JOURNAL OF BACTERIOLOGY, Jan. 1990, p. 80-85

Vol. 172, No. 1

0021-9193/90/010080-06$02.00/0 Copyright C) 1990, American Society for Microbiology

Cloning and DNA Sequence of the Gene Coding for the Major Sigma Factor from Myxococcus xanthus SUMIKO INOUYE

Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 Received 24 July 1989/Accepted 28 September 1989

The gene for a sigma factor (rpoD) was cloned from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon starvation for nutrients. The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide of 708 amino acid residues (Mr = 80,391) was identified. Except for the amino-terminal sequence consisting of 100 residues, the M. xanthus sigma factor (sigma-80) showed extensive similarity with Escherichia coli sigma-70 as well as BaciUus subtilis sigma-43. In particular, the carboxy-terminal sequence of 242 residues that is known to be required for promoter recognition and core recognition showed 78 and 72% amino acid sequence identity with the E. coli and B. subtilis sigma factors, respectively. The putative RpoD protein was detected at the position of an apparent molecular weight of 86,000 by Western blot (immunoblot) analysis by using antiserum against B. subtilis sigma-43, which agreed well with the position of a vegetative sigma factor of M. xanthus previously identified by Rudd and Zusman (K. Rudd and D. R. Zusman, J. Bacteriol. 151:89-105, 1982).

Myxococcus xanthus is a gram-negative bacterium that lives in soil and moves by gliding on solid surfaces. Upon nutritional starvation, cells aggregate and form fruiting bodies, in which rod-shaped vegetative cells change to round or ovoid myxospores (for a review, see reference 23). During fruiting body formation, dramatic changes in the pattern of protein synthesis have been reported (12). The most striking change in the pattern of protein synthesis is the appearance of protein S (12, 13), whose synthesis is induced early in development and increases until it reaches 15% of total protein synthesis at a later stage of development (13). In addition to the gene for protein S, several other genes expressed either developmentally or vegetatively were cloned and characterized (5, 7, 8, 16, 22, 30, 32, 33). Kroos and Kaiser (17, 18) constructed a TnS lac fusion, a transposon that was able to transpose the lacZ gene under an M. xanthus promoter (17, 18). Using this system, they were able to isolate various LacZ+ cells in which lacZ expression was developmentally regulated. These results suggest that fruiting body formation is controlled by expression of stagespecific developmental genes. The sigma subunit of RNA polymerase has been shown to play an important role both in the selective binding of polymerase to promoters and in the efficient initiation of transcription in bacteria (6, 11). It is known that one major and several minor sigma factors are present in Bacillus subtilis and that they play an important role in its differentiation (6, 11, 19). In M. xanthus, RNA polymerase has been purified from vegetative cells and its sigma factors have been identified (25). It has been also shown that M. xanthus and Escherichia coli RNA polymerases are able to recognize common promoters (25). Furthermore, two promoters of M. xanthus have been identified by S1 nuclease mapping (14, 16). These promoter sequences showed significant sequence homology to the E. coli consensus promoter sequence. In this study the rpoD gene of M. xanthus was cloned by using the E. coli rpoD gene as a probe and the M. xanthus rpoD gene DNA sequence was determined.

