Variation in scorpion metabolic rate and rate-temperature relationships: implications for the fundamental equation of the metabolic theory of ecology.

The fundamental equation of the metabolic theory of ecology (MTE) indicates that most of the variation in metabolic rate are a consequence of variation in organismal size and environmental temperature. Although evolution is thought to minimize energy costs of nutrient transport, its effects on metabolic rate via adaptation, acclimatization or acclimation are considered small, and restricted mostly to variation in the scaling constant, b(0). This contrasts strongly with many conclusions of evolutionary physiology and life-history theory, making closer examination of the fundamental equation an important task for evolutionary biologists. Here we do so using scorpions as model organisms. First, we investigate the implications for the fundamental equation of metabolic rate variation and its temperature dependence in the scorpion Uroplectes carinatus following laboratory acclimation. During 22 days of acclimation at 25 degrees C metabolic rates declined significantly (from 127.4 to 78.2 microW; P = 0.0001) whereas mean body mass remained constant (367.9-369.1 mg; P = 0.999). In field-fresh scorpions, metabolic rate-temperature (MRT) relationships varied substantially within and among individuals, and therefore had low repeatability values (tau = 0.02) and no significant among-individual variation (P = 0.181). However, acclimation resulted in a decline in within-individual variation of MRT slopes which subsequently revealed significant differences among individuals (P = 0.0031) and resulted in a fourfold increase in repeatability values (tau = 0.08). These results highlight the fact that MRT relationships can show substantial, directional variation within individuals over time. Using a randomization model we demonstrate that the reduction in metabolic rate with acclimation while body mass remains constant causes a decline both in the value of the mass-scaling exponent and the coefficient of determination. Furthermore, interspecific comparisons of activation energy, E, demonstrated significant variation in scorpions (0.09-1.14 eV), with a mean value of 0.77 eV, significantly higher than the 0.6-0.7 eV predicted by the fundamental equation. Our results add to a growing body of work questioning both the theoretical basis and empirical support for the MTE, and suggest that alternative models of metabolic rate variation incorporating explicit consideration of life history evolution deserve further scrutiny.

Some aspects of the SOS response system--a critical survey.

The SOS system and SOS mutagenesis are frequently studied, or exploited to obtain an increase in mutagenicity of bacteria. Here a short survey is made of the phenomenon of SOS response with special attention to latest and less discussed data, especially the induction of the SOS system in response to cell starvation or mutation of certain genes and the role of inducible DNA polymerases.

A new look at adaptive mutations in bacteria.

This is a short survey of the adaptive mutation processes that arise in non- or slowly-dividing bacterial cells and includes: (i) bacterial models in which adaptive mutations are studied; (ii) the mutagenic lesions from which these mutations derive; (iii) the influence of DNA repair processes on the spectrum of adaptive mutations. It is proposed that in starved cells, likely as during the MFD phenomenon, lesions in tRNA suppressor genes are preferentially repaired and no suppressor tRNAs are formed as a result of adaptive mutations. Perhaps the most provocative proposal is (iv) a hypothesis that the majority of adaptive mutations are selected in a pre-apoptotic state where the cells are either mutated, selected, and survive, or they die.

Effect of Tn10/Tn5 transposons on the survival and mutation frequency of halogen light-irradiated AB1157 Escherichia coli K-12.

We show here that the Tn10/Tn5 transposon when inserted into the chromosome of strain AB1157 makes the bacteria more sensitive to and less mutable by halogen light irradiation. These effects are most probably caused by depletion of UmuD and UmuC proteins since: (i) transformation of the transposon-bearing bacteria with plasmids harbouring umuD'C (or umuDC) leads to recovery of the original survival and mutation frequencies; (ii) insertion of Tn10/Tn5 into chromosomal DNA has no effect on the level of mutation induced by ethyl methane-sulphonate treatment, a mutagen whose activity is umuDC-independent; (iii) the decline in survival is in about the same range for Tn10-bearing bacteria as for bacteria with deleted umuDC. However, whereas transformation of bacteria deleted in umuDC with plasmids carrying umuD'C/umuDC leads to full recovery of halogen light-induced mutability, recovery of survival is poor. This suggests that the mechanisms leading to umuDC-dependent mutagenesis and umuDC-dependent protection of cell survival are different. None of these effects occurs in bacteria bearing the Tn9 transposon in their DNA.

