Mechanism of bacitracin resistance in gram-negative bacteria that synthesize exopolysaccharides

Abstract

Four representative species from three genera of gram-negative bacteria that secrete exopolysaccharides acquired resistance to the antibiotic bacitracin by stopping synthesis of the exopolysaccharide. Xanthomonas campestris, Sphingomonas strains S-88 and NW11, and Escherichia coli K-12 secrete xanthan gum, sphingans S-88 and NW11, and colanic acid, respectively. The gumD gene in X. campestris is required to attach glucose-P to C55-isoprenyl phosphate, the first step in the assembly of xanthan. A recombinant plasmid carrying the gumD gene of X. campestris restored polysaccharide synthesis to bacitracin-resistant exopolysaccharidenegative mutants ofX. campestris and Sphingomonas strains. Similarly, a newly cloned gene (spsB) from strain S-88 restored xanthan synthesis to the same X. campestris mutants. However, the intergeneric complementation did not extend to mutants of E. coli that were both resistant to bacitracin and nonproducers of colanic acid. The genetic results also suggest mechanisms for assembling the sphingans which have commercial potential as gelling and viscosifying agents.The bacitracins are a mixture of structurally related cyclic polypeptides with clinically useful antibiotic properties and are produced by certain species of Bacillus (13). Bacitracins interfere indirectly with the biosynthesis of bacterial cell walls by inhibiting the dephosphorylation of C55-isoprenyl pyrophosphate (IPP) (30). Bacitracin forms a complex with IPP and a divalent cation and prevents phosphatase from acting on IPP (32-34). Bacteria require the C.5-isoprenyl phosphates (IP) as carriers during synthesis of the repeat subunits of peptidoglycan. IPP is released when a repeat subunit, a disaccharidepentapeptide, is transferred to a growing peptidoglycan chain, but the IPP must be dephosphorylated before reuse. Although binding to IPP is thought to be the primary mode of action, bacitracin may also interfere with additional cellular processes: the actions of certain hydrolytic enzymes (19), the formation of ubiquinone precursors (29), the biosynthesis of membranederived oligosaccharides (41), and the involvement of membranes in cell division (26).One mechanism of resistance to bacitracin seems to result from increased de novo synthesis of IP. Cain et al. (3) have recently shown that increased intracellular levels of a lipid kinase, the product of the bacA gene of Escherichia coli, confers resistance to bacitracin. The kinase appears to increase the level of the carrier IP by phosphorylating isoprenyl alcohol, thereby circumventing the sequestration of IPP by bacitracin. Sutherland (35) had earlier suggested that resistance to bacitracin in Klebsiella species might be due to an abundance of isoprenoid lipids; however, actual quantitation of the lipids was incomplete. Fiedler and Rotering (7) described mutants of E. coli that display several phenotypes during growth under conditions of low osmolarity: partial resistance to bacitracin, failure to synthesize membrane-derived oligosaccharides, increased production of capsular...

Linkage of genes essential for synthesis of a polysaccharide capsule in Sphingomonas strain S88

“…The order of assembly prior to polymerization is glucose, glucose, mannose, glucuronic acid, and finally mannose (24). Our genetic complementation tests also showed that the wild-type gumD gene of X. campestris could restore synthesis of sphingans in analogous Bac r Sps Ϫ (sphingan polysaccharide-negative) mutants of Sphingomonas strains (45). Since there are two glucose residues in the backbone of each of the sphingans, we reasoned that the Bac r Sps Ϫ mutants were probably also blocked in the transfer of glucose-P to IP.…”

“…Most of the gum genes required by this bacterium for synthesis and secretion of xanthan gum have been described (3,6,19,20,58). Previously we learned from genetic complementation tests that a special class of mutations in X. campestris which are simultaneously xanthan gum negative (Gum Ϫ ) and resistant to bacitracin (Bac r ) are in the gumD gene (45). This gene is required to transfer glucose-P from UDP-Glc to a carrier isoprenylphosphate (IP) to give glucoseisoprenylpyrophosphate (Glc-PPI) and is the first dedicated step in synthesis of xanthan gum (59).…”

Regulation of the Bacillus subtilis bcrC Bacitracin Resistance Gene by Two Extracytoplasmic Function σ Factors

“…BacA homologs were also identified in Streptococcus pneumoniae and Staphylococcus aureus and are important for resistance to bacitracin, presumably by increasing the synthesis of C 55 -isoprenyl phosphate (6). In some gramnegative bacteria, mutations that block the synthesis of exopolysaccharides also lead to bacitracin resistance, presumably by increasing the supply of the common C 55 -isoprenyl phosphate carrier (22).…”

Adhesion of Aureobasidium pullulans Is Controlled by Uronic Acid Based Polymers and Pullulan

“…A. pullulans strain NRRL Y-2331-1, which produces high yields of a nonpigmented form of pullulan, 9 was obtained from the United States Department of Agriculture (USDA). The growth media consisted of a complex nitrogen stock solution with sucrose as the carbon source, 5 containing (per 1 L of ultrapure water): 2.0 g of yeast extract, 0.5 g of (NH 4 ) 2 SO 4 , 1.0 g of NaCl, 0.2 g of MgSO 4 , 3.0 g of K 2 HPO 4 , 0.01 g of FeSO 4 , 0.01 g of MnSO 4 , 0.01 g of ZnSO 4 , and 50 g of sucrose. All ingredients, excluding ZnSO 4 , MnSO 4 , FeSO 4 , and MgSO 4 , were mixed and autoclaved for 45 min at 121°C.…”

“…2 However, naturally occurring and mutant strains of A. pullulans exist that produce reduced levels of melanin. 5,6 A. pullulans may produce other macromolecules, depending on culture conditions. For example, cells grown on sucrose produce pullulan and an acidic glucan with uronic acid components, whereas cells cultured on maltose produce a heteropolysaccharide containing glucose, galactose, and mannose.…”