Intergeneric Complementation of Anthranilate Synthase Subunits

Partially purified subunits of anthranilate synthase were prepared from Bacillus subtilis and Pseudomonas aeruginosa. The large component from B. subtilis (I(B)) complements well with the small component from P. aeruginosa (II(P)) to reconstitute a glutamine-reactive anthranilate synthase. This interaction can be demonstrated with crude extracts from a B. subtilis trpX mutant and a P. aeruginosa trpA mutant. Complementation was also observed with the large component from P. aeruginosa (I(P)) and the small subunit from B. subtilis (II(B)). At saturation the heterologous complex I(B)II(P) has 93% of the activity of the homologous complex I(B)II(B), whereas the hybrid I(P)II(B) is only 22% as active as the homologous complex I(P)II(P).     

Use of Rhizobacteria in the Control of Root Rot–Root Knot Disease Complex of Mungbean

Thirty-two isolates of Pseudomonas aeruginosa and a Bacillus subtilis strain were isolated from rhizosphere and rhizoplane of four wild and 15 cultivated plants. Biocontrol and growth-promoting potentials of the bacterial isolates were tested under laboratory, greenhouse and field conditions. The bacterial isolates not only exhibited nematicidal activity by killing the second stage larvae of Meloidogyne javanica to a varying degree but also produced inhibition zones by inhibiting the radial growth of Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani . Strain IE-2 and IE-6 of P. aeruginosa also lysed the fungal mycelium. P. aeruginosa and B. subtilis used as seed dressing or as soil drench significantly suppressed root rot–root knot infection and nematode population densities under greenhouse and field conditions and thereby enhanced plant growth and yield in mungbean.     

Degradation of Uric Acid by Certain Aerobic Bacteria

We have isolated and identified nine cultures of aerobic bacteria capable of growing on an elective medium containing uric acid as the only source of carbon, nitrogen, and energy. Four of these cultures were identified as Aerobacter aerogenes, two as Klebsiella pneumoniae, and the remainder as Serratia killiensis, Pseudomonas aeruginosa, and Bacillus species. Another culture identified as P. fluorescens required both glucose and uric acid for growth. When 23 laboratory stock cultures were inoculated into the uric acid medium, A. aerogenes, B. subtilis, Mycobacterium phlei, P. aeruginosa, and S. marcescens were able to grow. These five cultures also grew when the uric acid was replaced with adenine, guanine, hypoxanthine, xanthine, or allantoin, but growth was poor. In all of these media, including the uric acid medium, addition of glucose along with the nitrogenous compounds yielded good growth. Induction experiments demonstrated that the ability of A. aerogenes, K. pneumoniae, P. aeruginosa, P. fluorescens, S. kiliensis, S. marcescens, B. subtilis, and Bacillus sp. to degrade uric acid is an induced property. Of these organisms, only Bacillus sp. accumulated a small amount of intracellular uric acid.     

Comparative action of glyphosate as a trigger of energy drain in eubacteria.

Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa, each possessing a 5-enolpyruvylshikimate 3-phosphate synthase that is sensitive to inhibition by glyphosate [N-(phosphonomethyl)glycine], provide a good cross-section of organisms exemplifying the biochemical diversity of the aromatic pathway targeted by this potent antimicrobial compound. The pattern of growth inhibition, the alteration in levels of aromatic-pathway enzymes, and the accumulation of early-pathway metabolites after the addition of glyphosate were distinctive for each organism. Substantial intracellular shikimate-3-phosphate accumulated in response to glyphosate treatment in all three organisms. Both E. coli and P. aeruginosa, but not B. subtilis, accumulated near-millimolar levels of shikimate-3-phosphate in the culture medium. Intracellular backup of common-pathway precursors of shikimate-3-phosphate was substantial in B. subtilis, moderate in P. aeruginosa, and not detectable in E. coli. The full complement of aromatic amino acids prevented growth inhibition and metabolite accumulation in E. coli and P. aeruginosa where amino acid end products directly control early-pathway enzyme activity. In contrast, the initial prevention of growth inhibition in the presence of aromatic amino acids in B. subtilis was succeeded by progressively greater growth inhibition that correlated with rapid metabolite accumulation. In B. subtilis glyphosate can decrease prephenate concentrations sufficiently to uncouple the sequentially acting loops of feedback inhibition that ordinarily link end product excess to feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by prephenate. The consequential unrestrained entry is an energy-rich substrates into the aromatic pathway, even in the presence of aromatic amino acid end products, is an energy drain that potentially accounts for the inability of end products to fully reverse glyphosate inhibition in B. subtilis. Even in E. coli after glyphosate inhibition and metabolite accumulation were allowed to become fully established, a transient period where end products were capable of only partial reversal of growth inhibition occurred. The distinctive metabolism produced by dissimilation of different carbon sources also profound effects upon glyphosate sensitivity.     

