天山大峡谷原始森林黏细菌的分离鉴定及其抗菌活性

【背景】黏细菌是一类具有多细胞群体行为特征的高等原核生物,其对植物病原真菌和细菌的捕食特性使其在植物病害防治方面具有重要的应用潜力。【目的】探究乌鲁木齐天山大峡谷原始森林可培养黏细菌的多样性并分析其抗菌活性,为发掘黏细菌生防菌株奠定基础。【方法】以天山大峡谷原始森林采集的土样和腐木为分离材料,采用兔粪诱导法和被捕食菌诱导法从中分离纯化黏细菌菌株,结合形态学观察、生理生化测定和16S rRNA基因序列分析确定其分类地位,并以6种植物病原真菌[大丽轮枝菌(Verticillium dahliae)、尖孢镰刀菌萎蔫专化型(Fusarium oxysporum f. sp. vasinfectum)、拟轮枝链孢霉(Fusarium verticillioides)、立枯丝核菌(Rhizoctonia solani)、黄色镰刀菌(Fusarium culmorum)、细极链格孢菌(Alternaria tenuissima)]和1种植物病原细菌[梨火疫病菌(Erwinia amylovora)]为靶标菌,通过平板对峙法和菌苔捕食法测定其抗菌活性。【结果】从采集的样品中分离出70株菌株,经纯化后获得36株黏细菌纯培养物。经鉴定隶属于4个属,黏球菌属(Myxococcus) 30株、孢囊杆菌属(Cystobacter) 3株、珊瑚球菌属(Corallococcus) 2株和原囊菌属(Archangium) 1株。抗菌活性分析显示,本研究获得的36株黏细菌至少对2种植物病原真菌有抗菌活性,表现出广谱的抗真菌活性,初步筛选出一株菌株NSE37-1兼具广谱和高效抗真菌活性;供试的15株黏细菌对梨火疫病菌均具有捕食活性,初步筛选出一株对梨火疫病菌具有较强捕食能力的黏细菌菌株NSE25。【结论】天山大峡谷可培养黏细菌资源比较丰富,黏球菌属是该地区可培养黏细菌菌群中的优势菌。分离纯化出的黏细菌菌株均表现出广谱的抗植物病原菌活性,具有进一步研究和开发的潜在价值。     

不同分离方法对子实体形成和粘细菌分离的影响

【目的】基于模拟原位环境策略、可培养粘细菌的营养策略及细菌互作网络,改良分离培养基,以提高分离粘细菌的多样性。【方法】通过添加土壤浸提液、使用不同种类的诱导菌和改变诱导菌的接种方式设置分离方法,同时以传统的分离方法作对照。【结果】改良的分离方法比对照组诱导出了更多粘细菌子实体种类,采自4个地区的9份样品共分离纯化出40株粘细菌,按形态学和分子生物学,将其归类于原囊菌属(Archangium)、珊瑚菌属(Corallococcus)、软骨霉状菌属(Chondromyces)、粘球菌属(Myxococcus)、侏囊菌属(Nannocystis)、多囊菌属(Polyangium)、匣状球菌属(Pyxidicoccus)。【结论】与传统分离方法相比,添加土壤浸提液,诱导菌点接法能大大提高诱导出的粘细菌子实体种类的数目,革兰氏阳性菌和革兰氏阴性菌作为诱导菌对子实体种类影响较小,但是也发现革兰氏阳性菌特异性诱导出的子实体。虽然本研究通过对分离培养基的改良大大增加了子实体种类,但是纯化出的粘细菌种类远少于观察到的子实体种类,说明除改良分离方法外,还需进一步研究粘细菌的纯化方法,提高分离所得粘细菌的多样性,获取更多粘细菌新资源。     

Adaptation of Salt-tolerant <Emphasis Type="Italic">Myxococcus</Emphasis> Strains and their Motility Systems to the Ocean Conditions

More and more studies have indicated that myxobacteria are able to live in seawater conditions, which, however, can decrease the fruiting body formation ability and also the adventurous (A) and social (S) motility systems of the myxobacteria. To learn the adaptation mechanism of the salt-tolerant myxobacteria to marine conditions, we analyzed 10 salt-tolerant Myxococcus strains of their fruiting body formation and motility. The isolates were from marine samples and possessed different levels of salt tolerance. They had the dual motility system and formed fruiting bodies in the presence of suitable seawater concentrations. Some high salt-tolerant strains even lost their fruiting abilities in the absence of seawater. In response to the presence of seawater, the S-motility was found to be increased in the high salt-tolerants but decreased in the low salt-tolerants. The A-motility, on the other hand, was observed in all the salt-tolerant Myxococcus strains, but increased or decreased in response to the presence of seawater. Perceived shifts of fruiting body formation abilities and motilities discovered in the salt-tolerant Myxococcus strains suggested an ecological adaptation of myxobacterial social behaviors to the marine environments.     

