The Antituberculosis Drug Ethambutol Selectively Blocks Apical Growth in CMN Group Bacteria.

Members of the genus Mycobacterium are the most prevalent cause of infectious diseases. Mycobacteria have a complex cell envelope containing a peptidoglycan layer and an additional arabinogalactan polymer to which a mycolic acid bilayer is linked; this complex, multilayered cell wall composition (mAGP) is conserved among all CMN group bacteria. The arabinogalactan and mycolic acid synthesis pathways constitute effective drug targets for tuberculosis treatment. Ethambutol (EMB), a classical antituberculosis drug, inhibits the synthesis of the arabinose polymer. Although EMB acts bacteriostatically, its underlying molecular mechanism remains unclear. Here, we used Corynebacterium glutamicum and Mycobacterium phlei as model organisms to study the effects of EMB at the single-cell level. Our results demonstrate that EMB specifically blocks apical cell wall synthesis, but not cell division, explaining the bacteriostatic effect of EMB. Furthermore, the data suggest that members of the family Corynebacterineae have two dedicated machineries for cell elongation (elongasome) and cytokinesis (divisome). IMPORTANCE: Antibiotic treatment of bacterial pathogens has contributed enormously to the increase in human health. Despite the apparent importance of antibiotic treatment of bacterial infections, surprisingly little is known about the molecular functions of antibiotic actions in the bacterial cell. Here, we analyzed the molecular effects of ethambutol, a first-line antibiotic against infections caused by members of the genus Mycobacterium We find that this drug selectively blocks apical cell growth but still allows for effective cytokinesis. As a consequence, cells survive ethambutol treatment and adopt a pneumococcal cell growth mode with cell wall synthesis only at the site of cell division. However, combined treatment of ethambutol and beta-lactam antibiotics acts synergistically and effectively stops cell proliferation.

The Mycobacterium phlei Genome: Expectations and Surprises.

Mycobacterium phlei, a nontuberculosis mycobacterial species, was first described in 1898-1899. We present the complete genome sequence for theM. phlei CCUG21000(T)type strain and the draft genomes for four additional strains. The genome size for all five is 5.3 Mb with 69.4% Guanine-Cytosine content. This is ≈0.35 Mbp smaller than the previously reported M. phlei RIVM draft genome. The size difference is attributed partly to large bacteriophage sequence fragments in theM. phlei RIVM genome. Comparative analysis revealed the following: 1) A CRISPR system similar to Type 1E (cas3) in M. phlei RIVM; 2) genes involved in polyamine metabolism and transport (potAD,potF) that are absent in other mycobacteria, and 3) strain-specific variations in the number of σ-factor genes. Moreover,M. phlei has as many as 82 mce(mammalian cell entry) homologs and many of the horizontally acquired genes in M. phlei are present in other environmental bacteria including mycobacteria that share similar habitat. Phylogenetic analysis based on 693 Mycobacterium core genes present in all complete mycobacterial genomes suggested that its closest neighbor is Mycobacterium smegmatis JS623 and Mycobacterium rhodesiae NBB3, while it is more distant toM. smegmatis mc2 155.

The application of metabolite profiling to Mycobacterium spp.: determination of metabolite changes associated with growth.

In order to decipher the complex biological networks underlying biochemical and physiological processes, cellular regulation at all levels must be studied. The metabolites determined by metabolomics represent the end-point of cellular regulation and thus vital components of any integrative network. In the case of pathogenic agents such as Mycobacterium tuberculosis metabolomics offers an ideal opportunity to gain a better understanding of how this species adapts to environmental conditions and antimicrobial treatments. In the present study a metabolite profiling protocol for Mycobacterium including optimised quenching, extraction and analysis has been devised. These methods have been applied to three different Mycobacterium spp. demonstrating potential translation across the genus. Steady-state levels of metabolites during growth have been determined for Mycobacterium smegmatis, Mycobacterium phlei and Mycobacterium bovis BCG (Bacillus Calmette-Guérin). The changes of designated biomarkers emphasised phenotypical differences (e.g. nitrogen metabolism) and similarities (e.g. cysteine biosynthesis) between the bacteria. Each time point showed distinguishable metabolic characteristics from early lag to late stationary phase/beginning of non-replicating phase. The combination of the metabolic results with published "omics" data indicated that transcription appeared to be the most predominant mode of cellular regulation utilised by these bacteria studied.

