Expression of the toxin-antitoxin genes yefM(Lrh), yoeB(Lrh) in human Lactobacillus rhamnosus isolates.

Lactobacilli are important microorganisms in various activities, for example, diary products, meat ripening, bread and pickles, but, moreover, are associated directly with human skin and cavities (e.g., mouth, gut, or vagina). Some of them are used as probiotics. Therefore, the molecular biological investigation of these bacteria is important. Earlier we described several toxin antitoxin systems (type II) in lactobacilli. Here, we describe the structure and transcriptional regulation of genes, encoding TA system YefM-YoeB(Lrh) in three strains of Lactobacillus rhamnosus comparing stationary and exponential growth phases, the influence of stress factors and mRNA stability. The same TA system is responding to physiological and stress conditions differently in related strains. Using primer extension and RLM-RACE methods we determined three transcription start sites of RNAs in the operon. The promoter region of the operon is preceded by a conserved BOX element occurring at multiple positions in the genomes of L. rhamnosus strains. Downstream of and partially overlapping with the 3' end of the yoeB(Lrh) toxin gene, a divergently transcribed unexpected RNA was detected.

Cervimycin C resistance in Bacillus subtilis is due to a promoter up-mutation and increased mRNA stability of the constitutive ABC-transporter gene bmrA.

Two independent cervimycin C (CmC)-resistant clones of Bacillus subtilis were identified, each carrying two mutations in the intergenic region preceding the ABC transporter gene bmrA. In the double mutant, real-time PCR revealed an increased amount of bmrA mRNA with increased stability. Accordingly, isolation of membrane proteins yielded a strong band at 64 kDa corresponding to BmrA. Analyses showed that one mutation optimized the -35 box sequence conferring resistance to 3 µM CmC, while the +6 mutation alone had no effect, but increased the potential of the strain harboring the -35 mutation to grow at 5 µM CmC. Transcriptional fusions revealed an elevated bmrA promoter activity for the double mutant. Electrophoretic mobility shift assays (EMSAs) confirmed a 30-fold higher binding affinity of RNA polymerase for this mutant compared with the wild type, and the effect was due to the -35 box alteration of the bmrA promoter. In vitro transcription experiments substantiated the results of the EMSA. EMSAs in the presence of heparin indicated that the mutations did not influence the formation and/or the stability of open complexes. Half-life measurements demonstrated that the +6 mutation stabilized bmrA mRNA ≈ 2-fold. Overall, we found that an ABC transporter confers antibiotic resistance by the cumulative effects of two mutations in the promoter region.

A dual-function sRNA from B. subtilis: SR1 acts as a peptide encoding mRNA on the gapA operon.

Small non-coding RNAs (sRNAs) have been found to regulate gene expression in all three kingdoms of life. So far, relatively little is known about sRNAs from Gram-positive bacteria. SR1 is a regulatory sRNA from the Bacillus subtilis chromosome that inhibits by base-pairing translation initiation of ahrC mRNA encoding a transcriptional activator of the arginine catabolic operons. Here we present a novel target of SR1, the glycolytic gapA operon. Both microarray and Northern blot analyses show that the amount of gapA operon mRNA is significantly higher in the presence of SR1 when cells were grown in complex medium until stationary phase. Translational lacZ fusions and toeprinting analyses demonstrate that SR1 does not promote translation of gapA mRNA. By contrast, the half-life of gapA operon mRNA is strongly reduced in the sr1 knockout strain. SR1 does not act as a base-pairing sRNA on gapA operon mRNA. Instead, we demonstrate that the 39 aa peptide encoded by SR1, SR1P, is responsible for the effect of SR1 on the gapA operon. We show that SR1P binds GapA, thereby stabilizing the gapA operon mRNA by a hitherto unknown mechanism. SR1 is the first dual-function sRNA found in B. subtilis.

Transcriptional response to the neuroleptic-like compound Ampullosporin A in the rat ketamine model.

