Mycolic Acid-containing Bacteria Research Articles

Arcyriaflavin E, a new cytotoxic indolocarbazole alkaloid isolated by combined-culture of mycolic acid-containing bacteria and Streptomyces cinnamoneus NBRC 13823.

Arcyriaflavin E, a new cytotoxic indolocarbazole alkaloid isolated by combined-culture of mycolic acid-containing bacteria and Streptomyces cinnamoneus NBRC 13823

Hydrocarbon-oxidizing microorganisms of hot springs and their significance in the assessment of the biodiversity of microbial communities

A microbiological study of surface thermal manifestations of the alkaline hot spring of the Kizlyarskoye deposit has been performed for the first time. The following organisms were isolated: nocardiomorphic actinomycetes, i.e., mycolic acid-containing bacteria (Nocardia asteroidas, Nocardia transvalensis, Rhodococcus rhodochrous, Rhodococcus maris, Rhodococcus luteus, Rhodococcus terrae, Rhodococcus brouchialis), non-spore-forming Gram-positive bacilli of irregular shapes (Corynebacterium bovis, Corynebacterium diphtheria, Propionibacterium freudenreichis), and mycobacteria (Mycobacterium marinum). Their morphological and physiological-biochemical environmental peculiarities are discussed.

Production of unique natural products through micro-scale co-cultivation of marine bacteria

Nature continues to supply chemically diverse and biologically active compounds, or natural products, with historically successful potential as therapeutic leads. Unfortunately, many of the biosynthetic gene clusters remain silent, or cryptic, under laboratory culture conditions. To coax the expression of cryptic biosynthetic gene clusters in marine invertebrate-associated bacteria, the bacteria were co-cultured to facilitate interspecies interactions and metabolite production. In this study, we utilize LC/MS followed by principal component analysis (PCA) for rapid and sensitive analysis, resulting in a graphical representation of metabolic differences between co-cultures and mono-culture controls. Furthermore, culture volumes were reduced to 500µL and grown in 96-well low-evaporation plates. This system allows for the simultaneous co-cultivation and chemical profiling of hundreds of strains, greatly improving throughput. Previous studies suggested natural product biosynthesis within Streptomyces sp. was induced when co-cultured them with mycolic acid-containing bacteria1. As a preliminary study, we selected Streptomyces sp. strains with no antibiotic activity and have shown the induction of antibiotic production upon co-culturing with mycolic acid-containing bacteria.

First Isolation ofSegniliparus rugosusfrom a Patient with Radiologic Features Similar to Non-Tuberculous Mycobacteriosis

In 2005, a group of mycolic acid-containing bacteria was characterized as belonging to a novel genus, Segniliparus with species Segniliparus rugosus and S. rotundus. We report a case of the S. rugosus isolated from a 54-year-old woman with radiologic features mimicking that of non-tuberculous mycobacteriosis (NTM). When the patient first visited our hospital, an acid-fast bacteria (AFB) smear tested positive and Mycobacterium tuberculosis polymerase chain reaction (TB PCR) was negative in the bronchoalveolar lavage sample. After 2 months, the growing colonies were reported as NTM, but could not be identified because they had died. One year after the initial visit, induced sputum samples showed the same results, positive AFB smear and negative TB PCR. At this point, the growing colonies were identified as S. rugosus. Therefore, we should consider Segniliparus genus as a differential diagnosis for AFB in respiratory specimens in addition to the genus Mycobacterium.

Mycolic Acid-Containing Bacteria Induce Natural-Product Biosynthesis in Streptomyces Species

Natural products produced by microorganisms are important starting compounds for drug discovery. Secondary metabolites, including antibiotics, have been isolated from different Streptomyces species. The production of these metabolites depends on the culture conditions. Therefore, the development of a new culture method can facilitate the discovery of new natural products. Here, we show that mycolic acid-containing bacteria can influence the biosynthesis of cryptic natural products in Streptomyces species. The production of red pigment by Streptomyces lividans TK23 was induced by coculture with Tsukamurella pulmonis TP-B0596, which is a mycolic acid-containing bacterium. Only living cells induced this pigment production, which was not mediated by any substances. T. pulmonis could induce natural-product synthesis in other Streptomyces strains too: it altered natural-product biosynthesis in 88.4% of the Streptomyces strains isolated from soil. The other mycolic acid-containing bacteria, Rhodococcus erythropolis and Corynebacterium glutamicum, altered biosynthesis in 87.5 and 90.2% of the Streptomyces strains, respectively. The coculture broth of T. pulmonis and Streptomyces endus S-522 contained a novel antibiotic, which we named alchivemycin A. We concluded that the mycolic acid localized in the outer cell layer of the inducer bacterium influences secondary metabolism in Streptomyces, and this activity is a result of the direct interaction between the mycolic acid-containing bacteria and Streptomyces. We used these results to develop a new coculture method, called the combined-culture method, which facilitates the screening of natural products.