Isolation of Halotolerant Thermus spp. from Submarine Hot Springs in Iceland

Thermophilic, aerobic bacteria of the genus Thermus were isolated from submarine alkaline hot springs in Iceland. Five submarine hot springs were sampled, and all had viable counts of Thermus spp. of about 10³ CFU/ml. All submarine strains grew in the presence of NaCl at 3% or higher, but no strains from terrestrial hot springs would grow at concentrations higher than 1% NaCl. The growth rate of submarine Thermus strains was not stimulated by NaCl and was reduced at NaCl concentrations higher than 1%. The pattern of growth of these isolates on single carbon sources was similar to that of terrestrial isolates.     

秦岭地区羊肚菌根系土样黏细菌的分离

通过分离陕西秦岭地区羊肚菌根系土样中的黏细菌,建立黏细菌的分离方法,分析羊肚菌根系土样中黏细菌的多样性。采集自陕西商洛地区种植羊肚菌根系土样,采用不同诱导方法分离土样中的黏细菌,比较不同方法的出菌率,通过菌落形态、显微照片对黏细菌进行观察,并结合16S rRNA序列,分析得到的黏细菌种类。采用活的大肠埃希菌作为诱导底物分离出的黏细菌数量最多,本次实验从羊肚菌根系土样中共分离纯化出17株黏细菌,包括5个属,其中黏球菌属(Myxococcus)12株,珊瑚球菌属(Corallococcus)2株,孢囊杆菌属(Cystobacter)1株,原囊菌属(Archangium)1株,标桩菌属(Stigmatella)1株。结果显示羊肚菌根系土壤有黏细菌存在,其中以黏球菌属居多,为后续探讨黏细菌与羊肚菌之间是否存在互生关系提供参考。     

Effect of sodium chloride on growth of heterotrophic marine bacteria

The effect of NaCl on the growth rates and yields of 31 gram-negative, heterotrophic, marine bacteria was determined. The strains used were representative of aerobic genera (Alteromonas, Pseudomonas, Alcaligenes, Bdellovibrio) as well as genera comprised of facultative anaerobes (Beneckea, Photobacterium). Two media were used-the first, a medium designed for the cultivation of marine bacteria and, the second, a medium used for the cultivation of terrestrial strains. These two media differed in the concentrations of divalent cations; the terrestrial medium (TM) contained 2 mM Mg⁺⁺ and 0.55 mM Ca⁺⁺ while the marine medium (MM) contained 50 mM Mg⁺⁺ and 10 mM Ca⁺⁺. The amount of NaCl necessary for optimal growth varied in different strains and was usually considerably higher in TM (100 to 460 mM) than in MM (70 to 300 mM). Many strains which grew in MM and TM had a shorter generation time in the former than in the latter medium. In addition, four strains which grew well in MM usually failed to grow in TM. These results show that higher levels of divalent cations are either essential for growth or stimulate growth rate, indicating that for many marine strains a terrestrial medium modified by the addition of NaCl cannot support optimal growth. Fourteen terrestrial strains of the genera Pseudomonas, Alcaligenes, Acinetobacter, Salmonella, Aeromonas, and Vibrio did not have ionic requirements comparable to those of the marine strains. All of the terrestrial organisms grew in TM without added NaCl (0.068 mM Na⁺ was present as a contaminant). In some terrestrial organisms, growth was stimulated by the addition of NaCl, the highest stimulation being found in Vibrio cholerae. The optimal growth rates and yields for four strains of this species were observed at 2.5 to 5.0 mM NaCl while the growth rates and yields in TM with no added NaCl were 40 to 50% of the optimum.     

Cellular fatty acid composition ofCorallococcus coralloides

The fatty acid composition of five strains ofCorallococcus coralloides and three reference species ofMyxococcus were determined by gas-liquid chromatography. Methyl esters of fatty acid containing from 12 to 22 carbon atoms were identified. The major fatty acids present were C₁₅ and C₁₇ saturated branched chain, and both C₁₆ saturated and unsaturated straight chain acids. The C₁₇ saturated branched and straight chain acids, which were in valuable concentration in species ofMyxococcus, were not, however, detected in all strains ofC. coralloides. The application of these results in the distinction ofC. coralloides from the genusMyxococcus is discussed.     

