Diego de Mendoza

The synthesis of l -cysteine, the major mechanism by which sulfur is incorporated into organic compounds in microorganisms, occupies a significant fraction of bacterial metabolism. In Bacillus subtilis the cysH operon, encoding several proteins involved in cysteine biosynthesis, is induced by sulfur starvation and tightly repressed by cysteine. We show that a null mutation in the cysK gene encoding an O -acetylserine-(thiol)lyase, the enzyme that catalyzes the final step in cysteine biosynthesis, results in constitutive expression of the cysH operon. Using DNA microarrays we found that, in addition to cysH , almost all of the genes required for sulfate assimilation are constitutively expressed in cysK mutants. These results indicate that CysK, besides its enzymatic role in cysteine biosynthesis, is a global negative regulator of genes involved in sulfur metabolism.

Aims: To determine if live recombinant Lactococcus lactis strains expressing rotavirus VP7 antigen are immunogenic in mice. Methods and Results: Using the food-grade lactic acid bacterium L. lactis as a carrier, we expressed VP7, the major rotavirus outer shell protein and one of the main components of the infective particle, as a cytoplasmic, secreted or cell wall anchored forms. Our results showed that recombinant L. lactis strains secreting VP7 proved to be more immunogenic than strains containing the antigen in the cytoplasm or anchored to the cell wall. Conclusions: This is the first demonstration that recombinant L. lactis producing VP7 can induce the production of a neutralizing antibody response against rotavirus by the intragastric route. Significance and Impact of the Study: Rotaviruses are the single most important aetiological agents of severe diarrhoea of infants and young children worldwide and have been estimated to be responsible for 650 000–800 000 deaths per year of children younger than 5 years old in development countries. Thus, the development of a safe and effective vaccine has been a global public health goal. Although two of five mice orally inoculated with L. lactis strains secreting VP7 elicited a specific-antibody response, these strains could be very useful to be used as a prototype to develop a new generation of protective rotavirus vaccines.

Most fatty acid desaturases are members of a large superfamily of integral membrane, O₂-dependent, iron-containing enzymes that insert double bonds into previously synthesized fatty acyl chains. The cold shock-induced, membrane-bound desaturase from Bacillus subtilis (Δ5-Des) uses existing phospholipids as substrates to introduce a cis-double bond at the fifth position of the fatty acyl chain. While essentially no three-dimensional structural information is available for these difficult-to-purify enzymes, experimental analysis of the topology of Δ5-Des has provided a model that might be extended to most acyl-lipid desaturases. In addition, studies of the cold-induced expression of Δ5-Des led to the identification of a two-component system composed of a membrane-associated kinase, DesK, and a transcriptional regulator, DesR, which stringently controls the transcription of the des gene, coding for the desaturase. A model for sensing and transduction of low-temperature signals has emerged from our results, which we discuss in the context of transcriptional regulation of membrane lipid fluidity homeostasis.

The Bacillus subtilis DesK/DesR two-component system regulates the expression of the des gene coding for the Δ5 acyl lipid desaturase. It is believed that a decrease in membrane lipid fluidity activates the DesK/DesR signal transduction cascade, which results in synthesis of the Δ5 acyl lipid desaturase and desaturation of membrane phospholipids. These newly synthesized unsaturated fatty acids then act as negative signals of des transcription, thus generating a regulatory metabolic loop that optimizes membrane fluidity. We previously suggested that DesK is a bifunctional enzyme with both kinase and phosphatase activities that could assume different signaling states in response to changes in the fluidity of membrane lipids. However, no direct experimental evidence supported this proposed model. In this study, we show that the C-terminal fragment of the DesK protein (DesKC) indeed acts as an autokinase. Addition of the response regulator DesR to phosphorylated DesKC resulted in rapid transfer of the phosphoryl group to DesR. Further, phosphorylated DesR can be dephosphorylated in the presence of DesKC, thus demonstrating that the sensor kinase has the ability to covalently modify DesR through both kinase and phosphatase activities. We also present evidence that DesKC might be locked in a kinase-dominant state in vivo and that its activities are not affected either in vivo or in vitro by unsaturated fatty acids. These findings provide the first direct evidence that the transmembrane segments of DesK are essential to sense changes in membrane fluidity and for regulating the ratio of kinase to phosphatase activities of the cytoplasmic C-terminal domain.

Bacillus subtilis was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions Δ5, Δ7, and Δ9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the B . subtilis acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by B . subtilis is located exclusively at the Δ5 position, regardless of the growth temperature and the length chain of the fatty acids.

