PhoR/PhoP two component regulatory system affects biocontrol capability of Bacillus subtilis NCD-2

The Bacillus subtilis strain NCD-2 is an important biocontrol agent against cotton verticillium wilt and cotton sore shin in the field, which are caused by Verticillium dahliae Kleb and Rhizoctonia solani Kuhn, respectively. A mutant of strain NCD-2, designated M216, with decreased antagonism to V. dahliae and R. solani, was selected by mini-Tn10 mutagenesis and in vitro virulence screening. The inserted gene in the mutant was cloned and identified as the phoR gene, which encodes a sensor kinase in the PhoP/PhoR two-component system. Compared to the wild-type strain, the APase activities of the mutant was decreased significantly when cultured in low phosphate medium, but no obvious difference was observed when cultured in high phosphate medium. The mutant also grew more slowly on organic phosphate agar and lost its phosphatidylcholine-solubilizing ability. The suppression of cotton seedling damping-off in vivo and colonization of the rhizosphere of cotton also decreased in the mutant strain when compared with the wild type strain. All of these characteristics could be partially restored by complementation of the phoR gene in the M216 mutant.     

H2O2 pretreated rice seedlings specifically reduces arsenate not arsenite: difference in nutrient uptake and antioxidant defense response in a contrasting pair of rice cultivars

The study investigated the reduction in metalloid uptake at equimolar concentrations (~53.3 μM) of As(III) and As(V) in contrasting pair of rice seedlings by pretreating with H₂O₂ (1.0 μM) and SA (1.0 mM). Results obtained from the contrasting pair (arsenic tolerant vs. sensitive) of rice seedlings (cv. Pant Dhan 11 and MTU 7029, respectively) shows that pretreatment of H₂O₂ and H₂O₂ + SA reduces As(V) uptake significantly in both the cultivars, while no reduction in the As(III) uptake. The higher growth inhibition, higher H₂O₂ and TBARS content in sensitive cultivar against As(III) and As(V) treatments along with higher As accumulation (~1.2 mg g⁻¹ dw) than in cv. P11, unravels the fundamental difference in the response between the sensitive and tolerant cultivar. In the H₂O₂ pretreated plants, the translocation of As increased in tolerant cultivar against AsIII, whereas, it decreased in sensitive cultivar both against AsIII and AsV. In both the cultivars translocation of Mn increased in the H₂O₂ pretreated plants against As(III), whereas, the translocation of Cu increased against As(V). In tolerant cultivar the translocation of Fe increased against As(V) with H₂O₂ pretreatment whereas, it decreased in the sensitive cultivar. In both the cultivars, Zn translocation increased against As(III) and decreased against As(V). The higher level of H₂O₂ and SOD (EC 1.15.1.1) activity in sensitive cultivar whereas, higher, APX (EC 1.11.1.11), GR (EC 1.6.4.2) and GST (EC 1.6.4.2) activity in tolerant cultivar, also demonstrated the differential anti-oxidative defence responses between the contrasting rice cultivars.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-014-0255-1) contains supplementary material, which is available to authorized users.     

Repeated triggering of sporulation in Bacillus subtilis selects against a protein that affects the timing of cell division

Bacillus subtilis sporulation is a last-resort phenotypical adaptation in response to starvation. The regulatory network underlying this developmental pathway has been studied extensively. However, how sporulation initiation is concerted in relation to the environmental nutrient availability is poorly understood. In a fed-batch fermentation set-up, in which sporulation of ultraviolet (UV)-mutagenized B. subtilis is repeatedly triggered by periods of starvation, fitter strains with mutated tagE evolved. These mutants display altered timing of phenotypical differentiation. The substrate for the wall teichoic acid (WTA)-modifying enzyme TagE, UDP-glucose, has recently been shown to be an intracellular proxy for nutrient availability, and influences the timing of cell division. Here we suggest that UDP-glucose also influences timing of cellular differentiation.     