The TetR‐type transcriptional regulator FasR of Corynebacterium glutamicum controls genes of lipid synthesis during growth on acetate

The addition of fatty acids to either Escherichia coli or Bacillus subtilis elicits an elaborate cellular response of the lipid metabolism. We found that in Corynebacterium glutamicum the expression of accD1 encoding the β-subunit of the essential acetyl-CoA carboxylase is repressed in acetate-grown cells without the addition of fatty acids. The TetR-type transcriptional regulator NCgl2404, termed FasR, was identified and deleted. During growth on acetate, but not on glucose, 17 genes are differentially expressed in the deletion mutant, among them accD1, and fasA and fasB both encoding fatty acid synthases, which were upregulated. Determination of the 5' ends of accD1, fasA, fasB and accBC together with the use of isolated FasR protein identified the FasR binding site, fasO, which is located within the accD1 and fasA transcript initiation site thus blocking transcription by RNA polymerase binding directly. The identified fasO motif is present in C. efficiens or C. diphtheriae, too, and it is actually similarly positioned in these bacteria within the 5' ends of the accD1 and fasA transcripts, and a fasR orthologue is also present. The identification of the FasR-fasO system in Corynebacteriaceae might indicate a conserved transcriptional control of the unique lipid synthesis in these mycolic acid-containing bacteria.

Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes for Rapid Detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in Potable-Water Biofilms

Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply.

Immunomagnetic separation of scum-forming bacteria using polyclonal antibody that recognizes mycolic acids

Mycolic acid-containing bacteria (mycolata) are thought to be involved in scum formation in aeration basins of activated sludge plants due to their ability to produce biosurfactants and their cell surface hydrophobicity. To isolate these bacteria, immunomagnetic separation (IMS) using an anti-mycolic acid polyclonal antibody was investigated. IMS that targeted Gordonia amarae SC1 exhibited a 100% recovery at 5×10 3 CFU ml −1. At cell concentration of 7.8×10 6 CFU ml −1, the recovery was lowered, but 80% of cells were still captured. Effect of bead concentrations on the recovery of SC1 at 10 6 CFU ml −1 was examined. The results showed that addition of more than 6–7×10 6 beads for 1×10 6 CFU reached a maximum recovery (83%). Furthermore, the IMS procedure optimized with SC1 cells was tested with another mycolata. The results suggested that variation of the recovery for each mycolata is dependent on the specificity of the polyclonal antibody and that mycolata which are recognized by the antibody can be recovered by this procedure.

Production of Anti-Gordonia amarae Mycolic Acid Polyclonal Antibody for Detection of Mycolic Acid-Containing Bacteria in Activated Sludge Foam.

Mycolic acid-containing actinomycetes (mycolata) are considered the causative agents of foaming of activated sludge and scum formation in activated sludge treatment plants. In this study, the production of anti-Gordonia amarae mycolic acid polyclonal antibodies was investigated. Rabbits were immunized with a conjugate of keyhole limpet hemocyanin and mycolic acids of G. amarae, which contained 48 to 56 carbon atoms (average, 52.0). Enzyme-linked immunosorbent assay (ELISA) demonstrated that the polyclonal antibodies could recognize cells of G. amarae ranging from 0.1 to 10 microg. The antibodies also reacted with other tested mycolata strains belonging to the genera Nocardia, Rhodococcus, Dietzia, Mycobacterium and Tsukamurella. However, reactivities against other gram-positive and gram-negative bacteria not containing mycolic acid were negligible or much lower. The results indicate that the anti-G. amarae mycolic acid antibodies show a reactivity selective for a group of mycolata involved in the foaming of activated sludge.

Production of anti- Gordonia amarae mycolic acid polyclonal antibody for detection of mycolic acid-containing bacteria in activated sludge foam

Mycolic acid-containing actinomycetes (mycolata) are considered the causative agents of foaming of activated sludge and scum formation in activated sludge treatment plants. In this study, the production of anti- Gordonia amarae mycolic acid polyclonal antibodies was investigated. Rabbits were immunized with a conjugate of keyhole limpet hemocyanin and mycolic acids of G. amarae, which contained 48 to 56 carbon atoms (average, 52.0). Enzyme-linked immunosorbent assay (ELISA) demonstrated that the polyclonal antibodies could recognize cells of G. amarae ranging from 0.1 to 10 μg. The antibodies also reacted with other tested mycolata strains belonging to the genera Nocardia, Rhodococcus, Dietzia, Mycobacterium and Tsukamurella. However, reactivities against other gram-positive and gram-negative bacteria not containing mycolic acid were negligible or much lower. The results indicate that the anti- G. amarae mycolic acid antibodies show a reactivity selective for a group of mycolata involved in the foaming of activated sludge.

