cis Elements Necessary for Developmental Expression of a ...

JOURNAL OF BACTERIOLOGY, Feb. 2003, p. 1405–1414 0021-9193/03/$08.00⫹0 DOI: 10.1128/JB.185.4.1405–1414.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 185, No. 4

cis Elements Necessary for Developmental Expression of a Myxococcus xanthus Gene That Depends on C Signaling Poorna Viswanathan and Lee Kroos* Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 Received 19 July 2002/Accepted 20 September 2002

Cell contact-mediated C signaling coordinates morphogenesis and gene expression during development of Myxococcus xanthus. One promoter that depends on C signaling for transcription lies upstream of ⍀4403, the site of a Tn5 lac insertion in the genome. The ⍀4403 promoter has a C-box sequence centered at ⴚ49 bp that matches the consensus 5ⴕ-CAYYCCY-3ⴕ, which is found in several C-signal-dependent promoters. Mutational analysis of the ⍀4403 promoter region was performed to test the importance of the C box and to identify other cis-acting elements. A 6-bp change in the ⴚ10 region eliminated promoter activity, but a 6-bp change in the ⴚ35 region decreased activity only about twofold. Certain single-base-pair changes in the C box centered at ⴚ49 bp abolished promoter activity, establishing the importance of this sequence element. Single-base-pair changes in a C-box-like sequence centered at ⴚ77 bp also abolished promoter activity, but the pattern of mutational effects was different from that for the C box centered at ⴚ49 bp. Additional single-base-pair changes indicated that all 10 bp from ⴚ79 to ⴚ70 bp are important for ⍀4403 promoter activity. Mutations at ⴚ59, ⴚ61, ⴚ62, and ⴚ63 bp also abolished promoter activity, defining a 5-bp element from ⴚ63 to ⴚ59 bp. This 5-bp element is separated from the 10-bp element (i.e., ⴚ79 to ⴚ70 bp) by 6 bp that can be changed without loss of promoter activity. Likewise, the 5 bp between the 5-bp element and the C box can be changed without loss of activity, but deletion of these 5 bp abolished activity, indicating that spacing is important. Sequences similar to the 5- and 10-bp elements, as well as the C box, are present in other C-signal-dependent promoters, suggesting some similarity in the regulatory mechanisms, but there are also indications that these cis elements do not function identically in the different promoters. Myxococcus xanthus is a gram-negative soil bacterium that undergoes multicellular development (7). When starved at high cell density on a solid surface, cells move in a coordinated fashion into aggregation centers, where they form moundshaped fruiting bodies that each contain approximately 105 cells. Within the fruiting bodies, some of the rod-shaped cells differentiate into dormant, ovoid spores that are heat and desiccation resistant. Driving these morphological changes is a highly ordered program of gene expression controlled by cellcell interactions (35). At least five cell-cell signals, known as the A, B, C, D, and E signals, are required for normal development (5, 13). Mutants defective in the production of any one of these signals are arrested in development at a particular stage but can be rescued by codevelopment with wild-type cells or cells that are defective in the production of a different signal. To study the role of cell-cell interactions in controlling gene expression during M. xanthus development, Tn5 lac, a transposon containing a promoterless E. coli lacZ gene, has been used to identify developmentally regulated genes (28). Transposition of Tn5 lac into the M. xanthus chromosome can generate a transcriptional fusion between lacZ and an M. xanthus promoter. By examination of the expression of transcriptional fusions to lacZ created by Tn5 lac and the appearance of other developmental markers in signaling-defective mutants, it has been shown that A and B signaling is required at the onset of

development (11, 27, 29), D and E signaling is required 3 to 5 h into development (4–6), and C signaling is required at about 6 h for normal developmental gene expression (27). C signaling is unusual in that it involves a cell surfaceassociated protein, CsgA (14, 23, 24, 30, 36), and in that transmission of the signal requires motility (22, 26), apparently to allow cells to make end-to-end contacts (21, 33). A low level of C signaling is sufficient for rippling behavior in which cells become organized in parallel ridges that appear as traveling waves in movies made by time-lapse microscopy (31, 37). An intermediate level of C signaling is enough for aggregation, but a higher level is needed to trigger sporulation, and this high level may only be reached as cells become aligned and make many end-to-end contacts in the nascent fruiting body (18, 25, 31). Different levels of C signaling are also required for expression of different developmental genes (12, 25). Hence, a rising level of C signaling during development is believed to coordinate the program of gene expression with morphogenesis of the fruiting body and differentiation of cells into spores. To begin to understand the mechanism by which C signaling regulates developmental gene expression, the promoter regions of C-signal-dependent genes have been identified. These genes or operons were originally identified by Tn5 lac insertions ⍀4403 (10), ⍀4400 (3), and ⍀4499 (9). The promoter regions do not resemble promoters that are transcribed by M. xanthus ␴A RNA polymerase, the major vegetative RNA polymerase (2). However, both ⍀4403 and ⍀4400 have the sequence 5⬘-CATCCCT-3⬘ centered at ⫺49 bp (3). Similar sequences are also found at positions near ⫺50 bp in the ⍀4499 promoter region and in other promoters that have been reported to be C signal dependent (9). Hence, a sequence with

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824. Phone: (517) 355-9726. Fax: (517) 353-9334. E-mail: kroos @pilot.msu.edu. 1405