Structural characterization of the late competence protein ComFB from Bacillus subtilis

Many bacteria take up DNA from their environment as part of the process of natural transformation. DNA uptake allows microorganisms to gain genetic diversity and can lead to the spread of antibiotic resistance or virulence genes within a microbial population. Development of genetic competence (Com) in Bacillus subtilis is a highly regulated process that culminates in expression of several late competence genes and formation of the DNA uptake apparatus. The late competence operon comF encodes a small protein of unknown function, ComFB. To gain insight into the function of ComFB, we determined its 3D structure via X-ray crystallography. ComFB is a dimer and each subunit consists of four α-helices connected by short loops and one extended β-strand-like stretch. Each subunit contains one zinc-binding site formed by four cysteines, which are unusually spaced in the primary sequence. Using structure- and bioinformatics-guided substitutions we analyzed the inter-subunit interface of the ComFB dimer. Based on these analyses, we conclude that ComFB is an obligate dimer. We also characterized ComFB in vivo and found that this protein is produced in competent cells and is localized to the cytosol. Consistent with previous reports, we showed that deletion of ComFB does not affect DNA uptake function. Combining our results, we conclude that ComFB is unlikely to be a part of the DNA uptake machinery under tested conditions and instead may have a regulatory function.     

New inhibitors of colony spreading in Bacillus subtilis and Bacillus anthracis

We have recently characterized sliding motility in Bacillus subtilis strains that lack functional flagella, and here describe the discovery of inhibitors of colony spreading in these strains as well as the aflagellate pathogen, Bacillus anthracis. Aflagellate B. subtilis strains were used to screen for new types of antibacterials that might inhibit colony spreading on semi-solid media. From a diverse set of organic structures, p-nitrophenylglycerol (NPG), an agent used primarily in clinical laboratories to control Proteus swarming, was found to inhibit colony spreading. The four stereoisomers of NPG were synthesized and tested, and only the 1R,2S-(1R-anti) and 1R,2R-(1R-syn) NPG isomers had significant activity in a quantitative colony-spreading assay. Twenty-six NPG analogs and related structures were synthesized and tested to identify more active inhibitors. p-Methylsulfonylphenylglycerol (p-SPG), but not its ortho or meta analogs, was found to be the most effective of these compounds, and synthesis and testing of all four p-SPG stereoisomers showed that the 1R-anti-isomer was the most active with an average IC(50) of 16 μM (3-5 μg mL(-1)). For B. anthracis, the colony-spreading IC(50) values for 1R-anti-SPG and 1R-anti-NPG are 12 μM (2-4 μg mL(-1)) and >150 μM, respectively. For both Bacillus species tested, 1R-anti-SPG inhibits colony spreading of surface cultures on agar plates, but is not bacteriostatic or bacteriocidal in liquid cultures. Work is in progress to find the cellular target(s) of the NPG/SPG class of compounds, since this could lead to an understanding of the mechanism(s) of colony spreading as well as design and development of more potent inhibitors for the control of B. anthracis surface cultures.     

An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments

Background

The Bacillus subtilis genome (BGM) vector is a novel cloning system based on the natural competence that enables B. subtilis to import extracellular DNA fragments into the cell and incorporate the recombinogenic DNA into the genome vector by homologous recombination. The BGM vector system has several attractive properties, such as a megabase cloning capacity, stable propagation of cloned DNA inserts, and various modification strategies using RecA-mediated homologous recombination. However, the endogenous RecA activity may cause undesirable recombination, as has been observed in yeast artificial chromosome systems. In this study, we developed a novel BGM vector system of an inducible recA expression BGM vector (iREX), in which the expression of recA can be controlled by xylose in the medium.

Results

We constructed the iREX system by introducing the xylose-inducible recA expression cassette followed by the targeted deletion of the endogenous recA. Western blot analysis showed that the expression of recA was strictly controlled by xylose in the medium. In the absence of xylose, recA was not expressed in the iREX, and the RecA-mediated recombination reactions were greatly suppressed. By contrast, the addition of xylose successfully induced RecA expression, which enabled the iREX to exploit the same capacities of transformation and gene modifications observed with the conventional BGM vector. In addition, an evaluation of the stability of the cloned DNA insert demonstrated that the DNA fragments containing homologous sequences were more stably maintained in the iREX by suppressing undesirable homologous recombination.

