The DNA sequence of γ-glutamyltranspeptidase gene of Bacillus subtilis (natto) plasmid pUH1

Summary The -glutamyltranspeptidase (-GTP) gene of Bacillus subtilis (natto) plasmid designated pUH1, which is responsible for polyglutamate production, has been cloned and the nucleotide sequence determined. The sequence contains a single open-reading frame stretching for 1260 bp with a relative molecular mass of 49356. Putative -35 and -10 sequences, TTCAAA and TATTAT, were observed as the consensus sequence for the promoter recognized by the ⁴³ RNA polymerase of B. subtilis, and the ribosome binding site, the sequence of which was AACGAG, was complementary to the binding sequence of B. subtilis 16S rRNA except for one base. The amino acid sequence of the gene with the segment of putative protein C403 of staphylococcal plasmid pE194 indicates homology, whereas that with Escherichia coli and mammalian -GTPs does not show any similarity at all.     

Growth kinetics of 1–2 mm and 3–4 mm colonies of Nostoc sphaeroides (Cyanophyta) in outdoor culture

Nostoc sphaeroides Kützing was cultivated in paddlewheel-driven raceway ponds and the growth kinetics of 1-2 mm and 3-4 mm colonies of N. sphaeroides was studied. The biomass productivities in 2.5 m(2) raceway ponds inoculated with 1-2 mm and 3-4 mm colonies were 5.2 and 0.25 g dry wt m(-2) d(-1), respectively. Furthermore, differently sized colonies showed different relative water content, total soluble carbohydrates, chlorophyll a content and density of filaments. This is the first report on mass culture of N. sphaeroides under outdoor conditions.     

DNA Sequence of Bacillus subtilis (natto) NR-1 γ-Glutamyltranspeptidase Gene,ggt

The ggt encoding γ-glutamyltranspeptidase (GGT) from Bacillus subtilis (natto) was cloned and sequenced. The DNA sequence contains a single open reading frame of 1761 bp that might be translated to a protein of 587 amino acid residues, and indicates that B. subtilis (natto) GGT is synthesized as prepro-GGT and processed later into large and small subunits. The putative catabolite responsive element (CRE) was located upstream of the ggt coding region.     

Role of the Y-Family DNA Polymerases YqjH and YqjW in Protecting Sporulating Bacillus subtilis Cells from DNA Damage

The role played by the Y-family DNA polymerases YqjH and YqjW in protecting sporulating cells of Bacillus subtilis from DNA damage was determined. The absence of either yqjH and/or yqjW not only reduced sporulation efficiency but also sensitized the sporulating cells to hydrogen peroxide, tert-butylhydroperoxide (t-BHP), mitomycin-C (M-C), and UV-C radiation. Moreover, these DNA-damaging agents increased the mutation frequency of wild-type sporulating cells to 4-azaleucine, but the production of mutants was YqjH- and YqjW-dependent. In conclusion, the results presented here indicate that YqjH/YqjW-dependent-translesion synthesis (TLS) operates in sporulating B. subtilis cells and contributes in processing spontaneous and artificially induced genetic damage, which is apparently required for an efficient sporulation process.     

Factors Affecting Use and Delay (≥8 Weeks) of Adjuvant Chemotherapy after Colorectal Cancer Surgery and the Impact of Chemotherapy-Use and Delay on Oncologic Outcomes

Purpose

To evaluate factors affecting the use and delay ≥8 weeks of adjuvant chemotherapy and the impact of chemotherapy use and delay on survival.

Methods

Between 2005 and 2012, consecutive patients with stage II and III colorectal cancer who were treated with major curative resection were enrolled.