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the consensus 5⬘-CAYYCCY-3⬘ (known as the C box) has been proposed to be important for C-signal-dependent gene expression (9). Here, we report the first detailed mutational analysis of the promoter region of a C-signal-dependent gene. In addition to demonstrating the importance of the C box centered at ⫺49 bp and the ⫺10 region of the ⍀4403 promoter, we identified two new cis-acting elements, a 5-bp element centered at ⫺61 bp and a 10-bp element centered at ⫺74.5 bp. Interestingly, similar sequence elements can be found in the promoter regions of other C-signal-dependent genes. MATERIALS AND METHODS Bacterial strains and plasmids. Strains and plasmids that were used in this work are listed in Table 1. Growth and development. Escherichia coli was grown at 37°C in Luria-Bertani medium (34) containing 50 ␮g of ampicillin/ml. M. xanthus was grown at 32°C in CTT medium (16) (1% Casitone, 10 mM Tris-HCl [pH 8.0], 1 mM KH2PO4K2HPO4, 8 mM MgSO4 [final pH ⫽ 7.6]) in liquid cultures or on agar (1.5%) plates. Forty micrograms of kanamycin per ml was used when required for selective growth. Fruiting body development was performed on TPM (10 mM Tris-HCl [pH 8.0], 1 mM KH2PO4-K2HPO4, 8 mM MgSO4 [final pH ⫽ 7.6]) agar (1.5%) plates as described previously (28). Site-directed mutagenesis. The QuikChange site-directed mutagenesis kit (Stratagene) was used to create mutations in the ⍀4403 promoter region that, in most cases, resulted in single or multiple A7 C or G7 T substitutions. Briefly, pMF0100 (10) bearing the ⍀4403 promoter region from ⫺80 to ⫹382 bp was subjected to PCR with a combination of mutagenic primers. The M. xanthus DNA insert in each plasmid was sequenced at the Michigan State University Sequencing Facility to ensure the presence of only the desired mutation(s). Construction of plasmids. Each mutant derivative of pMF0100 was digested with BamHI and XhoI, the ⍀4403 promoter fragment was gel purified, and the fragment was ligated with pREG1727 (10) previously cut with the same restriction enzymes. The ligation products were introduced into E. coli DH5␣ by electroporation, and ampicillin-resistant transformants were selected. A transformant with a plasmid containing the mutant ⍀4403 promoter was identified by PCR and used to produce plasmid for introduction into M. xanthus. Construction of M. xanthus strains and determination of lacZ expression during development. Strains containing pREG1727 derivatives integrated at the Mx8 phage attachment site (designated attB in Table 1) were constructed by electroporation (20) of M. xanthus DK1622 and selection for kamamycin-resistant transformants. Based on previous experience in our laboratory (3, 9, 10), the majority of transformants have a single copy of plasmid integrated at attB. To avoid transformants with unusual developmental lacZ expression, we initially screened at least 10 transformants on TPM agar plates containing 40 ␮g of 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal)/ml. This was done for about half of the pREG1727 derivatives with ⍀4403 promoter mutations, and unusual color on TPM agar containing X-Gal was found to be rare, so we abandoned this screening step and randomly chose three transformants for quantitative ␤-galactosidase assays. In all cases, the three transformants gave similar results (Table 2) when developmental ␤-galactosidase specific activity was measured as described previously (28).

RESULTS Effects of mutations in the ⴚ35 or ⴚ10 region of the ⍀4403 promoter. Promoter recognition by RNA polymerase holoenzyme in bacteria often involves interaction between the sigma subunit and specific sequences centered at ⫺35 and ⫺10 bp relative to the transcription start site. To investigate whether this is the case for the ⍀4403 promoter, the sequence 5⬘-TTC ATG-3⬘ centered at ⫺33.5 bp was changed to 5⬘-GGACGT-3⬘, and separately, the sequence 5⬘-TACACC-3⬘ centered at ⫺9.5 bp was changed to 5⬘-GCACGG-3⬘. Each mutation, in the context of an otherwise wild-type promoter region spanning from ⫺80 bp to ⫹382 bp, was fused to the E. coli lacZ gene in a vector (pREG1727) that integrates efficiently by site-specific

J. BACTERIOL.

recombination into the M. xanthus chromosome. The resulting plasmids (Table 1) were introduced into M. xanthus strain DK1622 by electroporation, and transformants resistant to kanamycin (due to the aphII gene of the plasmid) were selected. Three independent transformants with each plasmid were induced to develop, and ␤-galactosidase specific activity was measured at the times indicated (Fig. 1). In parallel, developmental lacZ expression was measured for two control strains that were constructed in a similar way. One has the fully wild-type promoter region fused to the lacZ reporter, and the other has no promoter fused to lacZ. Table 2 lists the average maximum ␤-galactosidase specific activity during the 48 h developmental time course for all determinations made for each promoter construct. It also shows how that value compared to the maximum ␤-galactosidase specific activity observed for the wild-type promoter in the same experiment. The 6-bp change in the ⫺35 region reduced expression by about twofold compared to the wild-type promoter. The 6-bp change in the ⫺10 region abolished developmental promoter activity so that only a low basal level of lacZ expression remained, which was comparable to that observed for the vector without a promoter. These results demonstrate the importance of the ⫺10 region sequence for ⍀4403 promoter activity and show that the ⫺35 region can tolerate substitution with little loss in activity. Effects of mutations in a C box centered at ⴚ49 bp. It was noted previously that the ⍀4403 promoter has the sequence 5⬘-CATCCCT-3⬘ centered at ⫺49 bp, which matches the consensus sequence 5⬘-CAYYCCY-3⬘ for C boxes that are found in several C-signal-dependent promoters (9). To test whether this sequence is an important promoter element, single-basepair changes were made in each of the 7 bp of this C box. We chose to make drastic mutations: transversions that, for example, change a CG base pair to an AT base pair. Figure 2A shows the ␤-galactosidase specific activity during development of two mutants that exhibited significantly increased activity, and Fig. 2B summarizes the results for all the mutants. Changing C to A at ⫺47 bp or ⫺48 bp abolished promoter activity. In contrast, changing C to A at ⫺49 bp or ⫺52 bp increased lacZ expression relative to the wild-type promoter as early as 6 h into development and resulted in 2- to 3.6-fold-higher maximum ␤-galactosidase specific activity at 36 h of development. On the other hand, changing the C at ⫺49 bp to G abolished promoter activity. This mutation was made for a reason explained below. Changing T to G at ⫺50 bp also decreased promoter activity, in this case threefold, whereas the changes made at ⫺51 or ⫺46 bp had less than a twofold effect on developmental lacZ expression. Clearly, some changes can be tolerated in this C box with little effect on promoter activity, but most changes produce dramatic effects, usually abolishing promoter activity but in some cases increasing it significantly. Effects of mutations in a C-box-like sequence centered at ⴚ77 bp. Prior 5⬘ deletion analysis of the ⍀4403 promoter revealed a critical element between ⫺80 and ⫺72 bp (10). This region encompasses the sequence 5⬘-CATGCCA-3⬘ centered at ⫺77 bp in the opposite orientation relative to the C box centered at ⫺49 bp. Although this sequence matches the C-box consensus sequence (5⬘-CAYYCCY-3⬘) in only five out of seven positions, the two mismatched positions correspond to positions in the C box centered at ⫺49 that were shown to

