Genetic fidelity under harsh conditions: analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius.

Microbes whose genomes are encoded by DNA and for which adequate information is available display similar genomic mutation rates (average 0.0034 mutations per chromosome replication, range 0.0025 to 0.0046). However, this value currently is based on only a few well characterized microbes reproducing within a narrow range of environmental conditions. In particular, no genomic mutation rate has been determined either for a microbe whose natural growth conditions may extensively damage DNA or for any member of the archaea, a prokaryotic lineage deeply diverged from both bacteria and eukaryotes. Both of these conditions are met by the extreme thermoacidophile Sulfolobus acidocaldarius. We determined the genomic mutation rate for this species when growing at pH 3.5 and 75 degrees C based on the rate of forward mutation at the pyrE gene and the nucleotide changes identified in 101 independent mutants. The observed value of about 0.0018 extends the range of DNA-based microbes with rates close to the standard rate simultaneously to an archaeon and to an extremophile whose cytoplasmic pH and normal growth temperature greatly accelerate the spontaneous decomposition of DNA. The mutations include base pair substitutions (BPSs) and additions and deletions of various sizes, but the S. acidocaldarius spectrum differs from those of other DNA-based organisms in being relatively poor in BPSs. The paucity of BPSs cannot yet be explained by known properties of DNA replication or repair enzymes of Sulfolobus spp. It suggests, however, that molecular evolution per genome replication may proceed more slowly in S. acidocaldarius than in other DNA-based organisms examined to date.

Characterization of intragenic recombination in a hyperthermophilic archaeon via conjugational DNA exchange.

Sulfolobus acidocaldarius is so far the only hyperthermophilic archaeon in which genetic recombination can be assayed by conjugation and simple selections. Crosses among spontaneous pyr mutants were able to resolve closely spaced chromosomal mutations, identify deletions and rearrangements, and map mutations to a given deletion interval. Frameshift mutations in pyrE exerted polar effects that depressed orotidine-5'-monophosphate decarboxylase activity (encoded by pyrF), whereas base pair substitutions and an 18-bp deletion had no effect.

The question of DNA repair in hyperthermophilic archaea.

Hyperthermophilic archaea grow at temperatures that destabilize the primary structure of DNA and in evolutionary terms they are highly divergent from other well studied microorganisms. These prokaryotes should therefore require DNA damage repair to be unusually effective, and could employ novel mechanisms for this repair. Recent genome sequence analyses and biochemical and genetic assays suggest a distribution of DNA repair strategies that raises intriguing questions for future study.

UV stimulation of chromosomal marker exchange in Sulfolobus acidocaldarius: implications for DNA repair, conjugation and homologous recombination at extremely high temperatures.

The hyperthermophilic archaeon Sulfolobus acidocaldarius exchanges and recombines chromosomal markers by a conjugational mechanism, and the overall yield of recombinants is greatly increased by previous exposure to UV light. This stimulation was studied in an effort to clarify its mechanism and that of marker exchange itself. A variety of experiments failed to identify a significant effect of UV irradiation on the frequency of cell pairing, indicating that subsequent steps are primarily affected, i.e., transfer of DNA between cells or homologous recombination. The UV-induced stimulation decayed rather quickly in parental cells during preincubation at 75 degrees, and the rate of decay depended on the incubation temperature. Preincubation at 75 degrees decreased the yield of recombinants neither from unirradiated parental cells nor from parental suspensions subsequently irradiated. We interpret these results as evidence that marker exchange is stimulated by recombinogenic DNA lesions formed as intermediates in the process of repairing UV photoproducts in the S. acidocaldarius chromosome.

Hyperthermophiles and the problem of DNA instability.

Rates of chemical decomposition of DNA at the optimal growth temperatures of hyperthermophiles seem incongruent with the requirements of accurate genome replication. The peculiar physiology, ecology and phylogeny of hyperthermophiles combine to suggest that these prokaryotes have solved a molecular problem (spontaneous loss of native DNA structure) of a magnitude that well-studied microorganisms do not face. The failure of DNA base composition to correlate with optimal growth temperature among hyperthermophiles provides indirect evidence that other mechanisms maintain their chromosomal DNA in the duplex form. Studies in vitro indicate that DNA primary structure is more difficult to maintain at extremely high temperature than is secondary structure, yet hyperthermophiles exhibit only modest levels of spontaneous mutation. Radiation sensitivity studies also indicate that hyperthermophiles repair their DNA efficiently in vivo, and underlying mechanisms are beginning to be examined. Several enzymes of DNA metabolism from hyperthermophilic archaea exhibit unusual biochemical features that may ultimately prove relevant to DNA repair. However, genomic sequencing results suggest that many DNA repair genes of hyperthermophilic archaea may not be recognized because they are not sufficiently related to those of well-studied organisms.

