1a). Figure 1 Mutational beta-catenin phosphorylation analysis of the S. meliloti hfq gene. (a) Arrangement of the genomic hfq region, multiple amino acid sequence alignment of Hfq proteins
encoded by enterobacterial and α-proteobacterial genomes and details of the hfq mutants. The genetic map is drawn to scale. Numbering denotes the gene coordinates in the S. meliloti genome database. In the 1021Δhfq mutant the full-length Hfq ORF was replaced by a HindIII site. The DNA fragment cloned on complementation plasmid pJBHfq is indicated. In the alignment, Hfq sequences are denoted by the species abbreviation as follows: Ecol, E. coli; Stiph, Salmonella tiphymurium; Bsu, Brucella suis; Bmel, B. melitensis; Acaul, Azorhizobium caulinodans; Atum, Agrobacterium tumefaciens; Mlot, Mesorhizobium loti; Rleg, Rhizobium leguminosarum; Smel, S. meliloti. Species belonging to the α-subdivision of the proteobacteria are indicated to the left. Shadowed are the amino acid residues conserved in at least 80% sequences
and boxed are the conserved amino acids within the C-terminal extension of Hfq proteins encoded by enterobacteria. The two conserved Sm-like domains are indicated. Double arrowheads indicate the integration sites of pK18mobsacB in 2011-3.4 and 2011-1.2 derivatives. (b) Growth curves in TY broth of the S. meliloti wild-type strains 2011 (left panel) and 1021 (right panel) and their respective hfq mutant derivatives as determined by OD600 readings of triplicate cultures in 2 h intervals. Graphs legends: 2011, wild-type strain; 1.2, 2011-1.2 control strain; 3.4, 2011-3.4 derivative; 3.4(pJBHfq), 2011-3.4 complemented with plasmid pJBHfq; 1021, reference wild-type strain; Δhfq, selleck chemicals 1021 hfq deletion mutant; Δhfq(pJBHfq), Δhfq complemented with pJBHfq. The S. meliloti hfq gene seems to form a dicistronic operon with the downstream hflX-like gene coding for a putative GTP-binding protein. Upstream of hfq are SMc01047 and trkA coding mafosfamide for a D-alanine aminotransferase and a potassium transporter, respectively (Fig. 1a). Immediately upstream of trkA is the gene cluster specifying
the nitrogen assimilation system ntr (ntrB-ntrC-ntrY-ntrX). This genomic arrangement is essentially conserved in all the nitrogen-fixing endosymbionts of the order Rhizobiales. The exception is the absence of either the trkA or SMc01047 homologs between the ntr operon and hfq in a few species (i.e. M. loti, R. leguminosarum bv. viciae). In contrast, the S. meliloti hfq upstream region totally diverges from that of its related intracellular animal pathogens (i.e. Brucella sp.). Enterobacterial and α-proteobacterial genomes only conserve the hflX gene downstream of hfq in this chromosomal region. Construction and growth characteristics of the S. meliloti hfq mutants As a first approach to address the S. meliloti Hfq functions in vivo two independent hfq knock-out mutants were constructed in strains 2011 and 1021. These S. meliloti strains are derived from the same progenitor (S.