SummaryUpon iron limitation, Bacillus subtilis secretes the catecholic trilactone (2,3-dihydroxybenzoate-glycinethreonine)3 siderophore bacillibactin (BB) for ferric iron scavenging. Here, we show that ferri-BB uptake is mediated by the FeuABC transporter and that YuiI, a novel trilactone hydrolase, catalyses ferri-BB hydrolysis leading to cytosolic iron release. Among several Fur-regulated ABC transport mutants, only DfeuABC exhibited impaired growth during iron starvation. Quantification of intra-and extracellular (ferri)-BB in iron-depleted DfeuABC cultures revealed a fourfold increase of the extracellular siderophore concentration, confirming a blocked ferri-BB uptake in the absence of FeuABC. Ferri-BB was found to bind selectively to the periplasmic binding protein FeuA (K d = 57 Ϯ 1 nM), proving high-affinity transport of the iron-charged siderophore. During iron starvation, a DyuiI mutant displayed impaired growth and strong intracellular (30-fold) and extracellular (6.5-fold) (ferri)-BB accumulation. Kinetic studies in vitro revealed that YuiI hydrolyses both BB and ferri-BB. While BB hydrolysis led to strong accumulation of the tri-and dimeric reaction intermediates, ferri-BB hydrolysis yielded exclusively the monomeric reaction product and occurred with a 25-fold higher catalytic efficiency than BB single hydrolysis. Thus, ferri-BB was the preferred substrate of the YuiI esterase whose gene locus was designated besA.

Previous studies with two-dimensional gel electrophoresis techniques revealed that the cold shock response in Bacillus subtilis is characterized by rapid induction and accumulation of two classes of specific proteins, which have been termed cold-induced proteins (CIPs) and cold acclimatization proteins (CAPs), respectively. Only recently, the B. subtilis two-component system encoded by the desKR operon has been demonstrated to be essential for the cold-induced expression of the lipid-modifying desaturase Des, which is required for efficient cold adaptation of the membrane in the absence of isoleucine. At present, one of the most intriguing questions in this research field is whether DesKR plays a global role in cold signal perception and transduction in B. subtilis. In this report, we present the first genomewide transcriptional analysis of a cold-exposed bacterium and demonstrate that the B. subtilis two-component system DesKR exclusively controls the desaturase gene des and is not the cold-triggered regulatory system of global relevance. In addition to this, we identified a set of genes that might participate as novel players in the cold shock adaptation of B. subtilis. Two cold-induced genes, the elongation factor homolog ylaG and the L -dependent transcriptional activator homolog yplP, have been examined by construction and analysis of deletion mutants.Free-living prokaryotic organisms have the capacity to react rapidly to fluctuations of growth temperature. These responses are regulated at transcriptional and posttranscriptional levels and have been extensively characterized for heat shock, but only partially characterized for cold shock. In recent years, Bacillus subtilis has become a model organism for studies of the bacterial cold shock response representing the gram-positive branch of mesophilic soil bacteria (14).Many reports have dealt with the function of the cold shock proteins (CSPs), a widespread protein family representing a model for the nucleic acid binding cold-shock domain (CSD). The CSD is highly conserved from bacteria to humans (15,39,40) and is involved in coupling transcription to translation (36). Only recently the CSDBase database was established (http: //www.chemie.uni-marburg.de/ϳcsdbase), which includes detailed information about the CSD (37). This protein family has been identified in almost all psychrotrophic, mesophilic, thermophilic, and hyperthermophilic bacteria examined so far, and their presence in Thermotoga and Aquifex indicates an ancient origin (15). In B. subtilis, csp double-deletion strains show a variety of phenotypes, such as altered protein synthesis, aberrant nucleoid structure, cell lysis upon entry into the stationary growth phase, and impairment in sporulation (13, 39). The latter two defects were shown to be cured by heterologous expression of translation initiation factor IF1 from Escherichia coli (36).Other investigations have revealed how B. subtilis prevents rigidification of the membrane at low temperatures. The fluidity of the membrane is maintained by i...

