Cloning was performed using standard procedures, with plasmids transformed in E. coli strain DH5α or DH5αλpir, as described previously (Bose et al., 2008). Cloned PCR products were sequenced to ensure that unintended alterations were not incorporated. Sequencing was conducted at the University of Michigan DNA Sequencing Core Facility or at the University of Selleck GSK3 inhibitor Georgia Molecular Genetics Instrumentation Facility. Plasmids were mobilized into V. fischeri from E. coli by triparental mating using strain CC118λpir with pEVS104 as a helper (Stabb & Ruby, 2002), and mutations were placed on the chromosome by allelic exchange. Parent

strains and plasmids used for allelic exchange are listed in Table 1. Key plasmids and oligonucleotides are described in Table 1, and an overview of allele construction follows. To mutate fnr, an ∼3.3 kb region of the V. fischeri genome centered on fnr was PCR amplified with primers EVS97 and EVS98 using ES114

or MJ1 genomic DNA as a template, and the fragments were ultimately subcloned into pEVS136 and pJLB69, respectively (Table 1). We generated Δfnr∷tmpR alleles by replacing the ClaI to AvrII fragment of fnr with the trimethoprim-resistance gene (tmpR) from pJLB1 (Dunn et al., 2005) on a BstBI to AvrII fragment, resulting in tmpR replacing an Volasertib mw internal 255-bp fragment beginning in the middle of fnr, with tmpR in the same orientation as fnr. The ES114-derived Δfnr∷tmpR allele was placed in pJLB5 and pJLB70, and the MJ1-derived Δfnr∷tmpR allele was used in pCDW5. For complementation of E. coli with ES114 fnr, we ligated the fnr-containing BsrBI–PstI fragment from pEVS136 into SmaI- and PstI-digested pDMA5, generating pJLB6. To place lacZ under control of Sclareol the arcA promoter, we PCR amplified an ∼3.1-kb fragment containing an engineered lacZ (Tomich et al., 1988) using pVSV3 (Dunn et al., 2006) as a template and primers JBLACZ1

and JBLACZ2 (Table 1). We cloned this product into SmaI-digested pAJ4 and pJLB55 (Bose et al., 2007), which carry regions flanking arcA from ES114 and MJ1, respectively, with the sequence between the start and the stop codons of arcA replaced by a 6-bp SmaI recognition site. The ParcA-lacZ alleles contain the arcA start codon, followed by a 5′-CCC-3′ proline codon, and then the lacZ reporter (Tomich et al., 1988) from its second codon onward. These ES114- and MJ1-derived alleles were subcloned into pAS31 and pJLB139, respectively. Overnight cultures in LBS were diluted 1 : 1000 into SWTO and incubated at 24 °C with shaking (200 r.p.m.). Aerobic cultures contained 50 mL of SWTO in 250-mL flasks. For anaerobic cultures, aerobically grown overnight cultures were diluted 1 : 10 in LBS before inoculation of 0.2 mL into 20 mL SWTO in 165-mL sealed bottles with a headspace containing 5% CO2, 10% H2, and 85% N2.