DNA mutagenesis and repair in UV-irradiated E. coli K-12 under condition of mutation frequency decline.

This paper shows that mutation frequency decline (MFD) occurs in UV-irradiated and transiently starved Escherichia coli K-12 strain AB1157. This effect involves preferential repair of pre-mutagenic lesions situated on the transcribed strand, and is mfd-, and uvrA-dependent. Mutations were tested by measuring reversion of argE3(ochre) to Arg+ and the types of reversions and the mutational specificity were defined. UV-irradiation induces reversions to Arg+ predominantly by forming ochre suppressors, supB and, much more rarely, supE(ochre). In irradiated and then transiently starved cells, the ratio of supB/supE(ochre) is reversed, and supE(ochre) suppressors predominate. Back reversions at the argE3 site occurs only in bacteria defective in UvrABC-excinuclease. The introduction of a Tn10 transposon makes bacteria more sensitive to UV-irradiation and diminishes the frequency of reversions. Transformation with pGW2123, a umuD'C-bearing plasmid, recovers and enhances the frequency of reversion but has no effect on post-UV-survival of the bacteria.

Mutation frequency decline in MMS-treated Escherichia coli K-12 mutS strains.

It has been shown that the decline in mutant frequency (MFD) (argE3(ochre)-->Arg+) which occurs in MMS-treated and then transiently starved AB1157 Escherichia coli K-12 cells concerns revertants which arose by supL suppressor formation in a process which is umuDC dependent. Here we have examined whether MMS-induced Arg+ revertants are susceptible to decline when bacteria are deficient in mismatch repair. We show that there is an absence of MFD in MMS-treated M1 (mutS) and in EC2416 (mutS delta umuDC) cells defective in mismatch repair which is associated with a change in the spectrum of MMS-induced mutations formed. In contrast to AB1157, transformation of M1 (mutS) bacteria with plasmids harbouring various combinations of umuD(D')C genes does not enhance the level of MMS-induced mutations but may influence the proportion of supL mutations. These supL mutations show MFD. Repair processes under MFD conditions were confirmed by analysis of plasmid DNA isolated from MMS-treated bacteria at different stages of their starvation and digestion with Fpg protein.

Mutation induction and mutation frequency decline in halogen light-irradiated Escherichia coli K-12 AB1157 strains.

The effects of halogen light irradiation on reversion of argE3-->Arg+ in E. coli K12 strain AB1157 and its mfd- mutant, and on mutation frequency decline (MFD) after transiently incubating irradiated bacteria under non-growing conditions were studied. The induction of mutations, the mutational specificity, and the MFD effect had the same characteristic features as those seen in E. coli B strains after irradiation with 254 nm UV light. MFD which is due to repair of premutagenic lesion in the transcribed strand of glnU gene and prevents mutations leading to supB formation, was not observed in halogen light-induced mutations in the mfd-1 strain. Overproduction of UmuD'C proteins led to a large increase in mutation frequency, which was much greater in mfd- than in mfd+ strains. In bacteria irradiated with halogen light and incubated immediately in a rich medium to express mutations, the formation of supB predominated strongly over that of supE(ochre) in mfd- cells but was at a similar level in mfd+ cells. Introduction of zcf117::Tn10 to AB1157 strain makes cells more sensitive to halogen light irradiation, whereas introduction of mfd-1 does not.

MMS-induced mutagenesis and DNA repair in Escherichia coli dnaQ49: contribution of UmuD' to DNA repair.

dnaQ-encoded epsilon subunit of DNA polymerase III, possesses 3',5' exonuclease (proofreading) activity, and is a fidelity factor of polymerase III holoenzyme. It is assumed that during SOS-induced mutagenesis, UmuD', UmuC, and RecA may suppress DnaQ proofreading activity, and allow for translesional DNA synthesis at the cost of fidelity of replication. In this report SOS-dependent, MMS-induced mutagenesis and DNA repair were tested in E. coli dnaQ49 strains. Bacteria were transformed with various pDNAs harboring compilation of the umuD(D')C genes, and the influence of plasmids on mutagenesis (argE3-->Arg+) and DNA repair was tested. DNA damage and repair were tested in plasmid DNA grown in MMS-treated bacteria and isolated either immediately after MMS treatment, or after starving the cells (MFD conditions) for 30 and 60 min, then nicking activity of Fpg protein on plasmid DNAs was analyzed. It has been found that (i) repair of MMS-induced lesions depends on umuD'C, umuD' (and to much less degree, on umuDC) genes encoded in pDNA; (ii) MMS-induced mutations, in contrast to DNA repair, are highest in the cells transformed with pDNA harboring umuDC, and lowest or zero in cells with plasmids harboring umuD'C. It is postulated that UmuD'C or UmuD' proteins play a role in the repair of damaged DNA and/or in maintenance of DNA integrity. The kinetics of these processes (perhaps due to introducing too many of the lesions) seems to be different in E. coli dnaQ+ and dnaQ cells, and probably this is a reason that (iii) MMS-induced mutations in dnaQ49 strains are not subject to MFD.