MATERIALS AND METHODS Materials. Restriction enzymes were purchased from either Bethesda Research Laboratories, Inc. (Gaithersburg, Md.), or New England BioLabs, Inc. (Beverly, Mass.). T4 DNA ligase was from Bethesda Research Laboratories, and the DNA polymerase I Klenow fragment was from Boehringer Mannheim Biochemicals (Indianapolis, Ind.). [Co-32P] dCTP was from Amersham Corp. (Arlington Heights, Ill.). Polyvinylidene difluoride membrane was purchased from Millipore Corp. (Bedford, Mass.). Cells and growth conditions. M. xanthus FB (DZF1) was grown in CYE medium (4). E. coli JA221 lpp(F' lacIP) (20) and E. coli JM83 and JM105 (36) were used for cloning chromosomal DNA fragments. E. coli JA221 was used for Western blot (immunoblot) analysis (34). Plasmids and phage. pBR322 (21) was used to clone BamHI-digested M. xanthus chromosomal DNA, and pUC9 (35) was used to subclone various restriction enzyme-digested DNA fragments. In order to determine the DNA sequence, fragments generated by restriction enzyme digestion were cloned into M13mpl8 and M13mpl9 (36). DNA manipulations and DNA sequencing. The chromosomal DNA was prepared as described previously (37). Plasmid isolation was performed as originally described by Birnboim and Doly (1). Blot hybridization analysis was carried out by the method of Southern (29) by using the following conditions: 40% formamide, 5 x SSPE (1 x SSPE is 180 mM NaCl, 10 mM Na2HPO4 [pH 7.4], 1 mM EDTA [pH 7.4]), 0.5% sodium dodecyl sulfate, 5 x Denhardt solution, and 106 cpm of 32P-labeled probe per ml at 37°C. The DNA sequence was determined by the chain-termination method by using 7-deaza dGTP (27). Western blot analysis. M. xanthus DZF1, E. coli JA221, and B. subtilis BR16 were grown to the mid-log phase and harvested by microcentrifugation. Pelleted cells were suspended in loading buffer (80 mM Tris hydrochloride [pH 6.8], 2% sodium dodecyl sulfate, 0.1 M P-mercaptoethanol, 10% glycerol, 0.12% bromphenol blue), sonicated for 2 min, 80

VOL. 172, 1990

81

MAJOR SIGMA FACTOR OF M. XANTHUS 1 2 3 4

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20-

FIG. 1. Southern blot analysis of the chromosomal DNA from M. xanthus DZF1, with nick-translated E. coli rpoD used as a probe. The DNA was digested with EcoRI (lane 1), BamHI (lane 2), PstI (lane 3), Sall (lane 4), XhoI (lane 5), and SmaI (lane 6). For each lane, 3 jig of the DNA digest was applied, and blot hybridization analysis was carried out as described in the text. Numbers at the left represent A DNA digested with Hindlll as the molecular weight markers (in thousand bases).

and incubated in a boiling water bath for 5 min. Samples were applied to a sodium dodecyl sulfate-12.5% polyacrylamide gel. After electrophoresis, protein bands were electrotransferred onto polyvinylidene difluoride membranes by using a semidry apparatus (Sartoblot; Sartorious, Gottingen, Federal Republic of Germany), as described by the manufacturer. The immunodetection of sigma factors was accomplished by using B. subtilis polyclonal anti-sigma-43 antiserum (a gift from R. H. Doi, University of California, Davis) and the alkaline phosphatase-conjugated goat anti-rabbit antibody as described by the manufacturer (Promega Biotec, Madison, Wis.).

RESULTS AND DISCUSSION Identification and isolation of rpoD from M. xanthus. Chromosomal DNA from M. xanthus was digested individually with the restriction enzymes SalI, BamHI, EcoRI, PstI, and HindIII. After the digestions, DNA was separated by 0.7% agarose gel electrophoresis, denatured, and transferred to nitrocellulose filters as described by Southern (29). In order to identify the rpoD gene of M. xanthus, the 664-base PstI-ClaI fragment from the E. coli rpoD gene was used as a probe. This fragment corresponds to the sequence from amino acid residues 344 to 566 of the amino acid sequence of the E. coli rpoD gene (2). This region was chosen because it has a very high degree of a homology between E. coli and B. subtilis (10). For each digestion, a band of a unique size was found to hybridize with the probe (Fig. 1). The sizes of these bands were calculated to be >23 kilobases (kb) for EcoRI, 11.5 kb for BamHI, 5.7 kb for PstI, 4.1 kb for Sall, 2.7 kb for

