Fine-tuning of galactoglucan biosynthesis in Sinorhizobium meliloti by differential WggR (ExpG)-, PhoB-, and MucR-dependent regulation of two promoters

Abstract

Depending on the phosphate concentration encountered in the environment Sinorhizobium meliloti 2011 synthesizes two different exopolysaccharides (EPS). Galactoglucan (EPS II) is produced under phosphate starvation but also in the presence of extra copies of the transcriptional regulator WggR (ExpG) or as a consequence of a mutation in mucR. The galactoglucan biosynthesis gene cluster contains the operons wga (expA), wge (expE), wgd (expD), and wggR (expG). Two promoters, differentially controlled by WggR, PhoB, and MucR, were identified upstream of each of these operons. The proximal promoters of the wga, wge, and wgd transcription units were constitutively active when separated from the upstream regulatory sequences. Promoter activity studies and the positions of predicted PhoB and WggR binding sites suggested that the proximal promoters are cooperatively induced by PhoB and WggR. MucR was shown to strongly inhibit the distal promoters and bound to the DNA in the vicinity of the distal transcription start sites. An additional inhibitory effect on the distal promoter of the structural galactoglucan biosynthesis genes was identified as a new feature of WggR in a mucR mutant. A regulatory model of the fine-tuning of galactoglucan production is proposed.

FIG. 1.

Genomic structure of the exoP downstream region after integration of the promoter probe vector pSRPP18 carrying a promoter fragment inserted into the KpnI and BamHI restriction sites. The stem-loop symbol indicates the position of the terminator downstream of exoP. The restriction sites available to clone the promoter fragments into this vector are marked as N for NruI, K for KpnI, and B for BamHI. The thick line denotes the vector part of the integrated plasmid, whereas the incomplete exoP gene is shown in parentheses. The scheme is not drawn to scale.

FIG. 2.

Galactoglucan biosynthesis gene cluster (top) and DNA sequence of the intergenic regions between wgcA and wgaA (A), wgdB and wgeA (B), wggR and wgdA (C), and wgdA and wggR (D). Start codons are indicated by a short dashed line associated with the gene name. Stop codons are marked by three asterisks. Black and gray boxes represent potential PHO boxes and reverse-oriented PHO boxes, respectively. Open boxes mark the 21-bp conserved region of the WggR binding site (5). Thin arrows represent the start of the primers used to generate DNA fragments for insertion into pSRPP18. These primers anneal to 18 bp of the intergenic region starting from the base of the arrow. Transcription starts are indicated by +1 and thick arrows. The dashed lines represent the predicted −35 boxes, whereas the bold lines denote the predicted −10 boxes.

FIG. 3.

Alignment of 50 bp downstream of the different transcription starts found by 5′RACE upstream of the wge, wga, wgd, and wggR operons determined by using CLUSTALW (34). The distance of the transcription start to the ATG of the first gene of the operon is given on the right. The bases are colored as a function of the shared similarities, where dark boxes represent the highest homology between the different sequences. At the bottom, a consensus sequence is proposed. The −35 and −10 boxes are marked by open boxes. The TSS are indicated in boldface.

FIG. 4.

On the left, a schematic representation of the investigated intergenic region is shown for wgcA-wgaA (A), wgdB-wgeA (B), wggR-wgdA (C), and wgdA-wggR (D). Black and gray boxes represent potential PHO boxes and reverse-oriented PHO boxes, respectively. Open boxes indicate the 21-bp conserved region of the WggR binding site (5). Transcription starts are marked by +1 and arrows. In the center, β-galactosidase activities mediated by the DNA fragments in different mutant backgrounds are shown. β-Galactosidase activities were determined in the wild-type (WT), the phoB mutant (phoB), the mucR101-spc mutant (mucR), the wggR deletion mutant SmSRΔG (wggR), and the double mutant mucR101-spc/wggR (wggR/mucR) in low-phosphate medium (gray bars) and in high-phosphate medium (black bars). β-Galactosidase activity values are the average of at least five independent assays. The background β-galactosidase activity of pSRPP18 in the wild-type strain Sm2011 was 15 ± 1 Miller units. Error bars denote the standard deviation. On the right, results of EMSAs with His₆-WggR and Flag-MucR are shown using the DNA fragments indicated on the left.

FIG. 5.

Model for transcriptional regulation of the structural EPS II biosynthesis genes by MucR, WggR, and PhoB. Black boxes represent potential PHO boxes, the open box marks the WggR binding site, and the hatched box represents the putative MucR binding site. Arrows indicate the transcription starts. Small gray boxes represent the −10 and −35 boxes for each promoter. The thickness of the arrow beginning at the transcription start marked by +1 indicates the amount of transcript in each situation. The circled P indicates the phosphorylation status of PhoB.