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TABLE 1. Bacterial strains and plasmids used in this study Strain or plasmid

Relevant characteristic(s)

Source or reference

E. coli strain DH5␣

␾80 lacZ⌬M15 ⌬lacU169 recA1 endA1 hsdR17 supE44 thi-1 gyrA relA1

15

M. xanthus strains DK1622 DK4368 MPV1727-8 MPV100-2 MPV010R-5, -6, and -7 MPV035R-3, -6, and -8 MPV045-1, -2, and -3 MPV046-7, -8, and -10 MPV047-2, -6, and -9 MPV048-1, -5, and -8 MPV049-4, -6, and -10 MPV049A-5, -6, and -7 MPV050-1, -2, and -8 MPV051-2, -5, and -7 MPV052-1, -4, and -6 MPV053-2, -4, and -5 MPV05458-4, -5, and -8 MPV05458D-1, -6, and -7 MPV05963-2, -4, and -8 MPV059-1, -3, and -6 MPV060-1, -6, and -7 MPV061-2, -4, and -6 MPV062-1, -2, and -5 MPV063-4, -5, and -8 MPV06468-1, -3, and -5 MPV06972-3, -6, and -8 MPV069-1, -4, and -6 MPV070-1, -2, and -4 MPV071-1, -4, and -5 MPV072-1, -2, and -7 MPV073-2, -5, and -7 MPV074-5, -6, and -7 MPV075-4, -6, and -8 MPV076-1, -4, and -7 MPV077-2, -4, and -5 MPV077A-1, -6, and -7 MPV078-5, -7, and -8 MPV079-5, -7, and -8 MPV080-3, -5, and -6

Wild type Tn5 lac (Kmr) ⍀4403 attB::pREG1727 attB::pMF100 attB::pPV010R attB::pPV035R attB::pPV045 attB::pPV046 attB::pPV047 attB::pPV048 attB::pPV049 attB::pPV049A attB::pPV050 attB::pPV051 attB::pPV052 attB::pPV053 attB::pPV05458 attB::pPV05458D attB::pPV05963 attB::pPV059 attB::pPV060 attB::pPV061 attB::pPV062 attB::pPV063 attB::pPV06468 attB::pPV06972 attB::pPV069 attB::pPV070 attB::pPV071 attB::pPV072 attB::pPV073 attB::pPV074 attB::pPV075 attB::pPV076 attB::pPV077 attB::pPV077A attB::pPV078 attB::pPV079 attB::pPV080

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Plasmids pGEM-7Zf pREG1727 pMF100 pMF0100 pPV10R pPV010R pPV35R pPV035R pPV45 pPV045 pPV46 pPV046 pPV47 pPV047 pPV48 pPV048 pPV49 pPV049 pPV49A pPV049A pPV50 pPV050 pPV51 pPV051 pPV52

Apr lac␣ Apr Kmr P1-inc attP ⬘lacZ pREG1727 with 521-bp HaeII-BamHI fragment from pMF01 pGEM-7Zf with 537-bp XhoI-BamHI fragment from pMF100 pMF0100 with TACACC-to-GCACAA change in the ⫺10 region pREG1727 with 537-bp XhoI-BamHI fragment from pPV10R pMF0100 with TTCATG-to-GGACGT change in the ⫺35 region pREG1727 with 537-bp XhoI-BamHI fragment from pPV35R pMF0100 with T-to-G mutation at ⫺45 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV45 pMF0100 with T-to-G mutation at ⫺46 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV46 pMF0100 with C-to-A mutation at ⫺47 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV47 pMF0100 with C-to-A mutation at ⫺48 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV48 pMF0100 with C-to-A mutation at ⫺49 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV49 pMF0100 with C-to-G mutation at ⫺49 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV49A pMF0100 with T-to-G mutation at ⫺50 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV50 pMF0100 with A-to-C mutation at ⫺51 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV51 pMF0100 with C-to-A mutation at ⫺52 bp

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J. BACTERIOL. TABLE 1—Continued

Strain or plasmid

pPV052 pPV53 pPV053 pPV5458 pPV05458 pPV5458D pPV05458D pPV5963 pPV05963 pPV59 pPV059 pPV60 pPV060 pPV61 pPV061 pPV62 pPV062 pPV63 pPV063 pPV6468 pPV06468 pPV6972 pPV06972 pPV69 pPV069 pPV70 pPV070 pPV71 pPV071 pPV72 pPV072 pPV73 pPV073 pPV74 pPV074 pPV75 pPV075 pPV76 pPV076 pPV77 pPV077 pPV77A pPV077A pPV78 pPV078 pPV79 pPV079 pPV80 pPV080

