Infection with

SummaryTuberculosis and other mycobacterial infections are the most serious infectious diseases in terms of human fatalities. The high content of unique cellwall lipids helps these organisms to resist antimicrobial drugs and host defences. The biosynthesis of these lipids is discussed briefly. The recent advances in recombinant DNA technology have begun to help to elucidate the nature of some of the enzymes involved in this process and the genes that encode them. Gene disruption and other molecular genetic technologies are beginning to provide new approaches to test for the biological functions of these gene products and may lead to identification of new antimycobacterial drug targets.

An open reading frame, ORF3, first identified adjacent to the mycocerosic acid synthase gene in Mycobacterium bovis BCG encodes a protein with acyl-CoA synthase (ACoAS) activity. Genes homologous to acoas are found adjacent to other multifunctional polyketide synthase genes in the mycobacterial genome. To test whether these gene products are necessary to esterify the fatty acids generated by the adjacent polyketide synthase gene products, the acoas gene was disrupted in M. bovis BCG using a suicide vector containing the acoas gene with an internal deletion and the hygromycin-resistant gene as selection marker. Allelic exchange at the acoas locus was confirmed by Southern hybridization and polymerase chain reaction amplification of both flanking regions expected from homologous recombination. Immunoblot analysis indicated that the 65-kDa ACoAS protein product was absent in the mutant. Chromatographic analysis of lipids derived from [1-14 C]propionate showed that the mutant did not produce mycocerosyl lipids, although it produced normal levels of mycocerosic acid synthase. These results suggest that ACoAS is involved in the synthesis of mycocerosyl lipids of the mycobacterial cell wall.

The aim of this study was to test for expression of a 900-bp open reading frame (ORF), ORF3, located at the 5 end of the mycocerosic acid synthase gene in Mycobacterium bovis BCG and to determine the nature of the ORF3 protein. ORF3 was expressed as a 61-kDa C-terminal fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal rabbit antiserum, prepared against this fusion protein, cross-reacted with a 65-kDa protein in M. bovis BCG crude extracts. Since this protein was larger than that predicted from the nucleotide sequence (32 kDa), ORF3 was resequenced, revealing an ORF of 1,749 bp that encodes a 64.8-kDa protein containing 583 amino acids. Reverse transcription-PCR revealed that ORF3 is expressed in M. bovis BCG. The ORF3 product has a high degree of similarity to the acyladenylate family of enzymes. Immunoaffinity absorption chromatography was used to isolate the 65-kDa cross-reacting protein from M. bovis BCG. This purified protein catalyzed coenzyme A (CoA) ester synthesis of n-C 10 to n-C 18 fatty acids but not mycocerosic acids. ORF3 antibodies severely inhibited acyl-CoA synthase activities of the purified protein and extracts of M. bovis BCG, Mycobacterium smegmatis, and E. coli. They also showed immunological cross-reactivity with proteins in these extracts. Both the ORF3 protein and the acyl-CoA synthase activity were located in the cell cytosol or were loosely associated with the cell membrane. These results indicate that ORF3 encodes an acyl-CoA synthase-like protein.Tuberculosis is the world's foremost cause of death from a single infectious agent. More than 30 million tuberculosisrelated deaths and 90 million new infections are expected to occur in the last decade of this century (11). The major difficulty in treating mycobacterial infections is that the cell walls of these organisms have a high lipid content (50 to 60%) and consequently are highly resistant to attack by both the host's own immune system and antimicrobial agents (7). With the advent of multidrug-resistant mycobacterial strains, identification of potential new drug targets has become a critical need. Components unique to pathogenic mycobacterial cell wall synthesis (such as the mycolic acids, mycocerosic acids, and phthiocerols) (6) may be such selective targets, and inhibitors of such processes may be used in conjunction with conventional antimicrobial therapies to treat mycobacterial diseases (9).In spite of accumulating evidence which highlights the importance of the nature and quantity of lipids in the cell wall, much basic information remains to be elucidated about the metabolism of such lipids. Mycocerosic acids are produced by a multifunctional synthase, mycocerosic acid synthase (MAS), which elongates n-C 20 acids by successive reactions involving the incorporation of four C 3 units from methylmalonyl-coenzyme A (CoA) (34). When the mas gene was cloned and sequenced, an adjacent open reading frame (ORF), ORF3, was identified that showed homology to the acyladenylate family of enzymes, suggesting a role...