Conformation of plasmid DNA from Escherichia coli deficient in the repair systems protecting DNA from 8-oxyguanine lesions.

8-Oxyguanine (8ohG) is a major oxidation product of guanine and a biomarker of oxidative stress in mammal. We have attempted to estimate the level of 8ohG residues in plasmid DNA (pGW2123 and pBR322) grown in various bacterial strains (fpg, mutY, or mutT, plus mutT fpg and mutT mutY double mutants) differing in the system protecting cells against the mutagenic effects of 8ohG in DNA. The method was based on digestion of plasmid DNA with Fpg, and agarose gel electrophoresis. Fpg converts pDNA from covalently closed circular to the open circular (ccc-->oc) form of pDNA when there is at least one 8ohG, or apurinic site, per ccc pDNA molecule. It was found that: i) the content of 8ohG in pDNA grown in any of the tested bacteria is below one 8ohG per 10(4) base pairs; ii) a substantial part of pGW2123 is isolated from the bacteria in the oc form; iii) the ratio of oc/ccc in pGW2123 depends on the bacterial host and is the lowest when the plasmid was harvested from mutY- deficient cells; iv) pBR322, unlike pGW2123, is isolated predominantly in the ccc form; and v) of the pBR322 grown in the tested bacteria apparently the most resistant to Fpg digestion was pBR322 grown in the mutY strain. It is proposed that this reflects the compact structure of pDNAs when they are grown in bacteria deficient in mutY gene product.

The frequency of MMS-induced, umuDC-dependent, mutations declines during starvation in Escherichia coli.

It has been found that the level of methyl methanesulfonate (MMS)-induced mutation in Escherichia coli is dependent on the level of UmuD(D')C proteins. The frequency of argE(ochre)-->Arg+ mutations (which occur predominantly by AT-->TA transversions) and RifS-->RifR mutations is much higher when UmuDC or UmuD'C are overproduced in the cell. When MMS-treated bacteria were starved for progressively longer times and hence the expression of mutations delayed, the level of mutations observed progressively declined. This same treatment had no effect on the degree of SOS induction. Examination of plasmid DNAs, isolated from MMS-treated cells, for their sensitivity to the specific endonucleases Fpg and Nth revealed that MMS causes formation of abasic sites, which are repaired during cell starvation. It is assumed that, in non-dividing cells, apurinic sites are mostly repaired by RecA-mediated recombinational repair. This pathway, which is error-free, is compared with the processing pathway in metabolically active cells, where translesion synthesis by the UmuD'2C-RecA-DNA polymerase III holoenzyme complex occurs; this latter pathway is error-prone.

Some aspects of EMS-induced mutagenesis in Escherichia coli.

AB2497 and its mutS and umuDC derivatives were EMS-treated at the stationary phase and specificity of mutation measured. It was found that: (i) in mutS+ cells EMS induces predominantly GC-->AT transitions (by supB or supE(oc) formation) and in mutS- cells mainly AT-->TA transversions (by supL(NG) formation); (ii) transversions of AT-->TA are umuDC-dependent and mutational specificity is biased towards AT-->GC transitions in mutS- umuDC- strains. When mutS- umuDC- cells were transfected with plasmids bearing umuD'C or umuDC genes, mutational specificity was again biased towards AT-->TA transversions; (iii) experiments with bacteria bearing umuC::lacZ or recA::lacZ fusions suggest that processing of UmuD-->UmuD' might be poorer in EMS-treated mutS- than in mutS+ cells.

Effect of heat shock on expression of proteins not involved in the heat-shock regulon.

