Distinctive genetic features exhibited by the Y-family DNA polymerases in Bacillus subtilis

Translesional DNA polymerases form a large family of structurally related proteins, known as the Y-polymerases. Bacillus subtilis encodes two Y-polymerases, referred herewith as Pol Y1 and Pol Y2. Pol Y1 was expressed constitutively and did not mediate UV mutagenesis. Pol Y1 overexpression increased spontaneous mutagenesis. This effect depended on Pol Y1 polymerase activity, Pol Y1 interaction with the beta-clamp, and did not require the presence of the RecA protein. In addition, Pol Y1 overexpression delayed cell growth at low temperature. The growth delay was mediated by Pol Y1 interaction with the beta-clamp but not by its polymerase activity, suggesting that an excess of Pol Y1 in the cell could sequester the beta-clamp. In contrast, Pol Y2 was expressed during the SOS response, and, in its absence, UV-induced mutagenesis was abolished. Upon Pol Y2 overproduction, both UV-induced and spontaneous mutagenesis were stimulated, and both depended on the Pol Y2 polymerase activity. However, UV mutagenesis did not appear to require the interaction of Pol Y2 with the beta-clamp whereas spontaneous mutagenesis did. In addition, Pol Y2-mediated spontaneous mutagenesis required the presence of RecA. Together, these results show that the regulation and the genetic requirements of the two B. subtilis Y-polymerases are different, indicating that they fulfil distinct biological roles. Remarkably, Pol Y1 appears to exhibit a mutator activity similar to that of Escherichia coli Pol IV, as well as an E. coli UmuD-related function in growth delay. Pol Y2 exhibits an E. coli Pol V-like mutator activity, but probably acts as a single polypeptide to bypass UV lesions. Thus, B. subtilis Pol Y1 and Pol Y2 exhibit distinctive features from the E. coli Y-polymerases, indicating that different bacteria have adapted different solutions to deal with the lesions in their genetic material.     

Comparative genome analysis of Bacillus cereus group genomes with Bacillus subtilis

Genome features of the Bacillus cereus group genomes (representative strains of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis sub spp. israelensis) were analyzed and compared with the Bacillus subtilis genome. A core set of 1381 protein families among the four Bacillus genomes, with an additional set of 933 families common to the B. cereus group, was identified. Differences in signal transduction pathways, membrane transporters, cell surface structures, cell wall, and S-layer proteins suggesting differences in their phenotype were identified. The B. cereus group has signal transduction systems including a tyrosine kinase related to two-component system histidine kinases from B. subtilis. A model for regulation of the stress responsive sigma factor sigmaB in the B. cereus group different from the well studied regulation in B. subtilis has been proposed. Despite a high degree of chromosomal synteny among these genomes, significant differences in cell wall and spore coat proteins that contribute to the survival and adaptation in specific hosts has been identified.     

Biotransformation of nitric oxide in the cerebrospinal fluid of amyotrophic lateral sclerosis patients

Recent findings indicate that nitric oxide (NO*) over-production might be an important factor in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). We measured significantly higher concentrations of uric acid and thiol group-containing molecules (R-SH groups) in the cerebrospinal fluid (CSF) from SALS patients compared to controls. The above factors, together with a slightly increased free iron concentration found in the CSF, favour conditions necessary for the formation of the dinitrosyl iron complex, capable of NO* bio-transformation. Thus, we performed ex vivo saturation of CSF (from both SALS patients and controls) with NO*. A decrease in the level of R-SH was found. This was more pronounced in the CSF from SALS patients. In the CSF from SALS patients the production of nitrite and hydroxylamine was greater than that observed in the CSF from controls. Moreover, we also found increased Cu,Zn-SOD activity in the CSF from SALS patients (when compared to control subjects) but no activity corresponding to Mn-SOD in any CSF samples. As Cu,Zn-SOD can react with nitroxyl forming NO*, the conditions for a closed, but continuous, loop of NO* biotransformation are present in the CSF of ALS patients.     

The effect of 3-weeks stationary cardiac rehabilitation on plasma lipids level in 444 patients with coronary heart disease

