An operon encoding enzymes for synthesis of a putative extracellular carbohydrate attenuates acquired vancomycin resistance in Streptomyces coelicolor.

Actinomycete bacteria use polyprenol phosphate mannose as a lipid-linked sugar donor for extra-cytoplasmic glycosyl transferases that transfer mannose to cell envelope polymers, including glycoproteins and glycolipids. Strains of Streptomyces coelicolor with mutations in the gene ppm1, encoding polyprenol phosphate mannose synthase, and in pmt, encoding a protein O-mannosyltransferase, are resistant to phage ϕC31 and have greatly increased susceptibility to some antibiotics, including vancomycin. In this work, second-site suppressors of the vancomycin susceptibility were isolated. The suppressor strains fell into two groups. Group 1 strains had increased resistance to vancomycin, teicoplanin and ß-lactams, and had mutations in the two-component sensor regulator system encoded by vanSR, leading to upegulation of the vanSRJKHAX cluster. Group 2 strains only had increased resistance to vancomycin and these mostly had mutations in sco2592 or sco2593, genes that are derepressed in the presence of phosphate and are likely to be required for the synthesis of a phosphate-containing extracellular polymer. In some suppressor strains the increased resistance was only observed in media with limited phosphate (mimicking the phenotype of wild-type S. coelicolor), but two strains, DT3017_R21 (ppm1-vanR-) and DT3017_R15 (ppm1- sco2593-), retained resistance on media with high phosphate content. These results support the view that vancomycin resistance in S. coelicolor is a trade-off between mechanisms that confer resistance and at least one that interferes with resistance mediated through the sco2594-sco2593-sco2592 operon.

Disruption of the GDP-mannose synthesis pathway in Streptomyces coelicolor results in antibiotic hyper-susceptible phenotypes.

Actinomycete bacteria use polyprenol phosphate mannose as a lipid linked sugar donor for extra-cytoplasmic glycosyl transferases that transfer mannose to cell envelope polymers, including glycoproteins and glycolipids. We showed recently that strains of Streptomyces coelicolor with mutations in the gene ppm1 encoding polyprenol phosphate mannose synthase were both resistant to phage φC31 and have greatly increased susceptibility to antibiotics that mostly act on cell wall biogenesis. Here we show that mutations in the genes encoding enzymes that act upstream of Ppm1 in the polyprenol phosphate mannose synthesis pathway can also confer phage resistance and antibiotic hyper-susceptibility. GDP-mannose is a substrate for Ppm1 and is synthesised by GDP-mannose pyrophosphorylase (GMP; ManC) which uses GTP and mannose-1-phosphate as substrates. Phosphomannomutase (PMM; ManB) converts mannose-6-phosphate to mannose-1-phosphate. S. coelicolor strains with knocked down GMP activity or with a mutation in sco3028 encoding PMM acquire phenotypes that resemble those of the ppm1- mutants i.e. φC31 resistant and susceptible to antibiotics. Differences in the phenotypes of the strains were observed, however. While the ppm1- strains have a small colony phenotype, the sco3028 :: Tn5062 mutants had an extremely small colony phenotype indicative of an even greater growth defect. Moreover we were unable to generate a strain in which GMP activity encoded by sco3039 and sco4238 is completely knocked out, indicating that GMP is also an important enzyme for growth. Possibly GDP-mannose is at a metabolic branch point that supplies alternative nucleotide sugar donors.

Streptomyces coelicolor strains lacking polyprenol phosphate mannose synthase and protein O-mannosyl transferase are hyper-susceptible to multiple antibiotics.

Polyprenol phosphate mannose (PPM) is a lipid-linked sugar donor used by extra-cytoplasmic glycosyl tranferases in bacteria. PPM is synthesized by polyprenol phosphate mannose synthase, Ppm1, and in most Actinobacteria is used as the sugar donor for protein O-mannosyl transferase, Pmt, in protein glycosylation. Ppm1 and Pmt have homologues in yeasts and humans, where they are required for protein O-mannosylation. Actinobacteria also use PPM for lipoglycan biosynthesis. Here we show that ppm1 mutants of Streptomyces coelicolor have increased susceptibility to a number of antibiotics that target cell wall biosynthesis. The pmt mutants also have mildly increased antibiotic susceptibilities, in particular to ß-lactams and vancomycin. Despite normal induction of the vancomycin gene cluster, vanSRJKHAX, the pmt and ppm1 mutants remained highly vancomycin sensitive indicating that the mechanism of resistance is blocked post-transcriptionally. Differential RNA expression analysis indicated that catabolic pathways were downregulated and anabolic ones upregulated in the ppm1 mutant compared to the parent or complemented strains. Of note was the increase in expression of fatty acid biosynthetic genes in the ppm1- mutant. A change in lipid composition was confirmed using Raman spectroscopy, which showed that the ppm1- mutant had a greater relative proportion of unsaturated fatty acids compared to the parent or the complemented mutant. Taken together, these data suggest that an inability to synthesize PPM (ppm1) and loss of the glycoproteome (pmt- mutant) can detrimentally affect membrane or cell envelope functions leading to loss of intrinsic and, in the case of vancomycin, acquired antibiotic resistance.

