ATP Modulates the Growth of Specific Microbial Strains

The regulatory function of extracellular ATP (exATP) in bacteria is unknown, but recent studies have demonstrated exATP induced enhanced secondary metabolite production and morphological differentiation in Streptomyces coelicolor. The growth of Streptomyces coelicolor, however, was unaffected by exATP, although changes in growth are common phenotypes. To identify bacteria whose growth is altered by exATP, we measured exATP-induced population changes in fast-growing microbes and actinomycetes in compost. Compared with the water-treated control, the addition of 10 ml 100 μM ATP to 10 g of compost enhanced the actinomycetes population by 30% and decreased fast-growing microbial numbers by 20%. Eight microbes from each group were selected from the most populated colony, based on appearance. Of the eight isolated fast-growing microbes, the 16S rRNA sequences of three isolates were similar to the plant pathogens Serratia proteamaculans and Sphingomonas melonis, and one was close to a human pathogen, Elizabethkingia meningoseptica. The growth of all fast-growing microbes was inhibited by ATP, which was confirmed in Pseudomonas syringae DC3000, a pathogenic plant bacterium. The growth of six of eight isolated actinomycetes strains, all of which were identified as close to Streptomyces neyagawaensis, was enhanced by ATP treatment. This study suggests that exATP regulates bacterial physiology and that the exATP response system is a target for the control of bacterial ecology.     

Potential of natamycin in enhancing the antagonistic activity of Bacillus amyloliquefaciens BGP20 against post-harvest bacterial soft rot of green pepper

Bacillus amyloliquefaciens BGP20 is a promising antagonist in controlling post-harvest bacterial soft rot of vegetables caused by Erwinia carotovora subsp. carotovora (Ecc). The objective of this study was to screen a kind of natural and safe additive which could enhance the bio-control activity of BGP20 against post-harvest bacterial soft rot of green pepper. The results of this study indicated that the additive natamycin had stronger inhibition against the pathogen Ecc compared with bamboo vinegar and chitosan in the 2× Yeast extract and Tryptone (2YT) medium. However, natamycin had a slight negative effect on the growth of BGP20 in the 2YT medium. In preventative treatments, natamycin significantly improved the bio-efficacy of BGP20, and enhanced its competitive position against Ecc in the wounds of green pepper. Compared with the treatment with BGP20 alone, the viable count of BGP20 after 72?h of incubation increased by 115.8% in the wounds of green pepper treated with BGP20 and 0.1% natamycin, while that of Ecc decreased by 92.1%. In addition, natamycin remarkably promoted the flocculation of Ecc cells in the 2YT medium, while promoting the dispersion of BGP20. Natamycin had no negative effects on the spore germination of BGP20 and its shelf life. These results indicated that natamycin had perfect compatibility with the antagonist BGP20, and it had a great potential in enhancing the bio-control activity of BGP20 against post-harvest bacterial soft rot of green pepper in preventative treatments.     

放线菌中亮氨酸应答调控蛋白的生物学功能及其调控机理

放线菌是一类革兰氏阳性细菌,可产生氨基酸等初级代谢产物和抗生素等次级代谢产物,其广泛用于食品、医药、添加剂及化妆品行业。此外,还有少数放线菌,如分枝杆菌等,是可以引起人和动植物病害的病原菌。亮氨酸应答调控蛋白(Leucine-responsive regulatory protein,Lrp)是一类在氨基酸代谢及其相关代谢过程中的重要转录调控子,能够应答各种氨基酸,参与调控微生物细胞的多个生理过程,例如氨基酸代谢和转运、中心代谢、细菌的持久性和毒力等。本文总结了放线菌Lrp的生物学功能,并综述了放线菌中不同种属Lrp以及天蓝色链霉菌和红色糖多孢菌Lrp调控机理的研究进展。     

Phenotypic and Genetic Diversity of Erwinia carotovora ssp. carotovora Strains from Asia

A collection of 87 strains of the soft rot pathogen Erwinia carotovora ssp. carotovora (Ecc) isolated from various host plants in Japan, Korea and Thailand was characterized by bacteriological, pathological and genetic properties. On the basis of pathogenicity on the potato, tomato, onion and cucumber, strains were divided into four groups. They were also characterized by PCR‐restriction fragment length polymorphisms (RFLP) of 16S ribosomal DNA (rDNA), 16S‐23S rDNA intergenic spacer regions (ISRs) and a pel gene encoding pectate lyase. By analysis of 16S rDNA RFLP generated by Hinf I, Ecc strains were differentiated into two groups where it was discovered that most strains from Korea and Japan belonged to the same group. In the analysis of ISRs RFLP with MboI, two patterns were found. All Thai strains showed the same pattern. In the analysis of the pel gene RFLP with Sau3AI, all strains were separated into two independent patterns except for one strain. The strain (MAFF 301937) isolated from the mulberry showed a unique RFLP pattern of the pel gene. In cluster analysis based on 26 phenotypic characters, Ecc strains were composed of two groups, A and B. Group A contained typical Ecc strains which provided negative reactions in testing the production of reducing substances from sucrose and acids from α‐methyl glucoside. All Thai strains and most of the Korean strains belonged to group A, whereas group B contained atypical Ecc strains, which were isolated in Japan and Korea; the properties of this group were similar to those of E. carotovora ssp. atroseptica. The research reported here was undertaken to provide information on the strains of E. carotovora ssp. carotovora in Asia.     

