Siderophore biosynthesis genes ofRhizobiumsp. …
The regulatory logic of siderophore biosynthetic genes in bacteria ..
Genomic DNA from A. salmonicida ACR168.1 was partially digested with restriction enzyme Sau3AI and ligated into the SuperCos1 cosmid vector (Stratagene). Recombinant cosmids were packaged in vitro and transduced into E. coli XL1-Blue MR (Stratagene). The cosmid library was screened by performing colony PCR with pools of recombinant clones using primers targeted to genes encoding putative A. salmonicida siderophore biosynthesis genes encoded in plasmids pFMON24 and pFMON46 previously isolated using the FURTA (Table ).
Genes involved in siderophore biosynthesis ..
To determine the transcriptional organization of the siderophore biosynthesis gene cluster, A. salmonicida RSP74.1 was grown until exponential phase in low-iron CM9 (containing 40 μM EDDA), and total RNA was isolated using the RNAwiz isolation reagent (Ambion) by following the manufacturer's instructions. Reverse transcriptase PCR (RT-PCR) analyses were performed with 0.6 μg of RNA pretreated with RQ1 RNase-free DNase (Promega) by using the Moloney murine leukemia virus RT (Invitrogen). For operon mapping, a reverse transcription reaction was performed with primers RT-1 and RT-2, which are homologous to the 3′ ends of asbG and asbC, respectively (Fig. ; see Table S1 in the supplemental material). The resulting cDNAs were used as templates for PCR amplification with Taq polymerase (Bioline), using specific primer pairs for each gene (see Table S1 in the supplemental material). A negative control PCR was performed with total RNA without Moloney murine leukemia virus RT to confirm the lack of genomic DNA contamination in each reaction mixture, and genomic DNA was used as a positive control for PCR.