Summary In Bacillus subtilis, the competence transcription factor ComK activates its own transcription as well as the transcription of genes that encode DNA transport proteins. ComK is expressed in about 10% of the cells in a culture grown to competence. Using DNA microarrays representing ≈ 95% of the protein‐coding open reading frames in B. subtilis, we compared the expression profiles of wild‐type and comK strains, as well as of a mecA mutant (which produces active ComK in all the cells of the population) and a comK mecA double mutant. In these comparisons, we identified at least 165 genes that are upregulated by ComK and relatively few that are downregulated. The use of reporter fusions has confirmed these results for several genes. Many of the ComK‐regulated genes are organized in clusters or operons, and 23 of these clusters are preceded by apparent ComK‐box promoter motifs. In addition to those required for DNA uptake, other genes that are upregulated in the presence of ComK are probably involved in DNA repair and in the uptake and utilization of nutritional sources. From this and previous work, we conclude that the ComK regulon defines a growth‐arrested state, distinct from sporulation, of which competence for genetic transformation is but one notable feature. We suggest that this is a unique adaptation to stress and that it be termed the ‘K‐state’.

Two genes encoding insecticidal crystal proteins from Bacillus thuringiensis subsp. kurstaki HD-1 were cloned and sequenced. Both genes, designated cryBi and cryB2, encode polypeptides of 633 amino acids having a molecular mass of ca. 71 kilodaltons (kDa). Despite the fact that these two proteins display 87% identity in amino acid sequence, they exhibit different toxin specificities. The cryBI gene product is toxic to both dipteran (Aedes aegypti) and lepidopteran (Manduca sexta) larvae, whereas the cryB2 gene product is toxic only to the latter. DNA sequence analysis indicates that cryBI is the distal gene of an operon which is comprised of three open reading frames (designated orfi, orJ2, and cryBi). The proteins encoded by cryBi and orJ2 are components of small cuboidal crystals found in several subspecies and strains of B. thuringiensis; it is not known whether the orfl or cryB2 gene products are present in cuboidal crystals. The protein encoded by orJ2has an electrophoretic mobility corresponding to a molecular mass of ca. 50 kDa, although the gene has a coding capacity for a polypeptide of ca. 29 kDa. Examination of the deduced amino acid sequence for this protein reveals an unusual structure which may account for its aberrant electrophoretic mobility: it contains a 15-amino-acid motif repeated 11 times in tandem. Escherichia coli extracts prepared from cells expressing only orfi and orJ2 are not toxic to either test insect.The common soil bacterium Bacillus thuringiensis synthesizes proteinaceous crystalline inclusions that are lethal to a variety of insects. Several subspecies have been identified to date, and their host ranges can vary substantially. By far the greatest number produce large bipyramidal crystals which are toxic only to lepidopteran larvae (reviewed in reference 38). These crystals consist of one or more related protoxin polypeptides having a molecular mass of 130 to 140 kilodaltons (kDa); toxins of ca. 68 kDa are derived from the protoxins by proteolytic processing in the larval gut. Some strains that are lethal to lepidopterans also contain small cuboidal crystals (28,40). Bacillus thuringiensis subsp. kurstaki HD-1 is such a strain: it produces bipyramidal crystals composed of two or three protoxin polypeptides encoded by homologous crystal protein genes (16, 17) and cuboidal crystals containing a ca. 65-kDa polypeptide ("P2 toxin"; 41) which is toxic to both lepidopteran and dipteran larvae.We have cloned and sequenced four genes, designated cryBI, cryB2, orfl, and orf2, from a 225-kilobase (kb) plasmid doublet from B. thuringiensis subsp. klirstaki HD-1.Three of these genes (of1, orJ2, and cryBI) are part of an operon; ciyB2 is located separately. Based on immunoblot analyses and toxicity data, cuboidal crystals contain the cryBi and orJ2 gene products; we do not know whether the orfl and cryB2 gene products are present in cuboidal crystals. Interestingly, although the ciyBl and cryB2 genes are closely related, their gene products exhibit different host range specificities. The cryBl...

