Effect of growth temperature on the lipid composition of Mycobacterium smegmatis ATCC 607

The total lipid content of Mycobacterium smegmatis ATCC 607 was the same whether it was grown at 27 or 37 degrees C. The total phospholipid content, however, increased significantly at 27 degrees C. Phosphatidylethanolamine increased most markedly with a simultaneous decrease in phosphatidylinositol mannosides. Among individual phosphatidylinositol mannosides, tri- and tetra-acylated dimannophosphoinositides and tetra-acylated hexamannophosphoinositides all decreased at the lower growth temperature. Triacylglycerols and monoacylglycerols increased at the lower temperature but diacylglycerols were unaffected. Unsaturated fatty acids of total phospholipids increased as the temperature was lowered.     

Macromolecular biosynthesis in Mycobacterium smegmatis ATCC 607 in the presence of antibodies to mannophosphoinositides

The biosynthesis of DNA, proteins, RNA and phospholipids in Mycobacterium smegmatis ATCC 607 was investigated by studying the incorporation of radiolabelled components in the presence of antiserum to mannophosphoinositides. The antiserum had an inhibitory effect on the rate of synthesis of these macromolecules. However, the inhibition was greater when antibody was present together with complement.     

Effect of ethambutol on the phospholipids of ethambutol susceptible and resistant strains of Mycobacterium smegmatis ATCC 607

Effect of ethambutol (EMB) on phospholipid composition and metabolism of EMB-susceptible and EMB-resistant strains of M. smegmatis ATCC 607 was studied. Treatment with ethambutol had different effect in both the strains resulting in decreased total phospholipid and cardiolipin content in EMB-susceptible strain and increased content in EMB-resistant strain, with no effect on fatty acyl group composition. These changes were further corroborated by the use of [1-14C]sodium acetate in these studies.     

Ca2+/calmodulin dependent protein kinase from Mycobacterium smegmatis ATCC 607

A soluble Ca²⁺/calmodulin dependent protein kinase has been partially purified (~400 fold) from Mycobacterium smegmatis ATCC 607 using several purification steps like ammonium sulphate precipitation (30-60%), Sepharose CL-6B gel filtration, DEAE-cellulose and finally calmodulin-agarose affinity chromatography. On SDS-PAGE, this enzyme preparation showed a major protein band of molecular mass 35 kD and its activity was dependent on calcium, calmodulin and ATP when measured under saturating histone IIs (exogenous substrate) concentration. Phosphorylation of histone IIs was inhibited by W-7 (calmodulin inhibitor) and KN-62 (CaM-kinase inhibitor) with IC₅₀ of 1.5 and 0.25 m respectively, but was not affected by inhibitors of PKA (Sigma P5015) and PKC (H-7). All these results confirm that purified enzyme is Ca²⁺/ calmodulin dependent protein kinase of M. smegmatis. The protein kinase of M. smegmatis demonstrated a narrow substrate specificity for both exogenous as well as endogenous substrates. These results suggest that purified CaM-kinase must be involved in regulating specific function(s) in this organism.     

Biosynthesis of hexamannophosphoinositides inMycobacterium smegmatis ATCC 607

The biosynthesis of hexamannophosphoinositides inMycobacterium smegmatis ATCC 607 was examined using labelled tri- and tetraacylated dimannophosphoinositides (PIM₂-3F and PIM₂-4F) as precursors, byin vivo andin vitro incorporation. Tetraacylated dimannoside was metabolically more active as compared to triacylated dimannoside and seems to be the precursor for the synthesis of hexamannophosphoinositides.Abbreviations M. 607 Mycobacterium smegmatis ATCC 607 - PIMs Mannophosphoinositides - PIM₂-2F diacylated dimannoside - PIM₂-3F triacylated dimannoside - PIM₂-4F tetraacylated dimannoside - PIM₃-3F triacylated trimannoside - PIM₆-3F triacylated hexamannoside - PIM₆-4F tetraacylated hexamannoside - GDP-mannose Guanosine diphosphomannose     

Cell wall composition of ethambutol susceptible and resistant strains of Mycobacterium smegmatis ATCC 607

Changes in the cell wall that accompany acquisition of ethambutol (EMB) resistance ina single step mutant of Mycobacterium smegmatis ATCC 607 were analysed. Quantitative changes were seen in the chemical constituents of the cell wall of resistant cultures in comparison with EMB-susceptible M. smegmatis . Alterations in the binding of 1-anilinonaphthalene-8-sulphonate (ANS) were suggestive of structural changes in the cell surface.     

