Transcription profiling of Geobacter sulfurreducens wild type and pilR mutants with acetate as the electron donor and ferric citrate as the electron acceptor

ABSTRACT: G. sulfurreducens wild type and pilR mutant (GSU1495) were grown in chemostats for RNA extraction used for microarray analysis and qRT-PCR. The electron donor (acetate 5mM) was limiting at a dilution rate of 0.05 h-1 and ferric citrate(55mM) was used as the electron acceptor at 30C, as previously described (Esteve-Nunez et al., 2005). Analysis of acetate, Fe(II) and protein were performed as previously described (Esteve-Nunez et al., 2005). Disruption of the pilR gene (GSU1495) was made in G. sulfurreducens strain DL1 (ATCC 51573) by the recombinant PCR and single-step recombination method (Murphy et al., 2000), essentially as described (Lloyd et al., 2003). To disrupt the pilR gene a 2.25 kb DNA fragment was constructed by PCR in which 0.39 kb of the pilR coding sequence (codons 192 - 323) were replaced with the kanamycin resistance cassette (Knr) of pBBR1MCS-2 (Kovach et al., 1995). This fragment consisted of 29 bp of upstream sequence together with the first 575 bp of the pilR gene, followed by the kanr cassette (1.1 kb), and the last 409 bp of the pilR gene and 132 bp of downstream sequence. The mutant was selected in NBAF plates supplemented with kanamycin and incubated at 30C in an anaerobic chamber containing a mixture of 7% H2, 10% CO2, 83% N2. A single kanamycin-resistant colony was selected, tested for the insertion of the Knr cassette by PCR, and designated DLJK3.

ORGANISM(S): Geobacter sulfurreducens

SUBMITTER: Barbara Methe 

PROVIDER: E-JCVI-1 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress