Characterization of the regulatory region of a cell interaction-dependent gene in Myxococcus xanthus

Abstract

⍀4403 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C-signaling, are required for expression of Tn5 lac ⍀4403. We have cloned DNA upstream of the ⍀4403 insertion site, localized the promoter, and identified a potential open reading frame. From the deduced amino acid sequence, the gene disrupted by Tn5 lac ⍀4403 appears to encode a serine protease that is dispensable for development. The gene begins to be expressed between 6 and 12 h after starvation initiates development, as determined by measuring mRNA or ␤-galactosidase accumulation in cells containing Tn5 lac ⍀4403. The putative transcriptional start site was mapped, and sequences centered near ؊10 and ؊35 bp relative to this site show some similarity to the corresponding regions of promoters transcribed by Escherichia coli 70 RNA polymerase. However, deletions showed that an essential promoter element lies between ؊80 and ؊72 bp, suggesting the possible involvement of an upstream activator protein. DNA downstream of ؊80 is sufficient for C-signal-dependent activation of this promoter. The promoter is not fully expressed when fusions are integrated at the Mx8 phage attachment site in the chromosome. Titration of a limiting factor by two copies of the regulatory region (one at the attachment site and one at the native site) can, in part, explain the reduced expression. We speculate that the remaining difference may be due to an effect of chromosomal position. These results provide a basis for studies aimed at identifying regulators of C-signal-dependent gene expression.

Myxococcus xanthus Viability Depends on GroEL Supplied by Either of Two Genes, but the Paralogs Have Different Functions during Heat Shock, Predation, and Development

“…The generation time of the groEL1-complemented double-deletion mutant YL0308 was 6.92 Ϯ 0.29 h and that of the groEL2-complemented mutant YL0310 was 7.05 Ϯ 0.23 h in CTT medium, respectively. Several studies have re- ported reduced expression of genes at the Mx8 attB site (6,19,33). The quantitative-PCR results indicated that the levels of expression of the transposed groEL1 and groEL2 genes were approximately 30% to 50% and 75% to 90%, respectively, of that in the wild-type strain DK1622 at 24 h and 28 h in CTT cultures.…”

A cascade of coregulating enhancer binding proteins initiates and propagates a multicellular developmental program

“…Fragments carrying putative σ 54 promoters and enhancer elements were generated using PCR and purified using the Promega Wizard SV Gel and PCR Clean-Up System as described by the manufacturer. Purified promoter fragments were cloned into the promoterless lacZ vector pREG1727 (53), which is used to create lacZ transcriptional fusions, and transformed into M. xanthus WT strain DK1622. The in vivo activity of the promoter fragments in developing cells was determined by measuring β-galactosidase-specific activities at various times after starvation was induced as previously described (54).…”

Mutational Analysis of the Myxococcus xanthus Ω4406 Promoter Region Reveals an Upstream Negative Regulatory Element That Mediates C-Signal Dependence

“…Based on previous experience in our laboratory (1,6,7,12,32), the majority of transformants have a single copy of the plasmid integrated at attB. To eliminate colonies with unusual developmental lacZ expression, we screened at least 10 transformants on TPM agar plates containing 40 g of 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside per ml.…”

“…Genes that depend on C signaling for expression are activated at different times after 6 h into development, and some show partial expression in a csgA mutant, whereas others show no expression (23,29). Expression of all such genes examined thus far could be rescued by codeveloping the csgA mutant with wild-type cells (to supply C signal) (1,6,7,23,32) or by adding a 17-kDa fragment of CsgA (22). A higher level of the CsgA fragment was needed to rescue expression of a gene normally induced at the onset of sporulation than for a gene induced earlier, indicating different thresholds for response (21).…”

“…Insertions that generated a transcriptional fusion between a developmentally regulated M. xanthus promoter and the Escherichia coli lacZ gene have been tested for dependence of expression on csgA (23,29). The promoter regions upstream of two insertions that exhibited partial dependence on csgA (⍀4400 and ⍀4499) and two that exhibited absolute dependence on csgA (⍀4403 and ⍀4406) have been identified (1,6,7,32). Three of these promoter regions have been subjected to mutational analysis to identify important regulatory elements (46)(47)(48).…”