Haliangium ochraceum gen. nov., sp. nov. and Haliangium tepidum sp. nov.: novel moderately halophilic myxobacteria isolated from coastal saline environments

Phenotypic and phylogenetic studies were performed on two myxobacterial strains, SMP-2 and SMP-10, isolated from coastal regions. The two strains are morphologically similar, in that both produce yellow fruiting bodies, comprising several sessile sporangioles in dense packs. They are differentiated from known terrestrial myxobacteria on the basis of salt requirements (2-3% NaCl) and the presence of anteiso-branched fatty acids. Comparative 16S rRNA gene sequencing studies revealed that SMP-2 and SMP-10 are genetically related, and constitute a new cluster within the myxobacteria group, together with the Polyangium vitellinum Pl vt1 strain as the closest neighbor. The sequence similarity between the two strains is 95.6%. Based on phenotypic and phylogenetic evidence, it is proposed that these two strains be assigned to a new genus, Haliangium gen. nov., with SMP-2 designated as Haliangium ochraceum sp. nov. (= JCM 11303(T) = DSM 14365(T)), and SMP-10 as Haliangium tepidum sp. nov. (= JCM 11304(T)= DSM 14436(T)).     

Deoxyribonucleic Acid Homology Among the Fruiting Myxobacteria

Deoxyribonucleic acid similarities were determined by competition experiments. The several strains of fruiting myxobacteria tested showed from 23 to 89% homology with the reference strains Myxococcus xanthus (FB) and M. fulvus (M6).     

Characteristics and Living Patterns of Marine Myxobacterial Isolates

The growth, morphology, and life cycle of two marine myxobacterial isolates, halotolerant Myxococcus fulvus strain HW-1 and halophilic Haliangium ochraceum strain SMP-2, were studied as models to determine the living patterns of myxobacteria in the ocean. The growth, morphology, and development of halotolerant strain HW-1 shifted in response to salinity. The optimal seawater concentration for growth of HW-1 was 0 to 80% (salinity, 0.1 to 2.9%), and the strain grew poorly in media with a salinity of more than 4%. The cells became shorter as the seawater concentration increased. The fruiting body structure was complete only on agar prepared with low concentrations of seawater or salts (less than 60% seawater; salinity, 2.1%), and rudimentary structures or even simple cell mounds appeared as the seawater concentration increased. In contrast, the halophilic strain SMP-2 was unable to grow without NaCl. The cell length and the morphology of the fruiting body-like structure did not change in response to salts. In seawater liquid medium, the cells of both strains were confirmed to be able to form myxospores directly from vegetative cells, but they could not do so in medium containing a low seawater concentration (10% or less). HW-1 cells from medium containing a high concentration of seawater grew independent of cell density, while cells from medium containing a low concentration of seawater (10% or less) showed density-dependent growth. SMP-2 cells showed density-dependent growth under all salinity conditions. The results suggest that the halotolerant myxobacteria are the result of degenerative adaptation of soil myxobacteria to the marine environment, while the halophilic myxobacteria form a different evolutionary group that is indigenous to the ocean.     

Bacterial complexes of the fruiting bodies and hyphosphere of certain basidiomycetes

The surface and internal tissues of the fruiting bodies of basidiomycetes were shown to be specific bacterial habitats characterized by varying diversity and structure of bacterial complexes. On the surface of fruiting bodies, Gram-negative bacteria of the genera Pseudomonas, Xanthomonas, and Myxococcus prevailed, while in the internal tissues Gram-positive bacteria of the genera Streptomyces, Bacillus, Arthrobacter, and Micrococcus were identified in addition. Bacterial complexes from the surface and inner tissues of the fruiting bodies of the studied basidiomycetes showed significant similarity to each other and differed from those from the hyphosphere and the reference soil. On the surface and in the internal tissues of the fruiting bodies, representatives of the genus Myxococcus were identified for the first time, which could indicate initial decay of the fruiting body.     