The terminal inverted repeats of the linear plasmid SCP1 of Streptomyces coelicolor A3(2) possess a truncated copy of the transposon Tn 4811 of Streptomyces lividans 66

Streptomyces coelicolor A3(2), the best genetically studied streptomycete and Streptomyces lividans 66 are very closely related strains. This is further emphasized by our finding that a truncated copy of Tn4811 of S. lividans is present in the terminal inverted repeats of the S. coelicolor giant linear plasmid SCP1. The copy of Tn4811 in SCP1 lacks the first 1276 bp and shows only minor changes in the nucleotide sequence of the remaining 4.12 kb. Tn4811 exists in both ends of SCP1.     

西沙海藻共附生放线菌的分离鉴定及其抗菌活性评价

【目的】为了探究南海海藻共附生放线菌资源的多样性及潜在的应用价值,对中国西沙群岛来源的海藻进行共附生放线菌的分离鉴定与抗菌活性筛选。【方法】利用稀释涂布平板法,采用2种不同分离培养基对不同采样位点的6种海藻进行放线菌分离;通过16S rRNA基因序列分析、构建系统发育树对分离的放线菌进行鉴定;用打孔法对无乳链球菌(Streptococcus agalactiae)等10种敏感细菌进行抗菌活性筛选;对筛选得到的目标活性菌株HZ014进行全基因组测序,通过AntiSMASH在线工具分析其次级代谢产物生物合成基因簇,预测其产生新型活性物质的潜力。【结果】从6种海藻中分离得到36株共附生放线菌,基于16S rRNA基因序列比对和系统发育分析,鉴定结果为链霉菌属(Streptomyces) 2株、红球菌属(Rhodococcus) 2株、诺卡氏菌属(Nocardia)3株、小单孢菌属(Micromonospora) 5株和盐孢菌属(Salinispora) 24株;抗菌活性筛选结果表明,36株共附生放线菌发酵粗提物对至少1种敏感细菌表现出一定的抑制作用,不同菌株发酵粗提物的抗菌活性存在明显差异,...     

Genetic analysis of the φC31 -specific phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2)

The phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2) was shown to be specific to φC31 homo-immune phages, and to be absent from the closely related strain Streptomyces Iividans. A 16 kb fragment of S. coelicolor A3(2) DNA was isolated which complemented the Pgl^? phenotype of J1501, a pgl mutant derivative of the Pgl^tsS. coelicolor strain M130. The cloned DNA complemented only half of the available pgl mutants, which therefore represented at least two groups, designated Pgl class A and class B strains. It follows that more than one kind of high-frequency genetic event can lead to the Pgl^? phenotype. Crosses between class A and class B strains yielded high frequencies of Pgl⁺ recombinants. Crosses between strains of the same class gave no Pgl⁺ recombinants. The cloned DNA was altered by deletion or apparent point mutation upon passage through the two class B strains tested, such that it was no longer capable of complementing class A strains. This accumulation of mutations might suggest that the expression of the cloned DNA is toxic to at least some class B strains. The nature of the genetic instability associated with the Pgl system was not detectable by Southern blot analysis.     

邳州银杏内生放线菌分离、筛选及活性菌株鉴定

【目的】从银杏中分离、筛选得到具有抑菌作用的内生放线菌,为放线菌在生物防治上的应用提供新的菌种资源。【方法】采用组织贴片培养法进行分离,生长对峙法进行筛选。【结果】从银杏的根、茎、叶中分离得到98株、50株、8株内生放线菌(共计156株),47株放线菌具有拮抗植物病原真菌活性。菌株KLBMP 5501抗菌活性最好且具有广谱性,基于形态特征、培养特征、生理生化特征和16S rRNA基因序列的相似性分析等多项分类特征表明,菌株KLBMP 5501是一株浅紫链霉菌(Streptomyces violascens)。【结论】筛选得到了具有应用潜力的高活性菌株,并进行了菌种鉴定。     