XhoI, and 8.3 kb for SmaI. The BamHI fragments at the 11.5-kb region were purified from a preparative agarose gel and ligated into the unique BamHI site of pBR322. A total of 126 transformants were picked and grown in a microdilution plate. Plasmids were isolated in groups of six cultures. Plasmid DNAs from 21 groups were then digested with BamHI and electrophoresed on a 0.7% agarose gel. Southern blot hybridization was performed with the same probe described above. DNA fragments from two groups were found to be positive. Thus, Southern blot hybridization was carried out further on these positive groups to identify the individual transformants carrying the plasmids which hybridized with the probe. As a result, two positive transformants were finally identified. One of them, designated pSIG01, was characterized further. Restriction enzyme analysis revealed that pSIG01 contained an 11.5-kb fragment from M. xanthus. Its restriction map is shown in Fig. 2. Since pSIG01 did not contain a 5'-end portion of an open reading frame, as discussed below, this portion was cloned from the PstI digestion of total chromosomal DNA. When the 0.6-kb SalI(b)-SalI(c) fragment (Fig. 2) was used as a probe, a PstI band at 1.6 kb was identified to hybridize with the probe. The fragment was purified with a preparative agarose gel and cloned into the unique PstI site of pBR322. Colony hybridization was performed with the same probe. A plasmid which hybridized with the probe was isolated and designated pSIG02 (Fig. 2). In order to determine whether the DNA fragment cloned in pSIG01 contained the rpoD gene of M. xanthus, I first attempted to isolate a short DNA fragment from pSIG01 which hybridized with the E. coli rpoD probe. It was found that a 320-base-pair PstI-XhoI fragment [from P(c) to X in Fig. 2] strongly hybridized with the probe. Subsequently, the DNA sequence of this fragment was determined, and one of the reading frames of the DNA sequence was found to code for an amino acid sequence of 110 residues without any termination codons. Furthermore, this sequence (corresponding to the sequence from Leu-494 to Glu-603 in Fig. 3) showed an extensive similarity with the sequence from Leu-399 to Glu-508 of the E. coli sigma factor (sigma-70) (see Fig. 4). Of 110 residues, 96 were identical and 9 were functionally homologous. Thus, the overall similarity was 95%. On the basis of this similarity, it is most likely that the

J. BACTERIOL.

INOUYE

82

*

CAGGGAAATCATGGTGCGGCGCATCGACGAGCGGCTCGTTTATATAAAGCGGGCGACGGA *

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GluLysGluGluLeuValAlaArgAspValValAlaGluAlaThrGluLysLeuLysLys *

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240

AAGGAGTCGCGGGAGAACCCCAACATCGGCAAGAAGCTGCAGAAGCAGCTCAACCTCACG 1560

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300

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200

360

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GTCGAGGAGGAGGCCAACCTCCCCGTCGAGTCGCTGCGCCGCAACTACGACGCCATCCGT 1680

420

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CTGGGTGAGCGCCGCGCCGAACGCGCCAAGAGCGAGCTGGTGGAGGCCAACCTGCGCCTC 1740 LeuGlyGluArgArgAlaGluArgAlaLysSerGluLeuValGluAlaAsnLeuArgLeu 480

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GTGGTGTCCATCGCGAAGAAGTACACGAACCGCGGCCTGCAGTTCCTGGACCTCATCCAG 1800

*

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540

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GAGGGCAACATCGGCCTGATGAAGGCCGTGGACAAGTTCGAGTACAAGCGCGGCTACAAG 1860 600

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TTCTCGACCTACGCCACCTGGTGGATTCGTCAGGCCATCACCCGCGCCATCGCGGACCAG 1920 *

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GCCCGCACCATCCGCATCCCGGTGCACATGATCGAGACCATCAACAAGCTCATCCGCACC 1980 AlaArgThrIleArgIleProValHisMetIleGluThrIleAsnLysLeuIleArgThr 720

AGCCGCTACCTGGTGCAGGAGATTGGCCGCGAGCCGACGCCGGAGGAGATCGCGGAGAAG 2040 SerArgTyrLeuValGlnGluIleGlyArgGluProThrProGluGluIleAlaGluLys