Cyclopropane ring formation in membrane lipids of bacteria.

It has been known for several decades that cyclopropane fatty acids (CFAs) occur in the phospholipids of many species of bacteria. CFAs are formed by the addition of a methylene group, derived from the methyl group of S-adenosylmethionine, across the carbon-carbon double bond of unsaturated fatty acids (UFAs). The C1 transfer does not involve free fatty acids or intermediates of phospholipid biosynthesis but, rather, mature phospholipid molecules already incorporated into membrane bilayers. Furthermore, CFAs are typically produced at the onset of the stationary phase in bacterial cultures. CFA formation can thus be considered a conditional, postsynthetic modification of bacterial membrane lipid bilayers. This modification is noteworthy in several respects. It is catalyzed by a soluble enzyme, although one of the substrates, the UFA double bond, is normally sequestered deep within the hydrophobic interior of the phospholipid bilayer. The enzyme, CFA synthase, discriminates between phospholipid vesicles containing only saturated fatty acids and those containing UFAs; it exhibits no affinity for vesicles of the former composition. These and other properties imply that topologically novel protein-lipid interactions occur in the biosynthesis of CFAs. The timing and extent of the UFA-to-CFA conversion in batch cultures and the widespread distribution of CFA synthesis among bacteria would seem to suggest an important physiological role for this phenomenon, yet its rationale remains unclear despite experimental tests of a variety of hypotheses. Manipulation of the CFA synthase of Escherichia coli by genetic methods has nevertheless provided valuable insight into the physiology of CFA formation. It has identified the CFA synthase gene as one of several rpoS-regulated genes of E. coli and has provided for the construction of strains in which proposed cellular functions of CFAs can be properly evaluated. Cloning and manipulation of the CFA synthase structural gene have also enabled this novel but extremely unstable enzyme to be purified and analyzed in molecular terms and have led to the identification of mechanistically related enzymes in clinically important bacterial pathogens.

Genetic responses of the thermophilic archaeon Sulfolobus acidocaldarius to short-wavelength UV light.

The archaea which populate geothermal environments are adapted to conditions that should greatly destabilize the primary structure of DNA, yet the basic biological aspects of DNA damage and repair remain unexplored for this group of prokaryotes. We used auxotrophic mutants of the extremely thermoacidophilic archaeon Sulfolobus acidocaldarius to assess genetic and physiological effects of a well-characterized DNA-damaging agent, short-wavelength UV light. Simple genetic assays enabled quantitative dose-response relationships to be determined and correlated for survival, phenotypic reversion, and the formation of genetic recombinants. Dose-response relationships were also determined for survival and phenotypic reversion of the corresponding Escherichia coli auxotrophs with the same equipment and procedures. The results showed S. acidocaldarius to be about twice as UV sensitive as E. coli and to be equally UV mutable on a surviving-cell basis. Furthermore, UV irradiation significantly increased the frequency of recombinants recovered from genetic-exchange assays of S. acidocaldarius. The observed UV effects were due to the short-wavelength (i.e., UV-C) portion of the spectrum and were effectively reversed by subsequent illumination of S. acidocaldarius cells with visible light (photoreactivation). Thus, the observed responses are probably initiated by the formation of pyrimidine dimers in the S. acidocaldarius chromosome. To our knowledge, these results provide the first evidence of error-prone DNA repair and genetic recombination induced by DNA damage in an archaeon from geothermal habitats.

Rates of spontaneous mutation in an archaeon from geothermal environments.

To estimate the efficacy of mechanisms which may prevent or repair thermal damage to DNA in thermophilic archaea, a quantitative assay of forward mutation at extremely high temperature was developed for Sulfolobus acidocaldarius, based on the selection of pyrimidine-requiring mutants resistant to 5-fluoro-orotic acid. Maximum-likelihood analysis of spontaneous mutant distributions in wild-type cultures yielded maximal estimates of (2.8 +/- 0.7) x 10(-7) and (1.5 +/- 0.6) x 10(-7) mutational events per cell per division cycle for the pyrE and pyrF loci, respectively. To our knowledge, these results provide the first accurate measurement of the genetic fidelity maintained by archaea that populate geothermal environments. The measured rates of forward mutation at the pyrE and pyrF loci in S. acidocaldarius are close to corresponding rates reported for protein-encoding genes of Escherichia coli. The normal rate of spontaneous mutation in E. coli at 37 degrees C is known to require the functioning of several enzyme systems that repair spontaneous damage in DNA. Our results provide indirect evidence that S. acidocaldarius has cellular mechanisms, as yet unidentified, which effectively compensate for the higher chemical instability of DNA at the temperatures and pHs that prevail within growing Sulfolobus cells.

