and Michel Blot

Transposition of insertion sequences (IS) is an enzyme-mediated process that only occurs in a minority of cells within a bacterial culture. Transposition is thus a rare event, but transposition frequency may vary depending on experimental conditions. For instance in a rich broth, IS elements are known to transpose during stationary phase but not during exponential growth. Using a reporter system which involves the activation of the cryptic bgl operon in Escherichia coli, we show that the frequency of IS1 transposition is a function of glucose concentration in the growth medium, it is increased by streptomycin amounts that are below minimum inhibitory concentration (sub-MIC) and is inhibited in an rpsL150 strain with high translation accuracy. Since starved cells are known to enhance ribosome frameshifting, our data suggests that growth conditions applied in this study could affect IS1 transposition by increasing translation infidelity.


InTrodUCTIon
Transposable elements reside on the chromosomes of all sequenced bacteria and seem ubiquitous among many bacterial species (Mahillon & Chandler, 1998; http://www-is.biotoul.fr/is.html).Systematic studies on the spectrum of mutations have shown that insertion sequences (IS) provide a major contribution to spontaneous mutagenesis (Rodriguez et al., 1992;Hall, 1999a).The direct impact and the evolutionary role of IS elements have been extensively discussed because they were discovered as detrimental insertions, but they were also shown to generate mutations enhancing fitness (Chao et al., 1983;Chao & McBroom, 1985;Treves et al., 1998;Hall, 1999a;Cooper et al., 2001;Schneider et al., 2001).
Transposition of mobile genetic elements is an enzyme-mediated process requiring proteins expressed by the element itself (a transposase) and host genes as well.These host proteins include not only a polymerase and a ligase, but also other DNA binding proteins such as IHF, HU, HNS, Fis; the replication initiator DnaA; the protein chaperone/ protease ClpX, ClpP, and ClpA; the SOS control protein LexA; and the Dam DNA methylase (Mahillon & Chandler, 1998;Shiga et al., 2001), thus regulation of transposition depends on biochemical activities of the host.It has been proposed that transposition rates have evolved to be low enough to avoid a burden on the host genome so that IS elements may represent "not-too-harmful genetic parasites" (Orgel & Crick, 1980).This leads to a paradox at the population level as the transposition in itself is an enzymatically-driven process, but affects only a few individuals among billions of isogenic cells.How much this proportion is random or may depend on the biochemistry of the cells and their environment is a question of primary importance to understand the genetic adaptation to an environment.
Considering bacterial life styles, one can typically distinguish periods of abundance, when a bacterium grows as much as its genomic abilities allow, and periods of nutritional deprivation, when any mutation favouring continued growth would be advantageous.Thus, controlling mutation rates as a function of the growth regimen appears to be a reasonable strategy for the bacteria.For instance, it was shown that IS transposition increases with the length of bacterial starvation on agar plates (Hall, 1999b).IS transposition is not observed during exponential growth in rich media, but mutants become numerous when the medium is depleted or during exponential growth in a carbon-limited minimal medium (Noirclerc-Savoye & Blot, unpublished).
Utilization of β-glucosides in Escherichia coli is cryptic: most of the wild type strains exhibit the Arbutin/Salicin/Cellobiose negative (Arb -Sal -Cel -) phenotype although at least four genetic systems involved in β-glucoside utilization (including the arbT locus, the bgl, asc and cel operons) exist on the chromosome; surprisingly, they all are rendered silent.Spontaneous Arb + Sal + (Bgl + ) mutants are predominantly due to activation of the cryptic bgl operon (Reynolds et al., 1981).Mutational activation of the asc or cel operons also confers the Arb + Sal + phenotype, however, the Sal + phenotype conferred by asc is weak compared to that of bgl whereas the cel operon confers the Cel + phenotype in addition to Sal + .In contrast, mutations in arbT results only in the Arb + phenotype (Hall, 1998).The majority of the Bgl + mutants are due to transposition of IS elements into the bglR region, although mutations in unlinked loci such as hns, gyrA, bglJ and leuO can also activate the bgl operon (Defez & Felice, 1981;DiNardo et al., 1982;Giel et al., 1996;Ueguchi et al., 1998).
The main purpose of this study was to investigate the role of limiting and excess glucose concentrations in growth medium on transposition of E. coli K12 insertion sequences by using the bgl operon as a genetic reporter system.
In the study we shall show that (i) limiting or excess glucose concentration alters IS1 transposition; (ii) provoking translation infidelities increases the rate of IS1 transposition; and (iii) enhancing translation accuracy inhibits IS1 insertions into the reporter bgl operon, suggesting that growth conditions exert an effect on translation accuracy, and thus on mutability.
Construction of GBe150 strain carrying the rpsL150 mutation.Strain GBE150 was constructed by P1-transduction, in which the P1 phage was grown on the Ara -Str R donor MC4100 and P1 lysate obtained was transduced to the Ara + Str S recipient ZK126.Primary transductants were selected on LB agar plates supplied with 50 µg/ml streptomycin and 5 mM sodium citrate.One of the Str R Ara + transductant was referred to as GBE150 and used to confirm the presence of rpsL150 mutation by DNA nucleotide sequencing.In brief, oligonucleotide primers used to amplify the rpsL gene from the genomic DNA of MC4100, ZK126 and GBE150 were 5'acgttttattacgtgtttacg-3'; 5'-cgtttggccttacttaacgg-3'.PCR products were purified from the gel and sequenced from both ends.The strain was confirmed to carry rpsL150 mutation and was then used to study the role of the translation fidelity on transposition.
Isolation of Bgl + mutants and fluctuation test.In each experiment a set of 45 to 55 replicate cultures was made in M9 medium supplemented with 0.4% glucose (0.3 ml with about 10 3 cells) in capped microtitration plates (96 wells).The culture set was incubated at 37 o C with shaking (160 r.p.m.) until the OD 600 reached the value of 0.8 at which time 0.1 ml of each culture was plated on M9-arbutin.The cultures were then re-incubated for an additional period of 14.5 h during which the cultures entered in the stationary phase of growth.Another set of 0.1 ml aliquots were then removed from each culture and these were also plated on M9-arbutin.Plates from both the exponential and stationary phase were incubated at 37 o C for 48 h for the appearance of Bgl + mutants.Incubation of the plates was restricted to 48 h to prevent "post-plating" mutants (Hall, 1998).The plates from exponentially growing culture without any mutants were considered for studying the stationary phase-specific mutants, and the proportion of plates without colonies was used to estimate the mutation rate using the method of Lea and Coulson (1949).Nalidixic acid (Nal R ) mutants were isolated in the same manner as described above.They were used as controls to determine whether the altered glucose conditions had any effect on the rate of point mutations in the housekeeping gene gyrA.
Study of the bglR region.The primers used to amplify the bglR region were selected on the basis of the wild type E. coli bgl sequence (Schnetz et IS1 transposition at limiting and excess glucose concentrations al., 1987): 5'-cgatgagctggataaactgc-3';5'-cgctgttcatcatcaataacc-3'.The size of the PCR products distinguished insertion from non-insertion mutants.Insertion mutants were then processed for Southern analysis with internal probes of IS elements (Schneider et al., 2000).Probing with the IS1, IS2, and IS5 elements was sufficient to characterize all insertion mutants in this study.