The effect of heat shock on the expression of some genes of Escherichia coli was tested. To avoid side effects, promoters of the genes were fused to lacZ and their expression measured by the level of beta-galactosidase. The results show that expression of umuC, recA and polB, after induction of the SOS response, was somewhat higher in the heat-shocked than in the non-shocked cells, whereas expression of ada, alkB and alkA genes, after induction of the adaptive response, was about the same. Unexpectedly, it was found that expression of lacZ from its own promoter was drastically lowered in the heat-shocked cells. This effect, however, seems not to be dependent on the induction of heat-shock proteins.

Is the tRNA ochre suppressor supX derived from gltT?

Some of the argE3-->Arg+ revertants show supX suppressor activity. The genetic relationship of supX is not yet known but the evidences are presented, that supX does not derive from gltT encoding tRNA(Glu)UUG.

Mutagenesis of Escherichia coli: a method for determining mutagenic specificity by analysis of tRNA suppressors.

A method for estimating mutagenic specificity in Escherichia coli (argE3, hisG4, thr-1, supE44), based upon the isolation of Arg+ or His+ revertants and identification of tRNA suppressors, is described. The method gives an insight not only into mutagenic pathways but also into the functioning of tRNA. With N-methyl-N'-nitro-N-nitrosoguanidine, 98% of mutations are GC----AT transitions. With N4-hydroxycytidine, 100% are AT----GC transitions. With hydroxylamine, apart from GC----AT transitions, approximately 30% of Arg+ revertants are formed by GC (or AT)----TA transversions. When the chemistry of the mutagenic attack is known, the method allows us to discriminate whether mutations occur on the transcribed or non-transcribed strands of DNA. It has been found that reversion of argE3 to Arg+ is a better monitor of mutagenic pathways than reversion of hisG4 to His+.

Base-pairing models to account for the mutagenicity and specificity of the purine analog 2-amino-N6-hydroxyadenine.

Potential base-pairing mechanisms of the purine analog, 2-amino-N6-hydroxyadenine (AHA), with the natural bases of DNA are presented. Base-pairing properties of this analog indicate that AHA may induce transitions and transversions of base pairs in DNA.

Alternative pathways of methyl methanesulfonate-induced mutagenesis in Escherichia coli.

Methyl methanesulfonate (MMS) induced mutagenesis is known to be largely dependent on functional umuCD and recA genes. By phenotypic analysis of Arg+ (argE3, ochre) revertants according to their reversion of the mutations his-4 (ochre) and thr-1 (amber), we attempted to deduce the specificity and/or sites of MMS-induced mutations. It is shown that: (1) MMS-induced, umuC-dependent Arg+ revertants (which prevail in bacteria proficient in mismatch repair) result from a different mutational pathway from umuC-independent ones. UmuC-dependent Arg+ revertants belong to class 2 (Arg+His+Thr-), and umuC-independent ones to class 1 (Arg+His-Thr-). (2) The mismatch repair system very efficiently prevents mutations induced by MMS. We found that in the mutS strain, deficient in mismatch repair, class 1 Arg+ revertants are the most numerous, whereas class 2 Arg+ revertants occur at similar levels in MMS-treated mutS and mutS+ strains. Therefore the mismatch repair system very efficiently prevents formation of umuC-independent Arg+ revertants, but exerts negligible or no effect on umuC-dependent Arg+ revertants. (iii) Both mutS umuC and mutS recA strains, are highly mutable by MMS.

Influence of dam and mismatch repair system on mutagenic and SOS-inducing activity of methyl methanesulfonate in Escherichia coli.

In contrast to earlier reports (Mohn et al., 1980; Glickman, 1982), we show that E. coli dam- cells are able to mutate following MMS treatment. Since the mutagenicity of MMS has been regarded as largely dependent on induction of the SOS functions, E. coli strains bearing the recA::lacZ or umuC::lacZ fusions were used to determine the ability of MMS to induce the SOS functions in the various dam+ and dam- strains. The mutagenicity of MMS was also tested in several of these strains. The results show that (i) there is no direct correlation between SOS-inducing ability and mutagenicity potency of MMS; and (ii) most of the premutagenic lesions induced by MMS are removed from DNA of dam+ or dam- cells by the mismatch repair system. The role of strand breaks in repair of mismatches induced by alkylating agents is discussed.