Ethyl methanesulfonate mutagenesis in extremely halophilic archaebacteria: Isolation of auxotrophic mutants ofHaloferax mediterranei andHaloferax gibbonsii

The lethality and mutagenicity in ethyl methanesulfonate (EMS)-treated cells of five archaebacterial strains belonging to each of the three described genera of non-alkaliphilic halobacteria were investigated. In order to test the efficiency of the mutagenesis under a variety of experimental conditions, we chose the fast-growing halobacteriumHaloferax mediterranei as a model strain. A strong induced mutagenicity was found, since the spontaneous mutation rate (expressed as the rate of resistance to the antibiotic josamycin) increased up to 500-fold after mutagen exposure. The mutagenesis was also successfully used in obtaining auxotrophic mutants. Although a heterogeneous response to the induced effects caused after EMS exposure was detected for the other halophilic archaebacteria tested, a clear, efficient mutagenicity ofHalobacterium halobium andHaloferax gibbonsii was observed; auxotrophic mutants of this halobacterium were also produced. Optimal experimental conditions for EMS mutagenesis of some halobacteria were determined.     

Construction of a Shuttle Vector for, and Spheroplast Transformation of, the Hyperthermophilic Archaeon Pyrococcus abyssi

Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (10² to 10³ transformants per μg of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.     

Rapid extraction of plasmid pGT5 from the hyperthermophilic archaeonPyrococcus abyssi

Hyperthermophilic archaea, specificallyPyrococcus spp., are the target of current efforts in developing heterologous expression systems. However, the published plasmid purification and plasmid screening protocols are long and tedious. We describe a fast, simple protocol for plasmid purification fromPyrococcus spp. developed while extracting the plasmid pGT5 fromPyrococcus abyssi cells. The protocol is modified from the procedures for commercial plasmid minipreps and is completed in about 20 min. The DNA is easily digested by restriction enzymes and can be used in sequencing reactions without additional purification.     

Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough

A hyperthermophilic, anaerobic archaeon was isolated from hydrothermal fluid samples obtained at the Okinawa Trough vents in the NE Pacific Ocean, at a depth of 1395 m. The strain is obligately heterotrophic, and utilizes complex proteinaceous media (peptone, tryptone, or yeast extract), or a 21-amino-acid mixture supplemented with vitamins, as growth substrates. Sulfur greatly enhances growth. The cells are irregular cocci with a tuft of flagella, growing optimally at 98°C (maximum growth temperature 102°C), but capable of prolonged survival at 105°C. Optimum growth was at pH 7 (range 5–8) and NaCl concentration 2.4% (range 1%–5%). Tryptophan was required for growth, in contrast to the closely related strains Pyrococcus furiosus and P. abyssi. Thin sections of the cell, viewed by transmission electron microscopy, revealed a periplasmic space similar in appearance to the envelope of P. furiosus. The predominant cell membrane component was tetraether lipid, with minor amounts of diether lipids. Treatment of the cells by mild osmotic shock released an extract that contained a Zn²⁺-dependent alkaline phosphatase. Phylogenetic analysis of the sequences encoding 16S rRNA and glutamate dehydrogenase places the isolate with certainty within the genus Pyrococcus although there is relatively low DNA–DNA hybridization (<63%) with described species of this genus. Based on the reported results, we propose a new species, to be named Pyrococcus horikoshii sp. nov. Received: December 10, 1997 / Accepted: February 4, 1998     

The role of umuC gene product in mutagenesis by simple alkylating agents

Summary This paper describes studies to determine the role of the umuC gene product in the process of alkylation induced mutagenesis. An active umuC gene is necessary for most MMS induced mutagenesis but it is not essential for EMS nor for MNNG induced mutagenesis in either normal or adapted cultures. In this respect the umuC mutation differs from lexA mutations which have a striking effect on MNNG induced mutagenesis (Schendel, et al., 1978). These findings have prompted a re-evaluation of these previously published data and the advancement of an hypothesis which explains the lexA effect without evoking a role for error-prone repair in the process of alkylation induced mutagenesis.It was also observed that exposure to MNNG is capable of generating a small amount of W-reactivation and W-mutagenesis capacity in a umuC strain which is totally blocked for UV induced reactivation. In light of this result a possible function for the umuC gene product is discussed.     