Results

Among 750 patients with stage II (n = 318) and III (n = 432) disease, 153 (20.4%) did not receive chemotherapy. Among 597 patients with adjuvant chemotherapy, 31 (5.2%) began chemotherapy 8 weeks or more after surgery. Factors associated with not receiving chemotherapy were: age ≥80 years (hazard ratio [HR] = 5.2), American Society of Anesthesiologists score ≥3 (HR = 1.9), underlying cerebrovascular disease (HR = 1.7), stage II disease (HR = 2.0), presence of postoperative complications (HR = 2.2), or intensive care unit admission (HR = 2.4). Factors associated with chemotherapy delay ≥8 weeks were: male sex (HR = 4.2), rectal primary cancer (HR = 5.4), or presence of postoperative complications (HR = 2.5). Independent prognostic factors for overall survival included TNM III stage (HR = 2.04) and chemotherapy delay ≥8 weeks (HR = 0.39) or <8 weeks (HR = 0.22). Independent prognostic factors for recurrence-free survival were TNM III stage (HR = 2.26) and chemotherapy delay <8 weeks (HR = 0.35).

Conclusions

Postoperative complications were associated with both lack of and delayed chemotherapy. Timely initiation of chemotherapy, defined as <8 weeks, was a favorable prognostic factor for overall and recurrence-free survival. To increase the proportion of patients receiving chemotherapy and timely initiation of chemotherapy, surgical complications should be minimized after curative resection.     

Mutations suppressing the loss of DegQ function in Bacillus subtilis (natto) poly-γ-glutamate synthesis

The degQ gene of Bacillus subtilis (natto), encoding a small peptide of 46 amino acids, is essential for the synthesis of extracellular poly-gamma-glutamate (γPGA). To elucidate the role of DegQ in γPGA synthesis, we knocked out the degQ gene in Bacillus subtilis (natto) and screened for suppressor mutations that restored γPGA synthesis in the absence of DegQ. Suppressor mutations were found in degS, the receptor kinase gene of the DegS-DegU two-component system. Recombinant DegS-His(6) mutant proteins were expressed in Escherichia coli cells and subjected to an in vitro phosphorylation assay. Compared with the wild type, mutant DegS-His(6) proteins showed higher levels of autophosphorylation (R208Q, M195I, L248F, and D250N), reduced autodephosphorylation (D250N), reduced phosphatase activity toward DegU, or a reduced ability to stimulate the autodephosphorylation activity of DegU (R208Q, D249G, M195I, L248F, and D250N) and stabilized DegU in the phosphorylated form. These mutant DegS proteins mimic the effect of DegQ on wild-type DegSU in vitro. Interestingly, DegQ stabilizes phosphorylated DegS only in the presence of DegU, indicating a complex interaction of these three proteins.     

Selective Replication of Bacteriophage φ29 Deoxyribonucleic Acid in 6-(p-Hydroxyphenylazo)-Uracil-Treated Bacillus subtilis

Phage phi29 deoxyribonucleic acid (DNA) replicated under conditions where semiconservative DNA production in Bacillus subtilis host cells was blocked with 6-(p-hydroxyphenylazo)-uracil (HPUra). The time of initiation of phi29 DNA replication was not affected by HPUra, and normal quantities of viable phage were produced in the presence of the inhibitor. Studies with conditional lethal mutants of phage phi29 demonstrated the usefulness of HPUra for detection of viral-specific DNA production.     

Bacillus subtilis RNase J1 endonuclease and 5′ exonuclease activities in the turnover of ΔermC mRNA

RNase J1, a ribonuclease with 5′ exonuclease and endonuclease activities, is an important factor in Bacillus subtilis mRNA decay. A model for RNase J1 endonuclease activity in mRNA turnover has RNase J1 binding to the 5′ end and tracking to a target site downstream, where it makes a decay-initiating cleavage. The upstream fragment from this cleavage is degraded by 3′ exonucleases; the downstream fragment is degraded by RNase J1 5′ exonuclease activity. Previously, ΔermC mRNA was used to show 5′-end dependence of mRNA turnover. Here we used ΔermC mRNA to probe RNase J1-dependent degradation, and the results were consistent with aspects of the model. ΔermC mRNA showed increased stability in a mutant strain that contained a reduced level of RNase J1. In agreement with the tracking concept, insertion of a strong stem–loop structure at +65 resulted in increased stability. Weakening this stem–loop structure resulted in reversion to wild-type stability. RNA fragments containing the 3′ end were detected in a strain with reduced RNase J1 expression, but were undetectable in the wild type. The 5′ ends of these fragments mapped to the upstream side of predicted stem–loop structures, consistent with an impediment to RNase J1 5′ exonuclease processivity. A ΔermC mRNA deletion analysis suggested that decay-initiating endonuclease cleavage could occur at several sites near the 3′ end. However, even in the absence of these sites, stability was further increased in a strain with reduced RNase J1, suggesting alternate pathways for decay that could include exonucleolytic decay from the 5′ end.     

