The capacity of S. aureus to catabolize Neu5Ac. All S. aureus strains tested, and three other species of staphylococci, were shown to utilize Neu5Ac as a carbon source, and we identified a five-gene locus, known as nan, that may be accountable for Neu5Ac catabolism. The molecular architecture on the nan locus was examined, as well as the function of a transcriptional regulator encoded within the locus, NanR, was investigated.Materials AND METHODSStrains and development conditions. Bacterial strains made use of in this study are listed in Table 1. E. coli was grown on Luria-Bertani (LB) medium. S. aureus cultures were grown in tryptic soy broth (TSB) with no dextrose because the baseline wealthy medium, which was then supplemented with glucose orReceived 28 December 2012 Accepted four February 2013 Published ahead of print 8 February 2013 Address correspondence to Alexander R. Horswill, [email protected]. Supplemental material for this article may possibly be located at http://dx.doi.org/10.1128 /JB.02294-12. Copyright ?2013, American Society for Microbiology. All Rights Reserved. doi:ten.1128/JB.02294-April 2013 Volume 195 NumberJournal of Bacteriologyp. 1779 ?jb.asm.orgOlson et al.FIG 1 S. aureus Neu5Ac genetic locus and proposed catabolic pathway. (A) Schematic from the nan locus molecular organization in S. aureus (USA300 ORF numbering according to the FPR3757 genome) (32). (B) Schematic from the Neu5Ac catabolic pathway with putative assigned functions of proteins encoded in the nan locus. Neu5Ac enters the cell by way of a transporter (NanT), and N-acetylneuraminic lyase (NanA) removes a pyruvate group, yielding ManNAc. ManNAc kinase (NanK) phosphorylates at the C-6 position, yielding ManNAc-6P, as well as the N-acetylmannosamine-6-phosphate epimerase (NanE) converts ManNAc-6P into GlcNAc-6P. The final two methods are encoded by enzymes outside the nan locus. GlcNAc-6P deacetylase (NagA) removes the acetyl group from GlcNAc-6P, yielding glucosamine-6-phosphate (GlcN-6P), as well as the glucosamine-6-phosphate deaminase (NagB) removes an amine group to create fructose-6P (Fru-6P), which can enter central metabolism. TABLE 1 Strains and plasmids utilized within this studyStrain or plasmid Strains S. aureus RN4220 AH1263 AH1905 AH1956 AH1957 AH1957 AH2325 BK10296 UAMS-1 MRSA252 MW2 BK21157 COL Newman HG001 S. epidermidis 1457 RP62a ATCC 12228 S.MC-Val-Cit-PAB Chemical name carnosus S.4-Bromo-2-methylpyrimidine Chemscene lugdunensis S. intermedius S. saprophyticus E. coli AH3 TM300 AH2160 (N920 143) AH2776 (ATCC 15305) ER2566 Descriptiona Supply or referenceRestriction-deficient 8325-4 USA300 CA-MRSA Erms (LAC*) AH1263 nank AH1263 nanA AH1263 nanE AH1263 nanT AH1263 nanR USA100 USA200 USA200 USA400 USA600 HA-MRSA rsbU laboratory strain26 50 This perform This operate This operate This operate This perform Barry Kreiswirth 51 52 53 Barry Kreiswirth 54 55 56 57 54 58 59 60 Patrick Schlievert ATCC New England BiolabsPlasmids pCM11 pJB38 pMal-C2x pMO6 pMO7 pMO8 pMO13 pMO14 pMO15 pMO16 pMO17 pMO18 pSKErm-MCSsGFP expression vector, Ermr S.PMID:23509865 aureus gene knockout vector, Camr MBP tagged expression vector, Ampr nanA promoter sGFP fusion, Ermr nanR promoter sGFP fusion, Ermr nanK promoter sGFP fusion, Ermr nanE promoter sGFP fusion, Ermr nanA complementation plasmid, Camr nanE complementation plasmid, Camr nanK complementation plasmid, Camr nanR complementation plasmid, Camr nanR expression vector E. coli-S. aureus shuttle vector, Ermr61 62 New England Biolabs This work This operate This work This perform This operate This work This perform This function This work This worka Erm, erythromycin; Cam, chloramphenicol; A.