Relevant characteristic(s)

pREG1727 with 537-bp XhoI-BamHI fragment from pPV52 pMF0100 with T-to-G mutation at ⫺53 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV53 pMF0100 with CCGTC-to-AATGA change at ⫺58 to ⫺54 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV5458 pMF0100 with CCGTC from ⫺58 to ⫺54 bp deleted pREG1727 with 532-bp XhoI-BamHI fragment from pPV5458D pMF0100 with GACCG-to-TCAAT change at ⫺63 to ⫺59 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV5963 pMF0100 with G-to-T mutation at ⫺59 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV59 pMF0100 with C-to-A mutation at ⫺60 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV60 pMF0100 with C-to-A mutation at ⫺61 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV61 pMF0100 with C-to-A mutation at ⫺62 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV62 pMF0100 with G-to-T mutation at ⫺63 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV63 pMF0100 with TCACG-to-GACAT change at ⫺68 to ⫺64 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV6468 pMF0100 with TCAA-to-GACC change at ⫺72 to ⫺69 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV6972 pMF0100 with A-to-C mutation at ⫺69 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV69 pMF0100 with A-to-C mutation at ⫺70 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV70 pMF0100 with C-to-A mutation at ⫺71 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV71 pMF0100 with T-to-G mutation at ⫺72 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV72 pMF0100 with T-to-G mutation at ⫺73 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV73 pMF0100 with G-to-T mutation at ⫺74 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV74 pMF0100 with T-to-G mutation at ⫺75 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV75 pMF0100 with A-to-C mutation at ⫺76 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV76 pMF0100 with C-to-A mutation at ⫺77 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV77 pMF0100 with C-to-T mutation at ⫺77 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV77A pMF0100 with G-to-T mutation at ⫺78 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV78 pMF0100 with G-to-T mutation at ⫺79 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV79 pMF0100 with T-to-G mutation at ⫺80 bp pREG1727 with 537-bp XhoI-BamHI fragment from pPV80

tolerate certain substitutions (Fig. 2B). Indeed, the reason we changed the C at ⫺49 bp not only to A, which resulted in increased promoter activity, but also to G was that the C-boxlike sequence has G at ⫺77 bp, and we thought that perhaps a purine (A or G) at the center of the C box might be optimal rather than pyrimidine. This was not the case, because changing C at ⫺49 bp to G abolished promoter activity (Fig. 2B). Clearly, the C box centered at ⫺49 bp exhibits a different nucleotide preference at the central position from that exhibited by the C-box-like sequence centered at ⫺77 bp. To examine the role of individual nucleotides in the C-boxlike sequence, drastic single-base-pair changes were made at each position. Nearly every mutation between ⫺80 and ⫺74 bp reduced developmental lacZ expression dramatically (Table

Source or reference

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2). The only exception was the T-to-G change at ⫺80 bp, which increased promoter activity slightly. The results are summarized in Fig. 3, with the appropriate strand and orientation of the C-box-like sequence shown in order to facilitate comparison with the effects of mutations on the C box centered at ⫺49 bp (Fig. 2B). In addition to the strikingly different nucleotide preference at the central position, other differences in the pattern of mutational effects are evident. For example, changing the C at ⫺74 bp to A abolished promoter activity, but the same change at ⫺52 bp caused a twofold increase in activity. Likewise, changing A at ⫺75 bp to C abolished activity, but the same change at ⫺51 bp increased activity slightly. These differences suggest that the C box centered at ⫺49 bp and the C-box-like sequence centered at ⫺77 bp function differently.

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TABLE 2. Activities of mutant 4403 promoters Promoter assayed

Avg maximum ␤-galactosidase sp act during developmenta

Vector only 4403 wild type 4403 mutantsc TACACC ⫺12 to ⫺7 GCACAA TTCATG ⫺36 to ⫺31 GGACGT C ⫺45 A T ⫺46 G C ⫺47 A C ⫺48 A C ⫺49 A C ⫺49 G T ⫺50 G A ⫺51 C C ⫺52 A T ⫺53 G CCGTC ⫺58 to ⫺54 AATGA CCGTC ⫺58 to ⫺54 ⌬ GACCG ⫺59 to ⫺63 TCAAT G ⫺59 T C ⫺60 A C ⫺61 A A ⫺62 C G ⫺63 T TCACG ⫺68 to ⫺64 GACAT TCAA ⫺72 to ⫺69 GACC A ⫺69 C A ⫺70 C C ⫺71 A T ⫺72 G T ⫺73 G G ⫺74 T T ⫺75 G A ⫺76 C C ⫺77 A C ⫺77 T G ⫺78 T G ⫺79 T T ⫺80 G

% Wild-type activity measured in the same exptb

7.7 ⫾ 2.5 29.6 ⫾ 11.2 6.5 ⫾ 0.3 22.1 ⫾ 1.4 80.7 ⫾ 7.1 42.9 ⫾ 5.9 6.6 ⫾ 1.3 6.4 ⫾ 1.0 111.8 ⫾ 17.6 6.4 ⫾ 0.1 26.2 ⫾ 2.4 40.6 ⫾ 4.1 64.2 ⫾ 17.7 9.6 ⫾ 0.6 66.7 ⫾ 4.6 5.1 ⫾ 0.3 8.6 ⫾ 0.3 5.1 ⫾ 0.6 62.5 ⫾ 15.9 6.1 ⫾ 0.4 5.6 ⫾ 0.2 9.4 ⫾ 0.7 53.7 ⫾ 15.4 8.4 ⫾ 0.2 31.7 ⫾ 5.6 4.9 ⫾ 0.7 17.6 ⫾ 1.7 6.7 ⫾ 1.0 7.6 ⫾ 1.2 9.2 ⫾ 1.8 9.8 ⫾ 1.4 26.1 ⫾ 4.0 20.6 ⫾ 2.0 9.1 ⫾ 0.6 26.3 ⫾ 2.1 20.7 ⫾ 3.0 43.9 ⫾ 6.8