Cerulenin is a fungal mycotoxin that potently inhibits fatty acid synthesis by covalent modification of the active site thiol of the chain-elongation subtypes of β-ketoacyl-acyl carrier protein (ACP) synthases. The Bacillus subtilis fabF ( yjaY ) gene ( fabF _b ) encodes an enzyme that catalyzes the condensation of malonyl-ACP with acyl-ACP to extend the growing acyl chain by two carbons. There were two mechanisms by which B. subtilis adapted to exposure to this antibiotic. First, reporter gene analysis demonstrated that transcription of the operon containing the fabF gene increased eightfold in response to a cerulenin challenge. This response was selective for the inhibition of fatty acid synthesis, since triclosan, an inhibitor of enoyl-ACP reductase, triggered an increase in fabF reporter gene expression while nalidixic acid did not. Second, spontaneous mutants arose that exhibited a 10-fold increase in the MIC of cerulenin. The mutation mapped at the B. subtilis fabF locus, and sequence analysis of the mutant fabF allele showed that a single base change resulted in the synthesis of FabF _b [I108F]. The purified FabF _b and FabF _b [I108F] proteins had similar specific activities with myristoyl-ACP as the substrate. FabF _b exhibited a 50% inhibitory concentration (IC ₅₀ ) of cerulenin of 0.1 μM, whereas the IC ₅₀ for FabF _b [I108] was 50-fold higher (5 μM). These biochemical data explain the absence of an overt growth defect coupled with the cerulenin resistance phenotype of the mutant strain.

Both prokaryotes and eukaryotes respond to a decrease in temperature with the expression of a specific subset of proteins. Although a large body of information concerning cold shock‐induced genes has been gathered, studies on temperature regulation have not clearly identified the key regulatory factor(s) responsible for thermosensing and signal transduction at low temperatures. Here we identified a two‐component signal transduction system composed of a sensor kinase, DesK, and a response regulator, DesR, responsible for cold induction of the des gene coding for the Δ5‐lipid desaturase from Bacillus subtilis . We found that DesR binds to a DNA sequence extending from position −28 to −77 relative to the start site of the temperature‐regulated des gene. We show further that unsaturated fatty acids (UFAs), the products of the Δ5‐desaturase, act as negative signalling molecules of des transcription. Thus, a regulatory loop composed of the DesK–DesR two‐component signal transduction system and UFAs provides a novel mechanism for the control of gene expression at low temperatures.

The molecular mechanisms of regulation of the genes involved in the biosynthesis of cysteine are poorly characterized in Bacillus subtilis and other gram-positive bacteria. In this study we describe the expression pattern of the B. subtilis cysH operon in response to sulfur starvation. A 6.1-kb polycistronic transcript which includes the cysH , cysP , ylnB , ylnC , ylnD , ylnE , and ylnF genes was identified. Its synthesis was induced by sulfur limitation and strongly repressed by cysteine. The cysH operon contains a 5′ leader portion homologous to that of the S box family of genes involved in sulfur metabolism, which are regulated by a transcription termination control system. Here we show that induction of B. subtilis cysH operon expression is dependent on the promoter and independent of the leader region terminator, indicating that the operon is regulated at the level of transcription initiation rather than controlled at the level of premature termination of transcription. Deletion of a 46-bp region adjacent to the −35 region of the cysH promoter led to high-level expression of the operon, even in the presence of cysteine. We also found that O -acetyl- l -serine (OAS), a direct precursor of cysteine, renders cysH transcription independent of sulfur starvation and insensitive to cysteine repression. We propose that transcription of the cysH operon is negatively regulated by a transcriptional repressor whose activity is controlled by the intracellular levels of OAS. Cysteine is predicted to repress transcription by inhibiting the synthesis of OAS, which would act as an inducer of cysH expression. These novel results provide the first direct evidence that cysteine biosynthesis is controlled at a transcriptional level by both negative and positive effectors in a gram-positive organism.

In this study we describe the expression pattern of the Leuconostoc paramesenteroides citMCDEFGRP operon in response to the addition of citrate to the growth medium. An 8.8-kb polycistronic transcript, which includes the citMCDEFGRP genes, was identified; its synthesis was dramatically induced upon addition of citrate to the growth medium. We also found that expression of the cit operon is subjected to posttranscriptional regulation, since processing sites included in four complex secondary structures (I, II, III, and IV) were identified by Northern blot analysis and mapped by primer extension. Upstream of the citMCDEFGRP operon a divergent open reading frame, whose expression was also increased by citrate, was identified by DNA sequencing and designated citI . The start and end sites of transcription of the cit operon and citI gene were mapped. The start sites are separated by a stretch of 188 bp with a very high A+T content of 77% and are preceded by transcriptional promoters. The end sites of the transcripts are located next to the 3′ end of two secondary structures characteristic of ρ-independent transcriptional terminators. The effect of the citI gene on expression of the cit operon was studied in Escherichia coli . The presence of the citI gene in cis and in trans resulted in increased activity of the cit promoter. These data provide the first evidence that citrate fermentation in Leuconostoc is regulated at the transcriptional level by a transcriptional activator rather than by a repressor.