John Helmann invited presentations 

My invited seminars in 2008 have focused on one of the three following topics:

(i) Mechanisms of peroxide-sensing: The Bacillus subtilis PerR and OhrR proteins

Redox-sensing regulatory proteins coordinate the cellular responses to oxidative stress. The best characterized peroxide-sensors (e.g. E. coli OxyR) sense H2O2 by oxidation of cysteine ultimately leading to the formation of disulfide bonds. We demonstrate that Bacillus subtilis PerR, a member of the Fur family of Fe(II)-binding metalloregulatory proteins, senses peroxides by metal-catalyzed oxidation. Oxidation of PerR leads to the site-specific incorporation of oxygen atom into either His37 or His91, two ligands of the regulatory Fe(II). Inactivation of PerR leads to the expression of protective proteins such as catalase, alkylhydroperoxide reductase, the Fe(II)-binding mini-ferritin MrgA, and the Zn(II) uptake ATPase, ZosA. A second mechanism for sensing peroxides is exemplified by the organic peroxide sensor, OhrR. This protein senses organic peroxides by oxidation at its sole Cys residue. This leads to either the reversible formation of mixed disufide product (with low molecular mass thiols) or the protein sulfenamide. Stronger oxidants lead instead to formation of the irreversible oxidation product, the OhrR sulfinic and sulfonic acid derivative.

(ii) Regulation of cell envelope stress responses in Bacillus subtilis

Bacillus subtilis is a Gram positive soil bacterium and a noted producer of antibiotics. We have characterized the genetic responses of B. subtilis to cell envelope-targeting antibiotics produced by Bacilli, Streptomycetes, and other soil bacteria. Cluster analysis of transcriptome experiments indicates that each antibiotic induces a characteristic stimulon comprising one or more regulons. Antibiotics induce genes controlled by several extracytoplasmic function (ECF) sigma factors and two-component regulatory systems (TCS). We have also used genetic approches to identify endogenous antibiotics and toxic peptides produced by B. subtilis 168 that, in the absence of endogenous resistance determinants, can strongly induce one or more cell envelope stress regulons. The regulon controlled by SigX appears to function, in part, by modifying cell surface charge to provide protection against cationic peptides and antibiotics. The SigW regulon includes resistance genes that protect against both small molecule antibiotics (e.g. fosfomycin) and toxic proteins such as B. subtilis SdpC and sublancin. We have defined the regulon of genes controlled by SigM and demonstrate that this regulator controls genes important for cell envelope synthesis and cell division. Analysis of the regulons controlled by each of these three sigma factors reveals considerable overlap in promoter selectivity. In addition to ECF sigma factors, many cell envelope stress agents strongly induce the LiaRS TCS and its regulon. Other antibiotic loci include a newly identified regulator of autolysin activity, and a novel bipartite sigma factor. Current models for the signalling complexes controlling these stress-responsive regulons will be discussed.

(iii) Bacterial adaptations to metal ion limitation

Soil bacteria are, of necessity, highly adaptable to changing environmental conditions. We have defined the genetic and physiological responses of Bacillus subtilis to alterations in metal ion availability. Specific stress responses induced by metal ion limitation are here represented by the the Fur (iron) and Zur (zinc) regulons. Adaptation to iron limitation involves both the derepression of Fur-repressed genes and the indirect repression of numerous iron-utilizing pathways. Fur-repressed genes encode enzymes for the synthesis of bacillibactin (a catecholate siderophore), several ABC-type transporters for ferri-siderophore uptake, a ferric citrate uptake system, and an elemental iron uptake system. The uptake of ferric-bacillibactin is further regulated by substrate-dependent induction mediated by an AraC-family transcription factor designated Btr (bacillibactin transport regulator). Btr contains a fused FeuA-like substrate-binding domain that binds intracellular siderophore and enables transcription activation of the feuABC operon encoding the bacillibactin transporter. Three recently characterized Fur-regulated operons encode mediators of an iron-sparing response that leads to the repression of iron-utilizing enzymes. These include a Fur-regulated small RNA (FsrA) and three small, basic proteins that function as specific RNA chaperones (FrcA, FrcB, and FrcC). Together, the derepression of iron uptake pathways and the repression of iron utilizing enzymes facilitates adaptation to iron-limiting conditions. Adaptation to zinc limitation also involves the convergent efforts of proteins to (i) facilitate zinc uptake (a high affinity Zn-uptake ABC transporter), (ii) substitute zinc-dependent enzymes with enzymes not requiring zinc (functional substitution of MtrA by YciA), and (iii) mobilize zinc from intracellular stores (including principally ribosomal proteins). These responses to metal ion limitation are representative of a more general process in which cells adapt to limitations of specific elements by reducing their requirement for the corresponding element.