Investigation of sRNA-mRNA Interactions in Bacillus subtilis In Vivo.

In this chapter, we describe in vivo methods for the analysis of interactions between an sRNA and its target mRNA in B. subtilis. All these methods have been either established or significantly improved in our group and successfully employed to characterize a number of sRNA/target mRNA systems in Bacillus subtilis. Whereas in Chap. 8, we describe a combination of in vitro methods, e.g., EMSA and RNA secondary structure probing, we focus here on the investigation of RNA-RNA interactions in vivo using compatible plasmids or chromosomal insertions and deletions, the elucidation of the mechanisms of action of regulatory sRNAs employing transcriptional and translational reporter gene fusions, as well as the determination of expression profiles, half-lives of sRNA and mRNA, and their intracellular concentrations, and, finally, the investigation of RNA chaperones that promote the sRNA/mRNA interaction. For an in-depth analysis of sRNA-mRNA interactions in B. subtilis, a combination of in vivo and in vitro methods should be applied.

In Vitro Methods for the Investigation of sRNA-mRNA Interactions in Bacillus subtilis.

So far, in Bacillus subtilis, only four trans-encoded and 11 cis-encoded sRNAs and their targets have been investigated in detail, the majority of them in our group (rev. in 1, 2). Here, we describe in vitro methods for the analysis of sRNA/mRNA interactions. All these methods have been either elaborated or significantly improved in our group and successfully applied to characterize a number of sRNA/target mRNA systems in Bacillus subtilis for which we provide examples from our own work. The in vitro methods comprise the synthesis and purification of labeled and unlabeled RNA, the analysis of sRNA/mRNA interactions in electrophoretic mobility shift assays (EMSAs) including the calculation of their apparent binding rate constants (kapp) and equilibrium dissociation constants (Kd), the localization of minimal regulatory regions of an sRNA, the determination of the secondary structures of both interacting RNAs and their complex as well as the analysis of RNA chaperones that may promote the sRNA/mRNA interaction.

Small proteins in Gram-positive bacteria.

Small proteins comprising less than 100 amino acids have been often ignored in bacterial genome annotations. About 10 years ago, focused efforts started to investigate whole peptidomes, which resulted in the discovery of a multitude of small proteins, but only a number of them have been characterized in detail. Generally, small proteins can be either membrane or cytosolic proteins. The latter interact with larger proteins, RNA or even metal ions. Here, we summarize our current knowledge on small proteins from Gram-positive bacteria with a special emphasis on the model organism Bacillus subtilis. Our examples include membrane-bound toxins of type I toxin-antitoxin systems, proteins that block the assembly of higher order structures, regulate sporulation or modulate the RNA degradosome. We do not consider antimicrobial peptides. Furthermore, we present methods for the identification and investigation of small proteins.

A new role for SR1 from Bacillus subtilis: regulation of sporulation by inhibition of kinA translation.

SR1 is a dual-function sRNA from Bacillus subtilis. It inhibits translation initiation of ahrC mRNA encoding the transcription activator of the arginine catabolic operons. Base-pairing is promoted by the RNA chaperone CsrA, which induces a slight structural change in the ahrC mRNA to facilitate SR1 binding. Additionally, SR1 encodes the small protein SR1P that interacts with glyceraldehyde-3P dehydrogenase A to promote binding to RNase J1 and enhancing J1 activity. Here, we describe a new target of SR1, kinA mRNA encoding the major histidine kinase of the sporulation phosphorelay. SR1 and kinA mRNA share 7 complementary regions. Base-pairing between SR1 and kinA mRNA decreases kinA translation without affecting kinA mRNA stability and represses transcription of the KinA/Spo0A downstream targets spoIIE, spoIIGA and cotA. The initial interaction between SR1 and kinA mRNA occurs 10 nt downstream of the kinA start codon and is decisive for inhibition. The sr1 encoded peptide SR1P is dispensable for kinA regulation. Deletion of sr1 accelerates sporulation resulting in low quality spores with reduced stress resistance and altered coat protein composition which can be compensated by sr1 overexpression. Neither CsrA nor Hfq influence sporulation or spore properties.

Moonlighting in Bacillus subtilis: The Small Proteins SR1P and SR7P Regulate the Moonlighting Activity of Glyceraldehyde 3-Phosphate Dehydrogenase A (GapA) and Enolase in RNA Degradation.

Moonlighting proteins are proteins with more than one function. During the past 25 years, they have been found to be rather widespread in bacteria. In Bacillus subtilis, moonlighting has been disclosed to occur via DNA, protein or RNA binding or protein phosphorylation. In addition, two metabolic enzymes, enolase and phosphofructokinase, were localized in the degradosome-like network (DLN) where they were thought to be scaffolding components. The DLN comprises the major endoribonuclease RNase Y, 3'-5' exoribonuclease PnpA, endo/5'-3' exoribonucleases J1/J2 and helicase CshA. We have ascertained that the metabolic enzyme GapA is an additional component of the DLN. In addition, we identified two small proteins that bind scaffolding components of the degradosome: SR1P encoded by the dual-function sRNA SR1 binds GapA, promotes the GapA-RNase J1 interaction and increases the RNase J1 activity. SR7P encoded by the dual-function antisense RNA SR7 binds to enolase thereby enhancing the enzymatic activity of enolase bound RNase Y. We discuss the role of small proteins in modulating the activity of two moonlighting proteins.