Effect of fatty acid supplementation on the lipid composition of Mycobacterium smegmatis ATCC 607, grown at 27° and 37°C

Mycobacterium smegmatis ATCC 607 was grown at 27 and 37°C, with and without exogenous unsaturated fatty acids, viz. elaidic, oleic and palmitoleic acids, added to the growth medium. The total lipid content of M. smegmatis ATCC 607 was lower at 27°C, and with added oleic acid, when compared with the controls, but higher in presence of palmitoleic acid. At 37°C no significant differences were noted in the total lipid content. In general, the total lipid content was lower with all of the fatty acid supplementations at both 27 and 37°C. The phosphatidylethanolamine content was slightly higher at 27°C in the presence of elaidic or palmitoleic acid, but was markedly lower with oleic acid supplementation at 37°C. The cardiolipin content was lower in the presence of any of the fatty acids at 27°C, and higher in the medium supplemented with elaidic or oleic acid at 37°C. The unsaturated to saturated fatty acids ratio was higher with palmitoleic acid supplementation at 27°C, but remained unchanged in cells grown at 37°C. The modifications in mycobacterial lipids are a reflection of the organism's ability to adapt to changing growth conditions.     

Cloning and restriction analysis of ribosomal RNA genes from Mycobacterium smegmatis

A genomic library of Mycobacterium smegmatis DNA was constructed in phage EMBL3. A clone (gamma HB85) containing rRNA genes was isolated using as probes, fragments of E. coli rRNA cistron B. This cloned DNA fragment was mapped by restriction analysis and was shown to contain one complete set of rRNA genes (rRNA A). The physical mapping of the second set of rRNA genes of M. smegmatis (rRNA B) was done by restriction analysis of total chromosomal DNA. The two sets of rRNA genes showed highly conserved restriction sites within the respective sets but not in the flanking regions. The two rRNA sets of genes are organised like in the other eubacteria in the order 16S-23S-5S.     

Turnover of lipids in Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354

The rates of breakdown and renewal of individual lipids in cultures of Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354 were investigated by means of a pulse labelling technique using palmitate-1-¹⁴C. The results indicated that in growing cultures of both strains phospholipids were broken down, and cardiolipin had a very rapid turnover. In chase experiments, almost 45% and 40% of the radioactivity of this component were lost respectively from M. smegmatis and M. phlei during one generation time of the cell. The other two major components, phosphatidyl ethanolamine and phosphatidylinositol mannosides showed relatively low turnover. The loss of radioactivity from phosphatidylinositol mannosides was greater in M. phlei than in M. smegmatis but the loss of radioactivity from phosphatidyl ethanolamine was higher in M. smegmatis. The pattern of loss of radioactivity from lipids was almost the same in both strains, the difference being only in the extent of loss. The differences in the cellular localization of the phospholipids indicate their different roles within the cell. Results obtained with the glyceride fraction indicated a very rapid turnover of triglycerides in both strains.Abbreviations CL Cardiolipin - PE Phosphatidyl ethanolamine - PIMx phosphatidylinositol mannosides - PIM₂A phosphatidylinositol dimannoside tetra acylated - PIM₂B phosphatidylinositol dimannoside tri acylated - PIM₅ phosphatidylinositol pentamannoside tetra acylated     

MmpL11 Protein Transports Mycolic Acid-containing Lipids to the Mycobacterial Cell Wall and Contributes to Biofilm Formation in Mycobacterium smegmatis

