ABSTRACT
Bacteria respond to diverse growth-limiting stresses by producing a large set of general stress proteins. In Bacillus subtilis and related Gram-positive pathogens, this response is governed by the sigma(B) transcription factor. To establish the range of cellular functions associated with the general stress response, we compared the transcriptional profiles of wild and mutant strains under conditions that induce sigma(B) activity. Macroarrays representing more than 3900 annotated reading frames of the B. subtilis genome were hybridized to (33)P-labelled cDNA populations derived from (i) wild-type and sigB mutant strains that had been subjected to ethanol stress; and (ii) a strain in which sigma(B) expression was controlled by an inducible promoter. On the basis of their significant sigma(B)-dependent expression in three independent experiments, we identified 127 genes as prime candidates for members of the sigma(B) regulon. Of these genes, 30 were known previously or inferred to be sigma(B) dependent by other means. To assist in the analysis of the 97 new genes, we constructed hidden Markov models (HMM) that identified possible sigma(B) recognition sequences preceding 21 of them. To test the HMM and to provide an independent validation of the hybridization experiments, we mapped the sigma(B)-dependent messages for seven representative genes. For all seven, the 5' end of the message lay near typical sigma(B) recognition sequences, and these had been predicted correctly by the HMM for five of the seven examples. Lastly, all 127 gene products were assigned to functional groups by considering their similarity to known proteins. Notably, products with a direct protective function were in the minority. Instead, the general stress response increased relative message levels for known or predicted regulatory proteins, for transporters controlling solute influx and efflux, including potential drug efflux pumps, and for products implicated in carbon metabolism, envelope function and macromolecular turnover.
Subject(s)
Bacillus subtilis/drug effects , Bacillus subtilis/genetics , Bacterial Proteins/metabolism , Ethanol/pharmacology , Gene Expression Profiling , Gene Expression Regulation, Bacterial/drug effects , Genes, Bacterial/genetics , Sigma Factor/metabolism , Bacterial Proteins/genetics , Base Sequence , Genome, Bacterial , Markov Chains , Nucleic Acid Hybridization , Oligodeoxyribonucleotides , Oligonucleotide Array Sequence Analysis , Regulon/genetics , Sigma Factor/genetics , Transcription, Genetic/drug effectsABSTRACT
Two divergent reports have been published on the genetic complementation of rhizobial nod mutants using Frankia DNA. In 1991 putative Frankia cosmid library clones were reported to restore normal nodulation properties to Rhizobium leguminosarum biovar viciae nodD::Tn5, but no supporting sequence data were published. In 1992 a second group reported a failure to find any evidence of functional complementation of various rhizobial nod mutants by Frankia DNA (nodA, nodB and nodC). Complementation tests of nine NodR. leguminosarum bv. viciae or Sinorhizobium meliloti Tn5 mutants (nodA-, nodB-, nodC-, nodD-, nodF-, nodL-, nodH-) were thus performed using a Frankia gene library in pLAFR3 to clarify this situation. Rhizobial transconjugants obtained by tri-parental matings were screened for restoration of the nodulation phenotype on their host plants, Vicia sativa subsp. nigra or Medicago sativa. Nodulation was observed on plants inoculated with transconjugants of the R. leguminosarum bv. viciae nodC::Tn5 mutant. The Nod+ rhizobial transconjugants were isolated and analysed. The Nod+ phenotype of these transconjugants was found to be due to Tn5 excision/transposition. No functional complementation was found with any of the mutants used, suggesting that rhizobial complementation of nod mutants with Frankia DNA is unlikely to occur.
