Genetic technologies for extremely thermophilic microorganisms of Sulfolobus,the only genetically tractable genus of crenarchaea

Archaea represents the third domain of life, with the information-processing machineries more closely resembling those of eukaryotes than the machineries of the bacterial counterparts but sharing metabolic pathways with organisms of Bacteria, the sister prokaryotic phylum. Archaeal organisms also possess unique features as revealed by genomics and genome comparisons and by biochemical characterization of prominent enzymes. Nevertheless, diverse genetic tools are required for in vivo experiments to verify these interesting discoveries. Considerable efforts have been devoted to the development of genetic tools for archaea ever since their discovery, and great progress has been made in the creation of archaeal genetic tools in the past decade. Versatile genetic toolboxes are now available for several archaeal models, among which Sulfolobus microorganisms are the only genus representing Crenarchaeota because all the remaining genera are from Euryarchaeota. Nevertheless, genetic tools developed for Sulfolobus are probably the most versatile among all archaeal models, and these include viral and plasmid shuttle vectors, conventional and novel genetic manipulation methods, CRISPR-based gene deletion and mutagenesis, and gene silencing, among which CRISPR tools have been reported only for Sulfolobus thus far. In this review, we summarize recent developments in all these useful genetic tools and discuss their possible application to research into archaeal biology by means of Sulfolobus models.     

Spontaneous mutation in a thermoacidophilic archaeon: evaluation of genetic and physiological factors

We used direct selection of pyrE and pyrF mutants to estimate the rates of spontaneous mutation in Sulfolobus acidocaldarius as a function of genetic background and culture conditions. Fluctuation tests were applied to several genetically marked strains, including one isolated as a putative mutator strain, and to cultures grown over a wide range of temperature and other physiological conditions. The results suggested some impact of auxotrophic markers on the apparent rate of mutation, but no obvious pattern of effect of growth conditions, including those that gave evidence of being physiologically stressful. Received: 27 May 1997 / Accepted: 2 September 1997     

冰岛硫化叶菌遗传操作体系的研究进展

冰岛硫化叶菌是古菌研究中常用的模式菌株,为人们研究古菌复制、细胞周期以及CRISPR-Cas系统等作出了巨大贡献。冰岛硫化叶菌遗传操作体系的建立与完善对古菌学的全面深入研究起至关重要的作用。本文介绍了冰岛硫化叶菌遗传操作体系所使用的质粒载体、筛选标记和转化方法,论述了目前广泛使用的两类冰岛硫化叶菌基因敲除体系。最后提出了现有冰岛硫化叶菌基因操作体系存在的主要问题,并对其发展方向进行了展望。     

Genetic tools for <Emphasis Type="Italic">Sulfolobus</Emphasis> spp.: vectors and first applications

Sulfolobus species belong to the best-studied archaeal organisms but have lacked powerful genetic methods. Recently, there has been considerable progress in the field of Sulfolobus genetics. Urgently needed basic genetic tools, such as targeted gene knockout techniques and shuttle vectors are being developed at an increasing pace. For S. solfataricus knockout systems as well as different shuttle vectors are available. For the genetically more stable S. acidocaldarius shuttle vectors have been recently developed. In this review we summarize the currently available genetic tools and methods for the genus Sulfolobus. Different transformation protocols are discussed, as well as all so far developed knockout systems and Sulfolobus-Escherichia coli shuttle vectors are summarized. Special emphasis is put on the important vector components, i.e., selectable markers and Sulfolobus replicons. Additionally, the information gathered on different Sulfolobus strains with respect to their use as recipient strains is reviewed. The advantages and disadvantages of the different systems are discussed and aims for further improvement of genetic systems are identified.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

Importance of biochemical marker genes in inbred strains of mice. Some problems on checking genetic purity of inbred strains (author's transl)

In order to check genetic purity of inbred strains of mice, we surveyed five biochemical variants in certain 69 strains, some of which had established from Japanese fancy mice. The loci examined were Hemoglobin beta-chain, Malic enzyme (supernatant form), Isocitrate dehydrogenase (supernatant form), Serum esterase-1, and Serum esterase-2. These loci were all in homozygous states as far as examined, however some subline divergences were found in a few strains, that is to say, sublines were found to be fixed with different alleles as some loci, Important of biochemical marker genes to check genetic purity of strains and several derived problems are mentioned.     

Selectable mutant phenotypes of the extremely thermophilic archaebacterium Sulfolobus acidocaldarius.

As a first step toward developing the genetic potential of extremely thermophilic archaebacteria, mutant strains of Sulfolobus acidocaldarius were selected by plating cells directly on solid medium containing one of several growth inhibitors. Three spontaneous resistance phenotypes were observed (5-fluorouracil resistance, novobiocin resistance, and L-ethionine resistance), each at a different average frequency. Characterization of representative strains showed each of the three mutant phenotypes to provide a potentially useful genetic marker.     

