Microbial production host selection for converting second-generation feedstocks into bioproducts

Although many secondary metabolites with diverse biological activities have been isolated from myxobacteria, most strains of these biotechnologically important gliding prokaryotes remain difficult to handle genetically. In this study we describe the new fast growing myxobacterial thermophilic isolate GT-2 as a heterologous host for the expression of natural product biosynthetic pathways isolated from other myxobacteria. According to the results of sequence analysis of the 16S rDNA, this moderately thermophilic isolate is closely related to Corallococcus macrosporus and was therefore named C. macrosporus GT-2. Fast growth of moderately thermophilic strains results in shorter fermentation and generation times, aspects which are of significant interest for molecular biological work as well as production of secondary metabolites. Development of a genetic manipulation system allowed the introduction of the complete myxochromide biosynthetic gene cluster, located on a transposable fragment, into the chromosome of GT-2. Genetic engineering of the biosynthetic gene cluster by promoter exchange leads to much higher production of myxochromides in the heterologous host C. macrosporus GT-2 in comparison to the original producer Stigmatella aurantiaca and to the previously described heterologous host Pseudomonas putida (600 mg/L versus 8 mg/L and 40 mg/L, respectively).     

新疆阿克苏地区盐碱地粘细菌分离鉴定

【目的】采用传统的纯培养技术,分离新疆阿克苏地区典型的盐碱地中的粘细菌,并初步分析盐碱地土壤中可培养粘细菌资源的多样性。【方法】采用传统的水琼脂法、滤纸法和改良的土壤浸出液法分离新疆阿克苏地区25份盐碱地的粘细菌。结合分析土样的酸碱度、含盐量、地理位置及其植被分布情况分析新疆阿克苏地区盐碱地粘细菌资源多样性。【结果】共分离到58株粘细菌,它们被鉴定为:粘球菌属(Myxococcus)33株;珊瑚球菌属(Corallococcus)14株;孢囊杆菌属(Cystobacter)6株;堆囊菌属(Sorangium)2株;侏囊菌属(Nannocystis)2株;多囊菌属(Polyangium)1株。其中粘球菌抗逆性强,分离的菌株数最多,在pH值7.5-8.5范围的盐碱地中普遍存在;其次为珊瑚球菌属;而侏囊菌属、多囊菌属的菌株较少见。【结论】新疆阿克苏地区盐碱地粘细菌多样性不高,可能受分离纯化方法、含盐量以及土壤性质影响较大。     

Myxobacteria isolated in Israel as potential source of new anti-infectives

AIMS: To evaluate the patterns of the production of antimicrobial compounds by Israeli myxobacteria newly isolated from soil samples and barks by a battery of isolation and purification methods. METHODS AND RESULTS: A total of 100 myxobacteria belonging to five of the 12 described genera, were isolated from 48 soil and 45 tree bark samples collected in different areas inside the State of Israel. Four isolation methods based on the peculiar metabolic and cell cycle aspects of myxobacteria, were combined with purification procedures and optimization of cultivation conditions. Ninety-seven strains were fermented and screened for antimicrobial activities. Production of antimicrobial activities was detected in 62 isolates. More than 50% of the collection (54 strains) was able to inhibit Escherichia coli growth. CONCLUSIONS: The results of this study support the idea that myxobacterial strains can be isolated from particular habitats and then cultivated and screened for their capacity to produce secondary metabolites endowed with antibacterial and antifungal activities. Myxovirescin, a typical poliketide myxobacterial antibiotic, has been identified in one Israeli isolate. Althiomycin, a thiazolyl peptide, which inhibits prokaryotic protein synthesis, usually produced by actinomycetes, was detected in three strains selected in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm that myxobacteria are prolific producers of a variety of bioactive secondary metabolites including antibacterial and antifungal compounds, being their high frequency of anti-Gram-negative activities particularly appealing for the current anti-infective research. So far their screening has often been hampered because their isolation is time-consuming and are quite difficult to handle and cultivate. In this paper we demonstrate that a proper combination of isolation, purification and cultivation methods allow their pharmaceutical exploitation.     