Thermophilic biodesulfurization of various heterocyclic sulfur compounds and crude straight-run light gas oil fraction by a newly isolated strain Mycobacterium phlei WU-0103.

Various heterocyclic sulfur compounds such as naphtho[2,1-b]thiophene (NTH) and benzo[b]thiophene (BTH) derivatives can be detected in diesel oil, in addition to dibenzothiophene (DBT) derivatives. Mycobacterium phlei WU-0103 was newly isolated as a bacterial strain capable of growing in a medium with NTH as the sulfur source at 50 degrees C. M. phlei WU-0103 could degrade various heterocyclic sulfur compounds, not only NTH and its derivatives but also DBT, BTH, and their derivatives at 45 degrees C. When M. phlei WU-0103 was cultivated with the heterocyclic sulfur compounds such as NTH, NTH 3,3-dioxide, DBT, BTH, and 4,6-dialkylDBTs as sulfur sources, monohydroxy compounds and sulfone compounds corresponding to starting heterocyclic sulfur compounds were detected by gas chromatography-mass spectrometry analysis, suggesting the sulfur-specific desulfurization pathways for heterocyclic sulfur compounds. Moreover, total sulfur content in 12-fold-diluted crude straight-run light gas oil fraction was reduced from 1000 to 475 ppm S, with 52% reduction, by the biodesulfurization treatment at 45 degrees C with growing cells of M. phlei WU-0103. Gas chromatography analysis with a flame photometric detector revealed that most of the resolvable peaks, such as those corresponding to alkylated derivatives of NTH, DBT, and BTH, disappeared after the biodesulfurization treatment. These results indicated that M. phlei WU-0103 may have a good potential as a biocatalyst for practical biodesulfurization of diesel oil.

Thermophilic biodesulfurization of hydrodesulfurized light gas oils by Mycobacterium phlei WU-F1.

Recalcitrant organosulfur compounds such as dibenzothiophene (DBT) derivatives in light gas oil (LGO) cannot be removed by conventional hydrodesulfurization (HDS) treatment using metallic catalysts. The thermophilic DBT-desulfurizing bacterium Mycobacterium phlei WU-F1 grew in a medium with hydrodesulfurized LGO as the sole source of sulfur, and exhibited high desulfurizing ability toward LGO between 30 and 50 degrees C. When WU-F1 was cultivated at 45 degrees C with B-LGO (390 ppm S), F-LGO (120 ppm S) or X-LGO (34 ppm S) as the sole source of sulfur, biodesulfurization resulted in around 60-70% reduction of sulfur content for all three types of hydrodesulfurized LGOs. In addition, when resting cells were incubated at 45 degrees C with hydrodesulfurized LGOs in the reaction mixtures containing 50% (v/v) oils, biodesulfurization reduced the sulfur content from 390 to 100 ppm S (B-LGO), from 120 to 42 ppm S (F-LGO) and from 34 to 15 ppm S (X-LGO). Gas chromatography analysis with an atomic emission detector revealed that the peaks of alkylated DBTs including 4-methyl-DBT, 4,6-dimethyl-DBT and 3,4,6-trimethyl-DBT significantly decreased after biodesulfurization. Therefore, thermophilic M. phlei WU-F1, which could effectively desulfurize HDS-treated LGOs over a wide temperature range up to 50 degrees C, may be a promising biocatalyst for practical biodesulfurization of diesel oil.

Thermophilic biodesulfurization of naphthothiophene and 2-ethylnaphthothiophene by a dibenzothiophene-desulfurizing bacterium, Mycobacterium phlei WU-F1.

