Adaptation of model genetically engineered microorganisms to lake water: growth rate enhancements and plasmid loss.

When a genetically engineered microorganism (GEM) is released into a natural ecosystem, its survival, and hence its potential environmental impact, depends on its genetic stability and potential for growth under highly oligotrophic conditions. In this study, we compared plasmid stability and potential for growth on low concentrations of organic nutrients of strains of Pseudomonas putida serving as model GEMs. Plasmid-free and plasmid-bearing (NAH7) prototrophic isogenic strains and two amino-acid auxotrophs, all containing antibiotic resistance markers, were held physically separate from but in chemical contact with lake water containing the natural bacterium-sized microbial populations. Cells were reisolated at intervals over a 2-month period to determine the percent retaining the plasmid and the specific growth rate on various media. Plasmid stability in lake water was strongly strain specific; the NAH7 plasmid was stably maintained by the prototrophic strain for the duration of the test but was lost within 24 h by both of the auxotrophs. Specific growth rates of reisolates, compared with those of the corresponding non-lake water-exposed strains (i.e., parental strains), were not different when measured in rich medium (Luria-Bertani broth). However, specific growth rates were 42, 55, and 63% higher in reisolates of auxotrophs and the plasmid-free prototroph, respectively, when measured in 10-fold-diluted medium after exposure of 15 days or longer to lake water. Moreover, lake water-exposed strains grew actively when reintroduced into sterile lake water (28- to 33-fold increase in numbers over 7 days), while the corresponding unadapted parental strains exhibited no growth over the same period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Powerful mutator activity of the polA1 mutation within the histidine region of Escherichia coli K-12.

We examined 122 spontaneous histidine auxotrophs accumulated in overnight cultures of polA1 strains of Escherichia coli K-12 at approximate frequencies of 10(-3). One hundred and thirteen appeared to be minus frameshifts, and nine appeared to be deletions. Of the frameshift mutations, 109 affected the hisC gene, and 4 affected genes hisD, hisH, hisA, and hisI. The lack of base substitutions supported the idea that polymerase-defective polA is a minus frameshift- and deletion-type mutator. Contrary to a previous report, we did not observe superior growth of PolA auxotrophs over their prototrophic progenitors (15 auxotrophs tested). We conclude that the polA1 mutation exerts a powerful mutator activity in this specific genetic context.     

Occurrence of diploid strains of Cryptococcus neoformans.

A mating between niacin and pantothenate auxotrophs of Cryptococcus neoformans gave a few prototrophic progeny that were self-fertile. These were uninuclear but contained twice as much DNA as the parental strains. Segregation of nutritional markers was observed upon sporulation. We conclude that these self-fertile strains are diploids.     

Isolation and study of hybrid plasmids carrying Escherichia coli threonine operon genes

A fragment of Escherichia coli chromosome containing the intact threonine operon or its distinct genes has been cloned on the pBR322 plasmid. This fragment has been mapped using some restriction endonucleases. Cloning results in an increased level of appropriate enzyme activity in cells containing hybrid plasmids. Those carrying the complete threonine operon are capable of accumulating threonine up to 5 g/l in culture medium during 48 h. When multi-copy plasmids are used for gene cloning, interpretation of experiments aimed at transformation of auxotrophic bacterial strains, might be complicated. For example, transformation of appropriate threonine auxotrophs by a hybrid plasmid carrying mutation in the threonine gene, might result in prototrophic phenotype. It is possible that the great amount of mutant enzyme molecules compensated their low activity. On the contrary, the presence of a gene within the plasmid, as shown by restriction and biochemical analysis, did not always ensure the growth on a minimal medium of auxotrophs transformed by this plasmid.     

Genetic analysis of Staphylococcus aureus with Tn4001.