SR7 - a dual-function antisense RNA from Bacillus subtilis.

Here, we describe SR7, a dual-function antisense RNA encoded on the Bacillus subtilis chromosome. This RNA was earlier described as SigB-dependent regulatory RNA S1136 and reported to reduce the amount of the small ribosomal subunit under ethanol stress. We found that the 5' portion of SR7 encodes a small protein composed of 39 amino acids which we designated SR7P. It is translated from a 185 nt SigB-dependent mRNA under five different stress conditions and a longer SigB-independent RNA constitutively. About three-fold higher amounts of SR7P were detected in B. subtilis cells exposed to salt, ethanol, acid or heat stress. Co-elution experiments with SR7PC-FLAG and Far-Western blotting demonstrated that SR7P interacts with the glycolytic enzyme enolase. Enolase is a scaffolding component of the B. subtilis degradosome where it interacts with RNase Y and phosphofructokinase PfkA. We found that SR7P increases the amount of RNase Y bound to enolase without affecting PfkA. RNA does not bridge the SR7P-enolase-RNase Y interaction. In vitro-degradation assays with the known RNase Y substrates yitJ and rpsO mRNA revealed enhanced enzymatic activity of enolase-bound RNase Y in the presence of SR7P. Northern blots showed a major effect of enolase and a minor effect of SR7P on the half-life of rpsO mRNA indicating a fine-tuning role of SR7P in RNA degradation.

Intermolecular Communication in Bacillus subtilis: RNA-RNA, RNA-Protein and Small Protein-Protein Interactions.

In bacterial cells we find a variety of interacting macromolecules, among them RNAs and proteins. Not only small regulatory RNAs (sRNAs), but also small proteins have been increasingly recognized as regulators of bacterial gene expression. An average bacterial genome encodes between 200 and 300 sRNAs, but an unknown number of small proteins. sRNAs can be cis- or trans-encoded. Whereas cis-encoded sRNAs interact only with their single completely complementary mRNA target transcribed from the opposite DNA strand, trans-encoded sRNAs are only partially complementary to their numerous mRNA targets, resulting in huge regulatory networks. In addition to sRNAs, uncharged tRNAs can interact with mRNAs in T-box attenuation mechanisms. For a number of sRNA-mRNA interactions, the stability of sRNAs or translatability of mRNAs, RNA chaperones are required. In Gram-negative bacteria, the well-studied abundant RNA-chaperone Hfq fulfils this role, and recently another chaperone, ProQ, has been discovered and analyzed in this respect. By contrast, evidence for RNA chaperones or their role in Gram-positive bacteria is still scarce, but CsrA might be such a candidate. Other RNA-protein interactions involve tmRNA/SmpB, 6S RNA/RNA polymerase, the dual-function aconitase and protein-bound transcriptional terminators and antiterminators. Furthermore, small proteins, often missed in genome annotations and long ignored as potential regulators, can interact with individual regulatory proteins, large protein complexes, RNA or the membrane. Here, we review recent advances on biological role and regulatory principles of the currently known sRNA-mRNA interactions, sRNA-protein interactions and small protein-protein interactions in the Gram-positive model organism Bacillus subtilis. We do not discuss RNases, ribosomal proteins, RNA helicases or riboswitches.

Association of Vitamin D Receptor Gene Polymorphisms and Serum 25-Hydroxy Vitamin D Levels in Vitiligo - A Case-control Study.

