Sequence analysis and molecular characterization of the temperate lactococcal bacteriophage r1t

The temperate lactococcal bacteriophage r1t was isolated from its lysogenic host and its genome was subjected to nucleotide sequence analysis. The linear r1t genome is composed of 33 350 bp and was shown to possess 3' staggered cohesive ends. Fifty open reading frames (ORFs) were identified which are, probably, organized in a life-cycle-specific manner, Nucleotide sequence comparisons, N-terminal amino acid sequencing and functional analyses enabled the assignment of possible functions to a number of DNA sequences and ORFs. In this way, ORFs specifying regulatory proteins, proteins involved in DNA replication, structural proteins, a holin, a lysin, an integrase, and a dUTPase were putatively identified. One ORF seems to be contained within a self-splicing group I intron. In addition, the bacteriophage att site required for site-specific integration into the host chromosome was determined

COMK ACTS AS AN AUTOREGULATORY CONTROL SWITCH IN THE SIGNAL-TRANSDUCTION ROUTE TO COMPETENCE IN BACILLUS-SUBTILIS

The comK gene is a regulatory transcription unit which is essential for the development of genetic competence in Bacillus subtilis. The transcription of comK is under strict nutritional and growth phase-dependent control and has been shown to depend on the gene products of comA and srfA. In this report, we show that expression of comK is dependent on its own gene product as well as on the gene products of all other tested regulatory genes known to be involved in competence development (abrB, comA, comP, degU, sin, spoOA, spoOH, spoOK, and srfA). A mecA mutation is able to suppress the competence deficiency of mutations in any of these regulatory loci except for mutations in spoOA and, as we show here, in comK. Furthermore, we show that the presence of comK on a multiple copy plasmid leads to derepression of comK expression, causing an almost constitutive expression of competence in minimal medium as well as permitting competence development in complex medium. We infer from these results that the signals which trigger competence development, after having been received and processed by the various components of the competence signal transduction pathway, all converge at the level of comK expression. As soon as derepression of comK expression occurs, the positive autoregulation rapidly results in accumulation of the comK gene product, which subsequently induces competence

DIFFERENTIAL EXPRESSION OF 2 CLOSELY-RELATED DEOXYRIBONUCLEASE GENES, NUCA AND NUCB, IN BACILLUS-SUBTILIS

Despite the lack of involvement of the competence-specific, membrane-associated deoxyribonuclease (DNase) in competence development, the expression of the gene encoding this protein, nucA, was shown to be dependent on the competence signal transduction pathway, and in particular on ComK, the competence transcription factor, which was shown to bind to the DNA region upstream of nucA, The expression of nucB, specifying an extracellular DNase, which was cloned on the basis of its homology to nucA, was shown to be sporulation-specific and dependent on the gene products of spoOA and spollG, the latter constituting an operon responsible for the synthesis of the mother-cell-specific sigma factor sigma(E). The observed differential expression of nucA and nucB demarcates the appearance of DNase activities which are either associated with the cytoplasmic membrane or secreted into the medium during different post-exponential growth-phase processes

ISOLATION AND CHARACTERIZATION OF COML, A TRANSCRIPTION UNIT INVOLVED IN COMPETENCE DEVELOPMENT OF BACILLUS-SUBTILIS

Using the transformation-deficient mutant M465, which was previously isolated by means of insertional mutagenesis with plasmid pHV60, a transcription unit comL required for genetic competence of Bacillus subtilis was identified. A chromosomal DNA fragment flanking the inserted pHV60 was isolated and used to screen two different libraries of B. subtilis DNA in phage lambda EMBL4 and lambda EMBL13, respectively. With the aid of six recombinant phages that hybridize with this chromosomal fragment a restriction map of about 23 kb of B. subtilis chromosomal DNA was constructed. Using small adjoining pieces of this chromosomal DNA in Campbell integrations, the size of the transcription unit involved in competence development could be delimited to about 15 kb. By insertion of a promoterless lacZ gene into comL, the transcriptional regulation of comL was analysed and epistatic interactions among various other com genes were determined. The results of these experiments indicated that comL is optimally expressed in glucose-based minimal medium when the culture enters the stationary phase of growth and that the expression of late competence genes is dependent on previous transcription of comL, which in turn is dependent on the gene products of comA and comB

COMK ENCODES THE COMPETENCE TRANSCRIPTION FACTOR, THE KEY REGULATORY PROTEIN FOR COMPETENCE DEVELOPMENT IN BACILLUS-SUBTILIS

comK is a positive autoregulatory gene occupying a central position in the com petence-signal-transduction network. All regulatory routes identified in this network converge at the level of comK expression. The ComK protein is required for the transcriptional induction of comK and the late competence genes, which specify morphogenetic and structural proteins necessary for construction of the DNA-binding and uptake apparatus. In this report we demonstrate that ComK specifically binds to DNA fragments containing promoter and upstream sequences of the genes it affects (comC, comE, comF, comG and comK). Using portions of the region upstream of comC we show that the ComK-binding sequences are essential for the expression of competence. Moreover, we demonstrate that the presence of ComK stimulates the expression of comF-lacZ and comG-lacZ translational fusions in vivo in Escherichia coil. These results indicate that the gene product of comK is identical to the previously inferred competence transcription factor (CTF)