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).

The origins of 168, W23, and other Bacillus subtilis legacy strains.

Bacillus subtilis is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of B. subtilis genetics. For three--NCIB 3610T, PY79, and SMY--we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more "W23 islands" in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its trp and sac loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these B. subtilis legacy strains.

Transient expression and flux changes during a shift from high to low riboflavin production in continuous cultures of Bacillus subtilis.

At the onset of glucose-limited continuous cultures, riboflavin production in recombinant Bacillus subtilis declines significantly within 3 generations. This phenomenon was specific to riboflavin production and was not correlated with any other physiological parameter. Physiological analyses excluded genetic degeneration or co-metabolism of previously generated overflow metabolites as possible causes for the riboflavin transients. By developing a novel method for (13)C-based metabolic flux analysis under non-steady-state conditions, we showed that the pentose precursors of riboflavin were exclusively synthesized via the non-oxidative pentose-phosphate (PP) pathway as long as riboflavin production was high. The complete redirection of carbon flux to the oxidative branch of the PP pathway was achieved at unaltered PP pathway gene expression and correlated with the declining riboflavin production. With the possible exception of a slight down-regulation of the purine biosynthesis pathway, genome-wide expression analysis indicated that transcriptional regulation was not responsible for the production decline.

Genome-wide transcription profiling of Corynebacterium glutamicum after heat shock and during growth on acetate and glucose.

To monitor the global gene expression of Corynebacterium glutamicum we established two formats of DNA-arrays on nylon membranes. We produced an ordered DNA-array of PCR fragments from a shotgun library of C. glutamicum representing a threefold coverage of the genome. With this format we studied genome-wide transcriptional changes after heat shock. Sequence and subsequent BLAST analysis of PCR fragments with elevated expression after heat shock revealed PCR fragments harboring genes that encode several proteins of the heat shock family, proteins of the oxidative stress response and proteins with unknown function. DNA-arrays based on PCR fragments representing 2804 annotated ORFs of C. glutamicum were used to monitor the transcript levels during growth on acetate and glucose. We determined minimal detectable ratios and compared labeling approaches with random hexamers and ORF-specific primers. ORF-based DNA-array analysis with different labeling approaches showed similar results: e.g. increased mRNA levels of the pta-ack operon, aceA, aceB and genes encoding phosphoenolpyruvate carboxykinase and enzymes of the citric acid cycle during growth on acetate and elevated mRNA levels of some enzymes of the glycolytic pathway and lactate dehydrogenase upon growth on glucose. These results demonstrate that shotgun DNA-arrays and ORF-based DNA-arrays are appropriate tools to study physiology of microorganism.