Crude petroleum-oil biodegradation efficiency of Bacillus subtilis and Pseudomonas aeruginosa strains isolated from a petroleum-oil contaminated soil from North-East India

The efficiency of Bacillus subtilis DM-04 and Pseudomonas aeruginosa M and NM strains isolated from a petroleum contaminated soil sample from North-East India was compared for the biodegradation of crude petroleum-oil hydrocarbons in soil and shake flask study. These bacterial strains could utilize crude petroleum-oil hydrocarbons as sole source of carbon and energy. Bioaugmentation of TPH contaminated microcosm with P. aeruginosa M and NM consortia and B. subtilis strain showed a significant reduction of TPH levels in treated soil as compared to control soil at the end of experiment (120 d). P. aeruginosa strains were more efficient than B. subtilis strain in reducing the TPH content from the medium. The plate count technique indicated expressive growth and biosurfactant production by exogenously seeded bacteria in crude petroleum-oil rich soil. The results showed that B. subtilis DM-04 and P. aeruginosa M and NM strains could be effective for in situ bioremediation.     

Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production

Relatively little is known about the exact mechanisms used by Bacillus subtilis in its behavior as a biocontrol agent on plants. Here, we report the development of a sensitive plant infection model demonstrating that the bacterial pathogen Pseudomonas syringae pv tomato DC3000 is capable of infecting Arabidopsis roots both in vitro and in soil. Using this infection model, we demonstrated the biocontrol ability of a wild-type B. subtilis strain 6051 against P. syringae. Arabidopsis root surfaces treated with B. subtilis were analyzed with confocal scanning laser microscopy to reveal a three-dimensional B. subtilis biofilm. It is known that formation of biofilms by B. subtilis is a complex process that includes secretion of surfactin, a lipopeptide antimicrobial agent. To determine the role of surfactin in biocontrol by B. subtilis, we tested a mutant strain, M1, with a deletion in a surfactin synthase gene and, thus, deficient in surfactin production. B. subtilis M1 was ineffective as a biocontrol agent against P. syringae infectivity in Arabidopsis and also failed to form robust biofilms on either roots or inert surfaces. The antibacterial activity of surfactin against P. syringae was determined in both broth and agar cultures and also by live-dead staining methods. Although the minimum inhibitory concentrations determined were relatively high (25 microg mL(-1)), the levels of the lipopeptide in roots colonized by B. subtilis are likely to be sufficient to kill P. syringae. Our results collectively indicate that upon root colonization, B. subtilis 6051 forms a stable, extensive biofilm and secretes surfactin, which act together to protect plants against attack by pathogenic bacteria.     

