Thus, the present work aims to elucidate in vivo the capacity of the E. faecalis SUF operon to complement the ISC and SUF systems from the Proteobacteria representatives A. vinelandii and E. coli. The selleck chemicals llc Azotobacter vinelandii and Escherichia coli strains used in this study are listed in Table 1, and plasmids used for in vivo experiments in Table 2. Escherichia coli were grown in the following

media: Luria broth (10.0 g L−1 tryptone, 5.0 g L−1 yeast extract, 5.0 g L−1 NaCl), and M9-glycerol minimal medium, supplemented as needed with 5.0 mM adenine, 0.3 mM leucine, 0.3 mM isoleucine, 0.1 mM nicotinic acid, 0.3 mM thiamine, and 0.3 mM valine. Azotobacter vinelandii was grown in Burk’s minimal medium (BN) containing 2.0% sucrose as the carbon source and 13.0 mM ammonium acetate as nitrogen source (Strandberg & Wilson, 1968). The following antibiotics were used in this study: ampicillin (100 μg mL−1), rifampicin (100 μg mL−1), kanamycin (50 μg mL−1), gentamicin (50 μg mL−1), tetracycline (50 μg mL−1), and vancomycin (30 μg mL−1). Arabinose was used at 0.3% w/v for expression in E. coli and A. vinelandii under arabinose promoter (pBAD). X-gal at a final concentration of 0.6 mg mL−1

was used for cloning insertion determination. Recipient strains used in this work have been described previously (Table 1) and confirmed in terms of promoter region arrangements; modifications (either insertions or mutations) carried out in this work did not alter any characteristic of expression which could result in polar effects. Azotobacter check details vinelandii strains were constructed by transformation experiments in which homologous reciprocal

recombination occurred between cloned, isolated A. vinelandii DNA in a recombinant plasmid and a corresponding genome region. As an example, the vector pEFSC31 was constructed first using PCR (Epicentre’s Failsafe PCR kit) to isolate the sufU gene from the chromosomal DNA of E. faecalis. The PCR primers were designed to add an NdeI restriction enzyme site at the N-terminus of sufU and a BglII restriction enzyme site at the C-terminus. The 0.7-kb PCR product was ligated into the pCR4-TOPO vector (Invitrogen TOPO Abiraterone mw TA sequencing kit) or pCR-Blunt vector (Invitrogen). This plasmid was digested with NdeI and BglII and the resulting DNA fragment was ligated into the NdeI–BglII sites of pDB1568, putting expression of the SufU protein under control of the pBAD in a region of DNA containing the scrX gene. Other plasmids used in this study (Table 2) were constructed in a similar fashion. Incorporation of the SUF genes into the A. vinelandii genome was achieved as described by Jacobson et al. (1989a, b). DJ1418, used as the parent strain, contains the complete endogenous ISC operon and a lacZ:kanamycin resistance cartridge inserted into scrX.