Improvement of lincomycin production by mutant selection and metabolic regulation

Lincomycin is a lincosamide antibiotic produced by Streptomyces lincolnensis. Through mutagenesis by ethylmethansulfonate (EMS) and ultraviolet (UV) irradiation repeatedly, M2 was picked out in plate with glutamine and propylproline orderly. In 50-L stirred bioreactor, the production of lincomycin, fermented by M2, was increased to 8136?u/ml under the optimal condition as compared to original strain S. lincolnensis 07–5 (6634?u/ml). Two-dimensional gel electrophoresis (2-D GE) and mass spectrometry (MS)-shown LmbG, LmbI, and acetohydroxy acid isomeroreductase were remarkably synthesized in M2. The gene lmbG and lmbI are responsible for methylation in the lincomycin biosynthetic cluster, while acetohydroxy acid isomeroreductase contributes to stronger metabolic capability. Finally, we obtained a better strain for industrial production.     

Isolation and characterization of pyrimidine auxotrophs, and molecular cloning of the pyrE gene from the hyperthermophilic archaeon Pyrococcus abyssi

Uracil auxotrophic mutants of the hyperthermophilic archaeon Pyrococcus abyssi were isolated by screening for resistance to 5-fluoro-orotic acid (5-FOA). Wild-type strains were unable to grow on medium containing 5-FOA, whereas mutants grew normally. Enzymatic assays of extracts from wild-type P. abyssi and from pyrimidine auxotrophs demonstrated that the mutants are deficient in orotate phosphoribosyltransferase (PyrE) and/or orotidine-5′-monophosphate decarboxylase (PyrF) activity. The pyrE gene of wild-type P. abyssi and one of its mutant derivatives were cloned and sequenced. This pyrE gene could serve as selectable marker for the development of gene manipulation systems in archaeal hyperthermophiles. Received: 29 March 1999 / Accepted: 25 May 1999     

UV-induced mutability in repair-deficientrad6-1 strains ofSaccharomyces cerevisiae is caused by a suppressor gene

TheRAD6 gene is a multifunctional gene required for DNA repair, induced mutagenesis and sporulation. The survival and revertibility of two loci in fourrad6-1 mutant strains of different origin after UV irradiation were followed. As expected, all therad6-1 strains tested were more sensitive to UV radiation in comparison withRAD6 strains. The reversion frequency per survivor intrpl-289 andarg4–17 alleles was significantly higher in all fourrad6-1 mutant strains than in wild-type strains after equal doses of UV radiation. On the basis of genetic analysis we suggest that the phenomenon of increased frequency of induced mutagenesis is caused by a suppressor gene.     

Efficient hydroxylamine mutagenesis ofHaloferax mediterranei and other extremely halophilic archaebacteria

The lethal and mutagenic effects of hydroxylamine onHaloferax mediterranei and five other extremely halophilic archaebacteria are described for the first time. Although previous studies have shown thatH. mediterranei was very resistant to the lethal action of other DNA-damaging agents, this strain was found to be relatively sensitive to hydroxylamine, but also more successfully mutated by the latter. The efficiency of the mutagenicity obtained with the hydroxylamine treatment was tested under a variety of conditions, and optimal procedures are described that yielded a number of useful auxotrophic mutant strains ofH. mediterranei. Likewise, a strong induced mutagenicity after hydroxylamine mutagenesis was achieved for the majority of the other archaebacteria tested.     