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ATGGAGCTGCCGCTCGACAAGGTCCGCAAGGTCCTCAAGATTGCGAAGGAGCCCATCTCC 2100 MetGluLeuProLeuAspLysValArgLysValLeuLysIleAlaLysGluProIleSer 840

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CTCGAGACGCCGATTGGCGAGGAAGAGGACAGCCACCTGGGCGACTTCATCGAGGACAAG 2160

LeuGluThrProIleGlyGluGluGluAspSerHisLeuGlyAspPheIleGluAspLys 900

AGCCTCGTGTCGCCGGCGGACGCGGTCATCAACATGAACCTGGCGGAGCAGACCCGGAAG 2220 SerLeuValSerProAlaAspAlaValIleAsnMetAsnL.uAlaGluGlnThrArgLys 960

GTGCTCGCCACGCTGACGCCGCGCGAGGAGAAGGTGCTCCGCATGCGCTTCGGCATCGGC 2280

ValLeuAlaThrLeuThrProArgGluGluLysValLeuArgMetArgPheGlyIleGly

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AAGCGCATCGAGGATGGTGAGAAGGAAGTCCTCCGCGCGCTGCTGGCGTGCAAGGTGGCC LysArgI leGluAspGlyGluLysGluVal LeuArgAlaLeuLeuAlaCysLysValAl a *

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CCGGAGCAGTTGGAGGTCCTGGACCGCGACGTGCGCACGGCGGTGCGGAAGATCAAGAAG 1620

ValGluGluGluAlaAsnLeuProValGluSerLeuArgArgAsnTyrAspAlaIleArg

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TACCTGCGCAAGATGGGCAGCGTCAGCCTGCTCACCCGCGAGGGCGAGGTCGAAATCGCC TyrLeuArgLysMetGlySerValSerLeuLeuThrArgGluGlyGluValGluI leAla *

LysGluSerArgGluAsnProAsnIleGlyLysLysLeuGlnLysGlnLeuAsnLeuThr

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GAGGACGAGAAGGACGAGGACGACGAGCCGGGCGGCAAGTCGAACGACCCGGTGCGCCTG GluAspGluLysAspGluAspAspGluProGlyGlyLysSerAsnAspProValArgLeu *

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ProGluGlnLeuGluValLeuAspArgAspValArgThrAlaValArgLysIleLySLys

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GCTCAGAACAACGAAATCAAGCCCACCGTCACCGTCGAGGAAGAGAAGGAAGACGCCGAC *

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AlaGlnAsnAsnGluIleLysProThrValThrValGluGluGluLysGluAspAlaAsp *

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GACGACGTGATGAGCATGTTCGGCGACAACGACATCGAGATTGTCGACGCGCAGAAGGCC AspAspValMetSerMetPheGlyAspAsnAspIleGluIleValAspAlaGlnLysAla *

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Met *

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GAGCTGCGTGATTTGGAGCGCCGCTACGACTGCTCCATGAAGGAGCTGCGCCCGCAGCTG 1500

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AAAGGCGCCCATGCAACCGGTTTGAGTTCGCGTTTGTAAGAAACCCTGACTTTGCGGTAG *

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GGAACTGTTGGCGCTCAAGAAGCGTGTCCTGGAGGAGCTCAAGCCTGCCTCCTCGGGAAC

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GAGAAGTCCGACCACACGCTGGAAGAGGTGGGCCAGGACTTCGAGGTGACGCGCGAGCGC 2340

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GTCGAGGAGATACTCGACATCGGCAACAAGCTGAAGACGGCCAAGCTGCGCGTGCGCGAC 1080

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ATCCGTCAGATTGAGGCCAAGGCGCTGCGCAAGCTGCGCCACCCGAGCCGCTCCAAGCGC 2400

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GTCATCAAGGACGCGCCGGAGGAGACGCAGTCCGAAGGTGCCGAAGAGGCGCCCGAGGAG 1140

ValIleLysAspAlaProGluGluThrG1nSerGluGlyAlaGluGluAlaProGluGlu *

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ValGlyGluGlyAspAlaProAlaGlnLeuAlaGlnSerGluLeuAsnLysIleGluGln *