鲑点石斑鱼皮肤抗菌活性蛋白的纯化及抗菌活性(英文）

近年来在多种生物体中都发现有抗菌活性蛋白和多肽。由于其具有生物化学多样性,抗病毒、微生物、真菌、原生动物、肿瘤,促进伤口愈合等生物学活性,而引起研究者的极大兴趣。抗菌活性蛋白和多肽在动物的先天免疫中具有重要作用,它们直接作用于细菌,并将其杀死。鲑点石斑鱼(Epinephelusfario)是中国南方水产养殖中重要的海水鱼。近年来,由于细菌和病毒引发的病害造成鲑点石斑鱼大量死亡,但其抗菌活性蛋白及多肽目前还未见报道。本研究发现鲑点石斑鱼皮肤具有抗菌活性成分,鲑点石斑鱼皮肤匀浆物经胰蛋白酶水解后抗菌活性丧失,说明该活性是由蛋白质引起的。经离子交换层析及凝胶过滤层析,从鲑点石斑鱼皮肤中分离纯化到抗菌活性蛋白(Efap)。SDS-PAGE显示,Efap为单链蛋白,分子量约41kD。该成分能同时抑制革兰氏阳性菌,如金黄色葡萄球菌、滕黄微球菌、枯草牙胞杆菌和革兰氏阴性菌,如溶藻弧菌、副溶血弧菌、河流弧菌、多杀性巴氏杆菌、嗜水气单胞菌、大肠杆菌和铜绿假单胞菌。革兰氏阴性菌中,溶藻弧菌、副溶血弧菌、河流弧菌和多杀性巴氏杆菌对Efap较敏感,MIC<20mol/L,其他3种菌敏感性较差,MIC>20mol/L。另外,Efap显示出较强的抗金黄色葡萄球菌的活性,MIC为5—10mol/L。Efap的广谱抗菌性,说明其在鲑点石斑鱼免疫防御中具有一定的作用。     

羟基酪醇抑菌活性及抑菌稳定性研究

本研究探究了羟基酪醇对大肠杆菌、金黄色葡萄球菌、铜绿假单胞杆菌和枯草芽孢杆菌等四种供试菌的抑菌活性及抑菌稳定性。采用试管半倍稀释法确定MIC和MBC,并探讨羟基酪醇对供试菌的生长和细胞膜完整性的影响以及在不同介质下的抑菌稳定性。结果表明,羟基酪醇对大肠杆菌、金黄色葡萄球菌、铜绿假单胞杆菌和枯草芽孢杆菌的MIC分别为0.625、0.625、1.250、2.500 mg/mL,MBC分别为1.250、1.250、2.500、5.000 mg/mL。与对照组相比,四种供试菌核酸和可溶性蛋白泄漏显著,细胞膜的完整性被破坏。在不同NaCl浓度下,羟基酪醇对枯草芽孢杆菌的抑菌活性稳定;在1.0%和2.0%NaCl浓度下,羟基酪醇对大肠杆菌和铜绿假单胞杆菌的抑菌活性稳定;在2.0%NaCl介质下低浓度的羟基酪醇对金黄色葡萄球菌的抑菌活性稳定,在0.5%、1.5%和2.0%NaCl介质下高浓度的羟基酪醇对金黄色葡萄球菌的抑菌活性稳定。在蔗糖介质中,羟基酪醇对四种供试菌的抑菌活性均不稳定。因此,羟基酪醇可以作为一种新型的防腐剂。     

Sporulating bacteria prefers predation to cannibalism in mixed cultures

Predatory behavior, a property associated with ecosystems, is not commonly observed in microorganisms. However, cannibalistic tendencies have been observed in microorganisms under stress. For example, pure culture of Bacillus subtilis exhibits cannibalism under nutrient limitation. It has been proposed that a fraction of cells in the population produce Spo0A, a regulatory protein that is responsible for delaying sporulation. Cells containing spo0A would produce a killing factor by activating skf operon and an associated pump to export the factor. Cells that do not contain spo0A in the population are lysed. However in addition to the competition among the cells of B. subtilis, these cells also compete with other organisms for the limited nutrients. In this work, we report the cannibalistic behavior of B. subtilis in presence of Escherichia coli under severe nutritional limitation. We demonstrate that B. subtilis lyses cells of E. coli using an antibacterial factor under the regulation of Spo0A. Our experiments also suggest that B. subtilis prefers predation of E. coli to cannibalism in mixed cultures. B. subtilis also demonstrated predation in mixed cultures with other soil microorganisms, such as, Xanthomonas campestris, Pseudomonas aeruginosa and Acinetobactor lwoffi. This may offer B. subtilis a niche to survive in an environment with limited nutrients and under competition from other microorganisms.     