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and NTH derivatives can be detected in diesel oil following hydrodesulfurization treatment, in addition to DBT derivatives. Mycobacterium phlei WU-F1, which possesses high desulfurizing ability toward DBT and its derivatives over a wide temperature range (20-50 degrees C), could also grow at 50 degrees C in a medium with NTH or 2-ethylNTH, an alkylated derivative, as the sole source of sulfur. At 50 degrees C, the resting cells of WU-Fl degraded 67% and 83% of 0.81 mM NTH and 2-ethylNTH, respectively, within 8 h. By GC-MS analysis, 2-ethylNTH-desulfurized metabolites were identified as 2-ethylNTH sulfoxide, 1-(2'-hydroxynaphthyl)-1-butene and 1-naphthyl-2-hydroxy-1-butene, and it was concluded that WU-F1 desulfurized 2-ethylNTH through a sulfur-specific degradation pathway with the selective cleavage of carbon-sulfur bonds. Therefore, M. phlei WU-Fl can effectively desulfurize asymmetric organosulfur compounds, NTH and 2-ethylNTH, as well as symmetric DBT derivatives under high-temperature conditions, and it may be a useful desulfurizing biocatalyst possessing a broad substrate specificity toward organosulfur compounds.

Thermophilic biodesulfurization of dibenzothiophene and its derivatives by Mycobacterium phlei WU-F1.

Dibenzothiophene (DBT) derivatives can be detected in diesel oil following hydrodesulfurization treatment, and they are widely recognized as target compounds for more efficient desulfurization. The moderately thermophilic bacterium Mycobacterium phlei WU-F1 was isolated for its ability to grow at 50 degrees C in a medium with DBT as the sole source of sulfur. At 50 degrees C, resting cells of WU-F1 degraded 0.81 mM DBT within only 90 min to produce 2-hydroxybiphenyl as a desulfurized metabolite through the selective cleavage of carbon-sulfur bonds, and also degraded 0.81 mM of derivatives such as 2,8-dimethylDBT, 4,6-dimethylDBT and 3,4-benzoDBT within 8 h. In addition, the resting cells exhibited high DBT-desulfurizing ability over a wide temperature range from 20 to 50 degrees C. Because M. phlei WU-F1 possesses higher desulfurizing ability toward DBT and the derivatives over a wider temperature range than any other microorganisms previously reported, it may have useful practical applications for biodesulfurization.

Characterization of biofilm growth and biocide susceptibility testing of Mycobacterium phlei using the MBEC assay system.

The importance of non-tuberculosis mycobacterial biofilm species in medicine, industry and the environment has recently gained attention. Our objectives were to characterize biofilm growth of Mycobacterium phlei M4, as a model of rapidly growing mycobacteria using the minimal biofilm eradication concentration (MBEC) and to compare biocide susceptibility of planktonic and biofilm organisms. Scanning electron microscopy was also carried out to observe biofilm morphology. With the exception of Sporicidin and Virkon the minimum bactericidal concentration values for all biocides tested were lower than the MBEC values. The MBEC assay system was seen to produce multiple and reproducible biofilms of M. phlei and to be a useful tool for susceptibility studies.

Partial characterization of a major autolysin from Mycobacterium phlei.

Autolytic enzyme profiles of fast- and slow-growing mycobacteria were examined using SDS-PAGE zymography with incorporated mycobacterial peptidoglycan sacculi as substrate. Each species tested (Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium fortuitum and Mycobacterium kansasii) appeared to produce a different set of enzymes on the basis of differing number and molecular masses. A major autolysin from M. phlei was purified to apparent homogeneity by DEAE-cellulose chromatography, preparative gel electrophoresis and Mono Q FPLC. This enzyme had an estimated molecular mass of 38 kDa, an isoelectric point of 5.5 and a pH optimum of pH 7.5. Digestion of purified peptidoglycan by the enzyme resulted in the appearance of reducing sugars, suggesting that the 38 kDa autolysin is a beta-glycosidase. Partial internal amino acid sequence of the autolysin was determined and should facilitate identification, cloning and overexpression of the encoding gene.

Evaluation of methods for isolation of DNA from slowly and rapidly growing mycobacteria.