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GluLeuSerSerLysLysLysLeuThrGluValArgLysLysGluValLysGlnGluIle *

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700

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CGCGCATCCCGAGCCCCAGCATGGTTTCGCG

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2460

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ACGAAGACACCGAAGGCGAGAAGTGAAGAAGGCTCCCCTCCCCTTCTCCAAGCGTTGAAG *

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ATTCGTGGACCGGGGGCCTTCGTGTTTCTAGAGTCCGGGGCCGTGGCGCTCCGAGCGAAG

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ATCTGCAAGCAGATCGAGCGCTTCCGCAAGTTCGCCCAGGACTGCGACGTCTTGGAGGAG I leCysLysGlnI leGluArgPheArgLysPheAlaGInAsipCysAspVa lLeuGluGlu *

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CTGCGCTCCTTCGTGGAGAGCTGAGCCGGGCTTGAGCTGAGCTGAACGTGGGCCCCGTCC

2580

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CCTGGGGCGCCAGAGGCGTGGGCCGGGAGATGGCCA

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AAGGACCTCCGGACCAAGATGATGGAGGTCCTGGAGGAGATGCGGCTCAACAAGAAGCAG

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LysAspLeuArgThrLysMetMotGluValLeuGluGluMetArgteuAsnLysLysGln FIG. 3. Nucleotide sequence of the rpoD region and its deduced amino acid sequence. P- and X-labeled arrowheads indicate the locations of PstI(c) and XhoI from Fig. 2. The long arrow indicates the location of an inverted repeat sequence. Numbers on the right enumerate the nucleotide bases, and dots with numbers enumerate the amino acid residues.

reading frame is a part of the M. xanthus sigma factor corresponding to E. coli vegetative sigma-70. Nucleotide sequence and amino acid sequence of the M. xanthus sigma factor. When the nucleotide sequences of the regions flanking the open reading frame described above were determined, there was a long open reading frame which coded for a polypeptide of 708 amino acid residues, starting from the initiation codon at bases 298 to 300 (Fig. 3). The open

molecular weight of the product from this open reading frame consisting of 708 amino acid residues was calculated to be 80,391. It is unlikely that the open reading frame started further upstream, since there was a termination codon (bases 271 to 273) in the same phase as the initiation codon. For this initiation codon there was a possible ShineDalgarno sequence (GGAG) 10 bases upstream from the initiation codon. The M. xanthus sigma factor aligned par-

VOL. 172, 1990

MAJOR SIGMA FACTOR OF M. XANTHUS B.subtilis E.Coli M.xanthus

MADKQTHETELTFDQVKEQLTESGKKRGVLTYEEIAERMSSFEIESDQMDEYYEFLG-EQ MEQNPQSQLKLLVTRGKEQGYLTYAEVNDHLPEDIVDSDQIEDIIQMINDMGIQVME-EA DPVAERKEVKDLLAAGREKGFLTYDEVNDALPADIVSSDQIDDVMSMFGDNDIEIVDAQK * 0 0 0 000 0000o00000

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B.subtilis E.Coli

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EIDIAKRIEDGINQVQCSVAEYPEAITYLLEQYNRVEAEEARLSDLITGFVDPNAEEDLA

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FIG. 4. Amino acid sequence alignment of the rpoD sequence of M. xanthus, B. subtilis sigma-43, and E. coli sigma-70. Amino acid sequences were compared with matching residues which were assigned the following symbols: 0, amino acid residues shared by three proteins; 0, amino acid residues shared by the RpoD protein of M. xanthus and E. coli sigma-70; *, amino acid residues shared by the RpoD protein of M. xanthus and B. subtilis sigma-43.

