The cultures were incubated at 30°C with vigorous shaking (250 rpm). When the cultures reached mid-logarithmic phase, the cells were collected by centrifugation and flash frozen in liquid nitrogen. Cells were stored at -80°C prior to RNA extraction. For exogenous expression of Fur and RyhB, the fur and ryhB open reading frames (ORFs) were PCR amplified with primers fur-F1 and fur-R1, and ryhB-F1 and ryhB-R1, respectively (Table 2). The PCR products were digested with SalI and EcoRI, and cloned into the broad-range expression vector pBBR1MCS5-1 (Kmr), placing the ORFs under the transcriptional control of a strong lac promoter.

The resulting plasmids were verified by DNA sequencing and transferred into E. coli

WM3064, which is a diaminopimelic acid (DAP) auxotroph with plasmid RK4 integrated in the chromosome to mobilize plasmid in trans during conjugation [37]. Conjugation www.selleckchem.com/products/empagliflozin-bi10773.html was carried out by mating E. coli and S. oneidensis in 1:1 donor/recipient ratio for 8 hrs on a LB/DAP plate at 30°C followed by selection of S. oneidensis transconjugants on LB agar plates supplemented with 50 μg/ml kanamycin. The vector pBBR1MCS5-1 was also transformed into S. oneidensis for the purpose PF299804 cell line of comparison. Table 2 Oligonucleotide primers used in this study. Primer name Sequence strain construction   fur-F1 GGTCGACCAAGAGATTAGCAATGACAGATG fur-R1 GGAATTCGAGCAAGCTTATTCGTCGT ryhB-F1 GGTCGACAGGAGGAACTCTGATGACTGGTAATCTG Ruxolitinib mw ryhB-R1 GGAATTCAGTTAAATGTGGCGCAAAC Reverse Transcription-PCR ryhB-F2

TCTGACGTTGTTAAAGTGCTCC ryhB-R2 CCTAATGCGCCTATTCGCT Control 1-F TCAGGTTGTTTGGTATTGTGC check details Control 1-R CCATCAATCAAGGTTGTCG Control 2-F CTGTCAAATGGTGTGCTGC Control 2-R GTGTAACAGTGCTAAAGCCTGC Control 3-F TCTACTCAAATGACGAGCTGC Control 3-R GAAAAGCCGCCAAATGC Control 4-F TATGGTTTCCCGCTTTCG Control 4-R AACGCATCAGTGCTATTTGC Control 5-F TCACTCACAGAACGCTTCG Control 5-R GCAGCTACAGAATGTCACTACG Control 6-F TCTAGCAGGGATTAAATGAGC Control 6-R CCTTCGCCTTGTCTAAAGC 5′- and 3′-RACE assays   5′- RNA adapter GAUAUGCGCGAAUUCCUGUAGAACGAACACUAGAAGAAA ryhB-R3 AGAGTGTGTGAGCAATGTCG 3′- RNA adapter UUCACU GUUCUUAGCGGCCGCAUGCUC-idT Quantitative RT-PCR   RyhB-F TCTGACGTTGTTAAAGTGCTCC RyhB-R CCTAATGCGCCTATTCGCT SdhA-F GAGCAGTTAAAAGCCATCC SdhA-R GTTGTCCAATTCTAAACACTCG AcnA-F ACCAACAAACGCTAGACTACC AcnA-R ATCATCGCTCCACAAACC SodB-F TCTACTGGAACTGCTTAGCACC SodB-R TGAATGCATCGAATGAACC RecA-F AACCCAGAAACCACAACG RecA-R ACCAACCACCTCATCACC Primer sequences were derived from the S. oneidensis MR-1 genome sequence [25]. F and R stand for forward and reverse primers, respectively. HPLC analyses S. oneidensis wild-type (strain MR-1) and the fur mutant were grown to mid-logarithmic phase in M1 medium with 10 mM lactate as the sole carbon source.