Selective Extraction and Purification of a Mycobacterial Outer Membrane Protein

MspA forms water-filled channels in the mycolic acid layer of Mycobacterium smegmatis thereby allowing the diffusion of hydrophilic solutes through this permeability barrier into the periplasm. MspA is the first member of a new family of porins and is extremely stable against chemical and thermal denaturation. We developed a purification procedure based on selective extraction of MspA with detergents from whole cells of M. smegmatis at high temperatures. Anion-exchange and size-exclusion chromatography yielded about 230 μg apparently pure and highly active MspA per liter of culture. This was a 20-fold increased yield compared to previous purification protocols. Similar amounts of pure MspA were obtained with the detergents isotridecylpolyethyleneglycolether, lauryldimethylamine oxide, and octylpolyethylene oxide indicating that this purification procedure is not restricted to a specific detergent. This study will promote the structural and functional analysis of MspA and might be valuable for the isolation of porins from other mycolic acid-containing bacteria.

Macroamphiphilic cell envelope components of Rhodococcus equi and closely related bacteria

Recent progress towards an understanding of the architecture of the mycobacterial cell envelope (P.J. Brennan and H. Nikaido, Annual Review of Biochemistry 64 (1995) 29–63) provides a model with features more generally applicable to cell envelope organisation in other mycolic acid-containing bacteria. Using this archetype, a model for the organisation of the rhodococcal cell envelope is presented here, with particular reference to cell envelope composition in Rhodococcus equi. The likelihood that mycolic acids bound to the cell wall arabinogalactan contribute to the formation of a distinct outer lipid layer is emphasised. Furthermore, the model incorporates recent work which has characterised rhodococcal macroamphiphiles (lipoglycans and lipoproteins), including the VapA virulence-associated lipoproteins of R. equi.

Different rifampicin inactivation mechanisms in Nocardia and related taxa.

Mycolic acid-containing bacteria inactivate rifampicin in a variety of ways such as glucosylation, ribosylation, phosphorylation and decolorization. These inactivations were found to be a species-specific phenomena in Nocardia and related taxa. Gordona, Tsukamurella and fast-growing Mycobacterium modified rifampicin by ribosylation of the 23-OH group of the antibiotic. Such ribosylation was not observed in Rhodococcus and Corynebacterium, but phosphorylation of the 21-OH group of rifampicin was observed in one strain of Rhodococcus. Nocardia modified the antibiotic by glucosylation (23-OH group) and phosphorylation, but ribosylation was not observed.

Fatty acid composition of some mycolic acid-containing coryneform bacteria.

The fatty acid profiles of 74 strains of mycolic acid-containing coryneform bacteria were examined by gas-liquid chromatography. All of the strains contained major amounts of straight-chain and monounsaturated fatty acids although some also possessed substantial amounts of 10-methyloctadecanoic acid. Iso- and anteiso-branched acids were not present. Five distinct fatty acid patterns were evident: (i) Corynebacterium diphtheriae, C. pseudotuberculosis and 'C. ulcerans' strains contained major amounts of hexadecanoic and hexadecenoic acids; (ii) C. glutamicum, C. xerosis and related saprophytic and animal-associated strains, predominantly hexadecanoic and octadecenoic acids; (iii) C. bovis, major amounts of octadecenoic and 10-methyloctadecanoic acids; (iv) 'C. mycetoides', significant amounts of heptadecanoic acid as well as hexadecanoic and octadecenoic acids; and (v) strains related to Rhodococcus possessed significant quantities of 10-methyloctadecanoic acid in addition to straight-chain and monounsaturated acids.

Thin-layer chromatography of methanolysates of mycolic acid-containing bacteria

Whole-organism methanolysates of representative bacteria containing mycolic acids (long-chain 3-hydroxy 2-branched acids) were prepared by treating dry organisms with methanol-toluene-sulphuric acid (30:15:1) at 75° overnight. The mycolic acid esters from mycobacteria were not resolved satisfactorily by single dimensional thin-layer chromatographic analysis. Three distinct patterns of mycobacterial mycolates were produced by two-dimensional thin-layer chromatography, good examples of each pattern being the mycolates from Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium fortuitum.