ticularly well with E. coli sigma-70 (Fig. 4), except for an extra amino-terminal sequence of the M. xanthus sigma factor consisting of 100 residues. It should be noted that there was an inverted repeat structure 26 bases downstream from the termination codon (bases 2422 to 2424 in Fig. 3). This structure, which is underlined in Fig. 3, was able to form a stable stem-loop structure in the mRNA, with a AG calculated to be -26.2 kcal, as described by Salser (26). This secondary structure was followed by T residues (or U residues on the mRNA), which is characteristic of the procaryotic [rho]-independent transcription termination signal (24). Sequence similarity. Figure 4 shows the sequence alignment of the M. xanthus sigma factor with E. coli sigma-70 and B. subtilis sigma-43. The sequence alignment between sigma-70 and sigma-43 done by Gitt et al. (10) was used in Fig. 4. The M. xanthus sigma factor had an extra internal sequence (residues 228 to 466; a total of 239 residues), as did sigma-70 of E. coli (residues 127 to 371; a total of 245 residues), which was missing from sigma-43 of B. subtilis. Within this region, there were 48 identical residues (20%

identity; Fig. 4). In the amino-terminal domain, from residues 101 to 227 of the M. xanthus sigma factor, there were 17 identical residues with both sigma-70 and sigma-43 (Fig. 4). There were an additional 36 identical residues with sigma-70 and 11 identical residues with sigma-43 (Fig. 4); there were 42 and 22% identities with sigma-70 and sigma-43, respectively, in this region. In contrast to the amino-terminal region, the carboxyterminal region of 242 residues (residues 467 to 708) showed much higher similarities with both sigma-70 and sigma-43. There were 155 identical residues with both sigma-70 and sigma-43 (64% identity). In addition, there were 34 and 20 identical residues with sigma-70 and sigma-43, respectively, providing overall identities with sigma-70 and sigma-43 of 78 and 72%, respectively. The carboxy-terminal domain has been shown to be highly conserved among various procaryotic sigma factors and to contain three functionally distinct regions: regions 2, 3, and 4 (for a review, see reference 11). In particular, region 2 (residues 479 to 548; a total of 70 residues) is known to be highly conserved among these regions and is considered to

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FIG. 5. Western blot (immunoblot) analysis of the major sigma factors of B. subtilis, E. coli, and M. xanthus. Cells were solubilized in a loading buffer and analyzed as described in the text. Lane 1, total-cell extract of B. subtilis; lane 2, E. coli; lane 3, M. xanthus. Protein bands were detected by the immunoblot procedure by using B. subtilis anti-sigma-43 serum as a first antibody. Arrowheads with Ec and Bs represent positions of E. coli sigma-70 and B. subtilus sigma-43, respectively. Arrowheads marked a, b, and c are major bands in cell extracts of M. xanthus DZF1. Bars on the left indicate the positions of the following molecular weight markers, from top to bottom, respectively: ,B-galactosidase, bovine serum albumin, ovalbumin, carbonic anhydrase, and lysozyme.

be involved in core recognition and recognition of the conserved -10 region of promoter sequences. Indeed, there were only three residues different from sigma-70 in region 2; Val-483 for Ile, Lys-517 for Arg, and Ala-537 for Ser. Recently, an analysis of various mutations of sigma factors was carried out to characterize the interaction between the sigma factors and promoters (9, 28, 38). It should be noted that the M. xanthus sigma factor contained an extremely large number of charged amino acid residues: 151 acidic residues (Asp plus Glu) and 132 basic residues (Lys plus Arg). This indicates that 40% of the total residues of this protein are charged, which is one of the characteristic features of sigma factors. These charged residues exist as clusters in the protein, for acidic residues, in the sequences from residues 72 to 102, residues 129 to 190, residues 265 to 322, and residues 598 to 620 (Fig. 3). Similarly, basic residues are clustered in the sequences from residues 479 to 491, residues 588 to 597, and residues 679 to 703. As expected from the high G+C content of the M. xanthus chromosomal DNA (70% [15]), 94% of the codons used for the M. xanthus sigma factor had a G or a C residue at the third position. Identification of M. xanthus RpoD protein. In order to identify the M. xanthus rpoD gene product, Western blot (immunoblot) analysis (34) was performed by using a B. subtilis polyclonal anti-sigma-43 serum (a gift from R. H. Doi, University of California, Davis) as a first antibody. Three bands were detected with similar densities at positions with apparent molecular weights of 86,000 (band a), 80,000 (band b), and 51,000 (band c) (Fig. 5, lane 3). Sigma factors are well known to show abnormal motilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in the same gel, E. coli sigma-70 migrated slightly faster than band b (Fig. 5, lane 2). Rudd and Zusman (25) have identified two sigma factors from RNA polymerase prepared from vegetatively grown cells. These sigma factors were designated crI and oII, which probably correspond to band a and