Conclusions

We developed a novel BGM vector with inducible recA expression system, iREX, which enables us to manipulate large DNA fragments more stably than the conventional BGM vector by suppressing undesirable recombination. In addition, we demonstrate that the iREX can be applied to handling the DNA, which has several homologous sequences, such as multiple-reporter expression cassettes. Thus, the iREX expands the utility of the BGM vector as a platform for engineering large DNA fragments.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1425-4) contains supplementary material, which is available to authorized users.     

The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates

Ligninolytic enzyme complexes are involved in lignin degradation. Among them laccases are outstanding because they use molecular oxygen as a co-substrate instead of hydrogen peroxide as used by peroxidases. Bacterial laccase of Bacillus genus was first reported in Claus and Filip (Microbiol Res 152:209–216, 1997), since then more bacterial laccases have been found. In this research, laccase-producing bacteria were screened from pulp and paper industry wastewater, bagass and sugarcane rhizosphere. Nutrient agar medium containing 0.5 mM of guaiacol was used. It was observed that the laccase-producing strains developed brown colour from which 16 strains of Bacillus were identified. One of the isolated strains was identified as Bacillus subtilis WPI based on the results of biochemical tests and 16S rDNA sequence analysis. This strain showed laccase-like activity towards the oxidizing substrates ABTS and guaiacol. In this study guaiacol was used as the substrate of laccase activity assay. For determination of laccase activity of this isolate guaiacol was used as a substrate of assay for the first time in this study. SDS-PAGE and Native-PAGE confirmed the presence of laccase.     

Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity

Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data.     

A genetic resource for early-morning flowering trait of wild rice Oryza officinalis to mitigate high temperature-induced spikelet sterility at anthesis

Background and Aims

High temperatures over 32–36 °C at anthesis induce spikelet sterility in rice. The use of a germplasm with an early-morning flowering (EMF) trait has been hypothesized as a way of avoiding this problem. In this study, the effect of the EMF trait on avoiding high temperature-induced sterility at anthesis by flowering at a cooler temperature in the early morning was evaluated.

Methods

The EMF trait was introgressed from wild rice (Oryza officinalis) into the rice cultivar ‘Koshihikari’ (O. sativa). First, spikelets of the EMF line and Koshihikari were subjected to rising temperatures during the daytime in the greenhouse to test for differences in spikelet sterility. Secondly, spikelets of both plants were exposed to 26, 34 and 38 °C at anthesis and to 38 °C beginning at least 1 h after flowering, in the growth chambers at 70 % relative humidity, to test for differences in tolerance to high temperatures.

Key Results

Spikelets of the EMF line started and completed flowering a few hours earlier than Koshihikari. In a greenhouse experiment, spikelets of Koshihikari opened after the air temperature reached 35 °C, but those of the EMF line could open at cooler temperatures. Under these conditions, spikelet sterility significantly increased in Koshihikari, but did not in the EMF line. The number of sterile spikelets increased as their flowering time was delayed in Koshihikari. Furthermore, the chamber experiments revealed that 60 % of the spikelets from both lines were sterile when exposed to 38 °C at anthesis, indicating that tolerance of high temperature was similar in both genotypes.

Conclusions

Reduced sterility in the EMF line subjected to rising temperatures at anthesis in the greenhouse was attributed to an earlier flowering time compared with Koshihikari. The EMF trait of wild rice is effective in mitigating anticipated yield loss due to global warming by escaping high-temperature stress at anthesis during the daytime.     