枯草芽孢杆菌fmb60菌株非核糖体肽类化合物对铜绿微囊藻生长的抑制作用

铜绿微囊藻是一种分布广泛的水华微藻,可对人类健康和生态环境造成严重危害,而枯草芽孢杆菌作为一种生防微生物可通过非核糖体肽合成酶合成多种生物活性物质。因此,枯草芽孢杆菌fmb60非核糖体肽(Non-ribosomal peptide,NRP)类产物对铜绿微囊藻生长抑制作用的研究在水华治理方面具有重要的意义。利用基因组挖掘技术从枯草芽孢杆菌fmb60中分离鉴定出bacillibactin、表面活性素(Surfactin)和芬芥素(Fengycin)3种NRP类产物,进而通过添加不同浓度的NRP类产物研究其对铜绿微囊藻生长的影响,结果显示其对铜绿微囊藻4 d的半效应浓度值EC50.4 d为26.5 mg/L,且随着样品浓度的增加,枯草芽孢杆菌fmb60的NRP类产物对铜绿微囊藻的抑制作用增强。当向其分别加入50 mg/L的样品时,培养4 d的铜绿微囊藻Fv/Fm、Fv/Fo、Yield参数分别比对照组降低了2.8%、1.7%、2.0%。表明枯草芽孢杆菌fmb60 NRP类产物能够显著抑制铜绿微囊藻的光合作用强度及代谢合成,从而为枯草芽孢杆菌抑藻剂的开发奠定理论基础。     

RNase P cleaves the adenine riboswitch and stabilizes pbuE mRNA in Bacillus subtilis

RNase P from Bacillus subtilis cleaves in vitro the adenine riboswitch upstream of pbuE, which codes for an adenine efflux pump. The guanine riboswitch, encoded upstream of xpt-pbuX operon, is not cleaved. The cleavage sites do not occur at any predicted structures that should be recognized by RNase P in the theoretical model of the adenine riboswitch. However, it is possible to draw alternative secondary structure models that match the apparent requirements for RNase P substrates at these cleavage sites. Support for these models is provided by appropriate mutagenesis experiments. Adenine showed no effect on the cleavage in vitro of the pbuE adenine riboswitch by RNase P holoenzyme from B. subtilis. The results of genetic experiments performed in B. subtilis support the cleavage of adenine riboswitch by RNase P in vivo and suggest that it induces the stabilization of pbuE mRNA under normal conditions.     

昆虫病原斯氏和异小杆线虫对各种非共生微生物的信息反应(英文)

非线虫共生细菌 (Bacillussubtilis ,B .thuringiensis,Pseudomonasfluorescens ,Micromonosporapur purea,Rhizopusdelemar ,Pseudomonasaeruginosa ,Streptomycesvenezuelae ,Streptomycesantibioticus ,Penicilliumcitrnum ,Ganodermalucidum ,Agaricusbisporus,Pleurotusostreatus,Rhizobiumlegumi unosarum和Photobacteriumphosphoreum)的培养液以及其上清液、斜纹夜蛾 (Spodopteralitura)昆虫细胞系用于引诱无菌SteinernemacarpocapsaeA2 4和HeterorhabditisbacteriophoraH0 6发育。上述培养物均未能诱导H .bacteriophoraH0 6发育。虽然P .phosphoreum菌液可致死A2 4线虫 ,但是其上清液可诱导线虫发育。无菌S .carpocapsaeA2 4线虫可利用斜纹夜蛾昆虫细胞繁殖 ,产生下一代感染期线虫。结果进一步说明 ,引诱H .bacteriophoraH0 6发育的化学信息物质比S .carpocapsaeA2 4的专一。     

Antioxidant, radical-scavenging, anti-inflammatory, cytotoxic and antibacterial activities of methanolic extracts of some Hedyotis species

