具ACC脱氨酶活性的碱蓬内生细菌的分离、鉴定及其生物学特性

【目的】具有1-氨基环丙烷-1-羧酸(ACC)脱氨酶活性的盐生植物碱蓬内生细菌的分离及生物学特性的研究有助于探索内生细菌与宿主植物耐盐性的关系。【方法】采用研磨法从健康碱蓬植株的根、茎、叶中分离具有ACC脱氨酶活性的碱蓬内生细菌,根据形态特征、生理生化、API鉴定系统和16S rRNA对菌种进行鉴定,并分别测定了菌株产ACC脱氨酶、铁载体、吲哚乙酸、赤霉素、脱落酸、蛋白酶及溶磷、固氮和拮抗病原菌的特性。【结果】将分离得到的内生细菌LP11、SS12、TW1和TW2分别鉴定为栖稻假单胞菌(Pseudomonas oryzihabitans)、假单胞菌(Pseudomonas sp.)、成团泛菌(Pantoea agglomerans)和恶臭假单胞菌(Pseudomonas putida),4株菌除具有较高的ACC脱氨酶活力之外,还可不同程度地产生铁载体、吲哚乙酸、赤霉素和脱落酸,且均有溶磷作用,但无固氮能力及蛋白酶活力,唯有菌株SS12对萝卜枯萎病菌(Fusariumoxysporum f.sp.conglutinans)和黄瓜枯萎病菌(F.oxysporum f.sp.cucumerinum)具有拮抗作用。【结论】从盐生植物碱蓬中分离到的假单胞菌属和泛菌属内生细菌,具有丰富多样的生物学特性。     

Gene transfer from a bacterium injected into an aquifer to an indigenous bacterium

Two novel 3-chlorobenzoate-degrading bacteria were previously isolated from an aquifer in which no such bacteria could be enriched prior to the introduction of the 3-chlorobenzoate-degrading strain, Pseudomonas sp. B13. To understand the origin of 3-chlorobenzoate-degrading genes in the two novel isolates, the 16S ribosomal RNA, clcD (dienelactone hydrolase) and clcA (chlorocatechol oxygenase) genes from these bacteria were amplified and sequenced. The partial 16S rRNA gene sequences and REP-PCR patterns showed that these two novel isolates were identical but differed from strain B13. Phylogenetic analyses revealed that the novel isolates were closely related to Alcaligenes eutrophus in the beta subclass of the Proteobacteria, whereas strain B13 was related to Pseudomonas aeruginosa and P. mendocina in the gamma subclass of the Proteobacteria. In contrast, the clcD and clcA gene sequences were identical on strain B13 and these two isolates, indicating that the 3-chlorobenzoate-degrading genes were transferred from strain B13 to these isolates. What cannot be established is when this transfer occurred.     

荧光假单胞菌对食用菌的促生作用及其机理

从小麦根际分离到1株对食用菌有较强促生作用的荧光假单胞菌,命名为假单胞菌P2-10(Pseudomonas sp.P2-10)。该菌株与平菇天达300(Pleurotus ostreatus Td 300)、杏鲍菇杏6(Pleurotus eryngii X6)或鸡腿菇瑞7(Coprinus comatus R7)混合培养,可显著提高这些食用菌菌丝生长速度及诱导并促进子实体的形成和发育。假单胞菌P2-10对食用菌的促生作用来自其代谢产物,可能是通过其1-氨基环丙烷-1-羧酸(ACC)脱氨酶降解食用菌产生的ACC、减少食用菌乙烯的合成及乙烯对菌丝生长和子实体发育的抑制作用。     

Biodegradation of the sulfonylurea herbicide chlorimuron-ethyl by the strain Pseudomonas sp. LW3

Eleven carbazole (CAR)-degrading bacterial strains were isolated from seawater collected off the coast of Japan using two different media. Seven isolates were shown to be most closely related to the genera Erythrobacter, Hyphomonas, Sphingosinicella, Caulobacter , and Lysobacter . Meanwhile, strains OC3, OC6S, OC9, and OC11S showed low similarity to known bacteria, the closest relative being Kordiimonas gwangyangensis GW14-5 (90% similarity). Southern hybridization analysis revealed that only five isolates carried car genes similar to those reported in Pseudomonas resinovorans CA10 ( car _CA10) or Sphingomonas sp. strain KA1 ( car _KA1). The isolates were subjected to GC-MS and the results indicated that these strains degrade CAR to anthranilic acid.     

