Production of the Antibiotic Phenazine-1-Carboxylic Acid by Fluorescent Pseudomonas Species in the Rhizosphere of Wheat

Pseudomonas fluorescens 2-79 and P. aureofaciens 30-84 produce the antibiotic phenazine-1-carboxylic acid and suppress take-all, an important root disease of wheat caused by Gaeumannomyces graminis var. tritici. To determine whether the antibiotic is produced in situ, wheat seeds were treated with strain 2-79 or 30-84 or with phenazine-nonproducing mutants or were left untreated and then were sown in natural or steamed soil in the field or growth chamber. The antibiotic was isolated only from roots of wheat colonized by strain 2-79 or 30-84 in both growth chamber and field studies. No antibiotic was recovered from the roots of seedlings grown from seeds treated with phenazine-nonproducing mutants or left untreated. In natural soils, comparable amounts of antibiotic (27 to 43 ng/g of root with adhering soil) were recovered from roots colonized by strain 2-79 whether or not the pathogen was present. Roots of plants grown in steamed soil yielded larger bacterial populations and more antibiotic than roots from natural soils. In steamed and natural soils, roots from which the antibiotic was recovered had significantly less disease than roots with no antibiotic, indicating that suppression of take-all is related directly to the presence of the antibiotic in the rhizosphere.     

高产吩嗪-1-羧酸（PCA）假单胞菌株M18G发酵优化模型的构建

吩嗪-1-羧酸（phenazine-1-carboxylic acid, PCA）是促进根际生长假单胞菌分泌的重要抗菌物质。采用Plackett-Burman（PB）设计和响应面法（response surface method, RSM）对假单胞菌株M18（Pseudomonas sp. M18）的gacA基因突变株M18G的次级代谢产物PCA发酵的营养条件进行建模。运用Plackett-Burman（PB）设计试验,从12个营养成分中筛选出4个关键的组分,进而采用RSM 法对这4个因素进行中心组合设计试验,建立回归方程并进行统计学分析,绘制各营养因子之间的关系图。实验结果表明：建立的模型能合理地模拟并优化发酵中各参数及其浓度,确定发酵培养基的成分和浓度为：黄豆粉33.4g/L,葡萄糖12.7g/L,大豆蛋白胨10.9g/L和乙醇13.8 g/L, M18G菌株经60 h发酵培养,最高PCA产率能达到1.89 g/L,比优化前提高了6倍左右。各营养因子的等值线图表明黄豆粉和乙醇在PCA高产发酵中起到更为关键的作用,因此提供了提高PCA发酵产量的有效方法,并为其未来商业化应用奠定了基础。     

Population Structure of Clinical Pseudomonas aeruginosa from West and Central African Countries

Background

Pseudomonas aeruginosa (PA) has a non-clonal, epidemic population with a few widely distributed and frequently encountered sequence types (STs) called ‘high-risk clusters’. Clinical P. aeruginosa (clinPA) has been studied in all inhabited continents excepted in Africa, where a very few isolates have been analyzed. Here, we characterized a collection of clinPA isolates from four countries of West and Central Africa.

Methodology

184 non-redundant isolates of clinPA from hospitals of Senegal, Ivory Coast, Nigeria, and Central African Republic were genotyped by MLST. We assessed their resistance level to antibiotics by agar diffusion and identified the extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) by sequencing. The population structure of the species was determined by a nucleotide-based analysis of the entire PA MLST database and further localized on the phylogenetic tree (i) the sequence types (STs) of the present collection, (ii) the STs by continents, (iii) ESBL- and MBL-producing STs from the MLST database.

Principal Findings

We found 80 distinct STs, of which 24 had no relationship with any known STs. ‘High-risk’ international clonal complexes (CC155, CC244, CC235) were frequently found in West and Central Africa. The five VIM-2-producing isolates belonged to CC233 and CC244. GES-1 and GES-9 enzymes were produced by one CC235 and one ST1469 isolate, respectively. We showed the spread of ‘high-risk’ international clonal complexes, often described as multidrug-resistant on other continents, with a fully susceptible phenotype. The MBL- and ESBL-producing STs were scattered throughout the phylogenetic tree and our data suggest a poor association between a continent and a specific phylogroup.