The antioxidant, radical-scavenging, anti-inflammatory, cytotoxic and antibacterial activities of methanolic extracts of seven Hedyotisspecies were investigated. The antioxidant activity was evaluated by the ferric thiocyanate (FTC) and thiobarbituric acid (TBA) methods while the radical scavenging activity was measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. The anti-inflammatory activity related to NO inhibition of the plant extracts was measured by the Griess assay while cytotoxicity were measured by the MTT assay against CEM-SS cell line. The antibacterial bioassay (against 4 bacteria, i.e. Bacillus subtilis B28 (mutant), Bacillus subtilis B29 (wild-type), Pseudomonas aeruginosa UI 60690 and methicillin resistant Staphylococcus aureus, (MRSA) was also carried out using the disc-diffusion method. All tested extracts exhibited very strong antioxidant properties when compared to Vitamin E (alpha-tocopherol) with percent inhibition of 89-98% in the FTC and 60-95% in the TBA assays. In the DPPH method, H. herbacea exhibited the strongest radical scavenging activity with an IC50 value of 32 microg/ml. The results from the Griess assay showed that the tested extracts are weak inhibitors of NO synthase. However, all tested extracts exhibited moderate cytotoxic properties against CEM-SS cell line giving CD50 values in the range of 21-41 microg/ml. In the antibacterial bioassay, the stems and the roots of H. capitellata showed moderate activity against the 4 tested bacteria while the leaves showed moderate activity towards B. subtilis B28, MRSA and P. aeruginosa only. The roots of H. dichotoma showed strong antibacterial activity against all 4 bacteria. All other extracts did not exhibit any antibacterial activity.     

Antibacterial activity of the metabolic by-products of a Veillonella species and Bacteroides fragilis

A Veillonella species and Bacteroides fragilis were isolated from the cecal contents of adult chickens. When growth on an agar medium supplemented with 0.4% glucose and adjusted to pH 6.5, mixed cultures containing Veillonella and B. fragilis inhibited the growth of Salmonella typhimurium; Salmonella enteritidis, Escherichia coli 0157:H7 and Pseudomonas aeruginosa. Decreasing the glucose concentration of the agar decreased the inhibitory activity of the mixed culture. Mixed cultures grown on agar media supplemented with 0.5% glucose and adjusted to pH 6.5, 7.0 or 7.5 also inhibited the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 and P. aeruginosa. However, increasing the pH of the agar decreased the inhibitory activity of the mixed culture. Pure cultures of Veillonella or B. fragilis did not inhibit the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 or P. aeruginosa on any of the agar supplemented with different concentrations of glucose or on any of the agar adjusted to different pH levels. The inhibitory activity of the mixed culture was correlated with the concentration of volatile fatty acids that were formed as B. fragilis metabolized glucose to produce succinate and acetate and as the succinate produced by B. fragilis was decarboxylated by Veillonella to produce propionate.     

Analysis of essential oil composition of Thymbra spicata var. spicata: antifungal, antibacterial and antimycobacterial activities

The fresh leaves and brine of leaves of Thymbra spicata var. spicata (KARAKIZ) were analyzed by hydrodistillation, headspace and GC/MS techniques. The main components were determined as carvacrol, p-cymene, beta-myrcene, gamma-terpinene, a-terpinene and trans-caryophyllene. The essential oil and the main compounds, carvacrol and trans-caryophyllene, have been tested against E. coli, S. epidermidis, B. subtilis, S. aureus, S. typhimurium, K. pneumoniae, P aeruginosa, E. faecalis and C. albicans. While the essential oil and carvacrol showed strong activity against all microorganisms, except P. aeruginosa, trans-caryophyllene showed activity only against C. albicans. The essential oil and carvacrol also showed strong antimycobacterial activity.     

