Peter C. Loewen

In Escherichia coli, the transcription factor sigma s, encoded by rpoS, controls the expression of a large number of genes involved in cellular responses to a diverse number of stresses, including starvation, osmotic stress, acid shock, cold shock, heat shock, oxidative DNA damage, and transition to stationary phase. A list of over 50 genes under the control of rpoS has been compiled. The transcription factor sigma s acts predominantly as a positive effector, but it does have a negative effect on some genes. The synthesis and accumulation of sigma s are controlled by mechanisms affecting transcription, translation, proteolysis, and the formation of the holoenzyme complex. Transcriptional control of rpoS involves guanosine 3',5'-bispyrophosphate (ppGpp) and polyphosphate as positive regulators and the cAMP receptor protein-cAMP complex (CRP-cAMP) as a negative regulator. Translation of rpoS mRNA is controlled by a cascade of interacting factors, including Hfq, H-NS, dsrA RNA, LeuO, and oxyS RNA that seem to modulate the stability of a region of secondary structure in the ribosome-binding region of the gene's mRNA. The transcription factor sigma s is sensitive to proteolysis by ClpPX in a reaction that is promoted by RssB and inhibited by the chaperone DnaK. Despite the demonstrated involvement of so many factors, arguments have been presented suggesting that sensitivity to proteolysis may be the single most important modulator of sigma s levels. The activity of sigma s may also be modulated by trehalose and glutamate, which activate holoenzyme formation and promote holoenzyme binding to certain promoters.

The katF gene of Escherichia coli has been sequenced revealing a 1086 base pair open reading frame from which the sequence of a 362 amino acid protein has been deduced. The direction of transcription of katF was confirmed by expression of the gene cloned in both directions behind a T7 promoter. The KatF protein expressed in vitro migrates with an apparent size of 42 kDa. Comparison of the katF sequence to the sequence of rpoD, which encodes the sigma subunit of RNA polymerase, revealed a 181 bp region with 65% homology and a 38 bp segment that was 87% homologous. A 62 amino acid region of the predicted KatF protein sequence was found to be 85% homologous to the corresponding sequence of sigma 70, including a segment implicated in core polymerase binding. Homology was also observed with the heat shock regulatory protein encoded by htpR.

Pseudomonas chlororaphis strain PA23 is a biocontrol agent able to suppress growth of the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces an arsenal of exometabolites including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN), and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional levels by the Gac-Rsm system, RpoS, PsrA, and the Phz quorum-sensing system. Beyond pathogen-suppression, the success of a biocontrol agent is dependent upon its ability to establish itself in the environment where predation by bacterivorous organisms, including nematodes, may threaten persistence. The focus of this study was to investigate whether PA23 is able to resist grazing by Caenorhabditis elegans and to define the role played by exoproducts in the bacterial-nematode interaction. We discovered that both PRN and HCN contribute to fast- and slow-killing of C. elegans. HCN is well-established as having lethal effects on C. elegans; however, PRN has not been reported to be nematicidal. Exposure of L4 stage nematodes to purified PRN reduced nematode viability in a dose-dependent fashion and led to reduced hatching of eggs laid by gravid adults. Because bacterial metabolites can act as chemoattractants or repellents, we analyzed whether PA23 exhibited attractant or repulsive properties towards C. elegans. Both PRN and HCN were found to be potent repellents. Next we investigated whether the presence of C. elegans would elicit changes in PA23 gene activity. Co-culturing the two organisms increased expression of a number of genes associated with biocontrol, including phzA, hcnA, phzR, phzI, rpoS and gacS. Exoproduct analysis showed that PHZ and autoinducer signals were upregulated, consistent with the gene expression profiles. Collectively, these findings indicate that PA23 is able to sense the presence of C. elegans and it is able to both repel and kill the nematodes, which should facilitate environmental persistence and ultimately biocontrol.