Exchange of genetic markers at extremely high temperatures in the archaeon Sulfolobus acidocaldarius.

When cells of two auxotrophic mutants of Sulfolobus acidocaldarius are mixed and incubated on solid medium, they form stable genetic recombinants which can be selected, enumerated, and characterized. Any of a variety of auxotrophic markers can recombine with each other, and the phenomenon has been observed at temperatures of up to 84 degrees C. The ability to exchange and recombine chromosomal markers appears to be an intrinsic property of S. acidocaldarius strains. It occurs between two cell lines derived from the same parent or from different parents and also between a recombinant and its parent. This is the first observation of chromosomal marker exchange in archaea from geothermal environments and provides the first functional evidence of generalized, homologous recombination at such high temperatures.

Sulfolobus acidocaldarius synthesizes UMP via a standard de novo pathway: results of biochemical-genetic study.

A genetic approach was used to establish the route of UMP biosynthesis in Sulfolobus acidocaldarius, a member of the hyperthermophilic division (the Crenarchaeota) of the Archaea domain. Pyrimidine auxotrophs of S. acidocaldarius DG6 were isolated by direct selection and by brute-force methods. Enzymatic assay of extracts from wild-type S. acidocaldarius, from pyrimidine auxotrophs, and from phenotypic revertants demonstrated that S. acidocaldarius synthesizes UMP via orotate in six enzymatic steps corresponding to the de novo pathway of other organisms. The results also show that a single carbamoyl phosphate synthetase supplies both the pyrimidine and arginine pathways of this organism. To gain similar insight into pyrimidine salvage pathway(s), prototrophic mutants resistant to toxic pyrimidine analogs were also isolated and characterized. The results suggest that a single class of mutants which had acquired elevated resistance to four different 5-fluoropyrimidines had been isolated. These fluoropyrimidine-resistant mutants appear to have a regulatory defect leading to overproduction of one or more endogenous pyrimidine compounds.

Cyclopropane fatty acid synthase of Escherichia coli: deduced amino acid sequence, purification, and studies of the enzyme active site.

Cyclopropane fatty acid (CFA) synthase of Escherichia coli catalyzes a modification of the acyl chains of phospholipid bilayers. We report (i) identification of the CFA synthase protein, (ii) overproduction (> 600-fold) and purification to essential homogeneity of the enzyme, and (iii) the amino acid sequence of CFA synthase as deduced from the nucleotide sequence of the cfa gene. CFA synthase was overproduced by use of the T7 promoter/RNA polymerase system under closely defined conditions. The enzyme was readily purified by a two-step procedure requiring only ammonium sulfate fractionation and binding to phospholipid vesicles followed by flotation in sucrose density gradients. The deduced amino acid sequence predicts a protein of 43,913 Da (382 residues) that lacks long hydrophobic segments. The CFA synthase sequence has no significant similarity to known proteins except for sequences found in other enzymes that utilize S-adenosyl-L-methionine. We also report inhibitor studies of the enzyme active site.

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.

Phenotypic characterization of the archaebacterial genus Sulfolobus: comparison of five wild-type strains.

Though amenable to routine manipulation and a popular subject of molecular genetic and biochemical studies on archaebacteria, the genus Sulfolobus has remained poorly described in phenotypic terms. To delineate their physiological capabilities and diversity, five laboratory strains, including type strains of the described species Sulfolobus acidocaldarius and S. solfataricus, were compared with respect to a variety of growth and biochemical parameters, including component profile of the surface-layer cell wall, inhibitors of growth, growth rate as a function of temperature and pH, and compounds used as sole sources of carbon or nitrogen. Motility and photoregulated production of an orange pigment were detected in all five strains tested. The results provide new criteria for distinguishing Sulfolobus strains as well as potential tools for the physiological and genetic manipulation of these extreme thermophiles.

Temperature-sensitive murein synthesis in an Escherichia coli pdx mutant and the role of alanine racemase.