Effect of a Nickel-Tolerant ACC Deaminase-Producing Pseudomonas Strain on Growth of Nontransformed and Transgenic Canola Plants

Four bacterial strains were isolated from soils at nickel-contaminated sites based on their ability to utilize 1-aminocyclopropane-1-carboxylate (ACC) as a sole source of nitrogen. The four isolates were all identified as Pseudomonas putida Biovar B, and subsequent testing revealed that they all exhibited traits previously associated with plant growth promotion (i.e., indoleacetic acid and siderophore production and ACC deaminase activity). These four strains were also tolerant of nickel concentrations of up to 13.2 mM in the culture medium. The strain, HS-2, selected for further characterization, was used in pot experiments to inoculate both nontransformed and transgenic canola plants (expressing a bacterial ACC deaminase gene in its roots). Plants inoculated with the HS-2 strain produced an increase in plant biomass as well as in nickel (Ni) uptake by shoots and roots. The results suggest that this strain is a potential candidate to be used as an inoculant in both phytoremediation protocols and in plant growth promotion.     

A simple strategy for investigating the diversity and hydrocarbon degradation abilities of cultivable bacteria from contaminated soil

The use of indigenous bacterial strains is a valuable bioremediation strategy for cleaning the environment from hydrocarbon pollutants. The isolation and selection of hydrocarbon-degrading bacteria is therefore crucial for obtaining the most promising strains for site decontamination. Two different media, a minimal medium supplemented with a mixture of polycyclic aromatic hydrocarbons and a MS medium supplemented with triphenyltetrazolium chloride, were used for the isolation of bacterial strains from two hydrocarbon contaminated soils and from their enrichment phases. The hydrocarbon degradation abilities of these bacterial isolates were easily and rapidly assessed using the 2,6-dichlorophenol indophenol assay. The diversity of the bacterial communities isolated from these two soil samples and from their enrichment phases was evaluated by the combination of a bacterial clustering method, fluorescence ITS-PCR, and bacterial identification by 16S rRNA sequencing. Different PCR-based assays were performed in order to detect the genes responsible for hydrocarbon degradation. The best hydrocarbon-degrading bacteria, including Arthrobacter sp., Enterobacter sp., Sphingomonas sp., Pseudomonas koreensis, Pseudomonas putida and Pseudomonas plecoglossicida, were isolated directly from the soil samples on minimal medium. The nahAc gene was detected only in 13 Gram-negative isolates and the sequences of nahAc-like genes were obtained from Enterobacter, Stenotrophomonas, Pseudomonas brenneri, Pseudomonas entomophila and P. koreensis strains. The combination of isolation on minimal medium with the 2,6-dichlorophenol indophenol assay was effective in selecting different hydrocarbon-degrading strains from 353 isolates.     

Isolation and characterization of drought-tolerant ACC deaminase and exopolysaccharide-producing fluorescent Pseudomonas sp.

The enzyme 1-aminocyclopropane-1-carboxylate deaminase catalyzes the degradation of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of the plant hormone ethylene, into α-ketobutyrate and ammonia. The enzyme has been detected in a limited number of bacteria and plays a significant role in sustaining plant growth and development under biotic and abiotic stress conditions by reducing stress-induced ethylene production in plants. We have screened 32 fluorescent Pseudomonas sp. isolated from rhizosphere and non-rhizosphere soils of different crop production systems for drought tolerance using polyethylene glycol 6000 (PEG 6000). Nine of these isolates were tolerant to a substrate metric potential of ?0.30 MPa (15 % PEG 6000) and therefore considered to be drought-tolerant. All of these drought-tolerant isolates were screened for ACC deaminase activity using ACC as the sole nitrogen source, and one (SorgP4) was found to be positive for ACC, producing 3.71?±?0.025 and 1.42?±?0.039 μM/mg protein/h of α-ketobutyrate under the non-stress and drought stress condition, respectively. The isolate SorgP4 also showed other plant growth-promoting traits, such as indole acetic acid production, phosphate solubilization, siderophore and hydrogen cyanide production. The ACC deaminase gene (acdS) from the isolate SorgP4 was amplified, and the nucleotide sequence alignment of the acdS gene showed significant homology with acdS genes of NCBI Genbank. The 16S rRNA gene sequencing analysis identified the isolate as Pseudomonas fluorescens. Both sequences have been submitted to the NCBI GenBank under the accession numbers JX885767 and KC192771 respectively.     

Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase.