Yeast Catalysed Reduction of β-Keto Esters (1): Factors Affecting Whole-Cell Catalytic Activity and Stereoselectivity

Six yeasts were studied for their ability to reduce ethyl 4-chloroacetoacetate (ethyl 4-chloro-3-oxobutanoate) stereoselectively. Five species reduced the substrate to ethyl (S)-4-chloro-3-hydroxybutanoate of high (92–99%) optical purity. With glucose-grown cells, substrate reduction could only be demonstrated when growth was oxygen-limited, whereas xylose-grown Pichia capsulata could be grown under conditions of oxygen excess without losing its reducing ability. Zygosaccha-romyces rouxii exhibited high enantioselectivity (≥98% ee (S)-enantiomer) under all conditions tested, whilst in P. capsulata, a novel switch was observed from producing mainly the (R)-enantiomer using glucose as co-substrate to producing mainly the (R)-enantiomer using 2-propanol as co-substrate. This switch was correlated with a change in reduction predominantly from an NADPH-dependent dehydrogenase system to an NADH-dependent system. In the production of ethyl (R)-4-chloro-3-hydroxybutanoate with P. capsulata, the enantioselectivity was also found to depend upon growth conditions. With glucose-grown cells, higher enantioselectivity was observed using cells harvested in stationary phase (93–94% ee) compared with cells harvested in exponential phase (43–60% ee). Growing P. capsulata with xylose rather than glucose as the major source of carbon for growth resulted in an eight-fold increase in the specific rate of ethyl (R)-4-chloro-3-hydroxybutanoate production using 2-propanol as co-substrate, although enantioselectivity was slightly reduced (65–81% ee) compared with the maximum achieved with glucose-grown cells. The effect of growth on xylose could also be correlated with enhanced activity of an NADH-dependent (R)-selective dehydrogenase system.     

Highly efficient gene expression of a fibrinolytic enzyme (subtilisin DFE) in Bacillus subtilis mediated by the promoter of α-amylase gene from Bacillus amyloliquefaciens

Subtilisin DFE is a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. The promoter and signal peptide-coding sequence of alpha-amylase gene from B. amyloliquefaciens was cloned and fused to the sequence coding for pro-peptide and mature peptide of subtilisin DFE. This hybrid gene was inserted into the Escherichia coli/Bacillus subtilis shuttle plasmid vector, pSUGV4. Recombinant subtilisin DFE gene was successfully expressed in B. subtilis WB600 with a fibrinolytic activity of 200 urokinase units ml(-1).     

Alternation of Factors Affecting Bacterioplankton Abundance in the Danube River Floodplain (Kopački Rit, Croatia)

The purpose of this investigation was to determine the influence of different phases of the hydrological cycle on the abundance of colony-forming units (CFU) of bacteria in the Kopački Rit floodplain and to assess temporal and spatial variations in the proportion of eutrophic and oligotrophic bacteria (r- and K-strategists) as a biological indicator of dissolved organic carbon (DOC) availability. Determination of bacterial abundance was performed with abiotic variables (water temperature, depth, Secchi disc transparency, dissolved oxygen, pH, electrical conductivity, ammonia, nitrates+nitrites, total nitrogen and total phosphorus) and one biotic (chl-a) variable, and dimensionality reduced with principal component analysis (PCA). The component scores were used as independent variables in a multiple regression and the relationship between scores (indirect variables) and bacterial abundance was examined. An elevated bacterial abundance was observed during the limnophase (floodplain isolated from the river) and potamophase (flood). During the limnophase also the highest chl-a concentrations were found and in Sakadaš Lake during September 2003 significantly higher numbers of eutrophic than oligotrophic bacteria emerged (p = 0.026). During potamophase the opposite state was established. Elevated bacterial abundance occurred in parallel with minimal chl-a concentrations and during July 2004 in the Čonakut Channel a significantly higher number of oligotrophic than eutrophic bacteria emerged (p < 0.001). Differentiation in the number of eutrophic and oligotrophic bacteria suggests the existence of a higher concentration of labile DOC during September 2003 than during July 2004. Multiple regression analysis explains 13.6% of the variation in abundance of eutrophic bacteria. The regression model for oligotrophic bacteria is not significant. The results suggest two different mechanisms control bacterioplankton numbers to some extent in the Kopački Rit floodplain. In addition, two sources of DOC differentiating the quality of organic matter predominate under totally different hydrological regimes. Under these conditions, eutrophic bacteria may be partially bottom-up controlled, whereas it is not clear how the abundance of oligotrophs is controlled.     