Tn4001, a 4.5-kilobase composite transposon with IS256 ends that confers resistance to gentamicin (Gmr), tobramycin, and kanamycin in Staphylococcus aureus, can transpose to diverse chromosomal sites in S. aureus. Chromosomal insertions of Tn4001 were isolated either after UV irradiation of transducing lysates carrying pII147::Tn4001 or by selection for thermoresistant Gmr isolates with strains containing thermosensitive derivatives of plasmids pI258 and pII147 carrying Tn4001. Frequent integration of the entire delivery plasmid occurred under these selective conditions in recombination-proficient hosts. When selection for thermoresistant Gmr isolates was done with these plasmids in recombination-deficient hosts, 99% or more of the Gmr isolates resulted from transposition of Tn4001 in the absence of plasmid integration. Efficient isolation of Tn4001 insertions near markers of interest and the isolation of insertional auxotrophs were achieved. Reversion frequencies of insertional auxotrophs were between 10(-6) and 10(-7) (higher than those observed with Tn551 and Tn917). About 50% of the prototrophic revertants were Gms, and these are attributed to precise excision of Tn4001. The Gmr prototrophic revertants were due to intergenic suppression.     

Isolation of spontaneous mutant strains of Flavobacterium spp.

Flavobacterium sp. ATCC 27551 is prototrophic, streptomycin-resistant and contains plasmid DNA. Two strains of this Flavobacterium sp. with altered growth requirements and antibiotic sensitivity have been isolated. Unlike the parental strain, both isolates are sensitive to the antibiotic streptomycin. Isolate YE requires yeast extract supplementation of the medium and contains no plasmid DNA. Isolate AHC contains plasmid DNA and requires aspartic acid, histidine and cysteine for growth.     

Prototrophic hybrids of bakers yeast. I. Selection of haploids and diploids of prototrophic yeast

153 haploids, including 8 prototrophs, 12 biotin prototrophs and 4 biotin auxotrophs were isolated from Y, F and FB strains of the baker's yeast Saccharomyces cerevisiae. Remaining haploids were dependent on vitamins of B group other than biotin. Obtained haploids were characterized by large cell sizes, a or α mating type, the ability to fermentation of sugars and the assimilation of non-fermented carbon sources. Haploids obtained from the yeast of Y and FB strains a good growth in the synthetic medium. Prototrophic haploids, prototrophic haploids with biotin auxotrophs and biotin prototrophs with biotin auxotrophs were crossed. 89 prototrophic hybrids capable of growth in synthetic medium without vitamins were selected out of 178 hybrids obtained. Hybrids are characterized by features typical for baker's yeast; however, not all of them are capable of sporulation. As result of selection, prototrophic hybrids and 16 hybrids characterized by a good increase of biomass in molasses medium were chosen. The efficiency of the biomass of hybrids designated as YY 3040/2, YY 3040/3 and FFB 1910/2 is considerably higher than one obtained from the cultivation of the industrial strain French Mautner (Mf). All hybrids possess adequate enzymatic activity in anaerobic metabolism of saccharose and maltose. Selected prototrophic hybrids were sent out to two yeast factories in the country for experimental propagation.     

Effects of Ribonucleosides on Thymidine Incorporation: Selective Reversal of the Inhibition of Deoxyribonucleic Acid Synthesis in Thymineless Auxotrophs of Escherichia coli

Addition of certain ribonucleosides to exponentially growing cultures of Escherichia coli increased the extent of thymidine incorporation. The prolonged uptake of thymidine was correlative with the ability of these ribonucleosides to prevent the degradation of thymidine. In addition to protecting thymidine, uridine reversed partially (70 to 80%) the inhibition of deoxyribonucleic acid (DNA) synthesis in thymineless auxotrophs by cytosine arabinoside, hydroxyurea, and nalidixic acid. This reversal was selective for auxotrophic strains since no reversal of inhibition by uridine was observed in any of the prototrophic strains examined. In the presence of uridine, the rapid assimilation of thymidine by prototrophic and auxotrophic strains was prevented and the rate of DNA synthesis became a function of the available exogenous thymidine. Under these conditions, prototrophic strains accumulated equivalent amounts of thymidine into the acid-soluble (pool) and acid-insoluble (DNA) cell fractions. In contrast, 95 to 98% of the thymidine taken up by auxotrophs was found in the acid-insoluble (DNA) cell fraction. The results suggest that different mechanisms for DNA synthesis exist in auxotrophs and prototrophs. Based on these observed differences, some possible mechanisms for the selective reversal of the inhibition of DNA synthesis in auxotrophs are discussed.     

