Identification of the omega4400 regulatory region, a developmental promoter of Myxococcus xanthus

Abstract

Omega4400 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 normal expression of Tn5 lac omega4400. The DNA upstream of the omega4400 insertion has been cloned, the promoter has been localized, and a partial open reading frame has been identified. From the deduced amino acid sequence of the partial open reading frame, the gene disrupted by Tn5 lac omega4400 may encode a protein with an ATP- or GTP-binding site. Expression of the gene begins 6 to 12 h after starvation initiates development, as measured by beta-galactosidase production in cells containing Tn5 lac omega4400. The putative transcriptional start site was mapped, and deletion analysis has shown that DNA downstream of -101 bp is sufficient for C-signal-dependent, developmental activation of this promoter. A deletion to -76 bp eliminated promoter activity, suggesting the involvement of an upstream activator protein. The promoter may be transcribed by RNA polymerase containing a novel sigma factor, since a mutation in the M. xanthus sigB or sigC gene did not affect Tn5 lac omega4400 expression and the DNA sequence upstream of the transcriptional start site did not match the sequence of any M. xanthus promoter transcribed by a known form of RNA polymerase. However, the omega4400 promoter does contain the sequence 5'-CATCCCT-3' centered at -49 and the C-signal-dependent omega4403 promoter also contains this sequence at the same position. Moreover, the two promoters match at five of six positions in the -10 regions, suggesting that these promoters may share one or more transcription factors. These results begin to define the cis-acting regulatory elements important for cell-cell interaction-dependent gene expression during the development of a multicellular prokaryote.

FIG. 1

Physical map of the Ω4400 insertion region and summary of deletions tested for promoter activity. The upper schematic depicts the restriction sites in Tn5 lac and the upstream M. xanthus chromosome that were used in this study. The position of the 5′ end of the oligonucleotide used to construct the PCR-generated deletion fragment is also indicated (∗). Distances of restriction sites from the Tn5 lac Ω4400 insertion are given in kilobases. Ap, ApaLI; B, BamHI; Bx, BstXI; M, SmaI; R, EcoRI; Sp, SphI; X, XhoI. The lower diagram depicts the segments of Ω4400 upstream DNA fused to the promoterless lacZ gene of pREG1727 to test for promoter activity. The plasmid designation is indicated on the left (Table 1). The maximum β-galactosidase specific activities during a 48- to 72-h developmental time course of derivatives of wild-type M. xanthus DK1622 containing a single copy of each plasmid integrated at Mx8 attB are given as percentages of the maximum activity observed for Tn5 lac Ω4400-containing strain DK4292. In each case, the activity of at least three independent transductants was measured at least once as described previously (34) and the average is given.

FIG. 2

Developmental expression of lacZ under the control of the Ω4400 promoter. Developmental β-galactosidase specific activity was measured as described previously (34) for Tn5 lac-containing strain DK4292 (□) and for at least three independently isolated transductants of DK1622 containing a single copy of the 1.8-kb (◊), 1.1-kb (○), 0.64-kb (▵), or 0.4-kb (⧫) Ω4400 upstream DNA fused to promoterless lacZ within pREG1727 and integrated at Mx8 attB. The β-galactosidase specific activity of DK1622 containing the pREG1727 vector alone with no insert integrated at Mx8 attB (•) is also shown. The average β-galactosidase activity from six determinations for DK4292 or from at least one determination for each of three independent transductants is plotted. β-Galactosidase specific activity is expressed in nanomoles of o-nitrophenyl phosphate per minute per milligram of protein.

FIG. 3

Localization of a single mRNA 5′ end within the Ω4400 upstream region. Low-resolution S1 nuclease mapping of the Ω4400-associated transcript was performed by using RNA prepared from DK4292 cells grown vegetatively (lanes V) or cells that had undergone 24 h of development (lanes D). The RNA was hybridized to DNA probes that were radioactively labeled at either the SmaI site upstream of Ω4400 or the BamHI site near the left end of Tn5 lac prior to digestion by 25 U (left lanes of each set) or 50 U (right lanes) of S1 nuclease. The lengths (in base pairs) of some of the end-labeled, MspI-digested pBR322 molecular size standard fragments are indicated on the left.