Bacterial complexes of the fruiting bodies and hyphosphere of certain basidiomycetes

The surface and internal tissues of the fruiting bodies of basidiomycetes were shown to be specific bacterial habitats characterized by varying diversity and structure of bacterial complexes. On the surface of fruiting bodies, gram-negative bacteria of the genera Pseudomonas, Xanthomonas, and Myxococcus prevailed, while in the internal tissues gram-positive bacteria of the genera Streptomyces, Bacillus, Arthrobacter, and Micrococcus were identified in addition. Bacterial complexes from the surface and inner tissues of the fruiting bodies of the studied basidiomycetes showed significant similarity to each other and differed from those from the hyphosphere and the reference soil. On the surface and in the internal tissues of the fruiting bodies, representatives of the genus Myxococcus were identified for the first time, which could indicate initial decay of the fruiting body.     

First description of two moderately halophilic and psychrotolerant Mycoplasma species isolated from cephalopods and proposal of Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov

Two moderately halophilic and psychrotolerant new Mycoplasma species were isolated from common cephalopods. Three strains were isolated in pure culture from two individual European flying squid (Todarodes sagittatus), and two individual octopuses (Octopus vulgaris). The strains showed optimal growth at 25 °C and a salinity of 3% (w/v) NaCl. Molecular analyses revealed that the isolates belonged to two new, but phylogenetically related species, divergent from all previously described Mollicutes, representing the first marine isolates of the class, and also the first Mycoplasma strains for which NaCl requirement has been demonstrated. A genome search against all available marine metagenomes and 16S rRNA gene databases indicated that these two species represent a novel non-free-living marine lineage of Mollicutes, specifically associated with marine animals. Morphology and physiology were compatible with other members of this group, and genomic and phenotypic analyses demonstrated that these organisms represent two novel species of the genus Mycoplasma, for which the names Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov. are proposed; the type strains are PE^T (DSM 105487^T, CIP 111404^T) and 5H^T (DSM 105,488^T, CIP 111405^T), respectively.     

Characteristics of myxobacteria isolated from the surface of freshwater fish

A study was made of 32 nonpathogenic myxobacterial isolates obtained from a variety of fish taken in the Pacific Northwest. Morphological, cultural, biochemical, and serological studies were carried out on these strains. All were found to be members of the genus Cytophaga. Two myxobacterial strains pathogenic to fish were also included in this study for comparative purposes. These pathogenic organisms were found to be culturally and physiologically similar to some of the nonpathogenic strains. Antiserum against the pathogenic species, however, showed no cross agglutination when tested against the other myxobacterial isolates. As a result, serological procedures appear promising as a rapid means for distinguishing pathogenic myxobacteria from one another and from saprophytic myxobacteria commonly found on fish.     

Similarity of Tetracycline Resistance Genes Isolated from Fish Farm Bacteria to Those from Clinical Isolates

Tetracycline-resistant (Tet^r) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tet^r gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tet^r genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tet^r strains transferred Tet^r genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tet^r strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.     

Description of five novel thermophilic species of the genus Thermus: Thermus hydrothermalis sp. nov., Thermus neutrinimicus sp. nov., Thermus thalpophilus sp. nov., Thermus albus sp. nov., and Thermus altitudinis sp. nov., isolated from hot spring sediments

Biological denitrification is a significant process in nitrogen biogeochemical cycle of terrestrial geothermal environments, and Thermus species have been shown to be crucial heterotrophic denitrifier in hydrothermal system. Five Gram-stain negative, aerobic and rod-shaped thermophilic bacterial strains were isolated from hot spring sediments in Tibet, China. Phylogenetic analysis based on 16S rRNA gene and whole genome sequences indicated that these isolates should be assigned to the genus Thermus and were most closely related to Thermus caldifontis YIM 73026^T, and Thermus brockianus YS38^T. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the five strains and the type strains of the genus Thermus were lower than the threshold values (95% and 70%, respectively) recommended for bacterial species, which clearly distinguished the five isolates from other species of the genus Thermus and indicated that they represent independent species. Colonies are circular, convex, non-transparent. Cell growth occurred at 37–80 °C (optimum, 60–65 °C), pH 6.0–8.0 (optimum, pH 7.0) and with 0–2.0% (w/v) NaCl (optimum, 0–0.5%). Denitrification genes (narG, nirK, nirS, and norB genes) detected in their genomes indicated their potential function in nitrogen metabolism. The obtained results combined with those of morphological, physiological, and chemotaxonomic characteristics, including the menaquinones, polar lipids, and cellular fatty acids showed that the isolates are proposed as representing five novel species of the genus Thermus, which are proposed as Thermus hydrothermalis sp. nov. SYSU G00291^T, Thermus neutrinimicus sp. nov. SYSU G00388^T, Thermus thalpophilus sp. nov. SYSU G00506^T, Thermus albus sp. nov. SYSU G00608^T, Thermus altitudinis sp. nov. SYSU G00630^T.     

