Characterization of comE, a late competence operon of Bacillus subtilis required for the binding and uptake of transforming DNA

The binding and transport of DNA by competent Bacillus subtilis requires the assembly of a specialized apparatus. We present here the characterization of comE, an operon under competence control that is required for both DNA binding to the competent ceil surface, and for uptake. comE contains three open reading frames (0RF1‐3) read in the forward direction, preceded by a long untranslated leader sequence and an apparent EσA promoter. A minor promoter also is responsible for transcription of ORF2 and ‐3. A transcript containing a single ORF is produced in the reverse direction. The reverse ORF overlaps ORF1 and the untranslated comE leader. The comE transcript is present at a very low level during growth and at an elevated level in stationary‐phase cells. Converseiy, the reverse transcript is present during exponential growth and disappears during stationary phase. The reverse ORF resembles prokaryotic and eukaryotic pyrroiine‐5‐carboxylate reductases, while ORF2 is similar to several dCMP deaminases. 0RF1 and 0RF3 are predicted to be integral membrane proteins. The latter is specifically required for DNA uptake but not for binding.

[1]  C. Georgopoulos,et al.  The essential Escherichia coli msbA gene, a multicopy suppressor of null mutations in the htrB gene, is related to the universally conserved family of ATP‐dependent translocators , 1993, Molecular microbiology.

[2]  J. Mattick,et al.  Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria. , 1991, Gene.

[3]  D. Dubnau,et al.  Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex. , 1971 .

[4]  D. Cheo,et al.  Molecular characterization of regulatory elements controlling expression of the Bacillus subtilis recA+ gene. , 1992, Biochimie.

[5]  P. Deininger.  Molecular cloning: A laboratory manual , 1990 .

[6]  D. Dubnau,et al.  Nucleotide sequence and genetic organization of the Bacillus subtilis comG operon , 1989, Journal of bacteriology.

[7]  D. Dubnau,et al.  Expression of competence genes in Bacillus subtilis , 1987, Journal of bacteriology.

[8]  Rainer Fuchs,et al.  CLUSTAL V: improved software for multiple sequence alignment , 1992, Comput. Appl. Biosci..

[9]  D. Dubnau,et al.  comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP‐dependent RNA/DNA helicases , 1993, Molecular microbiology.

[10]  D. Dubnau,et al.  Suppression of early competence mutations in Bacillus subtilis by mec mutations , 1990, Journal of bacteriology.

[11]  R. Treisman,et al.  Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. , 1980, Methods in enzymology.

[12]  D. Dubnau,et al.  Cloning and characterization of the regulatory Bacillus subtilis competence genes comA and comB , 1989, Journal of bacteriology.

[13]  R. Dale,et al.  A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA. , 1985, Plasmid.

[14]  P. Sharp,et al.  Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids , 1977, Cell.

[15]  P. Kretschmer,et al.  Escherichia coli delta 1-pyrroline-5-carboxylate reductase: gene sequence, protein overproduction and purification. , 1982, Nucleic acids research.

[16]  M. Sarvas,et al.  Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector , 1985, Journal of bacteriology.

[17]  G. Maley,et al.  Cloning, sequence analysis, and expression of the bacteriophage T4 cd gene. , 1990, The Journal of biological chemistry.

[18]  D. Dubnau,et al.  Transformation and transduction in Bacillus subtilis: evidence for separate modes of recombinant formation. , 1969, Journal of molecular biology.

[19]  D. Valle,et al.  Cloning human pyrroline-5-carboxylate reductase cDNA by complementation in Saccharomyces cerevisiae. , 1992, The Journal of biological chemistry.

[20]  L. Hederstedt.  Krebs´ citric acid cycle , 1993 .

[21]  D. Dubnau,et al.  Sequence and properties of comQ, a new competence regulatory gene of Bacillus subtilis , 1991, Journal of bacteriology.

[22]  D. Dubnau,et al.  Molecular cloning and characterization of comC, a late competence gene of Bacillus subtilis , 1989, Journal of bacteriology.

[23]  D. Dubnau,et al.  Genetic competence in Bacillus subtilis. , 1991, Microbiological reviews.

[24]  D. Dubnau,et al.  Cloning and characterization of a cluster of linked Bacillus subtilis late competence mutations , 1989, Journal of bacteriology.

[25]  P. Piggot,et al.  Use of integrational plasmid vectors to demonstrate the polycistronic nature of a transcriptional unit (spoIIA) required for sporulation of Bacillus subtilis. , 1984, Journal of general microbiology.

[26]  D. Dubnau,et al.  Isolation and characterization of Tn917lac-generated competence mutants of Bacillus subtilis , 1987, Journal of bacteriology.

[27]  D. Haas,et al.  Comparison of proC and other housekeeping genes of Pseudomonas aeruginosa with their counterparts in Escherichia coli. , 1990, Gene.

[28]  D. Dubnau,et al.  Transcriptional regulation of comC: evidence for a competence-specific transcription factor in Bacillus subtilis , 1990, Journal of bacteriology.

[29]  R. Haynes,et al.  Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae , 1986, Molecular and cellular biology.

[30]  P. Lewis,et al.  DNA and protein sequence conservation at the replication terminus in Bacillus subtilis 168 and W23 , 1989, Journal of bacteriology.

[31]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[32]  T. Steitz,et al.  Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. , 1986, Annual review of biophysics and biophysical chemistry.

[33]  A. Pugsley.  The complete general secretory pathway in gram-negative bacteria. , 1993, Microbiological reviews.

[34]  E. Southern.  Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[35]  D. Dubnau,et al.  A Bacillus subtilis regulatory gene product for genetic competence and sporulation resembles sensor protein members of the bacterial two-component signal-transduction systems. , 1990, Genes & development.