The basis for disruption of morphogenesis by depletion of pyridoxine derivatives was studied using a pdxH null mutant of Escherichia coli K-12. Removal of pyridoxal from growing cultures severely inhibited murein synthesis in vivo, whereas simultaneous supplementation with D-alanine effectively prevented inhibition. Extractable alanine racemase was low following such starvation. Selection of mutants overcoming the glycine- or temperature-sensitivity imposed by pyridoxine limitation yielded a variety of phenotypes. The most effective of these extragenic suppressors conferred an elevated alanine racemase activity which was resistant to the effects of pyridoxal removal.

Characterization of Escherichia coli mutants completely defective in synthesis of cyclopropane fatty acids.

The synthesis of cyclopropane fatty acids (CFA) in bacteria represents a biochemically and physiologically unique membrane modification whose importance for the cell remains unknown, despite extensive study of a Cfa- mutant of Escherichia coli and of the cloned cfa gene. Recently we reported the isolation of new Cfa- mutants (D. W. Grogan and J. E. Cronan, Jr., Mol. Gen. Genet. 196:367-372, 1984). Molecular-genetic and biochemical analysis indicated that these were null mutants of the E. coli cfa locus which were formed by inversions of a chromosomal segment. Isogenic Cfa+ and Cfa- strains were constructed from one such mutant and subjected to various stress conditions. In nearly all cases, both strains responded equally, but certain treatments, such as repeated freezing and thawing, favored the survival of Cfa+ strains over Cfa- strains. Though not essential, CFA thus appeared to play some beneficial role (or roles) in the bacterial cell.

Cloning and manipulation of the Escherichia coli cyclopropane fatty acid synthase gene: physiological aspects of enzyme overproduction.

Like many other eubacteria, cultures of Escherichia coli accumulate cyclopropane fatty acids (CFAs) at a well-defined stage of growth, due to the action of the cytoplasmic enzyme CFA synthase. We report the isolation of the putative structural gene, cfa, for this enzyme on an E. coli-ColE1 chimeric plasmid by the use of an autoradiographic colony screening technique. When introduced into a variety of E. coli strains, this plasmid, pLC18-11, induced corresponding increases in CFA content and CFA synthase activity. Subsequent manipulation of the cfa locus, facilitated by the insertion of pLC18-11 into a bacteriophage lambda vector, allowed genetic and physiological studies of CFA synthase in E. coli. Overproduction of this enzyme via multicopy cfa plasmids caused abnormally high levels of CFA in membrane phospholipid but no discernable growth perturbation. Infection with phage lambda derivatives bearing cfa caused transient overproduction of the enzyme, although pL-mediated expression of cfa could not be demonstrated in plasmids derived from such phages. CFA synthase specific activities could be raised to very high levels by using cfa runaway-replication plasmids. A variety of physiological factors were found to modulate the levels of CFA synthase in normal and gene-amplified cultures. These studies argue against several possible mechanisms for the temporal regulation of CFA formation.

Genetic characterization of the Escherichia coli cyclopropane fatty acid (cfa) locus and neighboring loci.

The phenotypically silent cyclopropane fatty acid synthesis (cfa) gene of Escherichia coli K-12 has been located on the genetic linkage map. This was accomplished by integrating (via homologous recombination) the selectable marker of a recombinant plasmid into the host chromosome near the cfa locus. This integration allowed the subsequent isolation of a cfa-linked transposon Tn10 insertion. Genetic mapping of the Tn10 insertion, using conventional techniques, placed the cfa locus at min 36.5 on the linkage map in the vicinity of several other non-selectable markers. We ordered cfa and these other loci by three-factor transductional analyses. Selection for excision of the Tn10 element resulted in several types of mutants which harbor mutations of cfa and of neighboring genes, presumably as a consequence of Tn10-catalyzed chromosomal rearrangements.

Use of lambda phasmids for deletion mapping of non-selectable markers cloned in plasmids.

A nonselectable gene carried on a poorly selectable recombinant plasmid has been physically mapped by deletion analysis. Our method involved cloning the plasmid into a coliphage lambda vector and treating the recombinant phage with a chelator. Virtually all particles surviving this treatment carried large deletions within the plasmid insert. Further deletion analysis was done by inserting a selectable lambda sequence into one such deletion derivative and repeating the chelator selection. Chelator selection was also used to isolate deletions constructed in vitro. The deleted phage are readily characterized by restriction mapping, and the gene in question scored after infection of a mutant host strain. These techniques have enabled us to physically assign the cyclopropane fatty acid synthase gene of Escherichia coli to 0.8 kb of a 16-kb segment after characterizing only a small number of isolates. This approach should be generally useful in the mapping of plasmids for which no convenient method exists for selecting or scoring the gene in question.