Biological control of post-harvest late blight of potatoes

Introduction of US-8 genotypes of Phytophthora infestans has coincided with an increase in severity of potato late blight in North America. As alternatives to chemical fungicides, 18 bacterial strains patented as biological control agents (BCA) of both sprouting and Fusarium dry rot were cultivated in three liquid media and screened in wounded potato bioassays for their ability to suppress late blight incited by P. infestans (US-8, mating type A2). Washed or unwashed stationary-phase bacteria were mixed with fungal zoospores to inoculate potato wounds with 5µL containing ～10⁸ bacterial CFU/mL and 2×10⁴ zoospore count/mL. Disease suppressiveness was evaluated after tubers were stored a week at 15°C, 90% relative humidity. One-fifth of the 108 BCA treatments screened, reduced late blight by 25–60%, including among other strains Pseudomonas fluorescens S22:T:04 (showing most consistency), P22:Y:05, S11:P:12 and Enterobacter cloacae S11:T:07. Small-scale pilot testing of these four strains, alone and in combination, was conducted under conditions simulating a commercial application. Suspensions of 4×10⁴ P. infestans sporangia/mL were sprayed at a rate of 1.6 mL followed by 0.8 mL of bacteria treatment at ～5×10⁹ CFU/mL per each of 90 unwounded potatoes. Three replicate boxes per treatment (30 tubers per box) were randomized in storage and maintained 4 weeks at 7.2°C, 95% relative humidity. All BCA treatments significantly reduced disease; and unwashed bacteria outperformed those washed free of culture broth. Disease suppression ranged from 35% up to 86% the first test year and from 35 to 91% the second year. Highest overall performance rankings significantly above the control were achieved by the following strains in culture broth: four-strain mix > P. fluorescens S22:T:04> P. fluorescens S11:P:12. Combined with previous demonstrations of dry rot and sprout suppression, the consistent late blight control by these strains and strain mixtures suggests the commercial feasibility of a single treatment for broad spectrum suppression of post-harvest potato diseases and sprouting.     

Genetically Engineered Erwinia carotovora: Survival, Intraspecific Competition, and Effects upon Selected Bacterial Genera

Environmental use of genetically engineered microorganisms has raised concerns about potential ecological impact. This research evaluated the survival, competitiveness, and effects upon selected bacterial genera of wild-type and genetically engineered Erwinia carotovora subsp. carotovora to ascertain if differences between the wild-type and genetically engineered strains exist in soil microcosms. The engineered strain contained a chromosomally inserted gene for kanamycin resistance. No significant differences in survival in nonsterile soil over 2 months or in the competitiveness of either strain were observed when the strains were added concurrently to microcosms. For reasons that remain unclear, the engineered strain did survive longer in sterilized soil. The effects of both strains on total bacteria, Pseudomonas and Staphylococcus strains, and actinomycetes were observed. While some apparent differences were observed, they were not statistically significant. A better understanding of the microbial ecology of engineered bacteria, especially pathogens genetically altered for use as biological control agents, is essential before commercial applications can be accomplished.     

Heterologous expression of natural product biosynthetic gene clusters in Streptomyces coelicolor: from genome mining to manipulation of biosynthetic pathways

Heterologous gene expression is one of the main strategies used to access the full biosynthetic potential of actinomycetes, as well as to study the metabolic pathways of natural product biosynthesis and to create unnatural pathways. Streptomyces coelicolor A3(2) is the most studied member of the actinomycetes, bacteria renowned for their prolific capacity to synthesize a wide range of biologically active specialized metabolites. We review here the use of strains of this species for the heterologous production of structurally diverse actinomycete natural products.     

Effects of increased and deregulated expression of cell division genes on the morphology and on antibiotic production of streptomycetes

This paper describes the effects of increased expression of the cell division genes ftsZ, ftsQ, and ssgA on the development of both solid- and liquid-grown mycelium of Streptomyces coelicolor and Streptomyces lividans. Over-expression of ftsZ in S. coelicolor M145 inhibited aerial mycelium formation and blocked sporulation. Such deficient sporulation was also observed for the ftsZ mutant. Over-expression of ftsZ also inhibited morphological differentiation in S. lividans 1326, although aerial mycelium formation was less reduced. Furthermore, antibiotic production was increased in both strains, and in particular the otherwise dormant actinorhodin biosynthesis cluster of S. lividans was activated in liquid- and solid-grown cultures. No significant alterations were observed when the gene dosage of ftsQ was increased. Analysis by transmission electron microscopy of an S. coelicolor strain over-expressing ssgA showed that septum formation had strongly increased in comparison to wild-type S. coelicolor, showing that SsgA clearly influences Streptomyces cell division. The morphology of the hyphae was affected such that irregular septa were produced with a significantly wider diameter, thereby forming spore-like compartments. This suggests that ssgA can induce a process similar to submerged sporulation in Streptomyces strains that otherwise fail to do so. A working model is proposed for the regulation of septum formation and of submerged sporulation.     

