Members of this protein superfamily are typically single-polypeptide secondary carriers, comprising of 10–14 transmembrane α-helices which are able to transport small solutes such selleck inhibitor as sugars or toxins in response to chemiosmotic ion gradients [7, 8]. In this work, the role

of SMc02161 in bacterial resistance to toxics, nod gene expression and nodulation of alfalfa is described. Results and discussion S. meliloti ORF Smc02161 potentially codes for a transmembrane transporter with striking homology to MFS permeases To analyze the region find more surrounding the fadD gene of S. meliloti, the available sequence of S. meliloti 1021 [9] was used. The analysis using BLAST [10] revealed an ORF (SMc02163) downstream of fadD with homology to phosphoglucose isomerase (pgi) while upstream a divergently coding ORF (SMc02161) showed high identity to permeases of the Major Facilitator Superfamily (MFS). In this study, we characterize specifically ORF SMc02161. Putatively, this ORF encodes for a 411 amino acid protein with 11 transmembrane AZD2171 motifs typical of inner membrane proteins. This protein has an ATP/GTP binding motif, an alanine rich region (PROSITE [11]) and has the multi-domain of the MFS that covers most of the protein (from amino acid 73 to 331). The product shows the highest identity (66%) with a putative MFS protein in Beijerinckia indica subsp. indica ATCC9039, and shares most identity to MFS related permeases, transmembrane

proteins, sugar transporters and efflux proteins of bacteria belonging to the Rhizobiales and Burkholderiales orders. Unfortunately, the physiological functions of the closest SMc02161 homologs have not been experimentally tested. One of the few SMc02161 homologs with an experimentally assigned function is CmlR (P31141, 29% identity), a chloramphenicol resistance protein of Streptomyces lividans [12]. The S. meliloti SMc02161 mutant shows higher

sensitivity to chloramphenicol To functionally characterize SMc02161, we constructed the GR4T1 DOCK10 mutant in which the wild type locus was replaced with a mutated version. Considering the homology shown by SMc02161 with CmlR, we compared the sensitivity of the GR4T1 mutant with the wild type S. meliloti strain GR4 to different concentrations of antimicrobial compounds such as chloramphenicol, tetracycline, and salicylic acid. The influence of luteolin and plant root exudates on the growth of these strains was also compared. Only the presence of chloramphenicol reduced the growth of the mutant compared to that of the wild type strain (Figure 1). This suggests that the protein encoded by SMc02161 can function as an efflux pump, expelling the antibiotic chloramphenicol from the bacteria. As a result, we renamed ORF SMc02161 to tep1 for transmembrane efflux protein. To rule out possible polar effects of the created mutation in tep1 on downstream genes, complementation of the chloramphenicol sensitivity of the mutant was attempted with a plasmid construct.