GasPak™ Dry Anaerobic Indicator Strips were used to assure anaerobic condition (BD, Franklin Lakes, NJ, USA). Overnight liquid culture of the bacterial strains was harvested and washed by AUM using mini centrifuge, then serial-diluted to an initial optical density at 600 nm (OD600) of approximately 0.0005 (10,000~20,000× dilution) in AUM. Turbidity of the cultured

bacteria was monitored spectrophotometrically NVP-BGJ398 purchase at 600 nm. Gene disruption of the 13-kb genomic cluster Disruption of the citS together with the nearby regulatory region between the two divergently positioned operons in NK8 genome was done by a method facilitated by λ Red recombinase carried on pKD20 [26]. Two PCR primers (cits-HF: 5′-TTAAATCATC ATGCCGAACA CGATGCTGGC GATGACCAGA TTCCGGGGAT CCGTCGACC-3′, citc-HR: 5′-TTTTTTAGCG CTTCGTCATT TCAAAACGAA CTGTATTTCT GTAGGCTGGA GCTGCTTC-3′) were used to amplify an aac(3)IV (ApraR) apramycin resistance gene from pIJ773 [27] while creating the flanking homologous

sequence for recombination. As a result, 39-bp from the left end of the citS to the beginning of the citC2 (corresponding to location 34604-36125 of the MGH 78578) were disrupted by the apramycin resistant gene in NK8. The gene disruption was confirmed by PCR and DNA sequencing of the corresponding genomic region. Detection of citrate fermentation genes Comparative genomic hybridization (CGH) array (NimbleGen Systems, WI, USA) with probes designed according to the predicted coding sequences spanning the 13-kb genomic region of the Selleckchem Ku 0059436 K. pneumoniae strain NK8 (with 99% sequence identity in average compared to syntenic region of MGH 78578) was used to detect differences of this genomic region among the K. pneumoniae clinical isolates. A total of 687 probes were designed isothermally (Tm-balanced) with NimbleGen algorithms across these concatenated CDSs sequences in length of 50-mer with 33-nucleotide overlap between adjacent probe sequences. An intact ribosomal RNA Idoxuridine gene cluster (containing 16S-23S-5S

rRNAs) was included as a positive control. DNA labelling and hybridization methods of genomic DNA, and signal scanning procedure were performed according to manufacturer’s instructions. PCR detections of citrate fermentation genes among other clinical isolates were performed using specific primers listed in Table 1 following standard protocols. DNA sequence The complete genomic sequence of K. pneumoniae strain NTUH-K2044 has been deposited to the GenBank (accession no. AP006725)[12]. A fosmid clone, KPA-F06C06, containing the 13-kb citrate fermentation gene region, was selected from a fosmid library of K. pneumoniae strain NK8. Acknowledgements The project was funded by a grant from the National Science Council (NSC 96-3112-B-400-006) and an intramural grant from the National Health Research Institutes (MG-096-PP09). References 1. Schwarz E, Oesterhelt D: Cloning and expression of Klebsiella pneumoniae genes coding for citrate transport and fermentation. EMBO J 1985, 4:1599–1603.