Degradation of anthracene by bacteria isolated from oil polluted tropical soils

Four bacteria, identified as Pseudomonas aeruginosa, Alcaligenes eutrophus, Bacillus subtilis and Micrococcus luteus were isolated from crude oil polluted soils using anthracene as the sole carbon and energy source. All the organisms utilized n-hexadecane, n-tetradecane, diesel oil, engine oil and naphthalene as sole carbon sources. None could utilize hexane, cycloheptane, xylene, benzene, toluene, phenol, fluoranthene,and kerosene as carbon sources. Highest cell density obtained with 0.1% (w/v) anthracene were 4.5 x 10(7) (cfu/ml), 8.6 x 10(6) (cfu/ml), 5.4 x 10(6) and 2.4 x 10(6) (cfu/ml) respectively, for P. aeruginosa, A. eutrophus, B. subtilis and M. luteus after 30 days incubation. Growth of the organisms on a Nigerian crude oil resulted in a residual oil concentration of 22.2%, 33.3%, 39.3%, 44% and 91.7% respectively, for P. aeruginosa, A. eutrophus, B. subtilis, M. luteus and the noninoculated control on the 14 th day. Ring fission enzymes of the meta pathway were detected in induced cells of P. aeruginosa and A. eutrophus while ortho pathway enzymes were detected in B. subtilis and M. luteus. P. aeruginosa and A. eutrophus had specific catechol-2,3-dioxygenase activities of 3.8 +/- 0.183 and 0.64 +/- 0.032 micromol/min x mg protein respectively while catechol-1,2-dioxygenase activities of 1.95 +/- 0.029 and 1.89 +/- 0.026 micromol/min x mg protein were detected in B. subtilis and M. luteus respectively. This work, highlights the capability of these unreported tropical strains of A. eutrophus, B. subtilis and M. luteus as anthracene degraders.     

Rhamnolipid-dependent spreading growth of Pseudomonas aeruginosa on a high-agar medium: marked enhancement under CO2-rich anaerobic conditions

Anaerobiosis of Pseudomonas aeruginosa in infected organs is now gaining attention as a unique physiological feature. After anaerobic cultivation of P. aeruginosa wild type strain PAO1 T, we noticed an unexpectedly expanding colony on a 1.5% agar medium. The basic factors involved in this spreading growth were investigated by growing the PAO1 T strain and its isogenic mutants on a Davis high-agar minimal synthetic medium under various experimental conditions. The most promotive environment for this spreading growth was an O(2)-depleted 8% CO(2) condition. From mutational analysis of this spreading growth, flagella and type IV pili were shown to be ancillary factors for this bacterial activity. On the other hand, a rhamnolipid-deficient rhlA mutant TR failed to exhibit spreading growth on a high-agar medium. Complementation of the gene defect of the mutant TR with a plasmid carrying the rhlAB operon resulted in the restoration of the spreading growth. In addition, an external supply of rhamnolipid or other surfactants (surfactin from Bacillus subtilis or artificial product Tween 80) also restored the spreading growth of the mutant TR. Such activity of surfactants on bacterial spreading on a hard-agar medium was unique to P. aeruginosa under CO(2)-rich anaerobic conditions.     

Classification and genetic characterization of pattern-forming Bacilli

One of the more natural but less commonly studied forms of colonial bacterial growth is pattern formation. This type of growth is characterized by bacterial populations behaving in an organized manner to generate readily identifiable geometric and predictable morphologies on solid and semi-solid surfaces. In our first attempt to study the molecular basis of pattern formation in Bacillus subtilis , we stumbled upon an enigma: some strains used to describe pattern formation in B. subtilis did not have the phenotypic or genotypic characteristics of B. subtilis . In this report, we show that these strains are actually not B. subtilis , but belong to a different class of Bacilli , group I. We show further that commonly used laboratory strains of B. subtilis can co-exist as mixed cultures with group I Bacilli , and that the latter go unnoticed when grown on frequently used laboratory substrates. However, when B. subtilis is grown under more stringent semiarid conditions, members of group I emerge in the form of complex patterns. When B. subtilis is grown under less stringent and more motile conditions, B. subtilis forms its own pattern, and members of group I remain unnoticed. These findings have led us to revise some of the mechanistic and evolutionary hypotheses that have been proposed to explain pattern growth in Bacilli .