Production and characterization of partially purified extracellular thermostable α-amylase by Bacillus subtilis in submerged fermentation (SmF)

A Bacillus strain was isolated from soil samples from the campus area of Dicle University. Based on 16S ribosomal RNA sequencing, the microorganism was closely related to Bacillus subtilis. Effects of different culture medium, incubation time, carbon and nitrogen sources, and various starches, flours, and chemicals on α-amylase production were examined. Maximum enzyme production (7516 U/mL) was obtained in a basal medium A containing 0.05% Tween 40 in 24 h. Partially purified enzyme showed maximum activity at 60 °C with an optimum pH of 6.0. The effects of 0.2% detergents (sodium dodecyl sulfate [SDS], CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], and commercial detergent Omo Matic) on partially purified enzyme activity over a period of time (15-150 min) were examined and the order of inhibition effect from the most to the least was found as SDS > Omo Matic > CHAPS. Different metal ions inhibited α-amylase activity at low concentrations (1.5 mM). Co2? was a mild inhibitor and Hg2? and Cd2? were potent inhibitors, whereas Ca2? and Mg2? increased the enzyme activity. At 20 mM, Ca2? enhanced enzyme activity, and different Ca2? concentrations (10-300 mM) were studied.     

Study of the Antifungal Ability of Bacillus subtilis Strain PY-1 in Vitro and Identification of its Antifungal Substance （Iturin A）

A Bacillus strain,denoted as PY-1,was isolated from the vascular bundle of cotton.Biochemical,physiological and 16S rDNA sequence analysis proved that it should belong to Bacillus subtilis.The PY-1strain showed strong ability against many common plant fungal pathogens in vitro.The antibiotics producedby this strain were stable in neutral and basic conditions,and not sensitive to high temperature.From theculture broth of PY-1 strain,five antifungal compounds were isolated by acidic precipitation,methanolextraction,gel filtration and reverse-phase HPLC.Advanced identification was performed by mass spec-trometry and nuclear magnetic resonance spectroscopy.These five antifungal compounds were proved to bethe isomers of iturin A:A2,A3,A4,A6 and A7.In fast atom bombardment mass spectrometry/mass spec-trometry collision-induced dissociation spectra,fragmentation ions from two prior linear acylium ions wereobserved,and the prior ion,Tyr-Asn-Gln-Pro-Asn-Ser-βAA-Asn-CO~ ,was first reported.     

Analysis of the role of Bacillus subtilis σ(M) in β-lactam resistance reveals an essential role for c-di-AMP in peptidoglycan homeostasis

The Bacillus subtilis extracytoplasmic function (ECF) σ factor σ(M) is inducible by, and confers resistance to, several cell envelope-acting antibiotics. Here, we demonstrate that σ(M) is responsible for intrinsic β-lactam resistance, with σ(X) playing a secondary role. Activation of σ(M) upregulates several cell wall biosynthetic enzymes including one, PBP1, shown here to be a target for the beta-lactam cefuroxime. However, σ(M) still plays a major role in cefuroxime resistance even in cells lacking PBP1. To better define the role of σ(M) in β-lactam resistance, we characterized suppressor mutations that restore cefuroxime resistance to a sigM null mutant. The most frequent suppressors inactivated gdpP (yybT) which encodes a cyclic-di-AMP phosphodiesterase (PDE). Intriguingly, σ(M) is a known activator of disA encoding one of three paralogous diadenylate cyclases (DAC). Overproduction of the GdpP PDE greatly sensitized cells to β-lactam antibiotics. Conversely, genetic studies indicate that at least one DAC is required for growth with depletion leading to cell lysis. These findings support a model in which c-di-AMP is an essential signal molecule required for cell wall homeostasis. Other suppressors highlight the roles of ECF σ factors in counteracting the deleterious effects of autolysins and reactive oxygen species in β-lactam-treated cells.     

