Stress responses of Bacillus subtilis to membrane-active cationic antimicrobial peptides were studied. Global analysis of gene expression by DNA macroarray showed that peptides at a subinhibitory concentration activated numerous genes. A prominent pattern was the activation of two extracytoplasmic function sigma factor regulons, SigW and SigM. Two natural antimicrobial peptides, LL-37 and PG-1, were weak activators of SigW regulon genes, whereas their synthetic analogue poly-L-lysine was clearly a stronger activator of SigW. It was demonstrated for the first time that LL-37 is a strong and specific activator of the YxdJK two-component systems, one of the three highly homologous two-component systems sensing antimicrobial compounds. YxdJK regulates the expression of the YxdLM ABC transporter. The LiaRS (YvqCE) TCS was also strongly activated by LL-37, but its activation is not LL-37 specific, as was demonstrated by its activation with PG-1 and Triton X-100. Other strongly LL-37-induced genes included yrhH and yhcGHI. Taken together, the responses to cationic antimicrobial peptides revealed highly complex regulatory patterns and induction of several signal transduction pathways. The results suggest significant overlap between different stress regulons and interdependence of signal transduction pathways mediating stress responses.

The SBP-box gene family represents a group of plant-specific genes encoding putative transcription factors. Thus far, SBP-domain protein binding sites have been found in the promoters of Arabidopsis APETALA1 and Antirrhinum SQUAMOSA. A putative SBP-domain binding element has been observed in the promoter of BpMADS5, a close homologue of Arabidopsis FRUITFULL in silver birch (Betula pendula). A novel SBP-box gene from birch named BpSPL1 has been cloned and characterized. The nucleotide sequence of BpSPL1 is similar to Antirrhinum SBP2 and Arabidopsis SPL3, apart from the unique finding that BpSPL1 does not contain an intron typical to all other known SBP-box genes studied thus far. According to Northern blot analysis, BpSPL1 is expressed in birch inflorescences as well as in shoots and leaves. Studies using electrophoretic mobility shift assay demonstrate that there are nuclear proteins in birch inflorescences which specifically bind to the SBP binding element of the promoter of BpMADS5. BpSPL1 expressed in Escherichia coli also specifically binds to this element. According to Southern blot analysis, there are at least two SBP-box genes in birch. The results suggest that SBP-box genes are involved in the regulation of flower development in birch.

The Dlt system modulates the density of negative charge in the cell wall of Gram-positive bacteria by substituting anionic polymers (wall and lipoteichoic acids) with D-alanine. The htrA and htrB genes, regulated by the CssRS two-component system (TCS) and encoding membrane-associated protein quality control proteases, were expressed at strongly decreased levels in a mutant with defective Dlt (dltD : : miniTn10) as compared to the dlt + wild-type strain under a secretion stress condition (hypersecretion of AmyQ a-amylase). The level of HtrA protein in the extracellular proteome of the dltD mutant was decreased consistently. Expression from the promoter of the liaIHGFSR (yvqIHGFEC) operon (P liaI ) is dependent on the LiaRS TCS. The Dlt defect increased the expression from P liaI under two stress conditions, AmyQ hypersecretion and treatment with a cationic antimicrobial peptide (LL-37), but decreased the expression in vancomycin-treated cells. Furthermore, Dlt inactivation enhanced the expression of the YxdJK-regulated yxdL gene in LL-37-treated cells. The increased net negative charge of the cell wall seems to cause varied and opposite effects on the expression of CssRS-, LiaRS-and YxdJK-regulated genes under different stress conditions. The results suggest that TCSs which sense misfolded proteins or peptides are modulated by the density of negative charge in the cell wall. The density of negative charge on the outer surface of the cell membrane did not have a similar effect on TCSs.