A novel regulation on developmental gene expression of fruiting body formation in Myxobacteria

Myxobacteria are Gram-negative soil microorganisms that prey on other microorganisms. Myxobacteria have significant potential for applications in biotechnology because of their extraordinary ability to produce natural products such as secondary metabolites. Myxobacteria also stand out as model organisms for the study of cell–cell interactions and multicellular development during their complex life cycle. Cellular morphogenesis during multicellular development in myxobacteria is very similar to that in the eukaryotic soil amoebae. Recent studies have started uncovering molecular mechanisms directing the myxobacterial life cycle. We describe recent studies on signal transduction and gene expression during multicellular development in the myxobacterium Myxococcus xanthus. We provide our current model for signal transduction pathways mediated by a two-component His–Asp phosphorelay system and a Ser/Thr kinase cascade.     

Non-modular polyketide synthases in myxobacteria

Myxobacteria are prolific producers of a wide variety of secondary metabolites. The vast majority of these compounds are complex polyketides which are biosynthesised by multimodular polyketide synthases (PKSs). In contrast, few myxobacterial metabolites isolated to date are derived from non-modular PKSs, in particular type III PKSs. This review reports our progress on the characterisation of type III PKSs in myxobacteria. We also summarize current knowledge on bacterial type III PKSs, with a special focus on the evolutionary relationship between plant and bacterial enzymes. The biosynthesis of a quinoline alkaloid in Stigmatella aurantiaca by a non-modular PKS is also discussed.     

Characterization and description of Anaeromyxobacter dehalogenans gen. nov., sp. nov., an aryl-halorespiring facultative anaerobic myxobacterium

Five strains were isolated which form a physiologically and phylogenetically coherent group of chlororespiring microorganisms and represent the first taxon in the Myxobacteria capable of anaerobic growth. The strains were enriched and isolated from various soils and sediments based on their ability to grow using acetate as an electron donor and 2-chlorophenol (2-CPh) as an electron acceptor. They are slender gram-negative rods with a bright red pigmentation that exhibit gliding motility and form spore-like structures. These unique chlororespiring myxobacteria also grow with 2,6-dichlorophenol, 2,5-dichlorophenol, 2-bromophenol, nitrate, fumarate, and oxygen as terminal electron acceptors, with optimal growth occurring at low concentrations (<1 mM) of electron acceptor. 2-CPh is reduced by all strains as an electron acceptor in preference to nitrate, which is reduced to ammonium. Acetate, H(2), succinate, pyruvate, formate, and lactate were used as electron donors. None of the strains grew by fermentation. The 16S ribosomal DNA (rDNA) sequences of the five strains form a coherent cluster deeply branching within the family Myxococcaceae within the class Myxobacteria and are mostly closely associated with the Myxococcus subgroup. With the exception of anaerobic growth and lack of a characteristic fruiting body, these strains closely resemble previously characterized myxobacteria and therefore should be considered part of the Myxococcus subgroup. The anaerobic growth and 9.0% difference in 16S rDNA sequence from those of other myxobacterial genera are sufficient to place these strains in a new genus and species designated Anaeromyxobacter dehalogenans. The type strain is 2CP-1 (ATCC BAA-258).     

粘细菌基因组学研究进展

粘细菌(Myxobacteria)隶属于δ变形菌纲(Deltaproteobacteria)的粘球菌目(Myxococcales),是一类革兰氏阴性杆状细菌。它是继放线菌和真菌之后又一重要的活性次级代谢产物产生菌,尽管如此,由于分离纯化困难,粘细菌的研究进展一直较为缓慢。随着测序技术的进步和生物信息学的应用,大量粘细菌基因组被完成测序和报道。本文对粘细菌研究意义及该类资源开发价值、分离培养存在的困难进行了阐述,对粘细菌基因组注释及目前已测菌株的全基因组进行了归纳总结,同时介绍了基因组学在粘细菌生态、捕食机制、子实体形成以及次级代谢产物合成方面的研究进展。本文有助于了解基因组学在粘细菌研究中的重要价值,为联合应用多组学技术深入研究粘细菌代谢机制和社会性行为提供了参考,对粘细菌基础研究、资源发掘和开发利用具有重要意义。     

A brief tour of myxobacterial secondary metabolism

Myxobacteria are soil-dwelling, Gram-negative bacteria which are notable not only for their multi-cellular ‘social’ lifestyles, but for production of structurally diverse secondary metabolites with potential in clinical therapy. Here we briefly review the history of myxobacterial natural products research, provide an overview of their unique secondary metabolism, with an emphasis on assembly line biosynthesis of polyketide and non-ribosomal peptide metabolites, and look to the future of the field.     

Analysis of 16S rRNA and methane monooxygenase gene sequences reveals a novel group of thermotolerant and thermophilic methanotrophs, Methylocaldum gen. nov.