0 57 ⫾ 5 201 ⫾ 19 60 ⫾ 11 4⫾5 3⫾3 362 ⫾ 60 0 30 ⫾ 4 120 ⫾ 14 200 ⫾ 60 7⫾2 163 ⫾ 13 0 2 ⫾ 0.9 0 181 ⫾ 51 1⫾1 0 12 ⫾ 2 150 ⫾ 51 1 ⫾ 0.8 83 ⫾ 18 0 38 ⫾ 6 3⫾3 2⫾3 0 0 30 ⫾ 7 20 ⫾ 4 0 30 ⫾ 4 20 ⫾ 6 131 ⫾ 23

a The maximum ␤-galactosidase specific activity in nanomoles of o-nitrophenyl phosphate per minute per milligram of protein (average plus or minus 1 standard deviation) is listed for three independently isolated M. xanthus transformants (one determination each) in the case of mutant promoters and for one isolate (five determinations) in the case of the wild-type promoter (MPV100-2) or vector only (MPV1727-8). Samples were assayed at 0, 6, 12, 18, 24, 30, 36, and 48 h during development. b The wild-type promoter and vector-only strains were included in each experiment. The maximum for each mutant promoter is expressed as a percentage of the maximum observed for the wild-type promoter in the same experiment, after subtracting from both values the maximum observed for vector only in that experiment. The average percentage plus or minus 1 standard deviation is listed. A zero in this column indicates that expression from the mutant promoter was equal to or slightly less than that observed for the vector only control. c For example, mutant TACACC ⫺12 to ⫺7 GCACAA has a mutation changing TACACC at positions ⫺12 to ⫺7 bp to GCACAA, mutant C ⫺45 A has a mutation changing C at position ⫺45 bp to A, and mutant CCGTC ⫺58 to ⫺54 ⌬ has a mutation deleting CCGTC at positions ⫺58 to ⫺54 bp.

For example, they may be recognized by different proteins or by different parts of a single protein that activate(s) transcription. Some similarities were observed in the pattern of mutational effects for the two sequences (Fig. 2B and 3). Changing the T at ⫺76 or ⫺50 bp to G decreased developmental lacZ expression threefold. Changing C at ⫺78, ⫺79, ⫺48, or ⫺47 bp to A decreased promoter activity threefold or more. Changing the A at ⫺80 bp to C produced a closer match to the C-box consensus sequence and resulted in a slight increase in activity, whereas substitution of G for T at ⫺46 bp created a mismatch to the consensus, resulting in slightly decreased activity. If the two sequences are bound by one or more transcription factors, these similarities in the pattern of mutational effects suggest that part of the DNA-binding surfaces of the protein or proteins that interact with the two sequences may share some common features.

Mutational analysis of the region between the C box and the C-box-like sequence. To determine whether other sequences in the ⍀4403 region are important for promoter activity, additional mutations were made and tested as described above. The analysis proceeded in a stepwise fashion. In the first step, the base pairs adjacent to the C box centered at ⫺49 bp and the C-box-like sequence centered at ⫺77 bp were changed. As summarized in Fig. 4, changing the C at ⫺45 bp to A increased promoter activity twofold. Changing the T at ⫺53 bp to G or the T at ⫺73 bp to G abolished promoter activity. This indicated that some sequences between the C box and the C-boxlike element are important for developmental expression of ⍀4403. In the second step of the analysis, 5- or 4-bp changes were made in the region between the C box and the C-box-like sequence. The results are summarized in Fig. 4. Interestingly,

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FIG. 1. Effects of 6-bp changes in the ⫺35 or ⫺10 region of the ⍀4403 promoter. Developmental ␤-galactosidase specific activity was measured for three independent transformants of M. xanthus DK1622 with pPV035R or pPV010R bearing mutations in the ⫺35 (⽧) or ⫺10 region (■) region, respectively. Points show the average ␤-galactosidase specific activity in nanomoles of o-nitrophenyl phosphate per minute per milligram of protein. Error bars show 1 standard deviation of the data. MPV1727-8 with the vector bearing no insert (E) and MPV100-2 with the wild-type promoter (F) were included as controls.

the 5-bp changes at ⫺54 to ⫺58 bp and ⫺64 to ⫺68 bp increased promoter activity 1.6- and 1.5-fold, respectively, and in the case of the 5-bp change at ⫺54 to ⫺58 bp caused lacZ expression to increase significantly earlier during development than observed for the wild-type promoter (Fig. 5). In contrast, a 5-bp change at ⫺59 to ⫺63 bp or a 4-bp change at ⫺69 to ⫺72 bp abolished activity (Fig. 4). Clearly, sequences crucial for activity are present between the C box and the C-box-like elements. The results suggest that essential elements may be separated by nonessential elements with a 5-bp periodicity that would be consistent with a positive-acting transcription factor(s) recognizing one face of the DNA helix. In the third step of the analysis, we sought to identify critical base pairs in the regions from ⫺59 to ⫺63 bp and from ⫺69 to ⫺72 bp. Single-base-pair changes in the ⫺59 to ⫺63 bp region showed that every base pair was essential for promoter activity except the base pair at ⫺60, where a C-to-A change altered the timing of developmental lacZ expression and increased the maximum activity 1.8-fold (Fig. 6 and Table 2). Single-basepair changes in the ⫺69- to ⫺72-bp region had a variety of effects. Changing the T at ⫺72 bp to G or the A at ⫺70 bp to C abolished activity, while changing the C at ⫺71 bp to A reduced activity about twofold (Table 2). Changing A at ⫺69 bp to C had little effect on the maximum level of activity attained but allowed earlier expression of ⍀4403 during development (Fig. 6). To determine if spacing between elements is important in the ⍀4403 promoter region, the 5 bp from ⫺54 to ⫺58 bp was deleted. Promoter activity was abolished by this deletion (Table 2), demonstrating the importance of spacing between the C box centered at ⫺49 bp and promoter elements located farther upstream. Figure 7 summarizes the effects on ⍀4403 promoter activity of most of the mutations made in this study.