埃莎霉素Ⅰ组分高含量、高产量基因工程菌WSJ-IA及其原株的鉴定

【目的】利用调节基因acyB2激活异戊酰基转移酶(ist)基因表达的特点,将ist与调节基因acyB2在异戊酰螺旋霉素(埃莎霉素)Ⅰ产生菌菌株中共表达,获得埃莎霉素Ⅰ单组分的高含量及高产量菌株WSJ-IA。对其及原始螺旋霉素产生菌菌株Streptomyces spiramyceticus F21进行了初步鉴定。【方法】从形态学、培养和生理生化特征、细胞壁化学组成、16S rRNA基因序列、5个看家基因(atpD、gyrB、rpoB、recA和trpB)蛋白分析和系统发育树构建等方面对该菌株及其原株进行了鉴定。【结果】两株菌在形态培养特征、生理生化特征、细胞壁化学组成、16S rRNA基因序列和5个看家基因蛋白水平基本一致,在系统发育树分析中同处在一个分支中。而在16S rRNA基因序列和5个看家基因蛋白水平在系统发育上它们均与已知相近菌株处于不同的分支上,并且与不同基因的相近菌株各有不同,其中无一报道产生螺旋霉素。【结论】Streptomyces spira-myceticus F21可能是一个产生螺旋霉素的链霉菌新种,16S rRNA基因序列和5个看家基因蛋白序列分析可以作为埃莎霉素Ⅰ基因工程菌生产过程中进行鉴别的分子标志。     

Role of RNA polymerase sigma-factor (RpoS) in induction of glutamate-dependent acid-resistance of Escherichia albertii under anaerobic conditions

Escherichia albertii is a potential enteric food-borne pathogen with poorly defined genetic and biochemical properties. Acid resistance is perceived to be an important property of enteric pathogens, enabling them to survive passage through stomach acidity so that they may colonize the mammalian gastrointestinal tract. We analyzed glutamate-dependent acid-resistance pathway (GDAR) in five E. albertii strains that have been identified so far. We observed that the strains were unable to induce GDAR under aerobic growth conditions. Mobilization of the rpoS gene restored aerobic induction of this acid-resistance pathway, indicating that all five strains may have a dysfunctional sigma-factor. On the other hand, under anaerobic growth conditions where GDAR is induced in an RpoS-independent manner (i.e. in Shigella spp. and Escherichia coli O157:H7 strains), only three out of five E. albertii strains successfully induced GDAR. The remainder of the two strains exhibited dependence on functional RpoS even under anaerobic conditions to express GDAR, a regulatory function previously considered to be redundant. The data indicate that certain E. albertii strains may have an alternate RpoS-dependent pathway for acid-resistance under anaerobic growth conditions.     

Potential enhancement of photosynthetic energy conversion in algal mass culture

Actual laboratory data obtained from steady-state Dunaliella tertiolecta cultures grown under a wide range of photon flux densities were used in a simple model to calculate daily production in a conventional algal mass culture system. In spite of large physiological and biochemical variations between low-light- (LL) and high light- (HL) adapted cultures, the overall calculated daily productivity is almost identical for both strains grown at optimal conditions. When production of fine biochemicals is considered, however, a hypothetical HL strain, which cannot shade adapt, is advantageous. Based on biochemical and biophysical analysis of D. tertiolecta responses to growth irradiance levels, specific targets are defined for genetic manipulation to enhance productivity in algal mass culture systems. The targets identified are (1) amplification of the carboxylation enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase relative to the electron transport complexes, which should increase photosynthetic capacity at light saturation, and (2) enlargement of the light-harvesting complexes by varying their pigment composition in order to increase light harvesting at low photon flux densities.     

稻瘟病拮抗菌株的分离、筛选及鉴定

从水稻病健叶、茎和根组织以及稻田土壤中共分离得到细菌菌株321株。经发酵法初筛,对稻瘟病菌丝生长有抑制作用的有57株,再通过平板对峙法复筛,具有强烈拮抗作用的菌株有5株,其抑菌距离达16mm以上。分别对5个菌株进行形态学观察、生理生化指标进行鉴定,结果有1株(No.156)为Bacillus subtilis,2株(No.171和No.177)为Bacillus pumillus,2株(No.192和No.279)为Bacillus ploymyxa。     

桑黄菌株活力评价及优良菌株筛选

通过对菌株生长活力、菌株抗氧化能力等多种生理生化指标的测定分析,对7个“桑黄”菌株进行了菌株活力评价,筛选出1株优良菌株SH1,该菌株的菌丝生长活力、发酵生物量、抗氧化能力均优于其他6个菌株。并且通过相关性分析,建立了一种有效评价桑黄生物量高产菌株的快速筛选方法。     

Ubiquity of plasmids in coding for toluene and xylene metabolism in soil bacteria: evidence for the existence of new TOL plasmids.