Two methanotrophic bacteria with optimum growth temperatures above 40° C were isolated. Thermotolerant strain LK6 was isolated from agricultural soil, and the moderately thermophilic strain OR2 was isolated from the effluent of an underground hot spring. When compared to the described thermophilic methanotrophs Methylococcus capsulatus and Methylococcus thermophilus, these strains are phenotypically similar to Methylococcus thermophilus. However, their 16S rRNA gene sequences are markedly different from the sequence of Methylococcus thermophilus (∼ 8% divergence) and, together with Methylomonas gracilis, they form a distinct, new genus within the γ-subgroup of the Proteobacteria related to extant Type I methanotrophs. Further phenotypic characterisation showed that the isolates possess particulate methane monooxygenase (pMMO) but do not contain soluble methane monooxygenase. The nucleotide sequence of a gene encoding pMMO (pmoA) was determined for both isolates and for Methylomonas gracilis. PmoA sequence comparisons confirmed the monophyletic nature of this newly recognised group of thermophilic methanotrophs and their relationship to previously described Type I methanotrophs. We propose that strains OR2 and LK6, together with the misclassified thermophilic strains Methylomonas gracilis VKM-14L^T and Methylococcus thermophilus IMV-B3122, comprise a new genus of thermophilic methanotrophs, Methylocaldum gen. nov., containing three new species: Methylocaldum szegediense, Methylocaldum tepidum and Methylocaldum gracile. Received: 2 April 1997 / Accepted: 23 July 1997     

Discovery of the autonomously replicating plasmid pMF1 from Myxococcus fulvus and development of a gene cloning system in Myxococcus xanthus

Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G+C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies ( approximately 10(5) CFU/microg DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.     

Cultural and phylogenetic analysis of mixed microbial populations found in natural and commercial bioleaching environments.

A range of autotrophic and heterotrophic enrichment cultures were established to determine the cultural bacterial diversity present in samples obtained from the acidic runoff of a chalcocite overburden heap and from laboratory-scale (1- to 4-liter) batch and continuous bioreactors which were being used for the commercial assessment of the bioleachability of zinc sulfide ore concentrates. Strains identified as Thiobacillus ferrooxidans, Thiobacillus thiooxidans, "Leptospirillum ferrooxidans," and Acidiphilium cryptum were isolated from both the natural site and the batch bioreactor, but only "L. ferrooxidans," a moderately thermophilic strain of T. thiooxidans, and a moderately thermophilic iron-oxidizing bacterium could be recovered from the continuous bioreactor running under steady-state conditions. Sequence analysis of the 16S rRNA genes of 33 representative strains revealed that all of the strains were closely related to strains which have been sequenced previously and also confirmed the phylogenetic diversity of bacteria present in bioleaching environments.     

Improved methods of isolation and purification of myxobacteria and development of fruiting body formation of two strains

By using baiting techniques and different purification methods, a high number of myxobacterial strains have been isolated as pure cultures from soil of different regions of China. Because myxobacterial cells do not disperse easily in liquid media, a medium containing an enzymatic hydrolysate of casein (CEH) medium have been used for purification and purity tests combined in a single step. The key method, in which isolates are reintroduced to sterile rabbit dung to induce fruiting bodies formation, facilitates purification of myxobacteria. Sterile rabbit dung pellets are used to mimic the natural growth substance of these organisms which has the advantage that characteristic fruiting bodies emerge, which is a key characteristics in the taxonomy of myxobacteria. In this study, the optimum program of isolation and purification of some myxobacteria strains has been established which will facilitate screening programs. Moreover, the development of fruiting body formation of strain BD20 (Chondromyces) and strain BD54 (Cystobacter) have been recorded in this study.     

Leben und Überleben im Boden

Myxobacteria - survivalists in soil Myxobacteria like Myxococccus xanthus are soil-living microorganisms featuring a complex lifestyle, including movement by coordinated swarming on surfaces, predatory feeding on other microorganisms, and the formation of multicellular fruiting bodies when unfavorable environmental conditions are encountered. Bioinformatic analysis of the large myxobacterial genomes has enabled fascinating insights into the molecular basis for the biosynthesis of complex secondary metabolite structures by myxobacteria, and has set the stage for the discovery of novel natural products. Moreover, well-characterized myxobacteria like M. xanthus increasingly play a role as “biochemical factories” for the biotechnological production of bioactive molecules using synthetic biology approaches.     