FIG. 2. Effects of mutations in the C-box-centered ⫺49 bp in the ⍀4403 promoter region. (A) Developmental lacZ expression was determined for three independently isolated M. xanthus DK1622 transformants bearing each mutation. The single-base-pair changes were C to A at ⫺52 bp (⽧) or C to A at ⫺49 bp (Œ). Points show the average ␤-galactosidase specific activity in nanomoles of o-nitrophenyl phosphate per minute per milligram of protein. Error bars show 1 standard deviation of the data. MPV1727-8 with the vector bearing no insert (E) and MPV100-2 with the wild-type promoter (F) were included as controls. (B) Summary of the effects of single-base-pair changes. Upward- or downward-pointing arrows indicate increased or decreased developmental lacZ expression, respectively, caused by the given mutation. Numbers indicate the maximum average ␤-galactosidase specific activity observed for the mutant during 48 h of development with samples taken at the times indicated in panel A, expressed as a percentage of the maximum observed for the wild-type promoter in the same experiment (Table 2).

DISCUSSION Our mutational analysis of the ⍀4403 promoter region provides several important insights. First, the ⫺10 region sequence is essential for activity, but the ⫺35 region tolerated a 6-bp substitution with a less-than-twofold loss in activity. Second, the C box centered at ⫺49 bp contains certain base pairs essential for developmental promoter activity, confirming the idea proposed previously that it is a cis-acting regulatory element important for expression of this C-signal-dependent gene (9). Third, other sequences between the C box and ⫺79 bp are also important for developmental expression of ⍀4403. Below, we discuss these insights further and we compare sequences in

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FIG. 3. Effects of mutations in the C-box-like sequence centered at ⫺77 bp in the ⍀4403 promoter. The C-box-like sequence is in the opposite orientation relative to the C box centered at ⫺49 bp, so the noncoding DNA strand is shown between ⫺74 and ⫺80 bp, to facilitate comparison with Fig. 2B, in which the coding strand is shown between ⫺52 and ⫺46 bp. The meaning of arrows and numbers is explained in the Fig. 2B legend.

other M. xanthus developmental promoters that our detailed analysis of the ⍀4403 promoter suggests might be common cis-acting elements. It was noted previously that the ⍀4403 promoter differs considerably from vegetatively expressed promoters in the sequences centered at ⫺35 and ⫺10 bp (10). However, the ⫺10 region of the ⍀4403 promoter does match the developmentally regulated ⍀4400 promoter in five out of six positions (3). Our results show that the sequence in this region of the ⍀4403 promoter is essential for activity (Fig. 1). In contrast to most bacterial promoters, we found that the sequence of the ⫺35 region could be drastically altered with little loss in activity. One type of promoter characterized in E. coli does not require sigma factor recognition of the ⫺35 region. These “extended ⫺10 region” promoters have the sequence 5⬘-TG-3⬘ immediately upstream of the ⫺10 hexanucleotide (1). Interestingly, the ⍀4403 promoter has this sequence just upstream of its ⫺10 region, so it could fall into this class of promoters. Mutational analysis of the C box centered at ⫺49 bp in the ⍀4403 promoter demonstrated that it is an essential cis-acting element. The ⍀4400 promoter has the identical 7-bp sequence (5⬘-CATCCCT-3⬘) at the identical position (3). Changing this sequence to 5⬘-ACGAAAG-3⬘ abolished ⍀4400 promoter activity (G. Velicer and L. Kroos, unpublished data). It will be interesting to compare the effects of single-base-pair mutations in this C box in the two promoters. Single-base-pair changes in the C box centered at ⫺49 bp in the ⍀4403 promoter yielded some results that could have been

FIG. 4. Summary of the effects of single-base-pair changes adjacent to the C box and the C-box-like sequences, and multiple-base-pair changes between these two elements. The meaning of the arrows and numbers is the same as that given for Fig. 2B.

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FIG. 5. Developmental lacZ expression for M. xanthus DK1622 transformants with a multiple-base-pair mutation in the ⫺54 to ⫺58 bp region of the ⍀4403 promoter. The 5-bp change indicated in Fig. 4 was made, and ␤-galactosidase specific activity during development was determined for three independently isolated transformants (⽧). Points show the average ␤-galactosidase specific activity in nanomoles of o-nitrophenyl phosphate per minute per milligram of protein. Error bars show 1 standard deviation of the data. MPV1727-8 with the vector bearing no insert (E) and MPV100-2 with the wild-type promoter (F) were included as controls.

predicted based on the C box consensus sequence but also yielded other results that change our view of the C box. The consensus proposed previously, 5⬘-CAYYCCY-3⬘, was based on nine sequences found in the promoter regions of five different M. xanthus developmental genes (9). Based on this consensus sequence, one might have predicted that changing the T at ⫺50 bp (corresponding to Y in the consensus) to G would impair ⍀4403 promoter activity, and this was observed (Fig. 2 and summarized in Fig. 7). Likewise, the 5⬘-CC-3⬘ sequence at ⫺48 to ⫺47 bp (matching the 5⬘-CC-3⬘ sequence in the consensus) was shown to be crucial for activity. On the other hand, the 5⬘-CA-3⬘ sequence at ⫺52 to ⫺51 bp (matching the 5⬘CA-3⬘ sequence in the consensus) tolerated a C-to-A change at ⫺52 bp or an A-to-C change at ⫺51 bp. Also, changing the T

