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. 2007 May;189(10):3738-50.
doi: 10.1128/JB.00187-07. Epub 2007 Mar 16.

Regulation of dev, an operon that includes genes essential for Myxococcus xanthus development and CRISPR-associated genes and repeats

Poorna Viswanathan  1 , Kimberly Murphy, Bryan Julien, Anthony G Garza, Lee Kroos
Affiliations

Regulation of dev, an operon that includes genes essential for Myxococcus xanthus development and CRISPR-associated genes and repeats

Poorna Viswanathan et al. J Bacteriol. 2007 May.

Abstract

Expression of dev genes is important for triggering spore differentiation inside Myxococcus xanthus fruiting bodies. DNA sequence analysis suggested that dev and cas (CRISPR-associated) genes are cotranscribed at the dev locus, which is adjacent to CRISPR (clustered regularly interspaced short palindromic repeats). Analysis of RNA from developing M. xanthus confirmed that dev and cas genes are cotranscribed with a short upstream gene and at least two repeats of the downstream CRISPR, forming the dev operon. The operon is subject to strong, negative autoregulation during development by DevS. The dev promoter was identified. Its -35 and -10 regions resemble those recognized by M. xanthus sigma(A) RNA polymerase, the homolog of Escherichia coli sigma(70), but the spacer may be too long (20 bp); there is very little expression during growth. Induction during development relies on at least two positive regulatory elements located in the coding region of the next gene upstream. At least two positive regulatory elements and one negative element lie downstream of the dev promoter, such that the region controlling dev expression spans more than 1 kb. The results of testing different fragments for dev promoter activity in wild-type and devS mutant backgrounds strongly suggest that upstream and downstream regulatory elements interact functionally. Strikingly, the 37-bp sequence between the two CRISPR repeats that, minimally, are cotranscribed with dev and cas genes exactly matches a sequence in the bacteriophage Mx8 intP gene, which encodes a form of the integrase needed for lysogenization of M. xanthus.