The aim of this study was to investigate the effect of 3-weeks stationary cardiac rehabilitation on plasma lipids level in patients with CHD. The study included 444 consecutive patients (364 male and 80 female, mean age 58 +/- 9 year) with CHD who underwent 3-weeks stationary cardiac rehabilitation. Patients were divided into groups depending on their baseline levels of cholesterol and medication therapy: patients with normal (< 5 mmol/L, group I, 129 patients) and elevate plasma level of Total cholesterol (> 5 mmol/L, group II, 315 patients) and subgroups Ia and IIa (with statin in therapy), Ib and IIb (without statin in therapy). After 3-weeks cardiac rehabilitation, the levels of Total cholesterol 5.75 +/- 1.34 vs. 5.17 +/- 1.08 mmol/l; p < 0.001, triglycerides 2.04 +/- 1.33 vs. 1.81 +/- 1.06 mmol/L; p = 0.004, LDL-cholesterol 3.77 +/- 1.14 vs. 3.21 +/- 0.96 mmol/L; p < 0.001 were significantly lower while the level of HDL-cholesterol 0.94 +/- 0.28 vs. 0.99 +/- 0.27 mmol/L; p = 0.008 were significantly higher in comparison with the baseline values. Furthermore, we found significant changes in lipid profile at the end of rehabilitation in each group of patients compared with the baseline values. There were no significant differences in plasma lipids level between group of patients with or without statin in therapy at the end of rehabilitation. The results of this study suggest that moderate regular physical activity and diet alone or in combination with hypolipidemic drugs already after 3 weeks have a favourable effect on plasma lipids level and should be propagate in the prevention of CHD.     

A phage protein confers resistance to the lactococcal abortive infection mechanism AbiP

Phage bIL66M1 is sensitive to the lactococcal abortive infection mechanism AbiP. No spontaneous AbiP-resistant variant could be obtained at a frequency of <10(-10). However, AbiP-resistant variants were readily obtained during infection with both bIL66M1 and the highly homologous AbiP-resistant phage bIL170. Gain of AbiP resistance was due to the acquisition of the e6 gene from bIL170.     

The activity of antioxidant defence enzymes in the mussel Mytilus galloprovincialis from the Adriatic Sea

The activity of the antioxidant defence enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), glutathione peroxidase (GSH-Px, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2) and the phase II biotransformation enzyme glutathione-S-transferase (GST, EC 2.5.1.18) in whole mussels (Mytilus galloprovincialis) were studied. The mussels were collected in winter and in spring at two localities in the Adriatic Sea: Bar Port and Tivat Bay. Our results show that the activities of SOD, GSH-Px and GST were seasonally dependent with higher activities in winter. GR activity was also higher in winter, but only in mussels from Bar Port. In mussels from Tivat Bay, GR activity was lower in winter compared to spring. In addition, a decrease in CAT activity in mussels from Bar Port compared to those from Tivat Bay was found. It can be concluded that seasonal variations should be incorporated into interpretation of biomonitoring studies in mussels.     

v-Src SH3-enhanced interaction with focal adhesion kinase at beta 1 integrin-containing invadopodia promotes cell invasion

In viral Src (v-Src)-transformed cells, focal adhesion kinase (FAK) associates with v-Src by combined v-Src SH2 and gain-of-function v-Src SH3 domain binding to FAK. Here we assess the significance of the Arg-95 to Trp gain-of-function mutation in the v-Src SH3 domain through comparisons of Src-/- fibroblasts transformed with either Prague C v-Src or a point mutant (v-Src-RT) containing a normal (Arg-95) SH3 domain. Both v-Src isoforms exhibited equivalent kinase activity, enhanced Src-/- cell motility, and stimulated cell growth in both low serum and soft agar. The stability of a v-Src-RT.FAK signaling complex and FAK phosphorylation at Tyr-861 and Tyr-925 were reduced in v-Src-RT- compared with v-Src-transformed cells. v-Src but not v-Src-RT promoted Src-/- cell invasion through a reconstituted Matrigel basement membrane barrier and v-Src co-localized with FAK and beta(1) integrin at invadopodia. In contrast, v-Src-RT exhibited a partial perinuclear and focal contact distribution in Src-/- cells. Adenovirus-mediated FAK overexpression promoted v-Src-RT recruitment to invadopodia, the formation of a v-Src-RT.FAK signaling complex, and reversed the v-Src-RT invasion deficit. Adenovirus-mediated inhibition of FAK blocked v-Src-stimulated cell invasion. These studies establish that gain-of-function v-Src SH3 targeting interactions with FAK at beta(1) integrin-containing invadopodia act to stabilize a v-Src.FAK signaling complex promoting cell invasion.     

An Export-Specific Reporter Designed for Gram-Positive Bacteria: Application to Lactococcus lactis