粘细菌资源挖掘与多相分类研究进展

粘细菌(Myxobacteria)是一类具有复杂多细胞行为、能够广泛捕食各类细菌和真菌并能产生丰富次级代谢产物的药源微生物,具有重要研究和应用价值。然而,由于资源挖掘困难,多相分类研究进展缓慢,严重阻碍了粘细菌的开发利用。本文对粘细菌的特性、分离和纯化方法、目前存在的不足及改进措施进行阐述,并对粘细菌多相分类研究进展及存在的问题进行解析。另外,结合当前技术手段和发展现状,进一步对粘细菌的未来发展趋势进行展望,以期为相关研究提供参考。     

粘细菌次生代谢产物中大环类化合物的研究进展

粘细菌可以产生多种次生代谢产物,是继放线菌、芽胞杆菌之后的第3大类次级代谢产物产生菌。本文综述了粘细菌次生代谢产物中大环类化合物的研究进展。首先介绍了粘细菌中分离的主要大环类化合物种类、结构特征及来源菌株;然后分别从抗肿瘤、抗菌、抗病毒等几方面对其生物活性进行概述;最后对粘细菌次生代谢产物中大环类化合物的相关研究工作进行了展望。     

Evolutionary diversity of ketoacyl synthases in cellulolytic myxobacterium Sorangium

The diversity of type I polyketide synthases (PKSs) in cellulolytic myxobacterium Sorangium was explored by assaying the ketoacyl synthases (KSs) in 10 Sorangium strains with two degenerate primer sets and 64 different KS fragments were obtained. For their deduced amino acid sequences, eight were identical to three known KSs from Sorangium and Magnetospirillum, while the others showed 54-83% identities to the modular KS domains reported from various microorganisms. Parts of the Sorangium KSs tightly share the clade with Actinobacteria excluding any other analyzed myxobacterial KSs, or with Cyanobacteria /Myxobacteria. Parts are widely located in the three functional groups - "Loading", "NRPS/PKS" and "Trans-AT". Sorangium KSs in the Actinobacteria, Cyanobacteria/Myxobacteria, or "Loading" clade further evolved independently on its own genus. Notably, the modular KSs from other Myxobacteria genera, i.e. Myxococcus, Stigmatella, Melittangium, Cystobacter and Angiococcus are often distributed crosswise and form non-Sorangium blend subgroups. "NRPS/PKS" and "Trans-AT" are two rather diverse groups and the Sorangium KSs in these clades evolved crosswise with other taxa lineages. The results presented in this paper suggest that the inherent genetic strategies, together with frequent gene importing from many organisms (HGT) have contributed to the evolution of modular PKSs in Sorangium. These findings reinforce that Sorangium strains are really excellent creators for novel and diverse polyketides.     

Characterization of Novel Chitin-degrading laceyella spp. Strains from New Athos Cave (Abkhazia) Producing Thermostable Chitinases

Microbiology - Nine chitin-degrading isolates of moderately thermophilic bacteria with temperature optimum for growth and chitin destruction at 50–55°C were isolated from soils of the...     

两株采自惠州的细鞘丝藻亚科(Leptolyngbyaceae)嗜热蓝细菌的分离鉴定及细胞组分分析

【背景】随着CO_2排放增加,全球变暖愈发严峻,嗜热蓝细菌作为能够在45°C及以上环境中生长并实现生物固碳的微生物,具有重要的研究意义。【目的】对从广东惠州地区采集的藻种进行分离鉴定,并筛选出2株嗜热蓝细菌,研究其生长特性,为嗜热蓝细菌的后续应用提供依据。【方法】通过16SrRNA基因、藻蓝蛋白链A基因(PhycoA)序列分析确定从惠州地区采集到的菌株的分类学位置。对PKUAC-GDTS1-24和PKUAC-GDTS1-29两株嗜热蓝细菌进行形态观察和主要细胞成分(灰分、糖类、脂质、蛋白质和色素)分析。【结果】共分离出12株嗜热蓝细菌,其中PKUAC-GDTS1-24和PKUAC-GDTS1-29菌株,形态上呈蓝绿色球形毛状体,是由细胞形成密集的簇,彼此附着形成的。两株嗜热蓝细菌的主要细胞成分是糖类,分别占细胞干重的36.42%和28.46%。PKUAC-GDTS1-24的灰分、脂质和蛋白质含量分别为24.41%、21.40%和26.64%。PKUAC-GDTS1-29的细胞中,灰分、脂质和蛋白质含量分别为24.72%、23.92%和12.93%。藻蓝蛋白(Phycocyanin,PC)在PKUAC-GDTS1-24和PKUAC-GDTS1-29中的含量分别为157.29 mg/g DW和374.86 mg/g DW,类胡萝卜素分别为65.13 mg/g DW和18.87 mg/g DW。【结论】基于系统发育树研究,本实验的2个分离株属于细鞘丝藻亚科(Leptolyngbyaceae),与研究较少的纤发鞘丝蓝细菌属(Leptolyngbya)菌株相近,可能是广东和四川温泉中存在的一种新型丝状轻度嗜热蓝细菌属或Leptolyngbya新种。嗜热菌株PKUAC-GDTS1-24和PKUAC-GDTS1-29的形态和细胞组成相似,通过比较,2个菌株的藻胆蛋白含量远远高于其他研究报道的Leptolyngbya蓝细菌,尤其是PKUAC-GDTS1-29可以作为藻蓝蛋白生产的潜在菌株。     

