John B. Perkins

A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn917 insertions

SummaryA rapid and general procedure has been devised for the pBR322-mediated cloning in Escherichia coli of Bacillus subtilis chromosomal DNA extending in a specified direction from any Tn917 insertion. Derivatives of Tn917 have been constructed that contain a pBR322-derived replicon, together with a chloramphenicol-resistance (Cmr) gene of Gram-positive origin (selectable in B. subtilis), inserted by ligation in two orientations into a SalI restriction site located near the center of the transposon. When linearized plasmid DNA carrying such derivatives was used to transform to CmrB. subtilis bacteria already containing a chromosomal insertion of Tn917, the pBR322 sequences efficiently became integrated into the chromosomal copy of the transposon by homologous recombination. It was then possible to clone chromosomal sequences adjacent to either transposon insertion junction into E. coli, using a selection for ampicillin-resistance, by transforming CaCl2-treated cells with small amounts of insert-containing DNA that had been digested with various restriction enzymes and then ligated at a dilute concentration. Because pBR322 sequences may be inserted by recombination in either orientation with respect to the transposon arms, a single restriction enzyme (such as EcoRi or SphI) that has a unique recognition site in pBR322 DNA may be used to separately clone chromosomal DNA extending in either direction from the site of any transposon insertion. A family of clones generated from the region of an insertional spo mutation (spoIIH::Tn917) was used in Southern hybridization experiments to verify that cloned material isolated with this procedure accurately reflected the arrangement of sequences present in the chromosome. Strategies are discussed for taking advantage of certain properties inherent in the structure of clones generated in this way to facilitate the identification and study of promoters of insertionally mutated genes.

RNA Expression Analysis Using an Antisense Bacillus subtilis Genome Array

We have developed an antisense oligonucleotide microarray for the study of gene expression and regulation in Bacillus subtilis by using Affymetrix technology. Quality control tests of the B. subtilis GeneChip were performed to ascertain the quality of the array. These tests included optimization of the labeling and hybridization conditions, determination of the linear dynamic range of gene expression levels, and assessment of differential gene expression patterns of known vitamin biosynthetic genes. In minimal medium, we detected transcripts for approximately 70% of the known open reading frames (ORFs). In addition, we were able to monitor the transcript level of known biosynthetic genes regulated by riboflavin, biotin, or thiamine. Moreover, novel transcripts were also detected within intergenic regions and on the opposite coding strand of known ORFs. Several of these novel transcripts were subsequently correlated to new coding regions.

Genes for Small, Noncoding RNAs under Sporulation Control in Bacillus subtilis

The process of sporulation in the bacterium Bacillus subtilis is known to involve the programmed activation of several hundred protein-coding genes. Here we report the discovery of previously unrecognized genes under sporulation control that specify small, non-protein-coding RNAs (sRNAs). Genes for sRNAs were identified by transcriptional profiling with a microarray bearing probes for intergenic regions in the genome and by use of a comparative genomics algorithm that predicts regions of conserved RNA secondary structure. The gene for one such sRNA, SurA, which is located in the region between yndK and yndL, was induced at the start of development under the indirect control of the master regulator for entry into sporulation, Spo0A. The gene for a second sRNA, SurC, located in the region between dnaJ and dnaK, was switched on at a late stage of sporulation by the RNA polymerase sigma factor sigmaK, which directs gene transcription in the mother cell compartment of the developing sporangium. Finally, a third intergenic region, that between polC and ylxS, which specified several sRNAs, including two transcripts produced under the control of the forespore-specific sigma factor sigmaG and a third transcript generated by sigmaK, was identified. Our results indicate that the full repertoire of sporulation-specific gene expression involves the activation of multiple genes for small, noncoding RNAs.

New Ways to Study Developmental Genes in Spore-Forming Bacteria

The regulated activation of numerous sets of genes in multiple chromosomal locations is a hallmark of cellular differentiation in both eukaryotes and prokaryotes. Certain species of bacteria that experience complex developmental cycles are especially attractive as systems in which to study the mechanisms of this kind of gene regulation because they are highly amenable to both biochemical and genetic approaches. Bacillus subtilis, which undergoes extensive cellular differentiation when it sporulates, is one such system. Many new methods are now available in this Gram-positive species for identifying, manipulating, and studying the regulation of genes involved in spore formation, including the use of transposable genetic elements that create gene fusions in vivo as an automatic consequence of insertions into genes.

Small Genes under Sporulation Control in the Bacillus subtilis genome

Using an oligonucleotide microarray, we searched for previously unrecognized transcription units in intergenic regions in the genome of Bacillus subtilis, with an emphasis on identifying small genes activated during spore formation. Nineteen transcription units were identified, 11 of which were shown to depend on one or more sporulation-regulatory proteins for their expression. A high proportion of the transcription units contained small, functional open reading frames (ORFs). One such newly identified ORF is a member of a family of six structurally similar genes that are transcribed under the control of sporulation transcription factor σ(E) or σ(K). A multiple mutant lacking all six genes was found to sporulate with slightly higher efficiency than the wild type, suggesting that under standard laboratory conditions the expression of these genes imposes a small cost on the production of heat-resistant spores. Finally, three of the transcription units specified small, noncoding RNAs; one of these was under the control of the sporulation transcription factor σ(E), and another was under the control of the motility sigma factor σ(D).

Identification of the Two Missing Bacterial Genes Involved in Thiamine Salvage: Thiamine Pyrophosphokinase and Thiamine Kinase

The genes encoding thiamine kinase in Escherichia coli (ycfN) and thiamine pyrophosphokinase in Bacillus subtilis (yloS) have been identified. This study completes the identification of the thiamine salvage enzymes in bacteria.