Two uvs genes of Aspergillus nidulans with different functions in error-prone repair: uvsI , active in mutation-specific reversion, and uvsC , a recA homolog, required for all UV mutagenesis

Two genes of Aspergillus nidulans are known to function in UV mutagenesis, but have been assigned to different epistasis groups: uvsC, which is also required for meiosis and mitotic recombination, and uvsI, which may have no other function. To clarify their role in error-prone repair and to investigate their interaction, uvsI and uvsC single and uvsI;uvsC double mutant strains were further tested for mutagen sensitivities and characterized for effects on mutation. Spontaneous and induced frequencies were compared in forward and reverse mutation assays. All results confirmed that uvsI and uvsC are members of different epistasis groups, and demonstrated that these uvs mutants have very different defects in UV mutagenesis. The uvsI strains showed wild-type frequencies in all forward mutation tests, but greatly reduced spontaneous and UV-induced reversion of some, but not other, point mutations. In contrast, uvsC had similar effects in all assay systems: namely pronounced mutator effects and greatly reduced UV mutagenesis. Interestingly, the uvsI;uvsC double mutant strains differed from both single mutants; they clearly showed synergism for all types of reversion tested: none were ever obtained spontaneously, nor after induction by UV or EMS (ethylmethane sulfonate). Based on these results, we conclude that uvsI is active in a mutation-specific, specialized error-prone repair process in Aspergillus. In contrast, uvsC, which is now known to show sequence homology to recA, has a basic function in mutagenic UV repair in addition to recombinational repair, similar to recA of Escherichia coli. Received: 23 September 1996 / Accepted: 2 December 1996     

Salt-Sensitive and Auxotrophic Mutants of Halomonas elongata and H. meridiana by Use of Hydroxylamine Mutagenesis

The killing action and induced mutagenicity in hydroxylamine (HA)-treated cells of two moderately halophilic species of the genus Halomonas, H. elongata and H. meridiana, were investigated. A high sensitivity of H. elongata and especially of H. meridiana to HA was found. The efficiency of the mutagenicity obtained with the HA treatment was tested at different salinities. Optimal experimental conditions for HA mutagenesis of these two moderate halophiles were determined. A clear, efficient mutagenicity of both H. elongata and H. meridiana after HA mutagenesis was achieved. The optimal procedures yielded a number of useful auxotrophic mutants of H. elongata as well as different salt-sensitive mutants of H. elongata and H. meridiana for further studies. Some of these latter mutants appeared to be affected in the synthesis of compatible solutes. Received: 11 June 1996 / Accepted: 19 July 1996     

Isolation and characterization of pyrimidine auxotrophs, and molecular cloning of the pyrE gene from the hyperthermophilic archaeon Pyrococcus abyssi

Uracil auxotrophic mutants of the hyperthermophilic archaeon Pyrococcus abyssi were isolated by screening for resistance to 5-fluoro-orotic acid (5-FOA). Wild-type strains were unable to grow on medium containing 5-FOA, whereas mutants grew normally. Enzymatic assays of extracts from wild-type P. abyssi and from pyrimidine auxotrophs demonstrated that the mutants are deficient in orotate phosphoribosyltransferase (PyrE) and/or orotidine-5′-monophosphate decarboxylase (PyrF) activity. The pyrE gene of wild-type P. abyssi and one of its mutant derivatives were cloned and sequenced. This pyrE gene could serve as selectable marker for the development of gene manipulation systems in archaeal hyperthermophiles.     

Antibiotic production improvement in the rare actinomycete Planobispora rosea by selection of mutants resistant to the Aminoglycosides Streptomycin and Gentamycin and to Rifamycin

During a strain improvement program, spontaneous mutants with single or combined resistance to streptomycin (Str^r), gentamycin (Gen^r) or rifamycin (Rif^r) were selected from the industrial strain of Planobispora rosea, which is the producer of thiazolylpeptide GE2270. Among the mutants resistant to each single antibiotic, higher producers occurred more frequently (60%) among Gen^r than in Rif^r (10%) and Str^r (24%) populations. Two Gen^r mutants showed up to 1.5-fold improvement in GE2270 production while single resistant mutants Str^r and Rif^r produced slightly more than the parental strains. The combination of Str^r and Rif^r in the same strain improved GE2270 yield up to 1.7-fold. Finally, a higher GE2270 producing strain (1.8-fold improvement with respect to the parental strain) was selected among those mutants with triple resistance to streptomycin, rifamycin and gentamycin. A hierarchical increase in aerial mycelium and spore formation was observed which paralleled GE2270 production improvement.     

