Primer extension was carried out with the oligonucleotide primer PE-VMHR (5′-AACCGTGTCAATTGATGCCG-3′), which had been 5′-labeled with Texas Red. The labeled primer annealed to total RNA of 5 μg was extended with PrimeScript reverse transcriptase for 1 hr at 50oC. The extension products were separated with a SQ5500 DNA sequencer (Hitachi, Tokyo, Japan) on a sequencing gel together with the DNA sequence ladder of the control region as described previously (10). To construct deletion mutant strains, the following oligonucleotide primers were used: for the iucD deletion, D1 (5′-GGTTAACGCTCGAGGCTTGGCTCAGCAAACTG-3′),

D2 (5′-ccatggctatagtttggcgtTGTTAGTGTG-3′), D3 (5′-acgccaaactatagccatggTATTGCCGAG-3′), and D4 (5′-GATTCAAACTCGAGCTCTTGGCTTGTCG-3′); for the mhuA deletion, A1 (5′-GCCTCGTTTCTAGATAAGCTTACCTGCCTCG-3′), drug discovery selleck kinase inhibitor A2 (5′-agtagagtcgtgttatcgatGTCTTGAGCG-3′), A3 (5′-atcgataacacgactctactATTAGATACC-3′), and A4 (5′-TGGGTGAATCTAGAGTTACCGACTCACTGAG-3′); and for the mhuB deletion, B1 (5′-AAACCTCCTCGAGCGTCAGAACCGTAAAGG-3′), B2 (5′-caagacaatttaactcaaggAGCTAGGAGC-3′), B3 (5′-ccttgagttaaattgtcttgGCTTGGCGAC-3′), and B4 (5′-AAAACCGTCTAGATATCCGACCTTATCCAACCG-3′) (the underlined sequences in primers D1, D4 and B1, and primers A1, A4 and B4 are XhoI, and XbaI sites, respectively, and the small letter sequences in primers

D2 and D3, A2 and A3, and B2 and B3 are

each complementary to the corresponding gene sequences). To prepare a deletion fragment of iucD, two DNA fragments were amplified by PCR with V. mimicus 7PT chromosomal DNA as a template using primer pairs D1 and D2 (for amplification of the Fossariinae upstream region of iucD), and D3 and D4 (for amplification of the downstream region of iucD). The two amplicons were used as the templates in a second PCR using the primer pair D1 and D4, and a PCR fragment with a 1124-bp deletion in iucD was obtained. The deletion fragment was digested with XhoI, and the digested fragment was then ligated into the SalI site of an R6K-ori suicide vector, pXAC623 (18). The resulting hybrid plasmid, pXACΔiucD, was transformed into E. coliβ2155, crossed with V. mimicus 7PT, and the resulting merodiploids selected on LB agar plates with chloramphenicol at 10 μg/ml and without DAP. The merodiploids were then plated on LB agar plates containing 10% sucrose without NaCl and chloramphenicol, and grown at 25oC for 30 hr. Sucrose-resistant and chloramphenicol-sensitive colonies were selected, and the iucD deletion mutant, ΔiucD, was confirmed by PCR analysis using the primer pair D5 (5′-CTTCCTATCAGCTTGGACTC-3′) and D6 (5′-GTCGTCAGTGATGTCGTAAC-3′). Both the ΔiucDΔmhuA and ΔiucDΔmhuB deletion mutants were constructed in a similar manner to that described for the construction of the ΔiucD strain.