The nucleic acid binding cold shock proteins (CSPs) and the cold-induced DEAD box RNA helicases have been proposed separately to act as RNA chaperones, but no experimental evidence has been reported on a direct cooperation. To investigate the possible interaction of the putative RNA helicases CshA and CshB and the CSPs from Bacillus subtilis during cold shock, we performed genetic as well as fluorescence resonance energy transfer (FRET) experiments. Both cshA and cshB genes could be deleted only in the presence of a cshB copy in trans, showing that the presence of one csh gene is essential for viability. The combined gene deletion of cshB and cspD resulted in a cold-sensitive phenotype that was not observed for either helicase or csp single mutants. In addition to the colocalization of the putative helicases CshA and CshB with CspB and the ribosomes in areas surrounding the nucleoid, we detected a strong FRET interaction in vivo between CshB and CspB that depended on active transcription. In contrast, a FRET interaction was not observed for CshB and the ribosomal protein L1. Therefore, we propose a model in which the putative cold-induced helicases and the CSPs work in conjunction to rescue misfolded mRNA molecules and maintain proper initiation of translation at low temperatures in B. subtilis.A sudden decrease in temperature is a serious challenge for all living microorganisms. The cellular reaction required for efficient adaptation to low temperatures is termed cold shock response. The cell has to cope with changes at different physiological levels, such as reduced fluidity of the membrane and slowdown of protein synthesis and protein folding (39). Accordingly, a general decrease of protein synthesis was observed in Escherichia coli and Bacillus subtilis cells following a cold shock (9, 17). The stress arising from a decrease in temperature can in principle be traced back to a reduction in molecular dynamics. The initiation of translation was shown to be a limiting factor for bacterial growth at low temperatures (3, 6). In addition to the delicate interaction of the complex translation machinery, the formation of secondary structures of mRNA prevents efficient initiation of translation (12). The tendency of RNA to fold and become kinetically trapped was described as a general problem and interferes with proper biological function (14). The formation of unfavorable secondary structures is even more intricate at lower temperatures. In analogy to protein folding mechanisms, RNA chaperones have been proposed to resolve and prevent misfolding of RNA molecules by either destabilizing RNA duplexes or binding to singlestranded nucleic acids (21). In the cold shock response, DEAD box RNA helicases have already been identified to destabilize RNA duplexes and the major cold shock proteins (CSPs) to bind RNA. DEAD box RNA helicases belong to the large family of DExD/H-box proteins, which generally have an RNA-dependent ATPase activity in vitro. They are involved in various cellular processes, such as ribosome assembly, i...

Aryl acid adenylation domains are the initial enzymes for aryl‐capping of catecholic siderophores in a plethora of microorganisms. In order to overcome the problem of iron acquisition in host organisms, siderophore biosynthesis is decisive for virulence development in numerous important human and animal pathogens. Recently, it was shown that growth of Mycobacterium tuberculosis and Yersinia pestis can be inhibited in an iron‐dependent manner using the arylic acyl adenylate analogue 5′‐O‐[N‐(salicyl)‐sulfamoyl] adenosine that acts on the salicylate activating domains, MbtA and YbtE [Ferreras JA, Ryu JS, Di Lello F, Tan DS, Quadri LEN (2005) Nat Chem Biol1, 29–32]. The present study explores the behaviour of the 2,3‐dihydroxybenzoate activating domain DhbE (bacillibactin synthesis) and compares it to that of YbtE (yersiniabactin synthesis) upon enzymatic inhibition using a set of newly synthesized aryl sulfamoyl adenosine derivatives. The obtained results underline the highly specific mode of inhibition for both aryl acid activating domains in accordance with their natively accepted aryl moiety. These findings are discussed regarding the structure–function based aspect of aryl substrate binding to the DhbE and YbtE active sites.

The cold shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature and leads to a dramatic increase in the production of a homologous class of small, often highly acidic cold shock proteins. This protein family is the prototype of the cold shock domain (CSD) that is conserved from bacteria to humans. For B. subtilis it has been shown that at least one of the three resident cold shock proteins (CspB to D) is essential under optimal growth conditions as well as during cold shock. Analysis of the B. subtilis cspB cspC double deletion mutant revealed that removal of these csp genes results in pleiotropic alteration of protein synthesis, cell lysis during the entry of stationary growth phase, and the inability to differentiate into endospores. We show here that heterologous expression of the translation initiation factor IF1 from E. coli in a B. subtilis cspB cspC double deletion strain is able to cure both the growth and the sporulation defects observed for this mutant, suggesting that IF1 and cold shock proteins have at least in part overlapping cellular function(s). Two of the possible explanation models are discussed.