Genome shuffling of marine derived bacterium <Emphasis Type="Italic">Nocardia</Emphasis> sp. ALAA 2000 for improved ayamycin production

Genome shuffling is a recent development in microbiology. The advantage of this technique is that genetic changes can be made in a microorganism without knowing its genetic background. Genome shuffling was applied to the marine derived bacterium Nocardia sp. ALAA 2000 to achieve rapid improvement of ayamycin production. The initial mutant population was generated by treatment with ethyl methane sulfonate (EMS) combined with UV irradiation of the spores, resulting in an improved population (AL/11, AL/136, AL/213 and AL/277) producing tenfold (150 μg/ml) more ayamycin than the original strain. These mutants were used as the starting strains for three rounds of genome shuffling and after each round improved strains were screened and selected based on their ayamycin productivity. The population after three rounds of genome shuffling exhibited an improved ayamycin yield. Strain F3/22 yielded 285 μg/ml of ayamycin, which was 19-fold higher than that of the initial strain and 1.9-fold higher than the mutants used as the starting point for genome shuffling. We evaluated the genetic effect of UV + EMS-mutagenesis and three rounds of genome shuffling on the nucleotide sequence by random amplified polymorphic DNA (RAPD) analysis. Many differences were noticed in mutant and recombinant strains compared to the wild type strain. These differences in RAPD profiles confirmed the presence of genetic variations in the Nocardia genome after mutagenesis and genome shuffling.     

Uvm mutants of Escherichia coli K 12 deficient in UV mutagenesis

Summary Selection for defective reversion induction, after UV treatment of E. coli K 12, yielded uvm mutants. These mutants exhibited highly reduced or no UV mutability for all loci tested although they were moderately and normally mutable by X-rays and EMS, respectively. Uvm mutations confer only a slight sensitivity to killing by UV and X-rays and no clear sensitivity to the lethal effect of HN2, EMS or MMS. Growth and viability of untreated uvm cells were normal. The properties of uvm mutants are discussed in relation to those of other relevant mutant types and to some actual problems of induced mutagenesis.     

Selection of Ergot Alkaloid Producers by Induced Mutagenesis

Using the induced mutagenesis technique, a series of genetically modified Clavicepssp. VKM F-2609 strains that display high levels of agroclavine and elymoclavine synthesis were selected by induced mutagenesis. Compared to the parent strain, c106 displayed a 40-fold higher level of agroclavine synthesis, and c66 displayed an eightfold higher level of elymoclavine synthesis. The levels of synthesis of other alkaloids were decreased in these strains. The effects of various carbohydrates on the strain growth and ergot alkaloid biosynthesis was then investigated in both the parent strain and c106. The largest amount of agroclavine was synthesized by c106 strain growing on a medium with maltose.     

Mutagenic and error-free DNA repair in Streptomyces

Summary Two mutants of Streptomyces fradiae defective in DNA repair have been characterized for their responses to the mutagenic and lethal effects of several chemical mutagens and ultraviolet (UV) light. S. fradiae JS2 (mcr-2) was more sensitive than wild type to agents which produce bulky lesions resulting in large distortions of the double helix [i.e. UV-light, 4-nitroquinoline-1-oxide (NQO), and mitomycin C (MC)] but not to agents which produce small lesions [i.e. hydroxylamine (HA), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG)]. JS2 expressed a much higher frequency of mutagenesis induced by UV-light at low doses and thus appeared to be defective in an error-free excision repair pathway for bulky lesions analogous to the uvr ABC pathway of Escherichia coli. S. fradiae JS4 (mcr-4) was defective in repair of damage by most agents which produce small or bulky lesions (i.e., HA, NQO, MMS, MNNG, MC, and UV, but not EMS). JS4 was slightly hypermutable by EMS and MMS but showed reduced mutagenesis by NQO and HA. This unusual phenotype suggests that the mcr-4 ⁺ protein plays some role in error-prone repair in S. fradiae.     