Conclusions

ESBL- and MBL-encoding genes are borne by both successful international clonal complexes and distinct local STs in clinPA of West and Central Africa. Furthermore, our data suggest that the spread of a ST could be either due to its antibiotic resistance or to features independent from the resistance to antibiotics.     

Diversity of extremophilic purple phototrophic bacteria in Soap Lake, a Central Washington (USA) Soda Lake

Culture-based and culture-independent methods were used to explore the diversity of phototrophic purple bacteria in Soap Lake, a small meromictic soda lake in the western USA. Among soda lakes, Soap Lake is unusual because it consists of distinct upper and lower water bodies of vastly different salinities, and its deep waters contain up to 175 mM sulfide. From Soap Lake water new alkaliphilic purple sulfur bacteria of the families Chromatiaceae and Ectothiorhodospiraceae were cultured, and one purple non-sulfur bacterium was isolated. Comparative sequence analysis of pufM, a gene that encodes a key photosynthetic reaction centre protein universally found in purple bacteria, was used to measure the diversity of purple bacteria in Soap Lake. Denaturing gradient gel electrophoresis and subsequent phylogenetic analyses of pufMs amplified from Soap Lake water revealed that a significant diversity of purple bacteria inhabit this soda lake. Although close relatives of several of the pufM phylotypes obtained from cultured species could also be detected in Soap Lake water, several other more divergent pufM phylotypes were also detected. It is possible that Soap Lake purple bacteria are major contributors of organic matter into the ecosystem of this lake, especially in its extensive anoxic and sulfidic deep waters.     

Temporal Variation in Genetic Diversity and Structure of a Lotic Population of Burkholderia (Pseudomonas) cepacia

The genetic structure and temporal patterns of genetic diversity in a population of Burkholderia (Pseudomonas) cepacia, isolated from a southeastern blackwater stream, were investigated by multilocus enzyme electrophoresis. Allelic variation in seven structural gene loci was monitored at a single stream location at 0, 6, 12, and 24 h and at 2, 4, 8, 16, and 32 days. Over the length of the study, 217 isolates were collected, from which 65 unique electrophoretic types (ETs) were identified. Most of these ETs were present at only one or two time periods and were considered transients; however, one resident ET was particularly abundant (64 of the 217 isolates [29.4%]) and was found at all time points except day 32. The mean genetic diversity of the entire population was 0.520, and the index of association (a measure of multilocus linkage disequilibrium) was 1.33. These results, taken in conjunction with a previous study focusing on spatial patterns of genetic diversity in lotic B. cepacia, show that these bacterial populations exhibit greater variability among sites than within a site over time, suggesting relative stability over short time periods.     

Falcaustra washingtonensis n. sp. (Nematoda: Kathlaniidae) from Ambystoma tigrinum melanostictum (Caudata: Ambystomatidae) from Washington State, USA.

Falcaustra washingtonensis n. sp. (Nematoda: Kathlaniidae) from the intestine of the salamander Ambystoma tigrinum melanostictum is described and illustrated. Falcaustra washingtonensis n. sp. represents the 14th nearctic species assigned to this genus and is distinguished from other nearctic species by the distribution pattern of caudal papillae and number of posterior muscle groups of the male: 10 pairs of sessile caudal papillae--3 pairs precloacal, 1 pair adcloacal, 6 pairs postcloacal--and 5 posterior muscle groups.     

丹参ACC氧化酶基因(SmACO1)的克隆与序列分析

依据丹参转录组数据库序列信息,采用RT-PCR和染色体步移技术从丹参中首次克隆得到ACC氧化酶基因,命名为SmACO1(GenBank注册号为JQ026111)。该基因gDNA序列长1 347 bp,由3个外显子和2个内含子组成;cDNA全长1 117 bp,包含945 bp的开放阅读框,编码314个氨基酸残基。生物信息学分析显示SmACO1为无信号肽与跨膜结构域,且定位于细胞质的稳定亲水蛋白,含有Fe²⁺依赖的加氧酶结构域。实时荧光定量PCR结果表明,SmACO1基因在丹参不同组织器官中差异表达,花中表达量最高;其表达受到病原菌和茉莉酸甲酯的诱导,表明SmACO1基因可能在植物防御反应中发挥作用。     

