benjamin S. Schwartz. Isolation of a soluble resistance-enhancing factor from Mycobacterium phlei. J. Bacteriol. 92:285-290. 1966.-Extraction of a crude cell wall preparation from Mycobacterium phlei with 20% urea yielded a fraction which induced a state of enhanced resistance to microbial challenge. The resulting soluble extract, after removal of the urea, represented a 15% yield of solids with the separation of the biologically active component(s) and elimination of toxicity. Single oral or subcutaneous submicrogram doses of this material induced a prolonged state of increased resistance to subsequent challenge with Salmonella enteritidis in mice. This effect appeared as early as 2 hr after oral administration and persisted for at least 30 days. Protection against experimental infection with Staphylococcus aureus was also demonstrated. Resistance to viral challenge with influenza type A was observed after intranasal administration of the M. phlei extract to mice. The isolated material was found to contain carbohydrate, protein, nucleic acids, and lipids. The lipids represented 60% of the total solids, and were all short-chain fatty acids. No toxic effects, including pyrogenicity, could be demonstrated after oral or parenteral administration of this preparation.     

Mycobacterium phlei as an oral immunomodulator with Newcastle disease vaccine

Experiments were conducted in chickens to understand the effects of oral immunomodulation. Heat inactivated M phlei, a commensal Mycobacterium and a non-specific immunomodulator, was administered orally prior to live Newcastle disease F (ND F) strain vaccination. In experimental birds it lead to an enhanced cell mediated Immune response (CMI) against the vaccine. There was a reduction in the Haemagglutination inhibiting (HI) antibodies. However, it did not affect the protection against a virulent challenge, as the protection percentage was more or less same in vaccinated birds irrespective of the M.phlei administration. M. phlei administration could not enhance the immune response to inactivated ND F vaccine administered orally. The results indicate that M. phlei favours a CMI response to orally administered live ND F vaccine. It may be of potential use in enhancing CMI against vaccines and a cheaper alternative to costlier recombinant cytokines.     

Variations in the pathways of malate oxidation and phosphorylation in different species of Mycobacteria.

Mycobacterium tuberculosis H37Rv, the slow-growing human pathogenic strain of tubercle bacilli and Mycobacterium smegmatis and Mycobacterium phlei, the fast-growing saprophytes, have shown variations regarding the type of dehydrogenase that initiates malate oxidation in the respiratory chain. M. tuberculosis H37Rv is characterized by having a malate oxidase system (designated MALNAD pathway) in which malate oxidation is mediated by the NAD+-dependent malate dehydrogenase (EC 1.1.1.37) but not by FAD-dependent malate-vitamin K reductase. M. smegmatis possesses a different malate oxidase system (designated MALFAD pathway) in which malate oxidation is exclusively carried out by the FAD-dependent malate-vitamin K reductase because NAD+-dependent malate dehydrogenase is absent in this organism. M. phlei has a mixed system of malate oxidase (designated MALNAD+FAD pathways) in which both the NAD+-and FAD-dependent dehydrogenases take part. In all the three systems, the rest of the electron transport chain is common.     

Analyses of ribosomal proteins from mycobacteria with particular reference to the β antigen

Abstract The proteins of the ribosomal subunits of Mycobacterium phlei were analysed by sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) and by 2-dimensional PAGE. These techniques revealed that both the size and the charge of the mycobacterial ribosomal proteins were quite different from those of Escherichia coli . The divergent chemical properties of the mycobacterial ribosomes might be related to other exceptional properties of mycobacteria, e.g., their slow growth. An antigen designated β was furthermore revealed in both 30S and 50S subunits of M. phlei and Mycobacterium bovis BCG. 2 Proteins from each subunit migrated in a similar way in SDS-PAGE, being thus prime candidates as carriers of β-activity.     

Evidence for genetic divergence in ribosomal RNA genes in mycobacteria

DNA was isolated from Mycobacterium phlei and from M. smegmatis. Each DNA sample was restricted with endonucleases, the fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose film. Fragments of DNA containing rRNA sequences were identified by means of 125I-labelled rRNA of M. phlei or of M. smegmatis. The distributions of restriction endonuclease sites within the rRNA gene(s) and flanking sequences were found to be characteristic for each of the two species. Hybridizations with heterologous probes indicate that although M. phlei rRNA and M. smegmatis rRNA share regions of sequence homology, they are probably not identical in primary structure. The results suggest that the rRNA genes might prove to be useful taxonomic markers for mycobacteria.     

Quantitation of seven elements in Mycobacterium phlei and Mycobacterium bovis by neutron activation analysis.

Quantitative determination of the elements potassium, sodium, manganese, magnesium, iron, cobalt and zinc was performed in mycobacteria by neutron activation analysis. Mycobacterium phlei ATCC 19 249 at different phase of growth (4, 8, 13, 23 and 37 days old cultures), and 14 days old Mycobacterium bovis BCG cultures and uninoculated semi-synthetic Sauton culture media were examined. The elements studied could be divided into three groups; sodium, potassium and magnesium could be regarded as major, iron as minor, and zinc, manganese and cobalt as trace elements. M. phlei contained, with the exception of zinc, higher amounts of elements than M. bovis. Other metals (aluminium, antimony, rubidium) could also be detected.     

Influence of oxygen tension on the respiratory activity of Mycobacterium phlei

Growth of Mycobacterium phlei under low oxygen tension resulted in specific activities two to twenty times lower for formate dehydrogenase, malate dehydrogenase, beta-hydroxybutyrate dehydrogenase, lactate oxidase and NADH dehydrogenase than when cultures were grown under high aeration. An increase in fumarate reductase and succinate dehydrogenase occurred with M. phlei grown under low oxygen tension. Malate: vitamin K dehydrogenase and glucose-6-phosphate dehydrogenase activity were not significantly affected by the oxygen tension used to grow the bacteria, and neither culture contained a lactate dehydrogenase. With growth of M. phlei in conditions of low oxygen tension, cytochrome a was not detected, but cytochrome b was prominent in membranes and cytochrome c was present in the soluble fraction.     

Impurity in a common growth medium component: presence of an isoflavonoid in samples of L-asparagine

An impurity, present in some samples of l-asparagine, gave genistein on acid hydrolysis. The contaminant (probably the glucoside of genistein) was metabolized by Mycobacterium phlei to genistein and prunetin.     

In vitro antibacterial effect of the essential oil of Thymus longiflorus Boiss

The essential oil of Thymus longiflorus Boiss was tested for its in vitro antibacterial activity. The results showed antibacterial effects against Gram-positive and Gram-negative bacteria, especially against Pseudomonas fluorescens and Mycobacterium phlei.     

Purification and characterization of dihydrofolate reductase from Mycobacterium phlei.

The dihydrofolate reductase from Mycobacterium phlei was purified and characterized; it has an Mr of 15 000 and a pI of 4.8. It is competitively inhibited by both methotrexate and trimethoprim, although the affinity is less than for other bacterial dihydrofolate reductases.     

Anti-mycobacterial activity of biocides

The effects of different biocides on the growth and viability of Mycobacterium tuberculosis and other mycobacteria were studied. Mycobacterium phlei was the most sensitive of the test strains with a strain of the M. avium intracellulare (MAI) complex the most resistant. Chlorhexidine diacetate, quaternary ammonium compounds, a phenolic and esters of para (4)-hydroxybenzoic acid were inhibitory but not lethal to MAI, whereas 2% glutaraldehyde was bactericidal against all strains.