A growing body of evidence indicates that MmpL (mycobacterial membrane protein large) transporters are dedicated to cell wall biosynthesis and transport mycobacterial lipids. How MmpL transporters function and the identities of their substrates have not been fully elucidated. We report the characterization of Mycobacterium smegmatis MmpL11. We showed previously that M. smegmatis lacking MmpL11 has reduced membrane permeability that results in resistance to host antimicrobial peptides. We report herein the further characterization of the M. smegmatis mmpL11 mutant and identification of the MmpL11 substrates. We found that biofilm formation by the M. smegmatis mmpL11 mutant was distinct from that by wild-type M. smegmatis. Analysis of cell wall lipids revealed that the mmpL11 mutant failed to export the mycolic acid-containing lipids monomeromycolyl diacylglycerol and mycolate ester wax to the bacterial surface. In addition, analysis of total lipids indicated that the mycolic acid-containing precursor molecule mycolyl phospholipid accumulated in the mmpL11 mutant compared with wild-type mycobacteria. MmpL11 is encoded at a chromosomal locus that is conserved across pathogenic and nonpathogenic mycobacteria. Phenotypes of the M. smegmatis mmpL11 mutant are complemented by the expression of M. smegmatis or M. tuberculosis MmpL11, suggesting that MmpL11 plays a conserved role in mycobacterial cell wall biogenesis.     

Effect of IS900 Gene of Mycobacterium paratuberculosis on Mycobacterium smegmatis

Mycobacterium paratuberculosis is mycobactin dependent and contains multiple copies of the IS900 gene that encodes for p43 (46.5K protein). The correlation between the two characteristics has been investigated. A 3.2-kb BamHI fragment from M. paratuberculosis containing the 1.451 kb IS900 gene was cloned in Escherichia coli and Mycobacterium smegmatis with pcDNA II and pNEZ6.3 plasmids, respectively. Surprisingly, the recombinant M. smegmatis grew poorly and slower in 7H9 broth supplemented with OADC (12 day) compared with M. smegmatis wild type or to M. smegmatis transformed with pNEZ6.3 (2 day). The growth rate of the recombinant M. smegmatis was restored by the addition of 2.4 μM ferric mycobactin J to the media. There was no effect on the growth rate of E. coli recombinants. Western blot analysis with p43-specific anti-peptide antibodies resulted in the expression of 46.5K and a cleaved form of 33.5K protein bands in the recombinant E. coli. There was no expression in the recombinant M. smegmatis. A lower expression of 33.5K protein band was detected in the native M. paratuberculosis protein. The nucleotide sequence of the 3.2-kb fragment confirmed the presence of p43-encoded ORF. There was no additional encoding sequence in the fragment. This suggests that the IS900 gene and/or its encoding products are involved in mycobactin dependency and possibly the slow growth rate of M. paratuberculosis. Received: 12 May 1998 / Accepted: 6 July 1998     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Transfection of Mycobacterium smegmatis SN2 with mycobacteriophage I3 DNA

Mycobacterium smegmatis SN2 does not exhibit natural competence for the uptake of phage I3 DNA. Competence can artificially be induced by treatment with glycine or CaCl₂, and the combination of both is even more effective. The efficiency of transfection can be improved by inclusion of protamine sulphate and heterologous RNA in the system. From ³²P DNA uptake studies the major barrier for the entry of DNA has been found to be the complex cell wall. The efficiency of transfection calculated on the basis of fraction of DNA which has entered the cell is comparable to that of other bacterial systems. The phage development takes a longer time (7 h for one cycle) after transfection, as compared to infection (4 h).     

Comparison of stain reactions in different cyclical stages of haemoproteids and haemogregarines

Cytochemical studies showed that haemoproteids, represented by Haemoproteus columbae and Parahaemoproteus danilewskii, differ in their reactivity for Feulgen, Unna Pappenheim stains and glycogen from haemogregarines, represented by Haemogregarina boueti and Haemogregarina aegyptia. On the other hand, there was no difference in the cytochemical make-up of gametocytes between H. columbae and P. danilewskii. In its stain reactions, Haemoproteus appeared to behave in a manner similar to that of Plasmodium; they both exhibited fluctuations in DNA and RNA in different cyclical stages. All stages of the parasites studied were negative for lipids, but all showed a positive reaction for proteins.