Thirteen bacteria have been isolated from nine different soil samples by selective enrichment culture on m-toluate (m-methylbenzoate) minimal medium. Eight of these were classified as Pseudomonas putida, one as a fluorescent Pseudomonas sp., and four as nonfluorescent Pseudomonas sp. All 13 strains appeared to carry TOL plasmids superficially similar to that previously described in P. putida mt-2 in that: (i) all the wild-type strains could utilize toluene, m-xylene, and p-xylene as sole carbon and energy sources, (ii) these growth substrates were metabolized through the corresponding alcohols and aldehydes to benzoate, m-toluate, and p-toluate, respectively, and thence by the divergent meta (or alpha-ketoacid) pathway, and (iii) the isolates could simultaneously and spontaneously lose their ability to utilize the hydrocarbons, alcohols, aldehydes, and acids, particularly during growth on benzoate, giving rise to cured strains which could grow only on benzaldehyde and benzoate of the aromatic substrates by the alternative ortho (or beta-ketoadipate) pathway. Eight of the isolates were able to transfer their TOL plasmids into their own cured strains, but only five were able to transfer them in interstrain conjugation into the cured strains, but only five were able to transfer them in interstrain conjugation into the cured derivative of P. putida mt-2. However, P. putida mt-2 was able to transfer its TOL plasmid into 11 of the cured isolates, and eight of these were able to retransmit this foreign plasmid in intrastrain conjugation with their own cured derivatives. Three of the isolates, MT 14, MT 15, and MT 20, differed significantly from the others in that the wild-type strains dissimilated the p-methyl-substituted substrates poorly, and also, during growth on benzoate, in addition to the cured derivatives, they gave rise to derivatives with a phenotype intermediate between the cured and wild-type strains, the biochemical and genetic nature of which has not been elucidated.     

A Note on the Limitations of Identifying Soft Rot Erwinias by Temperature Tolerances and Sensitivity to Erythromycin on a Pectate Medium

Forty-five erwinia strains from 14 different hosts have been tested lor growth on a crystal violet pectate medium with or without 70 μ/ml erythromycin at 27, 33.5 and 37°C. On the basis of positive data in the literature it was tried to identify the strains as Erwnia carotovora subsp. atroseptica, E. carotovora subsp. carotovora or E. chrysanthemi using this method. It appeared that relatively few strains reacted differentially, apparently due to strain variation (all strains were identified also by biochemical and physiological tests). The conclusion is that for a reliable identification of field isolates of soft rot crwinias other (biochemical, physiological and serological) methods have to be applied.     

工业微生物中NADH的代谢调控

NADH是微生物代谢网络中的一种关键辅因子。调节微生物胞内NADH的形式与浓度是定向改变和优化微生物细胞代谢功能, 实现代谢流最大化、快速化地导向目标代谢产物的重要手段之一。以下在详尽总结了NADH生理功能的基础上, 从生化工程(添加外源电子受体、不同氧化还原态底物及NAD合成前体物, 调节培养环境和氧化还原电势)和代谢工程(过量表达NADH代谢相关酶、缺失NADH竞争途径及引入NADH外源代谢途径)两方面分析、归纳了NADH代谢调控策略, 进而凝练出调控NADH/NAD+比率调节微生物细胞代谢功能研究方面亟待解决的3个科学问题及可能的解决途径。     

Secreted euryarchaeal microhalocins kill hyperthermophilic crenarchaea

Few antibiotics targeting members of the archaeal domain are currently available for genetic studies. Since bacterial antibiotics are frequently directed against competing and related organisms, archaea by analogy might produce effective antiarchaeal antibiotics. Peptide antibiotic (halocin) preparations from euryarchaeal halophilic strains S8a, GN101, and TuA4 were found to be toxic for members of the hyperthermophilic crenarchaeal genus Sulfolobus. No toxicity was evident against representative bacteria or eukarya. Halocin S8 (strain S8a) and halocin R1 (strain GN101) preparations were cytostatic, while halocin A4 (strain TuA4) preparations were cytocidal. Subsequent studies focused on the use of halocin A4 preparations and Sulfolobus solfataricus. Strain TuA4 cell lysates were not toxic for S. solfataricus, and protease (but not nuclease) treatment of the halocin A4 preparation inactivated toxicity, indicating that the A4 toxic factor must be a secreted protein. Potassium chloride supplementation of the Sulfolobus assay medium potentiated toxicity, implicating use of a salt-dependent mechanism. The utility of halocin A4 preparations for genetic manipulation of S. solfataricus was assessed through the isolation of UV-induced resistant mutants. The mutants exhibited stable phenotypes and were placed into distinct classes based on their levels of resistance.     