Experimental silicification of the extremophilic Archaea Pyrococcus abyssi and Methanocaldococcus jannaschii: applications in the search for evidence of life in early Earth and extraterrestrial rocks

Hydrothermal activity was common on the early Earth and associated micro‐organisms would most likely have included thermophilic to hyperthermophilic species. 3.5–3.3 billion‐year‐old, hydrothermally influenced rocks contain silicified microbial mats and colonies that must have been bathed in warm to hot hydrothermal emanations. Could they represent thermophilic or hyperthermophilic micro‐organisms and if so, how were they preserved? We present the results of an experiment to silicify anaerobic, hyperthermophilic micro‐organisms from the Archaea Domain Pyrococcus abyssi and Methanocaldococcus jannaschii, that could have lived on the early Earth. The micro‐organisms were placed in a silica‐saturated medium for periods up to 1 year. Pyrococcus abyssi cells were fossilized but the M. jannaschii cells lysed naturally after the exponential growth phase, apart from a few cells and cell remains, and were not silicified although their extracellular polymeric substances were. In this first simulated fossilization of archaeal strains, our results suggest that differences between species have a strong influence on the potential for different micro‐organisms to be preserved by fossilization and that those found in the fossil record represent probably only a part of the original diversity. Our results have important consequences for biosignatures in hydrothermal or hydrothermally influenced deposits on Earth, as well as on early Mars, as environmental conditions were similar on the young terrestrial planets and traces of early Martian life may have been similarly preserved as silicified microfossils.     

Interstrain crosses enhance excision of Tc1 transposable elements in Caenorhabditis elegans

Summary We have studied spontaneous and UV mutagenesis of the glyU gene in Escherichia coli trpA461 (GAG) strains carrying the pIP11 plasmid, in which the dnaQ gene encoding the 3–5 exonuclease subunit (epsilon) of DNA polymerase III is fused to the tac(trp-lac) promoter. We have used a pair of M13glyU phage in which the gene encoding the glycyl-tRNA is cloned in opposite orientations, consequently the phage present either GGG or CCC anticodon triplets for mutagenesis. The presence of IPTG, the inducer of the tac-dnaQ fusion, results in about 100-fold decrease in frequency of spontaneous Su⁺ (GAG) mutations arising in the CCC phage. The enhanced expression of tac-dnaQ reduces 10-fold the frequency of UV-induced Su⁺ (GAG) mutations in the CCC phage and nearly completely prevents generation by UV of Su⁺ (GAG) mutations in the GGG phage, in which UV-induced pyrimidine photoproducts can be formed only in the vicinity of the target triplet. These results suggest that both locally and regionally targeted mutagenesis is affected by overproduction of the epsilon subunit. By delayed photoreversal mutagenesis we have shown that UV-induced chromosomal mutagenesis of the umuC36 trpA461 strain harboring pIP11 is completely abolished in the presence of IPTG. This result seems to indicate that the misinocorporation step of DNA translesion synthesis is affected by excess of the epsilon subunit. Finally, we have introduced the pIP13 plasmid carrying the dnaQ gene into the recA1207 strain, which is deficient in the recombinase activity of RecA but constitutive in the protease activity. We demonstrate that the transformant shows much higher UV sensitivity than recA1207 carrying the vector plasmid pBR325, indicating that translesion synthesis significantly contributes to DNA repair capacity of cells deficient in recombination.