Psychotic disorders affecting up to 1% of the human population represent pathological changes to the metabolic homeostasis of the brain. Increasing evidence in the literature suggests complex biochemical and/or transcriptional alterations accompanying schizophrenia-like phenomena. Sub-chronic treatment with sub-anaesthetic doses of ketamine induces schizophrenia-related psychotic alterations that can be used as an animal model in the study of this disorder. Ampullosporin A belongs to a specific group of pore-forming fungal peptides, peptaibols. We focused on the analysis of molecular events occurring in the brain of ketamine-pre-treated rats after administration of Ampullosporin A with neuroleptic-like activity. The complex experimental approach allowed us to correlate the use of low molecular weight substances with a transcriptome fingerprint in the prefrontal cortex. We found 63 genes to be up-regulated and 22 genes suppressed, with transthyretin, syndecan-1 and NeuroD1 showing the highest degree of up-regulation. Our results suggest the possibility that Ampullosporin A belongs to the group of neuroleptic-like compounds, inducing massive changes in neurotransmitter receptor composition, calcium signalling cascades and second messenger systems, and leading to the plastic reorganization of brain tissue, metabolic pathways and synapses.

Brevican-containing perineuronal nets of extracellular matrix in dissociated hippocampal primary cultures.

Perineuronal nets (PNN) are specialized extracellular matrix structures enwrapping CNS neurons, which are important regulators for neuronal and synaptic functions. Brevican, a chondroitin sulfate proteoglycan, is an integral component of PNN. Here, we have investigated the appearance of these structures in hippocampal primary cultures. The expression profile of brevican in mixed cultures resembles the in vivo pattern with a strong upregulation of all isoforms during the second and 3rd weeks in culture. Brevican is primarily synthesized by co-cultured glial fibrillary acidic protein (GFAP-)-positive astrocytes and co-assembles with its interaction partners in PNN-like structures on neuronal somata and neurites as identified by counterstaining with the PNN marker Vicia villosa lectin. Both excitatory and inhibitory synapses are embedded into PNN. Furthermore, axon initial segments are strongly covered by a dense brevican coat. Altogether, we show that mature primary cultures can form PNN, and that basic features of these extracellular matrix structures may be studied in vitro.

A beta-lysine adenylating enzyme and a beta-lysine binding protein involved in poly beta-lysine chain assembly in nourseothricin synthesis in Streptomyces noursei.

Nourseothricins (syn. Streptothricins), a group of nucleoside peptides produced by several streptomycete strains, contain a poly beta-lysine chain of variable length attached in amide linkage to the amino sugar moiety gulosamine of the nucleoside portion. We show that the nourseothricin-producing Streptomyces noursei contains an enzyme (NpsA) of an apparent M(r) 56,000 that specifically activates beta-lysine by adenylation but does not bind to it as a thioester. Cloning and sequencing of npsA from S. noursei including its flanking DNA regions revealed that it is closely linked to the nourseothricin resistance gene nat1 and some other genes on the chromosome possibly involved in nourseothricin biosynthesis. The deduced amino-acid sequence revealed that NpsA is a stand-alone adenylation domain with similarity to the adenylation domains of nonribosomal peptide synthetases (NRPS). Further analysis revealed that S. noursei contains a beta-lysine binding enzyme (NpsB) of about M(r) 64,100 which can be loaded by NpsA with beta-lysine as a thioester. Analysis of the deduced amino-acid sequence from the gene (npsB) of NpsB showed that it consists of two domains. The N-terminal domain of approximately 100 amino-acid residues has high similarity to PCP domains of NRPSs whereas the 450-amino-acid C-terminal domain has a high similarity to epimerization (E)-domains of NRPSs. Remarkably, in this E-domain the conserved H-H-motif is changed to H-Q, which suggests that either the domain is nonfunctional or has a specialized function. The presence of one single adenylating beta-lysine activating enzyme in nourseothricin-producing streptomycete and a separate binding protein suggests an iteratively operating NRPS-module catalyses synthesis of the poly beta-lysine chain.