Transposome mutagenesis of an integral membrane transporter in Corynebacterium matruchotii.
A transposon-5 insertion library of Corynebacterium matruchotii ATCC14266 was generated and screened for mutants with altered corynomycolic acid content. One of these designated 319 mutants showed an interruption of a gene encoding an integral membrane protein. MALDI mass spectra of trehalose monocorynomycolate (TMCM), trehalose dicorynomycolate, and methyl corynomycolates derived from cell wall arabinogalactan-corynomycolate showed that these lipids from the mutant contained a lower amount of short-chain (C24 to C34) and much greater amount of long-chain (primarily C(36:2)) corynomycolic acids than the wild type. An analysis of mRNA demonstrated that the integral membrane protein and ATP-binding cassette transporter are transcriptionally coupled. These results suggested that the proteins/enzymes encoded by the membrane transporter gene locus preferably move short-chain corynomycolic acids from the cytoplasm across the membrane bilayer to the periplasmic space where the synthesis of TMCM is thought to occur. This is the first evidence linking corynomycolic acid to a transporter gene locus.
Wang
Cindy
C
Mycobacteriology Research Laboratory, William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA.
Hayes
Barry
B
Vestling
Martha M
MM
Takayama
Kuni
K
D001692:Biological Transport; D015139:Blotting, Southern; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D002473:Cell Wall / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003311:Cord Factors / Q000737:chemistry; D003352:Corynebacterium / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004251:DNA Transposable Elements; D008055:Lipids / Q000737:chemistry; D013058:Mass Spectrometry; D008957:Models, Genetic; D016296:Mutagenesis; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000378:metabolism; D009171:Mycolic Acids / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D019895:Transposases / Q000378:metabolism; D014199:Trehalose / Q000737:chemistry
D003311:Cord Factors; D004251:DNA Transposable Elements; D008055:Lipids; D009171:Mycolic Acids; C473503:Tn5 transposase; C021099:trehalose monomycolate; C013898:corynomycolic acid; D012313:RNA; D014199:Trehalose; D019895:Transposases