Metabolomic Analysis of Campylobacter jejuni by Direct-Injection Electrospray Ionization Mass Spectrometry.

Direct-injection mass spectrometry (DIMS) is a means of rapidly obtaining metabolomic phenotype data in both prokaryotes and eukaryotes. Given our generally poor understanding of Campylobacter metabolism, the high-throughput and relatively simple sample preparation of DIMS has made this an attractive technique for metabolism-related studies and hypothesis generation, especially when attempting to analyze metabolic mutants with no clear phenotype. Here we describe a metabolomic fingerprinting approach with sampling and extraction methodologies optimized for direct-injection electrospray ionization mass spectrometry (ESI-MS), which we have used as a means of comparing wild-type and isogenic mutant strains of C. jejuni with various metabolic blocks.

Metabolomic analysis of the food-borne pathogen Campylobacter jejuni: application of direct injection mass spectrometry for mutant characterisation.

Campylobacter jejuni is the most frequent cause of human food-borne bacterial gastroenteritis but its physiology and biochemistry are poorly understood. Only a few amino-acids can be catabolised and these are known to be important for host colonization. Here we have established methods for rapid high throughput analyses of global metabolism in C. jejuni using direct injection mass spectrometry (DIMS) to compare metabolite fingerprints of wild-type and mutant strains. Principal component analyses show that the metabolic fingerprint of mutants that have a genomic deletion in genes for key amino-acid catabolic enzymes (either sdaA, serine dehydratase; aspA, aspartase or aspB, aspartate:glutamate transaminase) can easily be distinguished from the isogenic parental strain. Assignment of putative metabolites showed predictable changes directly associated with the particular metabolic lesion in these mutants as well as more extensive changes in the aspA mutant compared to the sdaA or aspB strains. Further analyses of a cj0150c mutant strain, which has no obvious phenotype, suggested a role for Cj0150 in the conversion of cystathionine to homocysteine. Our results show that DIMS is a useful technique for probing the metabolism of this important pathogen and may help in assigning function to genes encoding novel enzymes with currently unknown metabolic roles.

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent.

Campylobacter jejuni is a human pathogen of worldwide significance. It is commensal in the gut of many birds and mammals, where hydrogen is a readily available electron donor. The bacterium possesses a single membrane-bound, periplasmic-facing NiFe uptake hydrogenase that depends on the acquisition of environmental nickel for activity. The periplasmic binding protein Cj1584 (NikZ) of the ATP binding cassette (ABC) transporter encoded by the cj1584c-cj1580c (nikZYXWV) operon in C. jejuni strain NCTC 11168 was found to be nickel-repressed and to bind free nickel ions with a submicromolar K(d) value, as measured by fluorescence spectroscopy. Unlike the Escherichia coli NikA protein, NikZ did not bind EDTA-chelated nickel and lacks key conserved residues implicated in metallophore interaction. A C. jejuni cj1584c null mutant strain showed an approximately 22-fold decrease in intracellular nickel content compared with the wild-type strain and a decreased rate of uptake of (63)NiCl(2). The inhibition of residual nickel uptake at higher nickel concentrations in this mutant by hexa-ammine cobalt (III) chloride or magnesium ions suggests that low-affinity uptake occurs partly through the CorA magnesium transporter. Hydrogenase activity was completely abolished in the cj1584c mutant after growth in unsupplemented media, but was fully restored after growth with 0.5 mM nickel chloride. Mutation of the putative metallochaperone gene slyD (cj0115) had no effect on either intracellular nickel accumulation or hydrogenase activity. Our data reveal a strict dependence of hydrogenase activity in C. jejuni on high-affinity nickel uptake through an ABC transporter that has distinct properties compared with the E. coli Nik system.