Expression of dibenzothiophene-degradative genes in two Pseudomonas species

The genes encoding dibenzothiophene (DBT) degradation in Pseudomonas alcaligenes strain DBT2 were cloned into plasmid pC1 by other workers. This plasmid was conjugally transferred into a spontaneous variant of Pseudomonas sp. HL7b (designated HL7bR) incapable of oxidizing DBT (Dbt- phenotype). Acquisition of plasmid pC1 simultaneously restored oxidation of DBT and naphthalene to the transconjugant, although the primary DBT metabolite produced by transconjugant HL7bR(pC1) corresponded to that produced by wild-type strain DBT2 rather than that from wild-type strain HL7b. Inducers of the naphthalene pathway (naphthalene, salicylic acid, and 2-aminobenzoate) stimulated DBT oxidation in transconjugant HL7bR(pC1) when present at 0.1 mM concentrations but had no effect on wild-type strain HL7b. Higher concentrations (5 mM) of salicylic acid and naphthalene were inhibitory to DBT oxidation in all strains. DNA-DNA hybridization was not observed between plasmid pC1 and genomic DNA from strains HL7b or HL7bR, nor between authentic naphthalene-degradative genes (plasmid NAH2) and either plasmid pC1 or strain HL7b, despite the observation that the degradative genes encoded on plasmid pC1 functionally resembled broad-specificity naphthalene-degradative genes. Transconjugant HL7bR(pC1) is a mosaic of the parental types regarding DBT metabolite production, regulation, and use of carbon sources.     

Nonparental progeny resulting from protoplast fusion inTrichoderma in the absence of parasexuality

The purpose of this study was to characterize a number of progeny from intra- and interstrain protoplast fusion within the genusTrichoderma. We wished to determine whether parasexuality or other genetic mechanisms occur in these fungi. When two different auxotrophs of the same strain were fused, rapidly growing prototrophic progeny were obtained in high frequencies. When single spore isolates of these strains were prepared, equal numbers of strains indistinguishable from the two parental auxotrophic strains were obtained, even though 10⁹–10¹⁰ conidia were tested per strain. Thus, progeny from intrastrain fusions all appeared to be balanced heterokaryons, and no evidence of recombination between the two parental strains was obtained. When 16 separate interstrain fusions were conducted, very different results were obtained, regardless of whether fusions were within or between species. Following interstrain fusions, presumptive somatic hybrids developed very slowly and in low numbers as compared with hybrids from intrastrain fusions. Most were weakly prototrophic. These slow-growing progeny were unstable and sectors developed from them. Such sectors themselves were unstable and gave rise to other progeny. Usually sectors were more strongly prototrophic and more rapid growing than the original progeny strain. Sectoring gave rise to a very wide range of morphotypes. Most of these morphotype variants were stable through conidiation; thus, these types did not occur as a consequence of heterokaryosis. Isozyme analysis was conducted on over 1000 progeny strains. Nearly all progeny were identical to one or the other parental isozyme phenotypes. A few progeny, when tested as soon as possible after fusion, exhibited the isozyme phenotypes of both parents, but such biparental banding patterns were rapidly lost upon subsequent reculturing. Isozyme banding patterns of multimeric enzymes never gave band patterns indicative of heterokaryosis or heterozygosis. Banding patterns indicative of heterozygous diploids or recombinants were never detected. Despite the extreme variation in morphotype and nutritional requirements among progeny, isozyme banding patterns of derived progeny from any fusion were invariably identical to one or the other parental strains. From these results, we conclude that protoplast fusion in the genusTrichoderma gives rise to great variability, but that the classical parasexual cycle is not required for variation to occur.     