The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bacteria, in part due to drawbacks of available export reporters. In this work, we demonstrate the export specificity and use of the Staphylococcus aureus secreted nuclease (Nuc) as a reporter for gram-positive bacteria. Nuc devoid of its export signal (called Δ_SPNuc) was used to create two fusions whose locations could be differentiated. Nuclease activity was shown to require an extracellular location in Lactococcus lactis, thus demonstrating the suitability of Δ_SPNuc to report protein export. The shuttle vector pFUN was designed to construct Δ_SPNuc translational fusions whose expression signals are provided by inserted DNA. The capacity of Δ_SPNuc to reveal and identify exported proteins was tested by generating an L. lactis genomic library in pFUN and by screening for Nuc activity directly in L. lactis. All Δ_SPNuc fusions displaying a strong Nuc⁺ phenotype contained a classical or a lipoprotein-type signal peptide or single or multiple transmembrane stretches. The function of some of the predicted signals was confirmed by cell fractionation studies. The fusions analyzed included long (up to 455-amino-acid) segments of the exported proteins, all previously unknown in L. lactis. Homology searches indicate that several of them may be implicated in different cell surface functions, such as nutrient uptake, peptidoglycan assembly, environmental sensing, and protein folding. Our results with L. lactis show that Δ_SPNuc is well suited to report both protein export and membrane protein topology.Most exported proteins are targeted for transport by a primary export signal comprising a hydrophobic domain. The signal can be present at the protein N terminus and cleaved during transport (i.e., signal peptide), but it can also remain embedded in the membrane (i.e., transmembrane segment) (63). Exported proteins are estimated to represent about 20% of total cellular proteins in gram-negative bacteria (39, 44), and contribute to various essential processes like nutrient uptake, macromolecular transport and assembly, envelope biogenesis and integrity, motility, cell division, energy generation, scavenging and detoxification, signal transduction, stress resistance, cell communication, and virulence in the case of pathogens.Several years ago, the elegant strategy of translational fusion to an export-specific reporter protein was designed to specifically isolate genes encoding exported proteins. This kind of reporter is translocation competent but unable to direct its own export (it corresponds to a signal peptideless form of an exported protein), and its activity requires an extracytoplasmic location. Among a library of proteins N-terminally fused to such a reporter, only fusions having the proper signal are exported and active. This strategy was first described for Escherichia coli using alkaline phosphatase (PhoA) as a reporter (16, 36); since then it has been applied to many gram-negative bacteria, particularly pathogens (for reviews, see references 24 and 35 and references therein).Export-specific reporters have a potentially important use in gram-positive bacteria, not only for protein identification and structural analyses, but also for technological applications. Most studies directly adopted the gram-negative reporters available, PhoA and the E. coli TEM β-lactamase (BlaM) (5). The Bacillus licheniformis α-amylase, AmyL, has also been used (17). Surprisingly, relatively few fusion studies allowed identification and characterization of the exported proteins (32, 42). In many cases, only the export signal was characterized (17, 18, 43, 51, 54, 55), possibly because only very short polypeptides (60 amino acids) were fused to the reporter.The rather limited results obtained by using reporter fusions may reveal that the reporters used are not fully adapted for use in gram-positive bacteria. (i) Fusions to gram-negative reporters PhoA and BlaM seem to display little activity and/or to be less stable in gram-positive bacteria, probably because of improper folding (42, 54). Both PhoA (active as a dimer) and BlaM folding require disulfide bond formation, which is catalyzed by DsbA in various gram-negative bacteria (3, 22); it is not yet clear whether such a process exists in gram-positive bacteria (19). Furthermore, altered codon usage and GC content may decrease expression of reporter genes. (ii) Selection of BlaM fusions has been routinely performed in E. coli, possibly due to difficulties of direct ampicillin resistance selection in gram-positive bacteria (43, 51, 54). Such preselection may create a bias due to species specificity of export signals, which, for signal peptides, are significantly longer in gram-positive bacteria (65). (iii) AmyL, a reporter of gram-positive origin, may be the best suited for use in gram-positive bacteria. However, the plate detection test results in loss of cell viability (18a), and thus its use requires replica plating (17, 18).The above-mentioned considerations led us to design a protein export reporter which would be suitable for use in a broad host range of gram-positive bacteria. The reporter we chose is based on the Staphylococcus aureus secreted nuclease (Nuc), a small, stable, monomeric, extensively studied enzyme (EC 3.1.31.1 [9]), having a mature form devoid of cysteine residues (50). Nuc is efficiently secreted by various gram-positive bacteria as an active 168-amino-acid polypeptide which may undergo subsequent proteolytic cleavage of the N-terminal 19- to 21-amino-acid propeptide to give rise to another active form, called NucA (27, 30, 31, 38, 58). The enzymatic activity test for Nuc is sensitive and nontoxic to colonies (28, 29, 50). Several features of Nuc thus make it a potentially optimal candidate for reporting protein export in gram-positive bacteria.In this study, we show that a truncated form of Nuc lacking its export signal (called Δ_SPNuc) is an export-specific reporter. A shuttle vector, pFUN (for fusion to nuclease), was designed to specifically identify genes encoding exported proteins as translational fusions to Δ_SPNuc. pFUN was developed and used to study protein export in Lactococcus lactis, a gram-positive microaerophilic industrial microorganism used in dairy fermentations (37). Despite the technological importance of surface and extracellular proteins in this organism, export of relatively few proteins (excluding plasmid- or transposon-encoded proteins) has been reported to date (4, 6, 12, 13, 15, 26, 40, 60–62). In this work, we characterize 16 previously unknown exported L. lactis proteins. Our results confirm that Δ_SPNuc is a sensitive and specific export reporter for L. lactis and potentially for other gram-positive bacteria.