BACKGROUND: Vitamin D has stimulatory and protective effects on melanocytes and acts through its nuclear vitamin D receptor (VDR) on target cells. Various single-nucleotide polymorphisms (SNPs) in VDR genes have been described. AIMS: The aim was to study and compare the association of SNP of BsmI/Apa-I/TaqI/FokI/Cdx2 in VDR gene as well as the plasma vitamin D levels in vitiligo patients and healthy controls. METHODS: This was a case-control study, in which 100 patients of vitiligo and an equal number of healthy individuals were studied. The VDR polymorphisms of Bsm I, Apa-I, TaqI, fok I, and cdx2 were investigated, after extraction of genomic DNA by rapid capillary polymerase chain reaction with melting curve analysis, and 25 hydroxy vitamin D levels were measured in cases and controls. RESULTS: The frequency of genotypes (SNP FokI and cdx2) was higher in the patient group versus controls (P = 0.002). The genotype frequency (TaqI and Apa-I) was higher in the patients than the controls for the Tt genotype, but not significantly higher (48% vs. 39%, P = 0.1431). The difference between the groups in frequency of the genotype Aa(TaqI and Apa-I) was statistically significant (P = 0.0001 and P = 0.033). Statistically significant difference was also observed in Apa-I-evaluated alleles in cases when compared to controls (P = 0.0001). There was no significant difference in serum vitamin D levels between various genotypes among cases and controls. Out of 100 cases, 10 were found to have vitamin D levels of >30 ng/ml, 15 had levels between 20 and 30 ng/ml, 52 had ≤20 ng/ml, and 23 ≤ 10 ng/ml, respectively. LIMITATIONS: Since the skin biopsies were not taken from the lesions of vitiligo, the correlation of serum levels with tissue levels of VDR gene was not possible and the role of vitamin D supplementation was not evaluated. CONCLUSION: The single nucleotide gene polymorphisms of various VDR genes as found in the cases might lead to vitamin D deficiency, due to VDR dysfunction, which in turn could increase the susceptibility to develop vitiligo.

Topoisomerase IV can functionally replace all type 1A topoisomerases in Bacillus subtilis.

DNA topoisomerases play essential roles in chromosome organization and replication. Most bacteria possess multiple topoisomerases which have specialized functions in the control of DNA supercoiling or in DNA catenation/decatenation during recombination and chromosome segregation. DNA topoisomerase I is required for the relaxation of negatively supercoiled DNA behind the transcribing RNA polymerase. Conflicting results have been reported on the essentiality of the topA gene encoding topoisomerase I in the model bacterium Bacillus subtilis. In this work, we have studied the requirement for topoisomerase I in B. subtilis. All stable topA mutants carried different chromosomal amplifications of the genomic region encompassing the parEC operon encoding topoisomerase IV. Using a fluorescent amplification reporter system we observed that each individual topA mutant had acquired such an amplification. Eventually, the amplifications were replaced by a point mutation in the parEC promoter region which resulted in a fivefold increase of parEC expression. In this strain both type I topoisomerases, encoded by topA and topB, were dispensable. Our results demonstrate that topoisomerase IV at increased expression is necessary and sufficient to take over the function of type 1A topoisomerases.

Patients' Satisfaction with a Psychiatric Day Hospital in the West Galway Catchments Area.

AIM: To evaluate patients' satisfaction with a psychiatric day hospital in the West Galway Catchments area. STUDY DESIGN: This is a cross-sectional study. METHODS: This study was undertaken by using a 33-items questionnaire for 44 patients who attended the day hospital. SPSS version-12 was used for collecting and applying tests to the data, Fisher's exact test was used to analyse the data as sample size is small and P<0.05 was considered statistically significant. Results are also presented in the form of descriptive analysis including percentages and frequencies. RESULTS: Only 37 out of the 44 patients returned the questionnaires. Among the participants were 61% (n=22) male and 38% (n=15) female. The patients were mostly satisfied with the services available at the day hospital, except the waiting period for a first appointment. There was no significant (P=0.11) gender difference in terms of patients' satisfaction with the day hospital service. The age group, 45 years and above, were less satisfied (P=0.02) with the availability of a comfortable room for counselling, as compared to the younger age group. The patients did not like to tell others that they were attending the day hospital. The results were comparable with previous published patient satisfaction surveys using the same questionnaire by the Health Research Board, except that the patients in the present survey were more satisfied with the waiting area and availability of counselling. CONCLUSIONS: Patients are mostly satisfied with the West Galway day hospital services. The older patients (45 years and above) are not satisfied with the counselling room, and therefore further investigations are needed to ascertain their requirements for a counselling room. Patients are more satisfied with the waiting area and availability of counselling compared to the findings of other similar studies conducted in Ireland. Resources are needed to be allocated to the day hospital services.

Serum levels of homocysteine in mice with malignant tumours.

BACKGROUND: Oxygen radicals and malondialdehyde (MDA) are tumourigenic. Homocysteine generates oxygen radicals. The possibility exists that hyperhomocysteinemia is a risk factor for cancer. OBJECTIVE: To investigate if serum levels of homocysteine and MDA are elevated in mice with malignant tumours. METHODS: Levels of serum homocysteine and MDA were estimated in 22 control and 22 tumour-bearing Balb/c mice. RESULTS: Serum homocysteine levels in control and tumour-bearing mice were 3.01+/-0.26 mumol/L and 4.05+/-0.46 mumol/L, respectively. The serum levels of MDA were 6.23+/-0.72 nmol/mL and 11.60+/-1.72 nmol/mL, respectively, in control and tumour-bearing mice. CONCLUSION: These results suggest that cancer in mice is associated with an increase in serum levels of homocysteine and the lipid peroxidation product MDA. It is, however, not known if this rise in homocysteine and MDA is due to cancer or if this rise causes cancer.