Mycobacteria generally have thick cell walls and contain large amounts of lipid, making them resistant to DNA extraction. Five methods, namely, extensive enzymic digestion method (M1), 2-min mechanical glass-bead disruption method (M2), thermal shock method (M3), modified conventional enzymic digestion method (M4), and manual disruption with modified conventional enzymic digestion method (M5), were used to compare their effectiveness and simplicity in extracting DNA from slowly growing mycobacteria (Mycobacterium leprae, M. lepraemurium and M. bovis BCG), and a rapidly growing mycobacterium (M. phlei). The highest DNA yield was obtained by M2 from M. lepraemurium which produced 2.82 micrograms DNA/mg wet weight of cells, representing a theoretical yield of 78%. M3 gave the lowest DNA yield; 0.01 microgram DNA/mg wet weight of cells of M. lepraemurium was obtained. M4, in which proteinase K was used, is more effective than M1, in which subtilisin and pronase were used. M5 yielded a higher amount of DNA, but it required more manipulations to extract DNA as compared to M4. Extraction of DNA of M. leprae from nude mice is more difficult than that of M. leprae from armadillos by all of the methods used. These results suggest that the biosynthetic capabilities of these two forms of M. leprae may vary, depending on their cultural conditions and/or strain differences. Our results have shown that both M2 and M4 are the simplest, most effective and time-saving methods which are suitable for every routine laboratory to extract DNA from slowly and rapidly growing mycobacteria.

Water soluble palmitic acid in media for cultivation of leprosy derived psychrophilic mycobacteria from Mycobacterium leprae infected tissues

Cultivation trials for Mycobacterium leprae resulted in growth of Mycobacterium psychrophilum (L). Media were inoculated with host grown Mycobacterium leprae cells from armadillo tissues, Nu mice foot pads or human lepromata. Cultures were obtained in liquid and on semisolid multifactoria 1 media containing water soluble palmitic acid or its salts. Ammonium thioglycolate and Napalmitate served as carbon and energy sources. The water soluble palmitic acid remained in perfect solution following sterilization in the autoclave, thus easily accessible to the cells. The cyclodextrin-Fe complex served as a siderophore to grow the obtained leprosy derived psychrophilic cells. The leprosy derived cultures and subcultures grew opimally at+10 degrees Celssius but deteriorated rapidly at + 32 degrees Celsius, in the multifactorial media. No growth occurred in 7H9 media. Cultures were not identified for classification.

Water soluble complexes of C14 and C16 fatty acids and alcohols in media for cultivation of leprosy-derived psychrophilic mycobacteria.

Host-grown Mycobacterium leprae cell suspensions oxidized water-soluble complexes of palmitic acid, myristic acid, cetyl alcohol, and myristyl alcohol prepared with randomly methylated-beta-cyclodextrin as host molecules. Gas chromatography analysis showed that the water-soluble complexes retained their chemical structure following sterilization in the autoclave. Bioavailability of the two long-chain fatty acids and the corresponding long-chain alcohols was confirmed by Warburg manometric techniques with host-grown M. leprae cell suspensions. Inoculated with host-grown M. leprae cells in chemically well-defined, simple liquid and agar media, acid-fast bacilli were cultivable in primary cultures and subcultures at 10 degrees C with (NH4)2SO4 as the N source and water-soluble palmitic acid, myristic acid, cetyl alcohol or myristyl alcohol as the C and potent energy sources. M. phlei oxidized the complexed palmitic acid and myristic acid but not cetyl alcohol or myristyl alcohol. On agar media with any of these four carbon sources and (NH4)2SO4 but not ammonium thioglycolate as the N source, M. phlei grew abundantly at 36 degrees C. In liquid media only myristyl alcohol supported growth of M. phlei without any growth with palmitic acid, cetyl alcohol or myristic acid. The leprosy-derived, cold-loving cultures ("M. psychrophilum") were not fully tested for classification and identification. The cells are strongly acid-fast facultative psychrophiles, adapted in subcultures to mesophilic growth. They grow in chemically well-defined media with 14 and 16 C long-chain fatty acids or alcohols as the C and energy sources. None of the cultures grow on Low-enstein or 7H9 media. Heat-killed suspensions of the 4th and 6th subcultures provoke Mitsuda-type late skin reactions in tuberculoid, borderline and borderline-tuberculoid but not in lepromatous leprosy volunteers. When grown with (NH4)2SO4 as the N source (but not with the reducing agent ammonium thioglycolate) the subcultures multiplied abundantly in the foot pads of mice. It became evident that leprosy-derived, facultative psychrophilic mycobacteria really exist. Mycobacteria of this cluster do not distinguish between 14 or 16 C long chains with COOH or CH2OH as terminal bindings. Cells are quite aerophilic and grow preferentially on agar slant surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

Utilization of beet molasses for riboflavin production by Mycobacterium phlei.