Distribution of Genes for Synthesis of Trehalose and Mannosylglycerate in Thermus spp. and Direct Correlation of These Genes with Halotolerance

In this study we correlate the presence of genes leading to the synthesis of trehalose and mannosylglycerate (MG) in 17 strains of the genus Thermus with the ability of the strains to grow and accumulate these compatible solutes in a defined medium containing NaCl. The two sets of genes, namely, otsA/otsB for the synthesis of trehalose and mpgS/mpgP for the synthesis of MG, were necessary for the growth of Thermus thermophilus in a defined medium containing up to 6% NaCl. Strains lacking a complete otsA gene did not grow in defined medium containing >2% NaCl. One strain of T. thermophilus lacking the genes for the synthesis of MG did not grow in a medium with >1% NaCl. We did not identify any of these genes in the type strains of the other seven species of Thermus, and none of those strains grew in defined medium with 1% NaCl. The results strongly indicate that the combined accumulation of trehalose and MG is required for optimal osmotic adjustment.     

Investigations on ultraviolet light and nitrous acid induced mutations of halotolerant bacterial strains for the treatment of tannery soak liquor

The objective of this research work is to study the effect of physical and chemical mutagenesis on biological treatment of tannery saline wastewater (soak liquor) employing halotolerant bacterial strains. Four halotolerant bacterial strains isolated from saline sources were used. The strains were identified as Pseudomonas aeruginosa, Bacillus flexus, Exiguobacterium homiense and Staphylococcus aureus, respectively. The isolates were found to grow well in medium containing 0–10% NaCl. At high saline concentration (>5%), the identified strains and their mixed consortia showed a low degrading efficiency of soak liquor (35–45%). UV light and nitrous acid mutagenesis were performed over the strains and the mutated strains were employed for degradation of soak liquor at high salinity level (6% by wt). Comparison of Chemical Oxygen Demand (COD) removal rates for both pure mutant isolates and mixed mutated consortia showed that nitrous acid mutagenesis resulted in degradation of 71% COD removal. Ultraviolet (UV) mutagenesis has no effect on degradation effectiveness. Biomass sludge (Mixed Liquor Volatile Suspended Solids) growth was also found to be high in nitrous acid treated strains.     

Sedimenticola hydrogenitrophicus sp. nov. a chemolithoautotrophic bacterium isolated from a terrestrial mud volcano,and proposal of Sedimenticolaceae fam. nov. in the order Chromatiales

Chemolithoautotrophic microorganisms can play a significant role in the biogeochemical cycling of elements in deep-subsurface-associated environments. A novel facultatively anaerobic lithoautotrophic bacteria (strains SB48^T and SN1189) were isolated from terrestrial mud volcanoes (Krasnodar Krai, Russia). Cells of the strains were straight motile rods. Growth was observed at temperatures up to 35 °C (optimum at 30 °C), pH 6.0–8.5 (optimum at pH 7.5) and NaCl concentrations of 0.5–4.0% (w/v) (optimum at 1.5–2.0% (w/v)). The isolates grew chemolithoautotrophically with molecular hydrogen or thiosulfate as an electron donor, nitrate as an electron acceptor and CO₂/HCO₃⁻ as a carbon source. They also grew with organic acids, ethanol, yeast extract and peptone. The isolates were capable of either anaerobic respiration with nitrate or nitrous oxide as the electron acceptors or aerobic respiration under microaerobic condition. The total size of the genome of strains SB48^T and SN1189 was 4.71 and 5.13 Mbp, respectively. Based on phenotypic and phylogenetic characteristics, strains SB48^T and SN1189 represent a novel species of the genus Sedimenticola, S. hydrogenitrophicus (the type strain is SB48^T = KCTC 25568 ^T = VKM B-3680 ^T). The new isolates are the first representatives of the genus Sedimenticola isolated from a terrestrial ecosystem. Based on phylogenomic reconstruction we propose to include the genus Sedimenticola and the related genera into a new family Sedimenticolaceae fam. nov. within the order Chromatiales.     

Stability of Bacterial Mutants in Saline

By storage in 1% NaCl, genetically characterized strains of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa were stable for more than 1 year at 4 C. This method of preservation was more effective than maintenance of the strains in stab culture.