, 2003b). The entF gene in Brucella is homologous with the vibH gene of Doxorubicin mw Vibrio cholera that is involved in the synthesis of the siderophore vibriobactin, but its role in Brucella is not clearly understood. The work presented here clearly suggests a role of the entF gene in iron acquisition and subsequently in erythritol metabolism by B. abortus 2308. Brucella abortus 2308 was grown in trypic soy broth or tryptic soy agar (TSA). Iron minimal media (IMM) was prepared as described previously (Lopez-Goni et al., 1992). The concentration of iron in minimal media was determined using atomic absorption spectrophotometry (flame method) and found to be < 0.099 μg mL−1. All other

chemicals were bought from Sigma-Aldrich Inc. (St. Louis, MO) unless specifically stated. An unmarked mutation was created in the entF gene of strain B. abortus 2308 using the cre-lox methodology as described previously (Rajasekaran et al., 2008). A segment containing 497 base pairs were deleted within the entF gene without incorporating any antibiotic-resistant marker in the mutant. To create the complemented mutant, the pNSGroE plasmid was used as the expression vector (Seleem et al., 2004). The entF gene was HSP inhibitor amplified from B. abortus 2308 using entF forward (5′-GGG GGA TCC TTG

GTC CCA ATT TGT CAA CCG GGT-3′) and reverse (5′-GGG TCT AGA TCA TGG CAA ACG GCG GCG AAG ATC-3′) primers and was cloned in to the vector between the BamHI and XbaI restriction sites. A BAN1 strain complemented with pNSGroE∷entF was named BAN2. Genetic deletion of entF in BAN1 and complementation

in BAN2 was confirmed by PCR using entF forward and reverse primers. Total RNA was isolated from each of the three strains, B. abortus 2308 (wild Dichloromethane dehalogenase type), ΔentF mutant (BAN1) and ΔentF complemented mutant (BAN2), at 72 h of growth in IMM using a Pure link kit (Invitrogen) and RNA Easy kit (Qiagen) as per the manufacturer’s protocol. Turbo DNAase (Ambion) was used to treat RNA samples for DNA contamination according to the manufacturer’s protocol. The absence of contaminating chromosomal DNA was confirmed by failure of the detectable cDNA gene amplification reactions, in the absence of reverse transcriptase. To synthesize cDNA, iScript cDNA synthesis kit (Bio-Rad) was used as per the manufacturer’s instructions and finally PCR was performed with entF internal primers (forward primer: 5′-GGCGGAGGTTCTTTCCAT-3′, reverse primer: 5′-CGTCCTCCTCATGAATG-3′) and entB-A intergenic primers (forward primer: 5′-CTACGGCCTCGATTCGCTA-3′, reverse primer: 5′-GATGACGGTTGCGCCTTCGG-3′) and products were checked on 1% agarose gel containing ethidium bromide under UV light. Cultures in IMM were started at 106 CFU mL−1 from a frozen culture of B. abortus 2308. All the supplements including FeCl3 (50 μM), erythritol (0.1% or 0.05%) and ethylenediamine-N.N′-diacetic acid (EDDA, 15 μM) were added 48 h before start of the growth to allow the homogenous distribution and binding of chemicals.