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.
Further studies that assess the prevalence of licD alleles between epidemiologically comparable collections AG-014699 manufacturer of virulent and commensal NT H. influenzae strains may highlight which alleles are important in NT H. influenzae disease. One ChoP genotype that may be associated with NT H. influenzae disease isolates is the possession of two lic1 loci in the same strain where each
locus contains a different licD allele, providing the bacteria with two independently phase-variable ChoP substitutions. Fox et al  demonstrated that 4/25 (16%) NT H. influenzae middle ear strains had dual lic1 loci. In the current study, only NT H. influenzae and not H. haemolyticus possessed dual lic1 loci. Although only 7 of 88 (8%) total NT H. influenzae strains had dual loci, six were present among 43 (14%) middle ear strains present in this collection (unpublished results). Fox et al.  also noted that the genome sequenced NT H. influenzae strain, R2846, possessed a complete and partial lic1 loci, each containing a different licD allele, raising the possibility that other strains may have a similar genotype. An extensive
search on the lic1-containing strains in this collection using licD-specific PCR and hybridization, however, did not identify any strains (apart from the seven dual lic1 locus strains) that contained more than one licD allele, suggesting that the NT H. influenzae population contains mainly complete copies of lic1 (unpublished results). Although NT H. influenzae LOS structural studies have identified ChoP modifications
selleck kinase inhibitor on oligosaccharides extending from the heptose II position , specific licD alleles mediating this arrangement have Palbociclib not been characterized. It is possible that one or more of the current LicD alleles may overlap in this process or that stochastic factors in LOS biosynthesis may play a role. In addition, the clustering analysis of LicD protein alleles present in Figure 2 suggests that sub-variants may exist within the major allelic groups, and it is possible that one of these variants may facilitate heptose II-associated ChoP substitutions. As reviewed by Moxon et al , strains that are genetically and epidemiologically unrelated vary widely in the lengths of SSR (including licA tetranucleotide repeats), while individual strains that transmit within an outbreak or are extensively subcultured over time maintain a central modality in repeat numbers [32, 33]. Using a larger number of samples from a phylogenetically defined collection of NT H. influenzae strains has allowed us to partially resolve distribution trends for the licA repeat region in the NT H. influenzae and H. haemolyticus populations (Figure 3) and make statistical comparisons between and within species (Table 3). We found statistically significant trends toward the increased length of licA tetranucleotide repeats in NT H. influenzae compared to H.