Sequence analysis and heterologous expression of the lincomycin biosynthetic cluster of the type strain <Emphasis Type="Italic">Streptomyces lincolnensis</Emphasis> ATCC 25466

A cosmid bearing an insert of 38 217 bp covering the gene cluster and its flanking regions of type strain Streptomyces lincolnensis ATCC 25466 was sequenced. Two relatively extensive sequence changes and several hundred point mutations were identified if compared with the previously published sequence of the lincomycin (Lin) industrial strain S. lincolnensis 78-11. Analysis of the cluster-flanking regions revealed its localization within the genome of the ATCC 25466 strain. The cluster-bearing cosmid was integrated into the chromosome of Lin non-producing strains S. coelicolor CH 999 and S. coelicolor M 145. The modified strains heterologously produced Lin but the level dropped to ≈1–3 % of the production in the ATCC 25466 strain.     

Induction of defence-related enzymes in onion by combined application of fungal and bacterial biocontrol agents against Fusarium oxysporum f. sp. cepae

Basal rot disease of onion is a major problem in different onion growing regions of Tamil Nadu, India. Fungal and bacterial cultures were isolated and tested their efficiency against Fusarium oxysporum f. sp. cepae under in vitro conditions. Effective bacterial and fungal antagonists were tested alone and in combinations for the control of F. oxysporum f. sp. cepae in glasshouse experiments. Defence-related enzymes such as peroxidase, polyphenol oxidase and phenylalanine ammonia-lyase were induced and accumulated in onion treated with fungal and bacterial antagonists. Defence-related enzymes were significantly higher in onion pretreated with consortial formulation of Pf12 + Pf27 + TH3 at 5 days after the challenge inoculation with F. oxysporum f. sp. cepae and gave resistance to onion against basal rot disease.     

Insecticidal Bacillus thuringiensis Silences Erwinia carotovora Virulence by a New Form of Microbial Antagonism, Signal Interference

It is commonly known that bacteria may produce antibiotics to interfere with the normal biological functions of their competitors in order to gain competitive advantages. Here we report that Bacillus thuringiensis suppressed the quorum-sensing-dependent virulence of plant pathogen Erwinia carotovora through a new form of microbial antagonism, signal interference. E. carotovora produces and responds to acyl-homoserine lactone (AHL) quorum-sensing signals to regulate antibiotic production and expression of virulence genes, whereas B. thuringiensis strains possess AHL-lactonase, which is a potent AHL-degrading enzyme. B. thuringiensis did not seem to interfere with the normal growth of E. carotovora; rather, it abolished the accumulation of AHL signal when they were cocultured. In planta, B. thuringiensis significantly decreased the incidence of E. carotovora infection and symptom development of potato soft rot caused by the pathogen. The biocontrol efficiency is correlated with the ability of bacterial strains to produce AHL-lactonase. While all the seven AHL-lactonase-producing B. thuringiensis strains provided significant protection against E. carotovora infection, Bacillus fusiformis and Escherichia coli strains that do not process AHL-degradation enzyme showed little effect in biocontrol. Mutation of aiiA, the gene encoding AHL-lactonase in B. thuringiensis, resulted in a substantial decrease in biocontrol efficacy. These results suggest that signal interference mechanisms existing in natural ecosystems could be explored as a new version of antagonism for prevention of bacterial infections.     

Production of environmentally safe biocides from essential oils having antimicrobial activity

Different liquid formulations of anise, coriander and black cumin essential oils were used for preparing some biocides. The prepared formulations were tested for their antimicrobial activity against some post-harvest pathogenic microorganisms. The tested microorganisms were Fusarium oxysporum (Dray rot of potato), Alternaria alternata (Black rot of tomato), Penicillium italicum, P. digitatum, (Blue and green rot of orange, respectively), Botryitus cinerea (Gray rot of strawberry) and Erwinia carotovora (Soft rot of potato).The results revealed that the different formulations showed a complete inhibition effect on the growth of most of the tested microorganisms. Also, the antimicrobial activity of the formulated essential oils did not affect the formulation process compared with the original oil.     

Exploration of geosmin synthase from <Emphasis Type="Italic">Streptomyces peucetius</Emphasis> ATCC 27952 by deletion of doxorubicin biosynthetic gene cluster

Thorough investigation of Streptomyces peucetius ATCC 27952 genome revealed a sesquiterpene synthase, named spterp13, which encodes a putative protein of 732 amino acids with significant similarity to S. avermitilis MA-4680 (SAV2163, GeoA) and S. coelicolor A3(2) (SCO6073). The proteins encoded by SAV2163 and SCO6073 produce geosmin in the respective strains. However, the spterp13 gene seemed to be silent in S. peucetius. Deletion of the doxorubicin gene cluster from S. peucetius resulted in increased cell growth rate along with detectable production of geosmin. When we over expressed the spterp13 gene in S. peucetius DM07 under the control of an ermE* promoter, 2.4 ± 0.4-fold enhanced production of geosmin was observed.