Gap, a mycobacterial specific integral membrane protein, is required for glycolipid transport to the cell surface

The cell envelope of mycobacteria is a complex multilaminar structure that protects the cell from stresses encountered in the environment, and plays an important role against the bactericidal activity of immune system cells. The outermost layer of the mycobacterial envelope typically contains species-specific glycolipids. Depending on the mycobacterial species, the major glycolipid localized at the surface can be either a phenolglycolipid or a peptidoglycolipid (GPL). Currently, the mechanism of how these glycolipids are addressed to the cell surface is not understood. In this study, by using a transposon library of Mycobacterium smegmatis and a simple dye assay, six genes involved in GPLs synthesis have been characterized. All of these genes are clustered in a single genomic region of approximately 60 kb. We show by biochemical analyses that two non-ribosomal peptide synthetases, a polyketide synthase, a methyltransferase and a member of the MmpL family are required for the biosynthesis of the GPLs backbone. Furthermore, we demonstrate that a small integral membrane protein of 272 amino acids named Gap (gap: GPL addressing protein) is specifically required for the transport of the GPLs to the cell surface. This protein is predicted to contain six transmembrane segments and possesses homologues across the mycobacterial genus, thus delineating a new protein family. This Gap family represents a new paradigm for the transport of small molecules across the mycobacterial envelope, a critical determinant of mycobacterial virulence

Five new genes are important for common polysaccharide antigen biosynthesis in <em>Pseudomonas aeruginosa</em>

ABSTRACT Common polysaccharide antigen (CPA) is a conserved cell surface polysaccharide produced by Pseudomonas aeruginosa. It contains a rhamnan homopolymer and is one of the two forms of O polysaccharide attached to P. aeruginosa lipopolysaccharide (LPS). Our laboratory has previously characterized an eight-gene cluster (pa5447-pa5454 in P. aeruginosa PAO1) required for biosynthesis of CPA. Here we demonstrate that an adjacent five-gene cluster pa5455-pa5459 is also involved. Using reverse transcriptase PCR (RT-PCR), we showed that the original eight-gene cluster and the new five-gene cluster are both organized as operons. We have analyzed the LPS phenotypes of in-frame deletion mutants made in each of the five genes, and the results verified that these five genes are indeed required for CPA biosynthesis, extending the CPA biosynthesis locus to contain 13 contiguous genes. By performing overexpression experiments of different sets of these biosynthesis genes, we were able to obtain information about their possible functions in CPA biosynthesis. IMPORTANCE Lipopolysaccharide (LPS) is an important cell surface structure of Gram-negative bacteria. The human opportunistic pathogen Pseudomonas aeruginosa simultaneously produces an O-antigen-specific (OSA) form and a common polysaccharide antigen (CPA) form of LPS. CPA, the focus of this study, is composed of α-1-2, α1-3-linked d-rhamnose sugars and has been shown to be important for attachment of the bacteria to human airway epithelial cells. Genome sequencing of this species revealed a new five-gene cluster that we predicted to be involved in CPA biosynthesis and modification. In this study, we have generated chromosomal knockouts by performing in-frame deletions and allelic replacements. Characterizing the function of each of the five genes is important for us to better understand CPA biosynthesis and the mechanisms of chain length termination and regulation of this unique D-rhamnan polysaccharide