The SKI2 gene is part of a host system that represses the copy number of the L-A double-stranded RNA (dsRNA) virus and its satellites M and X A system of six yeast chromosomal genes (SK12, -3, -4, -6, -7, and -8) lowers the copy numbers of the L-A, M, X, and L-BC dsRNA viruses and of the ssRNA replicon 20S RNA (3,17,44,54,65 (70), and the ski mutations also suppress mkt mutations (54).The SK13 and SK18 genes have been cloned and characterized. The SK13 product is a 163-kDa nuclear protein (52) with several copies of an amino acid repeat pattern, the TPR repeat, of unknown function (62). The SK18 protein has two copies of a different repeat amino acid sequence pattern first identified in ,B-transducin (45). Deletion mutations of either SK13 or SK18 had no effect on cell growth unless an M replicon was present. In that case, the cells were cold sensitive for growth as discussed above. For this reason, we view the SKI system as a dedicated antiviral system. However, the mechanism by which the SKI proteins interfere with the propagation of the RNA replicons is completely unknown.We report here that Ski2p resembles helicases and nucleolar proteins and present evidence that Ski2p acts by blocking the translation of viral mRNA. MATERIALS AND METHODSStrains and media. The yeast strains used in this study are shown in Table 1. Escherichia coli DHSa was used to propagate plasmid DNA, strain MV1190 and M13 helper phage K07 were used for the isolation of single-stranded plasmid DNA for DNA sequencing, and strain CJ236 was used for the production of uracil-containing single-stranded plasmid DNA for site-directed mutagenesis (40). Yeast strains were grown on YPAD, SD, H-His, H-Trp, H-Ura, or 4.7MB medium (70). E. coli strains were grown on LB, TB, or M9 medium (43).The strains in which M or X is supported by an L-A cDNA clone were constructed as follows: a strain derived from strain 3221 harboring L-A and either M1 or X (produced by cytoduction into strain 3221) was transformed with the L-A 4331

Two highly related crystal protein genes from Bacillus thuringiensis subsp. kurstaki HD-1, designated cryIIA and cryIIB (previously named cryBl and cryB2, respectively), were used to study host range specificity. Their respective gene products are 87% identical but exhibit different toxicity spectra; CryIIA is toxic to both mosquito and tobacco hornworm larvae, whereas CryIIB is toxic only to the latter. Hybrids of the crylIA and crylIB genes were generated, and their resultant gene products were assayed for toxicity. A short segment of CrylA-corresponding to residues 307 through 382 was shown to be sufficient for altering host range specificity-i.e., when this region replaced the corresponding segment of CryIIB, the resulting hybrid protein acquired toxicity against mosquitoes. The CryHA and CryHIB polypeptides differ by only 18 amino acds in this region, indicating that very few amino acid changes can have a substantial effect on the toxicity spectra of these proteins.

The plus strand of the L-A double-stranded RNA virus of Saccharomyces cerevisiae has two large open reading frames, ORF1, which encodes the major coat protein, and ORF2, which encodes a single-stranded RNA-binding protein having a sequence diagnostic of viral RNA-dependent RNA polymerases. ORF2 is expressed only as a Gag-Pol-type fusion protein with ORF1. We have constructed a plasmid which expresses these proteins from the yeast PGK1 promoter. We show that this plasmid can support the replication of the killer toxin-encoding M1 satellite virus in the absence of an L-A double-stranded RNA helper virus itself. This requires ORF2 expression, providing a potential in vivo assay for the RNA polymerase and single-stranded RNA-binding activities of the fusion protein determined by ORF2. ORF1 expression, like a host si-mutation, can suppress the usual requirement of M1 for the MAKI], MAK18, and MAK27 genes and allow a defective L-A (L-A-E) to support M1 replication. These results suggest that expression of ORF1 from the vector makes the cell a ski-phenocopy. Indeed, expression of ORF1 in a wild-type killer makes it a superkiller, suggesting that a target of the SKI antiviral system may be the major coat protein.