FIG. 4

Nucleotide sequence of the region upstream of the Ω4400 Tn5 lac insertion. The transcriptional start site is indicated by +1, and the primer used for the primer extension analysis is underlined. The putative translational start codon and a potential ribosome-binding site are overlined. The deduced amino acid sequence of the Ω4400 partial ORF is shown below the nucleotide sequence, and the amino acids comprising the putative ATP- or GTP-binding site motif are in boldface type. The sequence from the end of Tn5 lac to the BamHI site within Tn5 lac is in italics. Restriction sites and important deletion sites are also indicated.

FIG. 5

Primer extension analysis of Ω4400 mRNA. RNA was isolated from wild-type cells grown vegetatively (lane V) or cells that had undergone 24 h of development (lane D), and 60 μg was subjected to primer extension analysis. The same primer was used to sequence Ω4400 upstream DNA. A portion of the DNA sequence is indicated at the right. The arrow indicates the putative transcriptional start site as determined by the migrational position of the single primer extension product detected by using developmental RNA samples.

FIG. 6

Alignment of Ω4403 (14) and Ω4400 promoter DNA sequences. The sequences have been aligned at their respective +1 positions. The 7 bp of identical sequence in the −50 region is in boldface type, and the similar −10 sequences are underlined. Short repeat sequences in the critical upstream regions are overlined, and a 6-bp mirror repeat in the Ω4400 upstream region is italicized.

FIG. 7

Developmental expression of lacZ under control of the Ω4400 promoter in a csgA background in the absence (A) and presence (B) of extracellular complementation. (A) Developmental β-galactosidase specific activity was measured for Ω4400 Tn5 lac-containing csgA strain DK5247 (□) and for at least three independently isolated transductants of DK5208 (csgA) containing a single copy of Ω4400 upstream DNA extending to −101 (▪) or −76 (○) fused to lacZ and integrated at Mx8 attB. The β-galactosidase activity of DK5208 (csgA) containing the pREG1727 vector alone with no insert integrated at Mx8 attB (▵) is also shown. (B) The strains used for panel A were codeveloped with an equal number of wild-type DK1622 cells (which express csgA but not lacZ), and the β-galactosidase specific activity was determined as previously described (34). β-galactosidase specific activity is expressed in nanomoles of o-nitrophenyl phosphate per minute per milligram of protein.

FIG. 8

Developmental lacZ expression of C-dependent promoters in sigB and sigC-1 mutant backgrounds. In each panel, specific activities are given as percentages of the maximum activity observed for the corresponding wild-type (sigB⁺ sigC⁺), Tn5 lac (Tc^r)-containing parental strain. The developmental β-galactosidase curves of the parental strains (□) represent the average of two assays, and the curves of the Tn5 lac (Tc^r)-containing sigB (◊) and sigC-1 (○) mutant strains represent the average of three independently isolated transductants, each assayed twice. Panels: A, Ω4400 Tn5 lac (Tc^r); B, Ω4459 Tn5 lac (Tc^r); C, Ω4403 Tn5 lac (Tc^r); D, Ω4499 Tn5 lac (Tc^r).

FIG. 8

Developmental lacZ expression of C-dependent promoters in sigB and sigC-1 mutant backgrounds. In each panel, specific activities are given as percentages of the maximum activity observed for the corresponding wild-type (sigB⁺ sigC⁺), Tn5 lac (Tc^r)-containing parental strain. The developmental β-galactosidase curves of the parental strains (□) represent the average of two assays, and the curves of the Tn5 lac (Tc^r)-containing sigB (◊) and sigC-1 (○) mutant strains represent the average of three independently isolated transductants, each assayed twice. Panels: A, Ω4400 Tn5 lac (Tc^r); B, Ω4459 Tn5 lac (Tc^r); C, Ω4403 Tn5 lac (Tc^r); D, Ω4499 Tn5 lac (Tc^r).