Differential Regulation of rsmA Gene on Biosynthesis of Pyoluteorin and Phenazine-1-carboxylic Acid in Pseudomonas sp. M18

Summary Plant growth promoting rhizobacteria (PGPR) strain Pseudomonas sp. M18 can produce two different types of antibiotics, pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA). The global regulator RsmA is a translational repressor of secondary metabolism in many prokaryotes. A chromosomally rsmA inactivated mutant strain M18R was constructed to study the regulatory mechanism of Plt and PCA biosynthesis and enhancement of Plt or PCA production in Pseudomonas sp. M18. The accumulation of Plt increased six-fold over that of the wild-type strain whereas PCA production was not significantly affected in cultures of M18R. Plt production was inhibited completely but PCA biosynthesis was not altered after complementation with rsmA gene in trans in the strain of M18R. The differential activity of rsmA gene on these two operons was further confirmed by the analysis of β-galactosidase activities from translational phzA′-′lacZ and pltA′-′lacZ fusion, in which phzA is the first enzyme gene of the phenazine biosynthesis pathway and pltA is the first gene of the pyoluteorin biosynthesis pathway. The results indicate that RsmA can control Plt production negatively but not PCA production in M18, and show that the global regulator RsmA does not repress the biosynthesis of all secondary metabolites.     

假单胞菌gacA插入突变对藤黄绿脓菌素和吩嗪-1-羧酸合成代谢的差异性调控

假单胞菌株M18分泌藤黄绿脓菌素 (Pyoluteorin ,Plt )和吩嗪 1 羧酸 (Phenazine 1 carboxylicacid ,PCA)并抑制多种植物病菌的生长。从M18中克隆双基因调控系统gacS gacA的组成基因gacA ,并构建了该基因抗性插入突变株M18G。在KMB培养基中 ,M18G合成Plt的能力受到完全抑制 ,而PCA的积累约比野生型提高 31倍左右。Plt合成基因簇突变株M18T和在M18G基础上构建的PCA合成基因簇突变株M18GA的Plt和PCA合成的动力学变化表明 ,在M18G菌株中 ,Plt合成的抑制并不引起PCA的过量积累 ,PCA的过量积累也不引起Plt合成的抑制。由此推测 ,gacA在基因表达的水平上全局性地执行着调控功能     

Prevalence and Antimicrobial Resistance of Thermophilic Campylobacter spp. from Cattle Farms in Washington State

The prevalence of thermophilic Campylobacter spp. was investigated in cattle on Washington State farms. A total of 350 thermophilic Campylobacter isolates were isolated from 686 cattle sampled on 15 farms (eight dairies, two calf rearer farms, two feedlots, and three beef cow-calf ranches). Isolate species were identified with a combination of phenotypic tests, hipO colony blot hybridization, and multiplex lpxA PCR. Breakpoint resistance to four antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, and doxycycline) was determined by agar dilution. Campylobacter jejuni was the most frequent species isolated (34.1%), followed by Campylobacter coli (7.7%) and other thermophilic campylobacters (1.5%). The most frequently detected resistance was to doxycycline (42.3% of 350 isolates). Isolates from calf rearer facilities were more frequently doxycycline resistant than isolates from other farm types. C. jejuni was most frequently susceptible to all four of the antimicrobial drugs studied (58.8% of 272 isolates). C. coli isolates were more frequently resistant than C. jejuni, including resistance to quinolone antimicrobials (89.3% of isolates obtained from calves on calf rearer farms) and to erythromycin (72.2% of isolates obtained from feedlot cattle). Multiple drug resistance was more frequent in C. coli (51.5%) than in C. jejuni (5.1%). The results of this study demonstrate that C. jejuni is widely distributed among Washington cattle farms, while C. coli is more narrowly distributed but significantly more resistant.     

