The human malaria parasite Plasmodium falciparum harbors a relict, nonphotosynthetic plastid of algal origin termed the apicoplast. Although considerable progress has been made in defining the metabolic functions of the apicoplast, information on the composition and biogenesis of the four delimiting membranes of this organelle is limited. Here, we report an efficient method for preparing highly purified apicoplasts from red blood cell parasite stages and the comprehensive lipidomic analysis of this organelle. Apicoplasts were prepared from transgenic parasites expressing an epitope-tagged triosephosphate transporter and immunopurified on magnetic beads. Gas and liquid chromatography MS analyses of isolated apicoplast lipids indicated significant differences compared with total parasite lipids. In particular, apicoplasts were highly enriched in phosphatidylinositol, consistent with a suggested role for phosphoinositides in targeting membrane vesicles to apicoplasts. Apicoplast phosphatidylinositol and other phospholipids were also enriched in saturated fatty acids, which could reflect limited acyl exchange with other membrane phospholipids and/or a requirement for specific physical properties. Lipids atypical for plastids (sphingomyelins, ceramides, and cholesterol) were detected in apicoplasts. The presence of cholesterol in apicoplast membranes was supported by filipin staining of isolated apicoplasts. Galactoglycerolipids, dominant in plant and algal plastids, were not detected in P. falciparum apicoplasts, suggesting that these glycolipids are a hallmark of photosynthetic plastids and were lost when these organisms assumed a parasitic lifestyle. Apicoplasts thus contain an atypical melange of lipids scavenged from the human host alongside lipids remodeled by the parasite cytoplasm, and stable isotope labeling shows some apicoplast lipids are generated de novo by the organelle itself.

The aerobic electron transport chain in Mycobacterium smegmatis can terminate in one of three possible terminal oxidase complexes. The structure and function of the electron transport pathway leading from the menaquinol-menaquinone pool to the cytochrome bc ₁ complex and terminating in the aa ₃ -type cytochrome c oxidase was characterized. M. smegmatis strains with mutations in the bc ₁ complex and in subunit II of cyctochome c oxidase were found to be profoundly growth impaired, confirming the importance of this respiratory pathway for mycobacterial growth under aerobic conditions. Disruption of this pathway resulted in an adaptation of the respiratory network that is characterized by a marked up-regulation of cydAB , which encodes the bioenergetically less efficient and microaerobically induced cytochrome bd -type menaquinol oxidase that is required for the growth of M. smegmatis under O ₂ -limiting conditions. Further insights into the adaptation of this organism to rerouting of the electron flux through the branch terminating in the bd -type oxidase were revealed by expression profiling of the bc ₁ -deficient mutant strain using a partial-genome microarray of M. smegmatis that is enriched in essential genes. Although the expression profile was indicative of an increase in the reduced state of the respiratory chain, blockage of the bc ₁ - aa ₃ pathway did not induce the sentinel genes of M. smegmatis that are induced by oxygen starvation and are regulated by the DosR two-component regulator.

Mycolic acids are essential components of the cell walls of bacteria belonging to the suborder Corynebacterineae, including the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. Mycolic acid biosynthesis is complex and the target of several frontline antimycobacterial drugs. The condensation of two fatty acids to form a 2-alkyl-3-keto mycolate precursor and the subsequent reduction of this precursor represent two key and highly conserved steps in this pathway. Although the enzyme catalyzing the condensation step has recently been identified, little is known about the putative reductase. Using an extensive bioinformatic comparison of the genomes of M. tuberculosis and Corynebacterium glutamicum, we identified NCgl2385, the orthologue of Rv2509 in M. tuberculosis, as a potential reductase candidate. Deletion of the gene in C. glutamicum resulted in a slow growing strain that was deficient in arabinogalactan-linked mycolates and synthesized abnormal forms of the mycolate-containing glycolipids trehalose dicorynomycolate and trehalose monocorynomycolate. Analysis of the native and acetylated trehalose glycolipids by MALDI-TOF mass spectrometry indicated that these novel glycolipids contained an unreduced β-keto ester. This was confirmed by analysis of sodium borodeuteride-reduced mycolic acids by gas chromatography mass spectrometry. Reintroduction of the NCgl2385 gene into the mutant restored the transfer of mature mycolic acids to both the trehalose glycolipids and cell wall arabinogalactan. These data indicate that NCgl2385, which we have designated CmrA, is essential for the production of mature trehalose mycolates and subsequent covalent attachment of mycolic acids onto the cell wall, thus representing a focus for future structural and pathogenicity studies.

The complete nucleotide sequence of Tn 10 has been determined. The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn 10 constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis . The glutamate permease was fully functional when expressed, but Tn 10 did not protect Escherichia coli from the toxic effects of various metals.

The cell walls of the Corynebacterineae, which includes the important human pathogen Mycobacterium tuberculosis, contain two major lipopolysaccharides, lipoarabinomannan (LAM) and lipomannan (LM). LAM is assembled on a subpool of phosphatidylinositol mannosides (PIMs), whereas the identity of the LM lipid anchor is less well characterized. In this study we have identified a new gene (Rv2188c in M. tuberculosis and NCgl2106 in Corynebacterium glutamicum) that encodes a mannosyltransferase involved in the synthesis of the early dimannosylated PIM species, acyl-PIM2, and LAM. Disruption of the C. glutamicum NCgl2106 gene resulted in loss of synthesis of AcPIM2 and accumulation of the monomannosylated precursor, AcPIM1. The synthesis of a structurally unrelated mannolipid, Gl-X, was unaffected. The synthesis of AcPIM2 in C. glutamicum ΔNCgl2106 was restored by complementation with M. tuberculosis Rv2188c. In vivo labeling of the mutant with [³H]Man and in vitro labeling of membranes with GDP-[³H]Man confirmed that NCgl2106/Rv2188c catalyzed the second mannose addition in PIM biosynthesis, a function previously ascribed to PimB/Rv0557. The C. glutamicum ΔNCgl2106 mutant lacked mature LAM but unexpectedly still synthesized the major pool of LM. Biochemical analyses of the LM core indicated that this lipopolysaccharide was assembled on Gl-X. These data suggest that NCgl2106/Rv2188c and the previously studied PimB/Rv0557 transfer mannose residues to distinct mannoglycolipids that act as precursors for LAM and LM, respectively.