b sigma factors, respectively, on the basis of their molecular weight estimations (they assumed that aII has a molecular weight similar to that of E. coli sigma-70). Therefore, band a (urI) is probably sigma-80 and band b (aII) is either a degradation product of sigma-80, as proposed by Rudd and Zusman (25), or another putative sigma factor, sigma-73, which may be produced from the downstream in-phase initiation codon (bases 405 to 407; Fig. 3). The molecular weight of this sigma factor was calculated to 73,117. It is unknown whether band c is related to sigma-80 or whether it is derived from another gene. Conclusion. On the basis of the extensive amino acid sequence similarity between the open reading frame determined in this study and sigma-70 of E. coli, the open reading frame is concluded to be the gene for the M. xanthus vegetative sigma factor. In E. coli, the rpoD gene is shown to be cotranscribed with dnaG and rpsU (3). A topic of great interest is the possible cotranscription of the M. xanthus rpoD gene with other genes. By using the rpoD gene in the present study as a probe, several DNA fragments that hybridize with the probe can now be cloned from the M. xanthus chromosomal DNA. One of them has been characterized and has been shown to contain a gene for a developmental sigma factor (D. Apelian, M. Inouye, and S. Inouye, manuscript in preparation). Recently, genes for sigma factors of various enterobacteria have been identified by using a synthetic oligonucleotide probe corresponding to a consensus rpoD box sequence (31). ACKNOWLEDGMENTS I thank Teiichi Furuichi for assistance at an early stage of this work and Yale Jen for assistance in DNA sequencing. I also thank R. H. Doi for B. subtilis sigma-43 and polyclonal anti-sigma-43 serum and C. A. Gross for E. coli sigma-70. I am most grateful to Masayori Inouye for continuous support and encouragement throughout this work. I am also grateful to Bert C. Lampson and David Apelian for critical reading of the manuscript. This work was supported by Public Health Service grant GM26843 from the National Institutes of Health. LITERATURE CITED 1. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 2. Burton, Z., R. R. Burgess, J. Lin, D. Moore, S. Holder, and C. A. Gross. 1981. The nucleotide sequence of the cloned rpoD gene for the RNA polymerase sigma subunit from E. coli K-12. Nucleic Acids Res. 9:2889-2903. 3. Burton, Z. F., C. A. Grass, K. K. Watanabe, and R. R. Burgess. 1983. The operon that encodes the sigma subunit of RNA polymerase also encodes ribosomal protein S21 and DNA primase in E. coli K12. Cell 32:335-349. 4. Campos, J. M., J. Geisselsoder, and D. R. Zusman. 1984. Isolation of bacteriophage Mx4, a generalized transducing phage for Myxococcus xanthus. J. Mol. Biol. 119:167-178. 5. Cumsky, M., and D. R. Zusman. 1979. Myxobacterial hemagglutinin: a development-specific lectin of Myxococcus xanthus. Proc. Natl. Acad. USA 76:5505-5509. 6. Doi, R. H., and L. F. Wang. 1986. Multiple prokaryotic ribonucleic acid polymerase sigma factors. Microbiol. Rev. 50:227-

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