Prevalence and antimicrobial resistance of thermophilic Campylobacter spp. from cattle farms in Washington State

The prevalence of thermophilic Campylobacter spp. was investigated in cattle on Washington State farms. A total of 350 thermophilic Campylobacter isolates were isolated from 686 cattle sampled on 15 farms (eight dairies, two calf rearer farms, two feedlots, and three beef cow-calf ranches). Isolate species were identified with a combination of phenotypic tests, hipO colony blot hybridization, and multiplex lpxA PCR. Breakpoint resistance to four antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, and doxycycline) was determined by agar dilution. Campylobacter jejuni was the most frequent species isolated (34.1%), followed by Campylobacter coli (7.7%) and other thermophilic campylobacters (1.5%). The most frequently detected resistance was to doxycycline (42.3% of 350 isolates). Isolates from calf rearer facilities were more frequently doxycycline resistant than isolates from other farm types. C. jejuni was most frequently susceptible to all four of the antimicrobial drugs studied (58.8% of 272 isolates). C. coli isolates were more frequently resistant than C. jejuni, including resistance to quinolone antimicrobials (89.3% of isolates obtained from calves on calf rearer farms) and to erythromycin (72.2% of isolates obtained from feedlot cattle). Multiple drug resistance was more frequent in C. coli (51.5%) than in C. jejuni (5.1%). The results of this study demonstrate that C. jejuni is widely distributed among Washington cattle farms, while C. coli is more narrowly distributed but significantly more resistant.     

Carrier-Mediated Uptake of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Vacuoles Isolated from Catharanthus roseus Cells

The uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, into vacuoles isolated from Catharanthus roseus cells has been studied by silicone layer floatation filtering. The transport across the tonoplast of MACC is stimulated fourfold by 5 millimolar MgATP, has a K_m of about 2 millimolar, an optimum pH around 7, and an optimum temperature at 30°C. Several effectors known to inhibit ATPase (N,N′-dicyclohexylcarbodiimide) and to collapse the transtonoplastic H⁺ electrochemical gradient (carbonylcyanide m-chlorophenylhydrazone, gramicidin, and benzylamine) all reduced MACC uptake. Abolishing the membrane potential with SCN⁻ and valinomycin also greatly inhibited MACC transport. Our data demonstrate that MACC accumulates in the vacuole against a concentration gradient by means of a proton motive force generated by a tonoplastic ATPase. The involvement of a protein carrier is suggested by the strong inhibition of uptake by compounds known to block SH—, OH—, and NH₂— groups. MACC uptake is antagonized competitively by malonyl-d-tryptophan, indicating that the carrier also accepts malonyl-d-amino acids. Neither the moities of these compounds taken separately [1-aminocyclopropane-1-carboxylic acid, malonate, d-tryptophan or d-phenylalanine] nor malate act as inhibitors of MACC transport. The absence of inhibition of malate uptake by MACC suggests that MACC and malate are taken up by two different carriers. We propose that the carrier identified here plays an important physiological role in withdrawing from the cytosol MACC and malonyl-d-amino acids generated under stress conditions.     

Reduced Genetic Diversity and Effective Population Size in an Endangered Atlantic Salmon (Salmo Salar) Population from Maine, USA

Atlantic salmon (Salmo salar) populations in Maine, USA, are listed as a Distinct Population Segment under the U.S. Endangered Species Act due to reduced spawning runs and juvenile densities. Whenever possible, optimal conservation strategies for endangered populations should incorporate both present and historical knowledge of genetic variation. We assayed genetic diversity at seven microsatellite loci and at the mitochondrial ND1 gene in an endangered wild population of Atlantic salmon captured from the Dennys River from 1963 to 2001 using DNA’s extracted from archival scale and tissue samples. We examined temporal trends of genetic diversity, population structure, and effective population size (Ne). Overall temporal trends of diversity and Ne show significant reductions from 1963 to 2001 raising the possibility that current restoration efforts may be impacted by historical loss of diversity potentially critical to adaptation. Although our results suggest genetic stability in this population from 1963 to 1981, significant differentiation was observed for both the 1995 and 2001 samples compared with all other temporal samples. The presence of an ND1 mtDNA haplotype in this population, historically observed only in European and Newfoundland stocks, may represent previously unrecognized local wild diversity or, alternatively, may represent introgression from non-native fish.     

