Genetic and biochemical characterization of Bacillus subtilis 168 mutants specifically blocked in the synthesis of the teichoic acid poly(3-O- -D-glucopyranosyl-N-acetylgalactosamine 1-phosphate): gneA, a new locus, is associated with UDP-N-acetylglucosamine 4-epimerase activity

Abstract

Journal of General Microbiology (1991), 137, 943-950. Printed in Great Britain 943Genetic and biochemical characterization of Bacillus subtifis 168 mutants specifically blocked in the synthesis of the teichoic acid poly(3-O-p-~-glucopyranosyl-N-acetylgalactosamine 1-phosphate) : gneA, a new locus, is associated with UDP-N-acetylglucosamine 4-epimerase activity The resistance spectrum to bacteriophage 43T of different Bacillus subtilis 168/W23 strains hybrid for wall teichoic acids suggested that poly(3-O-~-D-glucopyranosyl-~-acety~galac~osam~e 1-phosphate), a so-called minor teichoic acid of strain 168, forms part of the receptor for this phage, and a serologically related group of phages. A representative sample of 25 mutants specifically resistant to 43T, obtained from a mutagenized culture by direct selection, were all found to have a greatly reduced galactosamine content. Relevant mutations in these strains were shown by PBSl transduction and transformation to belong to two linkage groups; a minority, associated with an atypical colony morphology, were localized between sacA and purA, whereas the majority mapped between gtaB and tagBl (formerly tag-1), a region containing all known genes involved in the synthesis of the major wall teichoic acid, poly(glycero1 phosphate). The former mutations mapped in a new locus, gneA, characterized by a deficiency in UDP-N-acetylglucosamine 4-epimerase, while the latter ones, as well as the previously identified pha-3 (Estrela et al., 1986, Journal of General Microbiology 132,411-415), map in a locus named gga. They are likely to affect membrane-bound enzymes involved in the synthesis of the galactosaminecontaining teichoic acid. A possible biological role of this polymer is discussed. IntroductionCell walls of many Gram-positive bacteria contain relatively large amounts of anionic polymers (Baddiley, 1970; Archi bald, 1974). In Bacillus subtilis 168, several such polymers have been identified (Burger & Glaser, 1964;Shibaev et al., 1973); the phosphate-rich poly(g1y-cerol phosphate) [poly(groP)], glucosylated poly(groP) and poly (3-O-fl-D-glucopyranosyl-N-ace tylgalac tosamine 1-phosphate) [poly(Glc-GalNAc 1-P)], present under conditions of saturating phosphate, are replaced by teichuronic acid (Janczura et al., 1961 ;Ward, 1981) Abbreviations: DMAB, p-dimethylaminobenzaldehyde; GalN, galactosamine; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; MMC, mitomycin C ; MNNG, N-methyl-N'-nitro-Nnitrosoguanidine; ND, nephelometric density; 43TR, 43T resistant; 43TS, 43T sensitive; poly(G1c-GalNAc 1-P), pOly(3-O-P-D-glUCOpyranosyl-N-acetylgalactosamine 1-phosphate) ; poly(groP), poly(glycero1 phosphate).under phosphate-limiting growth conditions. Genetic analysis of conditional lethal mutants deficient in poly(groP) synthesis (Boylan et al., 1972;Karamata et al., 1972;Pooley et al., 1987;Briehl et al., 1989;, and in particular the more recent nucleotide sequencing studies (Honeyman & Stewart, 1989;Mauel et al., 1991) have led to the identification of six genes likely ...

Defects in glycopeptidolipid biosynthesis confer phage I3 resistance in Mycobacterium smegmatis

“…Indeed, studies of interactions of bacteriophages with host cells have often led to discovery of bacterial cell surface receptors and transporters that play important roles in microbial physiology (Heller, 1992;Estrela et al, 1991;Tran et al, 1999 Phage I3 is a generalized transducing phage belonging to the Myoviridae family. It infects M. smegmatis, and it has found utility as a genetic tool for generalized transduction (Raj & Ramakrishnan, 1970).…”

Genes concerned with synthesis of poly(glycerol phosphate), the essential teichoic acid in Bacillus subtilis strain 168, are organized in two divergent transcription units

“…Bacterial strains are listed in Table 1. Fragments from the chromosomal DNA inserts in phages A53 and LE51 Young et al, 1989) were subcloned in either pJHlOl (ApR TcR CmR) (Ferrari et al, 1983) or pMTL20EC (ApR EmR CmR) (Chambers et al, 1988) and maintained in E . coli strain DH5, except for the insert in plasmid 5307 , which was cloned in pMTL5OOC (ApR CmR) (Oultram et al, 1988) and maintained in B. subtilis.…”

“…Plasmid insertion in unit V generated 43T resistance. This is almost certainly due to disruption of the genes concerned with biosynthe- sis of poly(g1ucose N-acetylgalactosamine 1 -phosphate) (Estrela et al, 1991). The previously identified gtaB locus (Young, 1967), denoted (VI) in Fig.…”

“…A collection of integrational plasmids carrying different chromosomal fragments covering this region was used to transform strain L5047, a derivative of B. subtilis 168, with selection for chloramphenicol resistance. Absence of transformants was attributed to the essentiality of the disrupted transcription units; when present, transformants were tested for susceptibility to phages 425, 429, #3T, PBSY and PBSZ, whose receptors are formed, in part at least, by anionic cell wall polymers (Young, 1967;Karamata et al, 1987;Estrela et al, 1991 ; Fig. 1).…”

“…Mutants impaired in biosynthesis of this secondary anionic polymer found in cell walls of strain 168 (Shibaev et al, 1973) have been isolated on the basis of their resistance to phage 43T (Estrela et al, 1991). A 60 kbp DNA segment encompassing all these genes has recently been cloned Young et al, 1989).…”