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Figures

FIG. 1.
FIG. 1.
Map of the dev locus, expanded view of the dev promoter region, depiction of fragments fused to lacZ, and results of measuring developmental lacZ expression in a devS mutant. The top part shows a map of the dev locus with boxes indicating genes (20). The right-angle arrow indicates the TSS. CRISPR is explained in the text. Below is an expanded map of a 1,768-bp fragment spanning the dev promoter region. Numbers below the map are relative to the TSS (+1) and indicate the ends of the fragment or the ends of genes. Below the expanded map is a depiction of fragments fused to lacZ in the correct orientation to detect dev promoter activity. Numbers indicate fragment ends relative to +1. To the right, the maximum β-galactosidase specific activity among samples harvested at 0, 6, 12, 18, 24, 30, 36, and 48 h into development is shown for each fusion in the M. xanthus ΔdevS mutant DK11209. The numbers are the average β-galactosidase specific activities for three independently isolated transformants (1 standard deviation of the data is indicated) expressed in nanomoles of o-nitrophenyl phosphate per minute per milligram of protein.
FIG. 2.
FIG. 2.
Developmental lacZ expression from the 1,768-bp fragment in wild-type M. xanthus and a devS mutant. The fragment spanning from −934 to +834 was inserted into pREG1727 in the correct orientation to fuse transcription of the dev promoter to lacZ. The resulting plasmid was transformed into M. xanthus wild-type DK1622 (○) and ΔdevS mutant DK11209 (□), and developmental lacZ expression was determined for three independent transformants. As a control, developmental lacZ expression was determined for three colonies of M. xanthus DK5279 (•), which harbors Tn5 lac Ω4414 inserted in devR. The graph shows the average β-galactosidase specific activity expressed in nanomoles of o-nitrophenyl phosphate per minute per milligram of protein, and error bars show 1 standard deviation of the data. The error bars are too small to be seen for some data points.
FIG. 3.
FIG. 3.
RT-PCR analysis of the dev transcript. RNA prepared from M. xanthus wild-type DK1622 at 18 h into development (lanes 3 to 20) or from the ΔdevS mutant DK11209 at 23 h (lanes 1 and 2) was subjected to RT-PCR (odd-numbered lanes) or, as a control, only to PCR (even-numbered lanes). The primers were designed to detect transcription across the following junctions (lanes and predicted PCR product size in parenthesis): the MXAN_7266 gene and cas6 (lanes 1 to 4; 510 bp), cas6 and cas3 (lanes 5 and 6; 260 bp), cas3 and devT (lanes 7 and 8; 420 bp), devT and devR (lanes 9 and 10; 280 bp), devR and devS (lanes 11 and 12; 180 bp), devS and cas4-cas1 (lanes 13 and 14; 220 bp), cas4-cas1 and cas2 (lanes 15 and 16; 200 bp), cas2 and the first repeat of CRISPR plus the first unique insert (lanes 17 and 18, 320 bp), and cas2 and the second repeat of CRISPR plus the second unique insert (lanes 19 and 20; 400 bp). Lane M contains the 100-bp ladder (New England Biolabs).
FIG. 4.
FIG. 4.
Mapping the 5′ end of the dev developmental transcript by primer extension analysis. RNA was isolated from M. xanthus wild-type DK1622 cells that had undergone 12 h of development in submerged culture. The primer OAG477 hybridized to +71 to +90 relative to the mRNA 5′ end mapped in this experiment. The extension product in lane PE is indicated by an arrow. The other lanes show DNA sequencing products generated with primer OAG477. A portion of the DNA sequence is shown to the left, with the inferred TSS indicated by a right-angle arrow.
FIG. 5.
FIG. 5.
Developmental lacZ expression from 5′ deletions. Fragments spanning from −535 (▪), −114 (▴), or +14 (⋄) to +834 were inserted into pREG1727, the resulting plasmids were transformed into M. xanthus ΔdevS mutant DK11209, and developmental lacZ expression was determined for three independent transformants. The data for the 1,768-bp fragment from −934 to +834 (same as in Fig. 2) are shown for comparison (□). The meaning of points and error bars is the same as described in the Fig. 2 legend. The error bars are too small to be seen for some data points.
FIG. 6.
FIG. 6.
Mutational analysis identifies the dev promoter. The top part shows the sequence upstream of the inferred TSS, which is indicated by the right-angle arrow. Sequences that match the E. coli σ70 consensus promoter −35 and −10 regions (62) are in boldface. The putative dev promoter −35 and −10 regions are underlined, and the 6-bp mutation made in each region is indicated. Below is shown developmental lacZ expression from fragments that span from −934 to +581, after insertion into pREG1727 and transformation into M. xanthus ΔdevS mutant DK11209. Expression from three independent transformants bearing the wild-type promoter (▪), the −35 region mutation (▴), or the −10 region mutation (○) was measured. Likewise, developmental lacZ expression was measured for a fragment spanning from −934 to +833 (•). The data for the 1,768-bp fragment from −934 to +834 (same as in Fig. 2) are shown for comparison (□). The meaning of points and error bars is the same as described in the Fig. 2 legend. The error bars are too small to be seen for some data points.
FIG. 7.
FIG. 7.
Developmental lacZ expression from 3′ deletions. Fragments spanning from −934 to +280 (▴), +219 (•), or +32 (▵) were inserted into pREG1727, the resulting plasmids were transformed into M. xanthus ΔdevS mutant DK11209, and developmental lacZ expression was determined for three independent transformants. The meaning of points and error bars is the same as described in the Fig. 2 legend. The error bars are too small to be seen for some of the points.
FIG. 8.
FIG. 8.
Developmental lacZ expression from a MXAN_7266 gene-lacZ translational fusion and a short fragment bearing the dev promoter. The fragment spanning from −934 to +71 was inserted into pMC1403KmattPTT (▵) and pREG1727 (▪), creating translational and transcriptional fusions to lacZ, respectively. The fragment spanning from −114 to +71 was also inserted into pREG1727 (○). The resulting plasmids were transformed into M. xanthus ΔdevS mutant DK11209, and developmental lacZ expression was determined for three independent transformants. The data for the fragment from −934 to +833 (same as in Fig. 6) are shown for comparison (•). The meaning of points and error bars is the same as described in the Fig. 2 legend.
FIG. 9.
FIG. 9.
Developmental lacZ expression from dev promoter fragments in wild-type M. xanthus. A fragment spanning from −114 to +71 (▪) was inserted into pREG1727, the resulting plasmid was transformed into M. xanthus wild-type DK1622, and developmental lacZ expression was determined for three independent transformants. The data for the 1,768-bp fragment from −934 to +834 (same as in Fig. 2) are shown for comparison (○). The meaning of points and error bars is the same as described in the Fig. 2 legend. The error bars are too small to be seen for some of the points.
FIG. 10.
FIG. 10.
Sequences upstream of the dev promoter predicted to be important for its activity. Mirror repeats are underlined in a 17-bp sequence centered at −91. A sequence shown in boldface and centered at −60 is similar to 5-bp elements in other developmentally regulated M. xanthus promoter regions (see Discussion for references). Likewise, two C-box-like sequences are shown in boldface with underlining, centered at −51 and −47.
FIG. 11.
FIG. 11.
Summary of regulatory elements inferred from 5′ and 3′ deletion analyses of the dev promoter region. Numbers indicate the difference (n-fold) in maximum developmental lacZ expression in a ΔdevS mutant (Fig. 1) attributable to the indicated segment. Arrows indicate positive effects, and the dashed line with a barred end indicates a negative effect. Upstream regulatory elements likely affect transcriptional initiation, whereas downstream elements could alternatively or in addition affect postinitiation events.
FIG. 12.
FIG. 12.
Matches between unique inserts in the CRISPR at the dev locus and other sequences. (A) The antisense strand (top) of the insert between the first two repeats downstream of cas2 (20) matches the sense strand (bottom) of the bacteriophage Mx8 intP gene (89). (B) Part of the insert between the 23rd and 24th repeats downstream of cas2 matches the MXAN_7283 gene (20). The insert between the 23rd and 24th repeats downstream of cas2 contains the MXAN_7258 gene in the same orientation as dev and cas genes. The top sequence is from upstream of the MXAN_7258 gene and would be the antisense strand if transcription from either the dev promoter or another promoter in the same orientation proceeds across this region. The bottom sequence is from the sense strand of the MXAN_7283 gene. Numbers are from the cited references.
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