Genetic Diversity and Population Structure of Acacia senegal (L) Willd. in Kenya

The level of genetic diversity and population structure of Acacia senegal variety kerensis in Kenya was examined using seven polymorphic nuclear microsatellite loci and two chloroplast microsatellite loci. In both chloroplast and nuclear datasets, high levels of genetic diversity were found within all populations and genetic differentiation among populations was low, indicating extensive gene flow. Analysis of population structure provided support for the presence of two groups of populations, although all individuals had mixed ancestry. Groups reflected the influence of geography on gene flow, with one representing Rift Valley populations whilst the other represented populations from Eastern Kenya. The similarities between estimates derived from nuclear and chloroplast data suggest highly effective gene dispersal by both pollen and seed in this species, although population structure appears to have been influenced by distributional changes in the past. The few contrasts between the spatial patterns for nuclear and chloroplast data provided additional support for the idea that, having fragmented in the past, groups are now thoroughly mixed as a result of extensive gene flow. For the purposes of conservation and in situ management of genetic resources, sampling could target a few, large populations ideally distributed among the spatial groups identified. This should ensure the majority of extant variation is preserved, and facilitate the investigation of variation in important phenotypic traits and development of breeding populations.     

Biological Control of Fire Blight of Hawthorn (Crataegus monogyna) with Fluorescent Pseudomonas spp. under Protected Conditions

Naturally-occurring epiphytic fluorescent pseudomonads were isolated and characterized in terms of their potential to control fire blight infection of hawthorn, caused by Erwinia amylovora. Preliminary testing and selection of antagonists using an immature pear fruit assay gave some inconsistency in the amount of pathogen suppression on the pear tissue and also in the prediction of biocontrol effectiveness on the intact plant. Selected antagonists provided significant but variable control of fire blight under protected (polythene tunnel and glasshouse) conditions, with isolates HL83 and HL99 giving control of both blossom-blight and shoot-blight. In some cases the degree of control was equal to that of chemical treatments, including agrimycin 17 and experimental bactericides, and was achieved without any numerical advantage of applied control agent over pathogen. The timing of pseudomonad application in relation to pathogen inoculation was found to have a significant effect on the level of control of blossom-blight.     

羚牛的遗传多样性及其种群遗传结构分析

羚牛是亚洲大陆一种特有的大型珍稀动物,目前正面临着栖息地丧失、片段化和人类活动的严重威胁。为了有效地保护这种濒危动物,全面了解羚牛的种群结构、进化历史和整个分布区内遗传多样性的分布是至关重要的。本研究以mtDNA D-loop330bp基因片段为分子标记,比较分析了来自陕西秦岭、甘肃南部、四川岷山、邛崃山和云南贡山的40个样品的序列差异,根据分布特点将所采集到的羚牛分为3个地理单元,即秦岭、四川和云南。结果表明,在3个地理单元中存在4种单倍型,且地理单元间不存在共享单倍型,相互单倍型之间的平均序列差异为1.66％。进一步分析表明,3个地理单元间的基因流较低,存在着显的遗传分化 ,说明羚牛具有明显的系统地理分布格局。同时提出应将分布于秦岭山区、唐家河青川地区、天全以及云南贡山地区作为独立的管理单元分别加以保护。     

The Diversity of Coolia spp. (Dinophyceae Ostreopsidaceae) in the Central Great Barrier Reef Region

Background

Dinoflagellates are important primary producers, crucial in marine food webs. Toxic strains, however, are the main causative agents of non-bacterial seafood poisoning, a major concern for public health worldwide. Despite their importance, taxonomic uncertainty within many genera of dinoflagellates is still high. The genus Coolia includes potentially harmful species and the diversity within the genus is just starting to become apparent.

Methodology/Principal Findings

In the current study, cultures were established from strains of Coolia spp. isolated from the central Great Barrier Reef (GBR). Cultures were identified based on thecal plate morphology and analyses of sequences (18S, ITS and 28S) from the nuclear rRNA operon. We report that the central GBR harbors a high diversity of Coolia species, including two species known to be capable of toxin production (C. tropicalis and C. malayensis), as well as the non-toxic C. canariensis. The strain of C. canariensis isolated from the GBR may in fact be a cryptic species, closely related but nevertheless phylogenetically distinct from the strain on which the holotype of C. canariensis was based. We also found evidence of the occurrence of a cryptic species morphologically very similar to both C. malayensis and C. monotis. The consequences of taxonomic confusion within the genus are discussed.

Conclusion/Significance

The central GBR region harbors a previously unreported high diversity of Coolia spp., including two species known to potentially produce toxins. The presence of a cryptic species of unknown toxicity highlights the importance of cryptic diversity within dinoflagellates.