Biochemical and structural exploration of the catalytic capacity of Sulfolobus KDG aldolases

Aldolases are enzymes with potential applications in biosynthesis, depending on their activity, specificity and stability. In the present study, the genomes of Sulfolobus species were screened for aldolases. Two new KDGA [2-keto-3-deoxygluconate (2-oxo-3-deoxygluconate) aldolases] from Sulfolobus acidocaldarius and Sulfolobus tokodaii were identified, overexpressed in Escherichia coli and characterized. Both enzymes were found to have biochemical properties similar to the previously characterized S. solfataricus KDGA, including the condensation of pyruvate and either D,L-glyceraldehyde or D,L-glyceraldehyde 3-phosphate. The crystal structure of S. acidocaldarius KDGA revealed the presence of a novel phosphate-binding motif that allows the formation of multiple hydrogen-bonding interactions with the acceptor substrate, and enables high activity with glyceraldehyde 3-phosphate. Activity analyses with unnatural substrates revealed that these three KDGAs readily accept aldehydes with two to four carbon atoms, and that even aldoses with five carbon atoms are accepted to some extent. Water-mediated interactions permit binding of substrates in multiple conformations in the spacious hydrophilic binding site, and correlate with the observed broad substrate specificity.     

Conjugation in Archaea: Frequent Occurrence of Conjugative Plasmids inSulfolobus

We describe five novel conjugative plasmids (CPs) and two subfamilies, each comprising several closely related variants of CPs isolated from colony-cloned strains of the extremely thermophilic, heterotrophic archaeonSulfolobus islandicus,which were obtained by plating of samples from Icelandic solfataras after liquid enrichment. They are related to each other and to the previously described CP pNOB8 from a JapaneseSulfolobusstrain in that they share essential functions and limited similarity of genomes as demonstrated by DNA cross-hybridization and sequences. All these plasmids thus form a family of highly efficient self-spreading elements directly transferred from donor into recipient cells. Conjugation is initiated by pair formation, followed by selective transfer of the plasmids into the recipient and expression of transfer functions. Some of these CPs exclude superconjugation of the transcipients with closely related CPs. The novel CPs are stable upon conjugative transfer, but vary upon growth of transcipients. The stability of the CPs is higher in their original hosts or in relatedS. islandicusstrains, than inSulfolobus solfataricusstrain PH1 as recipient. The deletion variant pING3 has lost the ability to transfer itself but is still subject to being transferred by the transfer apparatus of its complete relative, pING6. The dissection of genes and functions has been initiated by characterizing this incomplete variant.     

Biochemical and physiological tests as aids to identification of Verticillium section Nigrescentia

An account is given of several biochemical and physiological techniques which were evaluated as tools to assist in identification of different strains of five species in Verticillium section Nigrescentia, including the important pathogens V. albo-atrum and V. dahliae. Although many of the tests gave results that varied between individual strains of the same species certain enzymatic activity tests provide a means of characterising the individual species studied.     

Phylometabonomic patterns of adaptation to high fat diet feeding in inbred mice

Insulin resistance plays a central role in type 2 diabetes and obesity, which develop as a consequence of genetic and environmental factors. Dietary changes including high fat diet (HFD) feeding promotes insulin resistance in rodent models which present useful systems for studying interactions between genetic background and environmental influences contributing to disease susceptibility and progression. We applied a combination of classical physiological, biochemical and hormonal studies and plasma (1)H NMR spectroscopy-based metabonomics to characterize the phenotypic and metabotypic consequences of HFD (40%) feeding in inbred mouse strains (C57BL/6, 129S6, BALB/c, DBA/2, C3H) frequently used in genetic studies. We showed the wide range of phenotypic and metabonomic adaptations to HFD across the five strains and the increased nutrigenomic predisposition of 129S6 and C57BL/6 to insulin resistance and obesity relative to the other strains. In contrast mice of the BALB/c and DBA/2 strains showed relative resistance to HFD-induced glucose intolerance and obesity. Hierarchical metabonomic clustering derived from (1)H NMR spectral data of the strains provided a phylometabonomic classification of strain-specific metabolic features and differential responses to HFD which closely match SNP-based phylogenetic relationships between strains. Our results support the concept of genomic clustering of functionally related genes and provide important information for defining biological markers predicting spontaneous susceptibility to insulin resistance and pathological adaptations to fat feeding.