Pseudomonas sp. strain ACP is capable of growth on 1-aminocyclopropane-1-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase and the subsequent conversion of ACC to alpha-ketobutyrate and ammonia (M. Honma, Agric. Biol. Chem. 49:567-571, 1985). The complete amino acid sequence of purified ACC deaminase was determined, and the sequence information was used to clone the ACC deaminase gene from a 6-kb EcoRI fragment of Pseudomonas sp. strain ACP DNA. DNA sequence analysis of an EcoRI-PstI subclone demonstrated an open reading frame (ORF) encoding a polypeptide with a deduced amino acid sequence identical to the protein sequence determined chemically and a predicted molecular mass of 36,674 Da. The ORF also contained an additional 72 bp of upstream sequence not predicted by the amino acid sequence. Escherichia coli minicells containing the 6-kb clone expressed a major polypeptide of the size expected for ACC deaminase which was reactive with ACC deaminase antiserum. Furthermore, a lacZ fusion with the ACC deaminase ORF resulted in the expression of active enzyme in E. coli. ACC is a key intermediate in the biosynthesis of ethylene in plants, and the use of the ACC deaminase gene to manipulate this pathway is discussed.     

Molecular detection and diversity of polycyclic aromatic hydrocarbon-degrading bacteria isolated from geographically diverse sites

Nineteen polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from environmental samples in Kuwait, Indonesia, Thailand, and Japan by enrichment with either naphthalene or phenanthrene as a sole carbon source. Sequence analyses of the 16-S rRNA gene indicated that at least seven genera (Ralstonia, Sphingomonas, Burkholderia, Pseudomonas, Comamonas, Flavobacterium, and Bacillus) were present in this collection. Determination of the ability of the isolates to use PAH and its presumed catabolic intermediates suggests that the isolates showed multiple phenotypes in terms of utilization and degradation pathways. The large subunit of the terminal oxygenase gene (phnAc) from Burkholderia sp. strain RP007 hybridized to 32% (6/19) of the isolates, whilst gene probing using the large subunit of terminal oxygenase gene (pahAc) from Pseudomonas putida strain OUS82 revealed no pahAc-like genes amongst the isolates. Using three degenerated primer sets (pPAH-F/NR700, AJ025/26, and RieskeF/R), targeting a conserved region with the genes encoding the large subunit of terminal oxygenase successfully amplified material from 6 additional PAH-degrading isolates. Sequence analyses showed that the large subunit of terminal oxygenase in 4 isolates was highly homologous to the large subunit of naphthalene dioxygenase gene from Ralstonia sp. strain U2. However, we could not obtain any information on the oxygenase system involved in the naphthalene and/or phenathrene degradation by 7 other strains. These results suggest that PAH-degrading bacteria are diverse, and that there are still many unidentified PAH-degrading bacteria.     

Microbial utilization of the industrial wastewater pollutants 2-ethylhexylthioglycolic acid and <Emphasis Type="Italic">iso</Emphasis>-octylthioglycolic acid by aerobic Gram-negative bacteria

Industrial wastewater from the production of sulfur containing esters and the resulting products of this synthesis, 2-ethylhexylthioglycolic acid (EHTG) and iso-octylthioglycolic acid (IOTG), were deployed in this study to enrich novel bacterial strains, since no wastewater and EHTG or IOTG degrading microorganisms were hitherto described or available. In addition, nothing is known about the biodegradation of these thiochemicals. The effect of this specific wastewater on the growth behaviour of microorganisms was investigated using three well-known Gram-negative bacteria (Escherichia coli, Pseudomonas putida, and Ralstonia eutropha). Concentrations of 5% (v/v) wastewater in complex media completely inhibited growth of these three bacterial strains. Six bacterial strains were successfully isolated, characterized and identified by sequencing their 16S rRNA genes. Two isolates referred to as Achromobacter sp. strain MT-E3 and Pseudomonas sp. strain MT-I1 used EHTG or IOTG, respectively, as well as the wastewater as sole source of carbon and energy for weak growth. More notably, both isolates removed these sulfur containing esters in remarkable amounts from the cultures supernatant. One further isolate was referred to as Klebsiella sp. strain 58 and exhibited an unusual high tolerance against the wastewater’s toxicity without utilizing the contaminative compounds. If cultivated with gluconic acid as additional carbon source, the strain grew even in presence of more than 40% (v/v) wastewater. Three other isolates belonging to the genera Bordetella and Pseudomonas tolerated these organic sulfur compounds but showed no degradation abilities.     

Transformation and expression of plasmid from Nocardioides sp. to Pseudomonas putida

Nocardioides sp. isolated from contaminated soil showed the presence of sulphur oxidizing (SO) genes in the plasmid pSB1 (34·2 kb). The presence of SO genes was confirmed by transformation to a plasmid-free Pseudomonas putida strain followed by hybridization studies.     