Patterns of associations of clinical features in neurofibromatosis 1 (NF1)

Neurofibromatosis 1 (NF1) is a common, fully penetrant autosomal dominant disease. The clinical course is generally progressive but highly variable, and the pathogenesis is poorly understood. We studied statistical associations among 13 of the most common or important clinical features in data from four separate sets of NF1 patients: a "developmental sample" of 1,413 probands from the NNFF International Database, an independent "validation sample" of 1,384 probands from the same database, 511 affected relatives of these probands, and 441 patients from a population-based registry in northwest England. We developed logistic regressive models for each of the 13 features using the developmental sample and attempted to validate these models in the other three samples. Age and gender were included as covariates in all models. Models were successfully developed and validated for ten of the 13 features analysed. The results are consistent with grouping nine of the clinical features into three sets: (1) café-au-lait spots, intertriginous freckling and Lisch nodules; (2) cutaneous, subcutaneous and plexiform neurofibromas; (3) macrocephaly, optic glioma and other neoplasms. In addition, three-way interactions among café-au-lait spots, intertriginous freckling and subcutaneous neurofibromas indicate that the first two groups are not independent. Our studies show that some individuals with NF1 are more likely than others to develop certain clinical features of the disease. Some NF1 features appear to share pathogenic mechanisms that are not common to all features.     

Variation in the Ichnofauna of the Collio Formation (Lower Permian) in the South-Alpine Region (Northern Italy)

In the Collio Formation (Lower Permian) of the South-Apine region (North Italy) paleobiodiversity consists of tetrapod footprints, invertebrate traces and rarer plants, and is characterized by an absolute poverty of taxa that becomes more acute from the lower to the upper part of the Collio Formation. Regionally, the drop in diversity can be explained by joint tectonic and climatic change; humid evergreen environments became mainly dry. Examination of ichnofossil distribution supports this hypothesis.     

Dead or Alive? Factors Affecting the Survival of Victims during Attacks by Saltwater Crocodiles (Crocodylus porosus) in Australia

Conflicts between humans and crocodilians are a widespread conservation challenge and the number of crocodile attacks is increasing worldwide. We identified the factors that most effectively decide whether a victim is injured or killed in a crocodile attack by fitting generalized linear models to a 42-year dataset of 87 attacks (27 fatal and 60 non-fatal) by saltwater crocodiles (Crocodylus porosus) in Australia. The models showed that the most influential factors were the difference in body mass between crocodile and victim, and the position of victim in relation to the water at the time of an attack. In-water position (for diving, swimming, and wading) had a higher risk than on-water (boating) or on-land (fishing, and hunting near the water''s edge) positions. In the in-water position a 75 kg person would have a relatively high probability of survival (0.81) if attacked by a 300 cm crocodile, but the probability becomes much lower (0.17) with a 400 cm crocodile. If attacked by a crocodile larger than 450 cm, the survival probability would be extremely low (<0.05) regardless of the victim’s size. These results indicate that the main cause of death during a crocodile attack is drowning and larger crocodiles can drag a victim more easily into deeper water. A higher risk associated with a larger crocodile in relation to victim’s size is highlighted by children’s vulnerability to fatal attacks. Since the first recently recorded fatal attack involving a child in 2006, six out of nine fatal attacks (66.7%) involved children, and the average body size of crocodiles responsible for these fatal attacks was considerably smaller (384 cm, 223 kg) than that of crocodiles that killed adults (450 cm, 324 kg) during the same period (2006–2014). These results suggest that culling programs targeting larger crocodiles may not be an effective management option to improve safety for children.