Intergenic Complementation of Glucoamylase and Citric Acid Production in Two Species of Aspergillus

All auxotrophs of Aspergillus foetidus and all but two auxotrophs of A. niger which we isolated yield glucoamylase and citric acid, respectively, at levels below that of the prototrophic strain from which they were derived. Results of representative heterokaryon tests suggest that the nucleus was principally responsible for the inheritance of citric acid or glucoamylase production. Most somatic diploid strains of A. foetidus gave rise to higher yields of glucoamylase when compared to their haploid component strains. Both heterokaryons and somatic diploid strains of A. niger synthesized between auxotrophs which were simultaneously reduced in citric acid yields also gave rise to enhanced yields when compared with their haploid components. The yields of a heterokaryon and somatic diploid synthesized between two high producers of citric acid were not higher than those of respective haploid components. We concluded from these results that gene dosage (or ploidy) does not increase the yield of citric acid. The apparent enhancement in yields observed in diploids or heterokaryons synthesized between auxotrophs with reduced yields in both species can be interpreted as resulting from intergenic complementation.     

The nature of arginine auxotrophy in cutaneous populations of staphylococci.

L-Arginine was required for growth by a high percentage of strains of Staphylococcus species that were niche-specific and/or host-specific, but was usually not required for growth by species showing a wide host range. Growth stimulation patterns with arginine intermediates indicated that most of the auxotrophic strains had blocks in an early step(s) in arginine biosynthesis. These strains were designated phenotypically as Arg(CHG) according to the Salmonella typhimurium classification scheme. Staphylococcus simulans strains appeared to be either ArgA or Arg I. The ArgI strains of S. simulans and S. capitis had moderate to high ornithine carbamoyltransferase (EC 2.1.3.3) activities and therefore could not be designated as argI mutants. ArgI strains in other species had no or very low ornithine carbamoyltransferase activities. All of the natural Staphylococcus auxotrophs tested grew in the presence of L-citrulline and had moderate to high argininosuccinase (EC 4.3.2.1) activities. Arginine auxotrophs of species with a wide host range were often capable of reverting to arginine-independent or complete prototrophic growth, whereas auxotrophs of species that tended to be niche-specific and/or host-specific were incapable of reversion to arginine-independence, even in the presence of various mutagens. A relationship between the nature of arginine auxotrophy and habitat is suggested.     

Bacteriophage SPO2-mediated plasmid transduction in transpositional mutagenesis within the genus Bacillus.

A single copy of the Streptococcus faecalis transposon Tn917, located in the Bacillus subtilis chromosome, was able to transpose onto the SPO2 cos plasmid pPL1017, which codes for chloramphenicol resistance and contains the bacteriophage phi 105 immunity region. Selection for pPL1017::Tn917 chimeras was performed by SPO2-mediated plasmid transduction of transposon-borne resistance to macrolide-lincosamide-streptogramin B antibiotics (MLSr). The transposition of Tn917 onto plasmid pPL1017 occurred with a frequency of 10(-5) and was dependent on the presence of a subinhibitory dose of erythromycin. Twelve chimeras were subjected to genetic and physical analyses. Two Cams transductants harbored plasmids whose chloramphenicol acetyltransferase genes had been insertionally inactivated by Tn917. Several transpositions in the vicinity of the phi 105 immunity region were detected. However, all of the 300 MLSr, Camr transductants screened were immune to phi 105 infectious activity. One pPL1017::Tn917 chimera, pLK200, was transferred by SPO2 plasmid transduction into the Bacillus amyloliquefaciens prototrophic strain DSM7. Plasmid pLK200 was effective in the mutagenesis of the DSM7 chromosome and yielded auxotrophs at a frequency of 0.5 to 5.3%. Generation of auxotrophs was also dependent on the presence of a subinhibitory dose of erythromycin. Forty-four auxotrophs representing at least nine amino acid requirements were recovered.     

Isolation and preliminary characterization of auxotrophic and morphological mutants of the yeastlike form of Paracoccidioides brasiliensis.