PksS from Bacillus subtilis is a cytochrome P450 involved in bacillaene metabolism

As part of the pksX gene cluster of Bacillus subtilis strain 168, pksS has been preliminarily annotated as a cytochrome P450 homolog that hydroxylates the polyketide product of this cluster, which was recently shown to be involved in the biosynthesis of bacillaene and dihydrobacillaene. Here we report that there is a frame-shift error in the reported sequence for pksS, and that we have successfully cloned, overexpressed, and purified the protein encoded by the corrected sequence. By utilizing electronic absorption spectrophotometry, we have observed that the ferrous CO complex of PksS absorbs maximally near 450 nm, which confirms the annotation that this protein is a cytochrome P450. We have also established a cell-free system derived from crude cytosolic B. subtilis protein extracts which provides reductase activity essential to sustaining the putative catalytic cycle of PksS. Using LC-MS analysis we have collected data which suggests that the substrate for PksS is dihydrobacillaene.     

Thermostable Bacillus subtilis lipases: in vitro evolution and structural insight

In vitro evolution methods are now being routinely used to identify protein variants with novel and enhanced properties that are difficult to achieve using rational design. However, one of the limitations is in screening for beneficial mutants through several generations due to the occurrence of neutral/negative mutations occurring in the background of positive ones. While evolving a lipase in vitro from mesophilic Bacillus subtilis to generate thermostable variants, we have designed protocols that combine stringent three-tier testing, sequencing and stability assessments on the protein at the end of each generation. This strategy resulted in a total of six stabilizing mutations in just two generations with three mutations per generation. Each of the six mutants when evaluated individually contributed additively to thermostability. A combination of all of them resulted in the best variant that shows a remarkable 15 °C shift in melting temperature and a millionfold decrease in the thermal inactivation rate with only a marginal increase of 3 kcal mol⁻¹ in free energy of stabilization. Notably, in addition to the dramatic shift in optimum temperature by 20 °C, the activity has increased two- to fivefold in the temperature range 25-65 °C. High-resolution crystal structures of three of the mutants, each with 5° increments in melting temperature, reveal the structural basis of these mutations in attaining higher thermostability. The structures highlight the importance of water-mediated ionic networks on the protein surface in imparting thermostability. Saturation mutagenesis at each of the six positions did not result in enhanced thermostability in almost all the cases, confirming the crucial role played by each mutation as revealed through the structural study. Overall, our study presents an efficient strategy that can be employed in directed evolution approaches employed for obtaining improved properties of proteins.     

Mapping and analysis of the LINE and SINE type of repetitive elements in rice

Non-LTR retrotransposons comprise significant portion of the plants genome. Their complete characterization is thus necessary if the sequenced genome is to be annotated correctly. The long and short interspersed nucleotide repetitive elements (LINE and SINE) may be responsible for alteration in the expression mechanism of neighboring genes, the complete identification of these elements in the rice genome is essential in order studying their putative functional interactions with the plant genes and its role in genome composition. The main emphasis of this work is to assemble a comprehensive dataset of nonLTR (LINEs and SINEs) and the map of completely inserted LINEs and SINE type of retroelement by both intact ends (3' and 5' ends). The assembled information and work may help for further research in this direction.     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

Bioconversion of shellfish chitin wastes for the production of Bacillus subtilis W-118 chitinase

Bacillus subtilis W-118, a strain that produces antifungal materials, excreted a chitinase when cultured in a medium containing shrimp- and crab-shell powder as the major carbon source. This chitinase, purified by sequential chromatography, had a molecular mass of 20,600 Da and a pI of 6. The optimum pH, optimum temperature, and pH stability of the chitinase were pH 6, 37 degrees C, and pH 5-7, respectively. The unique characteristics of the purified chitinase include low molecular mass and acidic pI. In the investigation of the inhibitory activity, it was found that the growth of Fusarium oxysporum was 100% inhibited after incubation for 1 day with sterilized W-118 chitinase solution (5.6 units/mL). The chitinase hydrolyzates of chitin with low degrees of polymerization (DP 1-6) were analyzed by HPLC. Longer reaction times led to the generation of chitin oligosaccharides with lower DP. The chitin oligosaccharides were examined for their inhibitory effects on F. oxysporum and human leukemia cell lines.