Characterization of Strong Promoters from an Environmental Flavobacterium hibernum Strain by Using a Green Fluorescent Protein-Based Reporter System

Abstract

We developed techniques for the genetic manipulation of Flavobacterium species and used it to characterize several promoters found in these bacteria. Our studies utilized Flavobacterium hibernum strain W22, an environmental strain we isolated from tree hole habitats of mosquito larvae. Plasmids from F. hibernum strain W22 were more efficiently (ϳ1,250-fold) transferred by electroporation into F. hibernum strain W22 than those isolated from Escherichia coli, thus indicating that an efficient restriction barrier exists between these species. The strong promoter, tac, functional in proteobacteria, did not function in Flavobacterium strains. Therefore, a promoter-trap plasmid, pSCH03, containing a promoterless gfpmut3 gene was constructed. A library of 9,000 clones containing chromosomal fragments of F. hibernum strain W22 in pSCH03 was screened for their ability to drive expression of the promoterless gfpmut3 gene. Twenty strong promoters were used for further study. The transcription start points were determined from seven promoter clones by the 5 rapid amplification of cDNA ends technique. Promoter consensus sequences from Flavobacterium were identified as TAnnTTTG and TTG, where n is any nucleotide, centered approximately 7 and 33 bp upstream of the transcription start site, respectively. A putative novel ribosome binding site consensus sequence is proposed as TAAAA by aligning the 20-bp regions upstream of the translational start site in 25 genes. Our primary results demonstrate that at least some promoter and ribosome binding site motifs of Flavobacterium strains are unusual within the bacterial domain and suggest an early evolutionary divergence of this bacterial group. The techniques presented here allow for more detailed genetics-based studies and analyses of Flavobacterium species in the environment.

Comparison of Bacteroides thetaiotaomicron and Escherichia coli 16S rRNA gene expression signals

“…Perhaps the 27 upstream region (TA) is critical to the activity of BT4001 P1, but not to other promoters examined thus far, since those examined by Bayley et al (2000) do not all share the consensus 27 upstream region. Our findings are similar to those of studies of Flavobacterium, where deletion of the 233 region of the ompA promoter (Chen et al, 2007b) or changes in either of the 27 upstream bases (TA) almost completely abolish gene expression (Chen et al, 2007a). Indeed, most substitutions at bases in both the 233 and the 27 region of the Flavobacterium promoter lead to decreased GFP reporter expression in comparison with the wild-type promoter levels (Chen et al, 2007a).…”

“…One region, TTTG, is centred at 233, and the other, TAnnTTTG, is centred at 27 (Bayley et al, 2000). Interestingly, a promoter consensus sequence similar to that of Bacteroides has also been found in Flavobacterium hibernum, another member of the phylum Bacteroidetes, with TTG (233) and TAnnTTTG (27) (Chen et al, 2007b).…”

Enigmatic, ultrasmall, uncultivated Archaea

“…However, more common is the use of a TAA motif. This has previously only been seen in Bacteria (35), Crenarchaea (Sulfolobus tokodaii TAAA at 13-15 bp, Nitrosopumilus maritimus AATAA at 13-15 bp, and Caldivirga maquilingensis TAA at 13-15 bp, weak motif), and N. equitans (TAAAA at 5-10 bp and TATAA 13-15 bp weak motif). The TAA motifs were found 3 to 15 bp upstream of genes in ARMAN-4 but only 3 to 10 bp upstream in ARMAN-5.…”

Construction of two selectable markers for integrative/conjugative plasmids in Flavobacterium columnare

“…The conserved sequences at -7 and -33 regions of promoters in Flavobacterium and Bacteroides are TAnnTTTG and TTG, and the consensus motif of RBS is TAAAA (Bayley et al, 2000;Hunnicutt and McBride, 2001;Chen et al, 2007aChen et al, , 2007b. However, the corresponding conserved sequences in other Gram-negative bacteria are TATAAT and TTGACA (Hawley and McClure, 1983), and AGGAGG (Ringquist et al, 1992).…”

“…Over the last ten years, molecular genetic techniques have been developed for some members of Flavobacterium (Shoemaker et al, 1986;Guiney et al, 1989;McBride and Kempf, 1996;Salyers et al, 1999;Chen et al, 2007b). Gliding mechanisms have been elucidated in F. johnsoniae by transposon Tn4351-mediated mutagenesis (Agarwal et al, 1997;Kempf and McBride, 2000;McBride et al, 2003;.…”