Bacterial biosynthesis of 1-aminocyclopropane-1-caboxylate (ACC) deaminase,a useful trait to elongation and endophytic colonization of the roots of rice under constant flooded conditions

This study was conducted to investigate the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Pseudomonas fluorescens strain REN₁ and its ability to reduce ethylene levels produced during stress, endophytically colonize and promote the elongation of the roots of rice seedlings under gnotobiotic conditions. We isolated 80 bacteria from inside roots of rice plants grown in the farmers’ fields in Guilan, Iran. All of the isolates were characterized for plant growth promoting (PGP) traits. In addition, the colonization assay of these isolates on rice seedlings was carried out to screen for competent endophytes. The best bacterial isolate, based on ACC deaminase production, was identified and used for further study. 16S rDNA sequence analysis revealed that the endophyte was closely related to Pseudomonas fluorescens. The results of this study showed ACC deaminase containing P. fluorescens REN1 increased in vitro root elongation and endophytically colonized the root of rice seedlings significantly, as compared to control under constant flooded conditions. The trait of low amount of indole-3-acetic acid (IAA) production (<15 μg mL⁻¹) and the high production of ACC deaminase by bacteria may be main factors in colonizing rice seedling roots compared to other PGP traits (siderophore production and phosphate solubilization) in this study. Endophytic IAA and ACC deaminase-producing bacteria may be preferential selections by rice seedlings. Therefore, it may be suggested that the utilization of ACC as a nutrient gives the isolates advantages in more endophytic colonization and increase of root length of rice seedlings.     

Genetic characterization of the dibenzofuran-degrading Actinobacteria carrying the dbfA1A2 gene homologues isolated from activated sludge

Thirteen dibenzofuran (DF)-utilizing bacteria carrying the DF terminal dioxygenase genes homologous to those of Terrabacter sp. strain DBF63 (dbfA1A2) were newly isolated from activated sludge samples. The amplified ribosomal DNA restriction analysis and the hybridization analyses showed that these strains were grouped into five genetically different types of bacteria. The sequence analyses of the 16S rRNA genes and the dbfA1A2 homologues from these five selected isolates revealed that the isolates belonged to the genus Rhodococcus, Terrabacter or Janibacter and that they shared 99-100% conserved dbfA1A2 homologues. We investigated the genetic organizations flanking the dbfA1A2 homologues and showed that the minimal conserved DNA region present in all five selected isolates consisted of an approximately 9.0-kb region and that their outer regions became abruptly non-homologous. Among them, Rhodococcus sp. strain DFA3 possessed not only the 9.0-kb region but also the 6.2-kb region containing dbfA1A2 homologues. Sequencing of their border regions suggested that some genetic rearrangement might have occurred with insertion sequence-like elements. Also, within their conserved regions, some insertions or deletions were observed.     

Analysis of denitrification genes and comparison of nosZ, cnorB and 16S rDNA from culturable denitrifying bacteria in potato cropping systems

Bacterial denitrification in agricultural soils is a major source of nitrous oxide, a potent greenhouse gas. This study examined the culturable bacterial population of denitrifiers in arable field soils in potato (Solanum tuberosum L.) production and denitrification genes (nir, nor and nos) and 16S rDNA in those isolates. Enrichments for culturable denitrifiers yielded 31 diverse isolates that were then analysed for denitrification genes. The nitrous oxide reductase (nosZ) gene was found in all isolates. The majority of isolates ( approximately 90%) contained the cnorB nitric oxide reductase gene, with the remainder containing the qnorB gene. Nitrite reductase genes (nirS and nirK) were amplifiable from most of the isolates, and were segregated between species similar to previously isolated denitrifiers. Isolated strains were preliminarily identified using fatty acid methyl ester analysis and further identified using 16S rDNA sequencing. The majority of isolates (21) were classified as Pseudomonas sp., with smaller groups of isolates being most similar to Bosea spp. (4), Achromobacter spp. (4) and two isolates closely related to Sinorhizobium/Ensifer spp. Phylogenetic trees were compared among nosZ, cnorB and 16S rDNA genes for a subset of Pseudomonas strains. The trees were mostly congruent, but some Pseudomonas sp. isolates grouped differently depending on the gene analysed, indicating potential horizontal gene transfer of denitrification genes. Although Bosea spp. are known denitrifiers, to the best of our knowledge this is the first report of isolation and sequencing of denitrification genes from this bacterial genus.     