Transcriptional regulation of genes involved in the biosynthesis of cell wall lipids of Mycobacterium tuberculosis is poorly understood. The gene encoding mycocerosic acid synthase (mas) and fadD28, an adjoining acyl coenzyme A synthase gene, involved in the production of a virulence factor, dimycocerosyl phthiocerol, were cloned from Mycobacterium bovis BCG, and their promoters were analyzed. The putative promoters were fused to the xylE reporter gene, and its expression was measured in Escherichia coli, Mycobacterium smegmatis, and M. bovis BCG. In E. coli, the fadD28 promoter was not functional but the mas promoter was functional. Both fadD28 and mas promoters were functional in M. smegmatis, at approximately two-and sixfold-higher levels, respectively, than the BCG hsp60 promoter. In M. bovis BCG, the fadD28 and mas promoters were functional at three-and fivefold-higher levels, respectively, than the hsp60 promoter. Primer extension analyses identified transcriptional start points 60 and 182 bp upstream of the translational start codons of fadD28 and mas, respectively. Both promoters contain sequences similar to the canonical ؊10 and ؊35 hexamers recognized by the 70 subunit of RNA polymerase. Deletions of the upstream regions of both genes indicated that 324 bp of the fadD28 and 228 bp of the mas were essential for promoter activity. Further analysis of the mas promoter showed that a 213-bp region 581 bp upstream of the mas promoter acted as a putative transcriptional enhancer, promoting high-level expression of the mas gene when present in either direction. This represents the identification of a rare example of an enhancer-like element in mycobacteria.

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A Rhizobium meliloti mutant, CMF1 2:38, was isolated which was specifically defective in the degradation of glutamate as sole carbon and nitrogen source. Biochemical analysis of CMF1 2:38 revealed a reduction in succinic semialdehyde dehydrogenase (SSDH) activity, the third enzyme of the γ‐aminobutyrate (GABA) bypass. Evidence is presented which suggests that the Tn 5‐induced mutation in CMF1 2:38 exists in a regulatory gene governing the expression of both NAD and NADP‐linked SSDH activity. CMF1 2:38 nodulated alfalfa plants, but was reduced in its nitrogen fixation activity and biomass accumulating ability relative to the wild‐type strain. The results presented in this study indicate that the GABA bypass is a major mechanism of glutamate degradation in R. meliloti CMF1 and that glutamate catabolism via this pathway may play an important role in the symbiotic nitrogen fixation process.

1 0 000 g and 17 000 g for 30 min at 0°C. The pellet was resuspended in a small volume of 0.1 M-KCI buffered with 5 mM-Tris/maleate (pH 7.0) and was resedimented at 17 000 g. The final pellet was resuspended in the same medium at a protein concentration of 15-20 mg/ml. The HSR was loaded with Ca*+ in a medium containing 93 mM-KCI, 19 mM-Mops (potassium salt), 7.5 mhksodium pyrophosphate, 1 mM-MgATP, creatine kinase (20 pg/ml), 5 mwcreatine phosphate and 250 pM-antipyralazo 111, pH 7.0 at 35°C. Loading with Ca?+ was usually complete within 2 min after which halothane was added to the stoppered cuvette from appropriate concentrated stock solutions made up in absolute ethanol. Halothane was redistilled from a Fluothane stock. Control experiments were run with ethanol in the cuvette to allow for any release of CaZ + which it might cause. Ca" concentration changes were measured by the dual-wavelength principle at 710 nm and 790 nm at 1 s intervals on a Hewlett-Packard 8452A diode-array spectrophotometer controlled by a H-P 9000/300 computer. This instrument is fitted with a thermostatically controlled cuvette holder and magnetic cuvette stirrer. The Ca' ' concentration changes in the cuvette were calibrated by measuring the absorbance changes caused by the addition of small samples of a 1 0 mM standard CaClz solution. Figure 1( a ) shows that halothane concentrations greater than 160 pwinduced the release of CaZ+ from HSR preloaded with Cazf in the presence of pyrophosphate; the range of halothane concentrations examined was 0-620 which corresponds to 0-2.2%, (v/v). This is the range of halothane concentration used in human anaesthesia. The maximal rate of Ca2+ release was about one-hundredth the rate of Ca' ' uptake under the same assay conditions. Halothane-induced Ca" release was blocked by physiological concentrations of Mg?' (Fig. l b ) with half-maximal inhibition occurring at 1.6 mM-Mg?'. Release was completely blocked by 4 mM-Mg". Dantrolene (35 p~) , a well-known blocker of excitation-contraction coupling in skeletal muscle, did not inhibit halothane-induced Ca'+ release from HSR. Repcated additions of the same concentration of Ca'+ produced a progressively decreasing Ca' + efflux.These results indicate that halothane induces Ca?' release from HSR through the Ca?+-induced Ca'+ release channel, since the action of the anaesthetic was blocked by Mg'+ but was not affected by dantrolene. Furthermore, it is shown that clinically useful concentrations of halothane induce Ca? + release in vitro in the absence of free Mg2+; since this release was blocked by physiological concentrations of Mg'+ it is unlikely that the Ca' + -releasing action o f halothane described here is important in vivo. Fluothane was a gift from I.C.I. plc. I . Endo, M. ( I YXS) Citrr.