Characterization and radiation resistance of new isolates of Rubrobacter radiotolerans and Rubrobacter xylanophilus

In this study we characterized new strains of the slightly thermophilic species Rubrobacter radiotolerans and the thermophilic species Rubrobacter xylanophilus, both of which were previously represented only by the type strains isolated, respectively, from Japan and the United Kingdom. The new isolates were recovered from two hot springs in central Portugal after gamma irradiation of water and biofilm samples. We assessed biochemical characteristics, performed DNA–DNA hybridization, and carried out 16S rDNA sequence analysis to demonstrate that the new Rubrobacter isolates belong to the species R. radiotolerans and R. xylanophilus. We also show for the first time that the strains of R. xylanophilus and other strains of R. radiotolerans are extremely gamma radiation resistant. Received: October 16, 1998 / Accepted: April 25, 1999     

致病疫霉拮抗菌株B25-I-3的鉴定及其次级代谢产物

【背景】粘细菌是一类具有社会性行为的高等原核生物,能产生丰富、多样、新颖的具有生物活性的次级代谢产物,具有很大的研究开发价值。【目的】从土壤样品中筛选出对致病疫霉(Phytophthora infestans)具有拮抗作用的粘细菌,并对其次级代谢产物进行研究。【方法】对分离到的一株拮抗P. infestans菌株,结合形态观察和16S rRNA基因序列分析确定其分类地位,并通过单因素分析与正交优化相结合的方法对菌株的发酵参数进行研究。采用滤纸片法对其浓缩发酵液中活性物质的稳定性及抗菌活性进行检测,并通过薄层色谱法(thin layer chromatography,TLC)与高效液相色谱法(high performance liquid chromatography,HPLC)等初步分离后,将具有抗P. infestans活性的组分利用液相色谱-质谱联用技术(HPLC-MS)进行检测,最后通过离体叶片法测定活性物质对马铃薯晚疫病的防病作用。【结果】从土壤样品中分离得到的菌株B25-I-3对P. infestans表现出较强的拮抗活性,经鉴定为橙色粘球菌(Myxococcus fulvus)。其拮抗P. infestans的活性物质主要存在于胞外,产活性物质的最优发酵条件为：摇床转速180 r/min,接种量10%,培养温度30 °C,发酵周期7 d。该菌株产生的活性物质耐受蛋白酶K以及紫外线与自然光照射,对温度耐受性极强,而且易于在低温下保存,对枯草芽孢杆菌(Bacillus subtilis)、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、酿酒酵母(Saccharomyces cerevisiae)、立枯丝核菌(Rhizoctonia solani)、尖孢镰刀菌(Fusarium oxysporum)、向日葵核盘菌(Sclerotinia sclerotiorum)均有不同程度的抑制作用。其对P. infestans的拮抗组分中含有N-(3-氨基-2-羟丙基)-N-甲基硫酸二酰胺[N-(3-amino-2-hydroxypropyl)-N-methylsulfuric diamide]与甲基(2R)-2-叠氮基-3-羟基-2-甲基丙酸酯[methyl(2R)-2-azido-3-hydroxyl-2-methylpropanoate],该活性物质能明显抑制P. infestans侵染马铃薯叶片且对不同品种的叶片均无损伤作用。【结论】研究为M. fulvus B25-I-3活性物质的分离鉴定及抗马铃薯晚疫病生物农药的研发提供了基础数据。