具有ACC脱氨酶活性的杜仲内生细菌的分离鉴定及其抗菌活性

采用富集筛选法从杜仲根中分离到5株具有ACC脱氨酶活性的内生细菌, 利用纸片法测定它们的抑菌活性, 通过形态特征、生理生化试验和16S rRNA序列分析对分离菌株进行鉴定。结果显示, 5株杜仲内生细菌均具有较高的ACC脱氨酶活性, 其中4株菌对大肠杆菌CGMCC1.1103和枯草芽孢杆菌CGMCC1.769均有较好的抑菌活性, 通过生理生化试验和16S rRNA序列分析, 将菌株JDM-2、JDM-8、JDM-11、JDM-14和JDM-19分别鉴定为Pseudomonas koreensis、肺炎克雷伯氏菌(Klebsiella pneumoniae)、路德维希肠杆菌(Enterobacter ludwigii)、变栖克雷伯氏菌(Klebsiella variicola)和阿氏肠杆菌(Enterobacter asburiae)。     

Screening of cry gene contents of Bacillus thuringiensis strains isolated from avocado orchards in Mexico, and their insecticidal activity towards Argyrotaenia sp. (Lepidoptera: Tortricidae) larvae

Aims:  To screen for Bacillus thuringiensis strains from avocado orchards in two Mexican states with lepidopteran-specific cry gene content and evaluate their insecticidal activity against Argyrotaenia sp., an undescribed species present in avocado orchards.
Methods and Results:  Lepidopteran-active cry 1, cry 2 and cry 9 genes were detected by PCR analysis in 37 isolates. cry 1 genes were more frequent in Michoacán, but were undetected in Nayarit isolates. cry 9 and cry 2 genes were detected in isolates from both states, although cry 2 genes were less frequent. A variety of crystal shapes were observed among the isolates. According to gene profile, eight isolates were selected and tested against 2-day old Argyrotaenia sp. larvae. Standard strain HD-125 caused the highest mortality followed by strain MR-26 from Michoacán at a concentration of 500 μg ml⁻¹, respectively.
Conclusions:  Bacillus thuringiensis strains isolated from avocado orchards exhibit a low toxic activity towards Argyrotaenia sp. larvae, in spite of their specific cry gene content.
Significance and Impact of the Study:  Toxic activity of B. thuringiensis is not necessarily related to insect pest habitat and neither to specific cry gene content associated to other lepidopterans.     

Isolation and characterization of ACC deaminase-producing fluorescent pseudomonads, to alleviate salinity stress on canola (Brassica napus L.) growth

Salinity stress is of great importance in arid and semi-arid areas of the world due to its impact in reducing crop yield. Under salinity stress, the amount of 1-aminocyclopropane-1-carboxylate (ACC), a precursor for ethylene production in plants, increases. Here, we conducted research under the hypothesis that isolated ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida can alleviate the stressful effects of salinity on canola (Brassica napus L.) growth. The experiments were conducted in the Soil and Water Research Institute, Tehran, Iran. Seven experimental stages were conducted to isolate and characterize ACC deaminase-producing Pseudomonas fluorescens strains and to determine factors enhancing their growth and, consequently, their effects on the germination of canola seeds. Under salinity stress, in 14% of the isolates, ACC deaminase activity was observed, indicating that they were able to utilize ACC as the sole N-source. Bacterial strains differed in their ability to synthesize auxin and hydrogen cyanide compounds, as well as in their ACC deaminase activity. Under salinity stress, the rate of germinating seeds inoculated with the strains of ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida, and seedling growth was significantly higher. These results indicate the significance of soil biological activities, including the activities of plant growth-promoting bacteria, in the alleviation of soil stresses such as salinity on plant growth.     

Properties and roles of bacterial symbionts of polyvinyl alcohol-utilizing mixed cultures.

From several polyvinyl alcohol (PVA)-utilizing mixed cultures, two component bacterial strains essential for PVA utilization were isolated, and their properties and roles in PVA utilization were studied. Each pair of essential component strains consisted of a type I strain, which produced a PVA-degrading enzyme and constituted the predominant population of the mixed culture in PVA, and a type II strain, which produced a certain growth stimulant for the former strain. All of the type I strains were taxonomically identical and assigned as Pseudomonas sp. In contrast, type II strains were taxonomically different from each other, belonging to Pseudomonas spp. and Alcaligenes sp. PVA utilization occurred in each mixed culture of a type I strain with Pseudomonas putida VM15A as a substitute for the type II strain of the original pair and also in each mixed culture of a type II strain with Pseudomonas sp. VM15C. The growth rates of these substituted, mixed cultures differed from each other.