FIG. 6. Developmental lacZ expression for M. xanthus DK1622 transformants with single-base-pair changes at ⫺60 (■) or ⫺69 (⽧) bp of the ⍀4403 promoter. ␤-Galactosidase specific activity during development was determined for three independently isolated transformants. Points show the average ␤-galactosidase specific activity in nanomoles of o-nitrophenyl phosphate per minute per milligram of protein. Error bars show 1 standard deviation of the data. MPV1727-8 with the vector bearing no insert (E) and MPV100-2 with the wild-type promoter (F) were included as controls.

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FIG. 7. Summary of mutational effects on developmental expression of the ⍀4403 promoter. Boxed regions were subjected to multiple-basepair changes or deletion (‚) as indicated. Arrows and numbers have the same meaning as that given for those in Fig. 2B. The C box centered at ⫺49 bp, the 5-bp element centered at ⫺61 bp, and the 10-bp element centered at ⫺74.5 bp are underlined. Multiple-base-pair changes in the ⫺59 to ⫺63 and ⫺69 to ⫺72 regions also abolished promoter activity, as indicated in the Fig. 4 legend, but the effects of single-base-pair changes in these regions are summarized in this figure.

at ⫺46 bp (corresponding to Y in the consensus) to G caused only a small decrease (less than twofold) in promoter activity. Most surprising was the 3.6-fold increase in developmental promoter activity when the C at ⫺49 bp (corresponding to Y in the consensus) was changed to A. Yet when G was substituted for C at this position, promoter activity was lost completely. Based on this mutational analysis, a more degenerate consensus for the C box can be proposed: (C/A)(A/C)Y(C/A)CC(T /G). Further mutational analyses of this and other C boxes will be necessary to determine whether the consensus is even more degenerate and whether it is a reliable indicator of cis-acting regulatory sequences. The 3.6-fold increase in developmental promoter activity observed when the C at ⫺49 bp was changed to A deserves further comment. The change might increase the affinity of a positive regulatory transcription factor for the C box. The same interpretation could apply to the twofold increase in promoter activity observed when the C at ⫺52 bp was changed to A. In both cases, a more complex explanation would be that the C box is a site for binding of both positive and negative regulatory factors and that mutations that increase developmental promoter activity impair binding of the negative factor. The C-box-like sequence centered at ⫺77 bp in the ⍀4403 promoter showed a very different pattern of effects upon mutation from that shown by the C box centered at ⫺49 bp, suggesting that it should not be considered a C box. For example, the C box centered at ⫺49 bp tolerated A or C at ⫺52 or ⫺51 bp (Fig. 2B), but the corresponding changes in the C-box-like sequence abolished promoter activity (Fig. 3). Also, the two sequences were very different with respect to the effect of the base pairs at the center of the 7-bp sequence. These differences suggest that the two sequences may be recognized by different transcriptional activator proteins or by different parts of the same protein. If the two sequences are recognized by different activator proteins, binding of both must be required because single-base-pair changes in either sequence can eliminate developmental expression of ⍀4403. The C-box-like sequence might better be thought of as a

10-bp element, 5⬘-GGCATGTTCA-3⬘, extending from ⫺79 to ⫺70 bp, because single-base-pair changes at every position in this sequence decrease ⍀4403 promoter activity more than twofold (Fig. 7). This 10-bp sequence is not found elsewhere in the partial M. xanthus genome sequence in the Cereon Microbial Sequence Database (http://microbial.cereon.com). The database contains 9.1 Mbp of unique sequence. The 10-bp sequence would be expected to occur once every 2.1 Mbp in random sequence with 68% G⫹C content, which is the composition of the database sequence. Therefore, the 10-bp element appears to be present in the genome at slightly less than the frequency expected based on chance alone. However, most recognition sites in DNA can function with one or more base pair substitutions, and several single-base-pair changes in the 10-bp element did not abolish ⍀4403 promoter activity (Fig. 7). If we allow one mismatch to the 10-bp sequence, we find 737 sequences in the partial M. xanthus genome sequence. Only about 20 such sequences would be expected based on chance alone, so sequences closely related to the 10-bp element may be regulatory elements of many other genes. Other sequences important for developmental expression of ⍀4403 lie between the 10-bp element and the C box centered at ⫺49 bp. Immediately upstream of the C box at ⫺53 bp is a T that when changed to G resulted in almost complete loss of promoter activity (Fig. 7). Examination of the eight other C boxes noted previously (9) revealed only one with a T immediately upstream, so the importance of this base pair comes as a surprise. Also surprising was the finding of a 5-bp element, 5⬘-GACCG-3⬘, at ⫺63 to ⫺59 bp, in which single-base-pair changes at 4 positions abolished promoter activity (Fig. 7). This 5-bp element is 6 bp downstream of the 10-bp element and 6 bp upstream of the C box. The three essential elements are separated by 5-bp sequences that can be changed with little or no loss of promoter activity (Fig. 7). Hence, the three elements are spaced so that at least part of each is on the same face of the DNA helix. Altering the spacing between the 5-bp element and the C box by deleting 5 bp in the middle resulted in a complete loss of promoter activity (Fig. 7). This could