Mycobacterium phlei was tested for its ability to utilize beet molasses as the sole carbon source and produce riboflavin. The crude beet molasses was analyzed and treated in various ways to reduce its heavy element content and to remove the muddy residue. Promising amounts of riboflavin were produced when the organism was cultivated on decationized (resin-treated) beet molasses. The highest vitamin productivity was achieved by incubating the inoculated medium containing 9% molasses and initially adjusted to pH 6 under shacked condition for 6 days in the dark.

Effect of growth temperature on the fatty acid composition of Mycobacterium phlei.

In Mycobacterium phlei, fatty acid unsaturation increased with decreasing temperature. The 10-hexadecenoic acid content increased as the temperature was reduced from 35 degrees C to 26-20 degrees C. At lower temperatures tuberculostearic acid decreased while oleic and linoleic acids increased, the latter being found in M. phlei for the first time. Concomitantly palmitic acid content decreased, and the 6- and 9-hexadecenoic acids increased slightly on reducing the temperature from 35 to 10 degrees C. Thus, down to 26-20 degrees C palmitic acid was mainly replaced by 10-hexadecenoic acid. From this range down to 10 degrees C, palmitic and tuberculostearic acids were replaced by oleic and linoleic acids. Consequently, fatty acid branching decreased and mean chain length increased, as the temperature was reduced. These observations support the view that regulation of membrane fatty acid composition is part of microbial temperature adaptation, and that the mechanism behind the responses might be more complex than generally believed.

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.

Factors influencing the in vitro growth of Mycobacterium leprae: effect of oxygen.

In our efforts to evaluate factors responsible for in vitro growth of Mycobacterium leprae in DH medium and also to improve the system, effects of oxygen tension on in vitro growth of M. leprae were determined. This was achieved by varying the ratio between DH medium and free air space above the medium in the culture tubes. Growth-competent M. phlei (ATCC 11758) could tolerate all the oxygen it can get in the medium. On the other hand, M. leprae seemed to be of microaerophilic nature. In vivo-grown M. leprae cells were more sensitive than their counterparts that were adapted to in vitro environment. In vivo-grown cells grew better when 70% of the space in culture tube was occupied by DH medium. These in vitro-adapted cells gave optimum growth in subcultures when the air spaces in the culture tubes were 40-50%. The role of oxygen tensions in the development of lesions in leprosy patients and armadillos has been discussed.

Phenotypic changes in mycobacteria grown in oxygen-limited conditions.

Laboratory strains of Mycobacterium phlei, M. smegmatis, M. fortuitum, M. gordonae, M. kansasi, M. bovis, M. tuberculosis and M. intracellulare were adapted to grow in an anaerobic environment. Concomitant with the transition to anaerobic growth was loss of acid-fastness, loss or modification of colonial pigmentation, and loss of ability to grow on a malachite green-containing medium. The mycobacteria grown anaerobically produced acid from a greater range of carbohydrates than aerobically grown cultures, lost iron-uptake activity, and showed a reduction of urease, catalase and nitratase activity. Back adaption of mycobacteria from an anaerobic to an aerobic environment resulted in the acquisition of acid-fastness, pigmentation, and other characteristics used in the taxonomy of mycobacteria. These results suggest that mycobacterial cultures, if grown in an anaerobic environment, may be erroneously identified in clinical laboratories.

Influence of culture medium and incubation time on the fatty acid compositions of some rapid-growing mycobacteria as analysed by packed and capillary column gas chromatography.

Cellular fatty acids of four rapid-growing mycobacterial species (Mycobacterium chelonei, M. fortuitum, M. phlei, and M. smegmatis) were analysed by packed and capillary column gas chromatography after one, three, four, six, eight, and twelve days of incubation on Löwenstein-Jensen and Sauton agar media. Variations in incubation time did not affect the chromatograms except in the case of twelve-day incubated M. smegmatis. Mycobacteria cultivated on Sauton agar medium contained more tuberculostearic and less oleic acid compared with Löwenstein-Jensen. For informative and reproducible analytical results, we recommend using a chemically defined culture medium and splitless injection on a capillary column capable of separating not only the methyl esters of the cellular fatty acids but also the diagnostically important secondary alcohols containing 18 and 20 carbon atoms.