N-methyl-N'-nitro-N-nitrosoguanidine, which is known to be a very effective mutagen in many systems, was used to induce mutants in the yeastlike form of Paracoccidioides brasiliensis strain IVIC Pb9, an imperfect fungus. Forty-three auxotrophic and 27 prototrophic morphological mutants were isolated after treatment with 50 mug of nitrosoguanidine per ml in 0.1 M citrate buffer, pH 5.0. Auxotrophic mutants required primarily either amino acids, purines, or pyrimidines. Some auxotrophs were also morphological mutants. The main morphological difference from the parental strain was the texture or the color of the yeast-like colonies. Only one prototrophic morphological mutant differed in the size and form of the yeastlike cells when compared with the parental strain. Suxotrophic mutants were used in pairwise combination to attempt heterokaryon formation without success.     

Comparative characteristics of spore-forming and asporogenic strains of Bacillus thuringiensis

Comparative characteristics of sporogenous and asporogenous Bacillus thuringiensis strains is carried out. Asporogenous strains are found to differ from wild type strains in a number of criteria, including colony morphology, character of growth on rich and poor media and UV-sensitivity. Sporogenous strains form R colonies, they are more stable and more rare produce variants forming S colonies. S colonies are typical for asporogenous mutants, and under the cultivation in unfavourable conditions (elevated temperature, a shift of pH, a change of an incubation regime) asporogenous strains dissociate with a high frequency into R form. Initial strains, which are multiple auxotrophs, under certain conditions can form "prototrophic" revertants which are unstable when incubated on rich media. Suppressor mutation is supposed to be a possible mechanism of the origination of "prototrophs".     

Conditions for mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine and relationships of A. tumefaciens mutants to crown-gall tumor induction

Optimum mutagenesis of Agrobacterium tumefaciens by N-methyl-N′-nitro-N-nitrosoguanidine occurred at pH 6.5 using 250 μg/ml of the mutagen for 3 h at 30°. Antibiotic-resistant mutants and amino acid auxotrophs were selected and scored for crown-gall tumor-inducing ability on Helianthus annuus (sunflower). Mutants resistant to neomycin, kanamycin or rifampicin were not directly affected in their tumor-inducing ability. Mutants that were resistant to neomycin were also resistant to kanamycin and vice versa. Various amino acid auxotrophs varied in virulence. Some of the auxotrophs that required histidine, leucine or tryptophan had simultaneously lost their virulence. The alteration of virulence of the organism is not dependent on its growth since the avirulent auxotrophs when supplemented with the amino acid requirement grew in vivo almost as well as the prototrophic strains and yet remained avirulent.     

Genetic Instability in Auxotrophs of SALMONELLA TYPHIMURIUM Requiring Cysteine or Methionine and Resistant to Inhibition by 1,2,4-Triazole

Triazole-resistant (Trz(r)) derivatives of six cysteine- or methionine-requiring (Cym(-)) mutants of Salmonella typhimurium were isolated. Some of the derivatives of each mutant (CTS) were prototrophic, i.e., Cym(-) was suppressed. In every case suppression was initially unstable, Cym(-) auxotrophs being segregated at high frequency, although Trz(r) was stable. After several subcultures on selective medium, CTS strains were classified as either persistently unstable or stabilized. The unstable strains segregated Cym( -) auxotrophs at frequencies of 50-70%, whereas the stabilized strains segregated at frequencies of less than 1%. All suppressed strains had a stable Trz(r) marker co-transducible with cysA. However, there was a correlation between the class of CTS strain and Cym(- ) phenotype. The stabilized strains were Cym(+), whereas the unstable strains were Cym(-). Acriflavin and ethidium bromide increased segregation in the unstable strains, suggesting the involvement of a plasmid. The stabilized strains were refractory to the curing agents. There was no detectable change in the quantity or quality of the S. typhimurium cryptic plasmid. The Trz(r) phenotype of the CTS strains suggested that Trz(r) mutations were of the stable TrzA type. It is suggested that correction of the Cym(-) lesions in CTS strains results from an insertion within the cysCDHIJ gene cluster of a DNA species originating in the cysALKptsHI region of the S. typhimurium chromosome.     