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FIG. 8. Sequences similar to the 5- and 10-bp elements of the ⍀4403 promoter in other promoters that have been reported to be C signal dependent. (A) Alignment of the 5-bp element and the C box of the ⍀4403 promoter with similar sequences in the ⍀4400 (3), ⍀4499 (9), csgA (31), and fruA (32) promoters. Numbers indicate the position relative to the start site of transcription. The 5-bp elements and C boxes are in boldface type. (B) Alignment of 10-bp elements. None was found at a similar position in the fruA promoter. Note that the sequence shown for the ⍀4499 promoter is in the opposite orientation relative to the start site of transcription, compared to the others. Boldface type indicates base pairs that are perfectly conserved.

mean that a single protein binds to the two sequences on one face of the DNA helix or that two different proteins bind cooperatively to the two sequences and interact on the DNA. Examination of other promoters that have been reported to be dependent on C signaling for expression suggests that a 5-bp element located 5 to 7 bp upstream of a C box may be a conserved feature. Figure 8A shows a comparison of the ⍀4403 promoter region with other promoters. Although similarity of the 5-bp elements is not as high as for the C boxes, it is higher than for the regions between the two sequences at three out of five positions in the 5-bp element. Moreover, changing 5⬘-GAAC-3⬘ to 5⬘-TCCA-3⬘ in the 5-bp element of the ⍀4400 or ⍀4499 promoter abolished developmental expression (D. Yoder and L. Kroos, unpublished data). Even so, there is reason to believe that the function of the 5-bp elements in the different promoters is not identical. If it were, we would not expect the single-base-pair changes that we made in the 5-bp element of the ⍀4403 promoter to abolish promoter activity, as was observed (Fig. 7), because one or more of the 5-bp elements in the other promoters (Fig. 8A) has the base pairs that abolished ⍀4403 activity when present at the corresponding position. It is also worth noting that all of the 5-bp elements have a C at the fourth position from the 5⬘ end (Fig. 8A), yet changing that C (at ⫺60 bp) to A did not abolish promoter activity. Rather, it caused earlier expression during development and increased the maximum activity about twofold (Fig. 6). Figure 8B shows that other C-signal-dependent promoters exhibit sequences similar to the 10-bp element of the ⍀4403 promoter. These sequences are located 9 to 12 bp upstream of the 5-bp element, slightly farther upstream than the 10-bp element in the ⍀4403 promoter. The sequence in the csgA promoter matches that in the ⍀4403 promoter at the first seven positions from the 5⬘ end. The sequence in the ⍀4400 promoter matches that in the csgA promoter at six of the first eight positions from the 5⬘ end of the csgA sequence. The ⍀4400

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sequence is extended 2 bp farther 5⬘ to illustrate that it matches a sequence found in the opposite orientation in the ⍀4499 promoter in eight of the first nine positions from the 5⬘ end, as noted previously (9). The significance of these sequence similarities remains to be tested. In the case of the ⍀4400 promoter, a 5⬘ deletion to ⫺101 bp showed developmental expression and a 5⬘ deletion to ⫺76 bp eliminated expression (3), consistent with the idea that the sequence between ⫺84 and ⫺73 bp (Fig. 8B) might be an essential promoter element. The similar arrangement of the C box, 5-bp element, and 10-bp element in the ⍀4403, ⍀4400, ⍀4499, and csgA promoters suggests some similarity in the regulatory mechanisms employed. For example, similar or identical transcriptional activator proteins might recognize one or more of these sequences. If more than one protein is involved at the ⍀4403 promoter, the proteins must bind or activate transcription in a cooperative, rather than an additive, fashion because certain single-base-pair changes in any one of the 3 elements abolished promoter activity (Fig. 7). Despite the similar arrangement of cis elements in the promoters listed in Fig. 8, regulation of these promoters is not identical. Synthesis of FruA (17) or expression of a fruA-lacZ fusion (8) is only slightly reduced at 8 to 12 h during development of a csgA mutant, so transcription from the fruA promoter appears to be less dependent on C signaling than is transcription from the other promoters listed in Fig. 8. Unlike the other promoters, the fruA promoter does not appear to have the 10-bp element. As noted above, the function of the 5-bp element does not appear to be identical in the different promoters. Likewise, differences in the 10-bp element sequences listed in Fig. 8B and/or differences in the number, position, or sequence of C boxes (9) may explain why the ⍀4403 promoter is expressed later during development and depends more strongly on C signaling than the ⍀4400, ⍀4499, and csgA promoters (27, 31). Mutational analyses of other C-signal-dependent promoters, guided by the results presented here, should provide insight into how differential gene expression in response to C signaling is achieved. Our results also lay the foundation for identification of the proteins that regulate these promoters during development. ACKNOWLEDGMENTS We thank D. Yoder for valuable discussions and technical assistance. We are grateful to the Monsanto Company and its subsidiary Cereon Genomics for providing access to their Microbial Sequence Database. We thank R. Welch, J. Jakobsen, and D. Kaiser for providing a pseudogenome sequence, generated from sequence information supplied by Cereon Genomics, that was used to search for sequence similarities, and we thank R. Halgren of the Michigan State University Genomics Technology Support Facility for performing the searches. This research was supported by NSF grant MCB-0090478 and by the Michigan Agricultural Experiment Station. REFERENCES 1. Barne, K. A., J. A. Bown, S. J. W. Busby, and S. D. Minchin. 1997. Region 2.5 of the Escherichia coli RNA polymerase ␴70 subunit is responsible for the recognition of the ⬘extended -10⬘ motif at promoters. EMBO J. 16:4034– 4040. 2. Biran, D., and L. Kroos. 1997. In vitro transcription of Myxococcus xanthus genes with RNA polymerase containing ␴A, the major sigma factor in growing cells. Mol. Microbiol. 25:463–472. 3. Brandner, J. P., and L. Kroos. 1998. Identification of the ⍀4400 regulatory region, a developmental promoter of Myxococcus xanthus. J. Bacteriol. 180: 1995–2004.

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