Thermosensitivity of naphthalene biodegradation plasmids

The object of the work was to study the functional expression of naphthalene and salicylic acid catabolism systems and the stability of naphthalene biodegradation plasmids NAH, pBS2, pBS3 and NPL-41 in Pseudomonas aeruginosa PAO. The catabolic systems of the plasmids were shown to be thermosensitive, with a slight variation between one another. The plasmids became unstable at a high temperature; the temperature of effective elimination was 41 degrees C for plasmids NPL-41 and pBS3, and 42 degrees C for plasmids NAH and pBS2. NAH and pBS2 produced a weak inhibiting effect while NPL-41 and pBS3 caused a strong inhibition of the PAO strain growth at 42 degrees C. As a result, many anomalous filamentous cells (partly in the state of lysis) appeared in the cultural broth. Only PAO cells that had lost their plasmid were capable of normal growth in a medium with MPA at an elevated temperature; this creates a convenient system for selection of clones that have lost the plasmids of naphthalene biodegradation. Some of these plasmids can inhibit growth of Pseudomonas strains at an elevated temperature; this fact should be taken into account when the capability of Pseudomonas to grow at a high temperature is used as a taxonomic feature.     

Transmission and expression of mutations to nalidixic acid resistance among products of protoplast fusion crosses of Candida albicans

Earlier studies suggested that heritable resistance to nalidixic acid (Nal) induced in the asexual, pathogenic yeast Candida albicans by growth on Nal results from mitochondrial mutation. To determine conclusively whether mutations to Nal resistance are cytoplasmic or nuclear, several stable Nal-resistant (Nalr) mutants exhibiting distinctive differences in degrees of Nal resistance were obtained from each of two doubly auxotrophic strains (Ade-, Thr- and Arg-, His-), both derived from the same wild-type stock. Inheritance of Nal resistance was then assessed in a series of protoplast fusion crosses between complementing auxotrophs. The initial, intact cellular products of a fusion cross are prototrophic heterokaryons which frequently assort single parental nuclei into monokaryotic blastospores containing biparental cytoplasms. Occasional karyogamy within heterokaryons also yields prototrophic hybrid monokaryons which can undergo recombinations for chromosomal markers through spontaneous or induced mitotic crossing-over. Segregation and expression of Nal resistance among non-hybrid, parental-type monokaryons from Nalr X Nals heterokaryons showed that Nalr mutations are nuclear and that their expressions are not noticeably affected by admixture of cytoplasms of sensitive and resistant parental strains. Analyses of heterokaryons and hybrid monokaryons from Nalr X Nals and Nalr X Nalr crosses demonstrated that Nal resistance is recessive to sensitivity, and that independent Nalr mutations arise at one gene in the Ade-, Thr- strain and at a separate, complementing single gene in the Arg-, His- strain. Prior work demonstrated that induction of Nalr mutations in wild-type C. albicans depends profoundly on the (i) carbon and nitrogen, (ii) growth temperature, (iii) contact with particular metabolic inhibitors and (iv) division stage of cells during exposure to Nal. The present observations indicate that the character of cellular auxotrophies can determine the genetic loci at which Nalr mutations can be recovered.     

Biodegradation of petroleum hydrocarbons by psychrotrophic Pseudomonas strains possessing both alkane (alk) and naphthalene (nah) catabolic pathways.

Three hydrocarbon-degrading psychrotrophic bacteria were isolated from petroleum-contaminated Arctic soils and characterized. Two of the strains, identified as Pseudomonas spp., degraded C5 to C12 n-alkanes, toluene, and naphthalene at both 5 and 25 degrees C and possessed both the alk catabolic pathway for alkane biodegradation and the nah catabolic pathway for polynuclear aromatic hydrocarbon biodegradation. One of these strains contained both a plasmid slightly smaller than the P. oleovorans OCT plasmid, which hybridized to an alkB gene probe, and a NAH plasmid similar to NAH7, demonstrating that both catabolic pathways, located on separate plasmids, can naturally coexist in the same bacterium.