Bacterial growth in various glucose concentrations
The ZK126 strain was tested for growth in M9 medium supplemented with a range of concentrations of glucose as a sole carbon source.Growth at 0.4% glucose gave a generation time of 90 min.Although at 4.0% glucose the cells grew faster, with a generation time of 70 min, the viable cell number of this culture was found to drop during the stationary phase by an order of magnitude.For this rea-sonthe 0.4% glucose concentration is referred to as the "standard" and 4% as an "excess" glucose concentration in our studies.However, 4% glucose was still very useful for studies of transposition.
Based on 1790 independent cultures, mutation rates were estimated before (exponential phase) and after (stationary phase) the cultures had reached the OD 600 = 0.8.The average mutation rate was 2.7 × 10 -8 mutation per cell per generation during exponential growth and 2.0 × 10 -8 per cell generation during stationary phase.

The bgl operon reporter genetic system: isolation and characterization of Bgl + mutants
The bgl operon (Fig. 1) of E. coli is a well studied cryptic genetic system that contains the genetic machinery for the uptake and metabolism of aryl βglucosides such as arbutin and salicin.Wild type E. coli strains exhibit a Bgl -phenotype and spontaneous mutants with the Arb + Sal + phenotype can be obtained on MacConkey Arbutin or MacConkey Salicin plates.Activating mutations can be linked or unlinked to the bgl operon.The majority of the linked mutations are due to transposition of insertion sequences into a regulatory element, the bglR region (Reynolds et al., 1981;Schnetz & Rak, 1992) whereas mutations in unlinked loci such as hns, gyrase, bglJ and leuO result in activation of the bgl operon (Defez & Felice, 1981;DiNardo et al., 1982;Giel et al., 1996;Ueguchi et al., 1998).Since most of the Bgl + mutants are a result of transposition events the activation of bgl operon by IS elements is assumed to be a good index of their mobility (Reynolds et al., 1981;Rodriguez et al., 1992;Schnetz & Rak, 1992;Moorthy & Mahadevan, 2002).The Arb + mutations could be due to not only activation of the bgl operon but also to mutations in the arbT locus (Arb + Sal -), activation of the asc operon giving a weak Sal + phenotype, and activation of the cel operon giving the Arb + Sal + Cel + phenotype.Mutations that were Sal -(arbT locus), weak Sal + (asc operon) or Cel + (cel operon) were eliminated from the study and only Arb + Sal + (bgl operon) mutants were used in further analysis.The Bgl + mutants described above were isolated from the ZK126 and GBE150 strains of E. coli K12.
A total of 792 independent Bgl + mutants were identified from standard, limiting and excess glucose concentrations during exponential and stationary phase growth.As the aim of this study was to investigate the role of limiting and excess glucose concentrations on the rate of transposition of IS elements, mutants displaying wild type PCR product in the bglR region (398 bp) (i.e.PCR products without insertions) were not used in further investigation.Insertional mutants displaying larger PCR products (760 to 1450 bp) were used in Southern hy- bridizations and were identified as IS1, IS2 and IS5 insertions.Of the 792 Bgl + mutants, 223 were due to transposition of an IS element, either IS1, IS5 or IS2 whereas the remaining 569 mutants were from non-IS insertion and could be due to point mutations in the CAP-cAMP binding box or due to mutations in unlinked loci.Amongst the 223 mutants carrying an IS element within the bglR region, the great majority of these were insertions of IS1 and in a few cases they were either IS2 or IS5.

Glucose concentration and IS1 transposition
Bacterial growth in liquid M9 cultures is limited by the concentration of glucose (Fig. 2).After preliminary trials, three concentrations were selected for the fluctuation tests: 0.4% as the "standard" glucose concentration, 0.1% as "limiting" and 4%as "ex-cess" glucose concentration.When compared with the standard glucose concentration, the mutation rate during exponential growth increased for strain ZK126 by 2-3 fold in the presence of excess glucose and 8-9 fold in the presence of limiting glucose concentrations (Table 1).A Z-test analysis of mutation rates indicated that these data were statistically significant (P < 0.05).A similar statistical analysis of mutant frequencies indicated that only the data with the excess glucose concentration were significant (P < 0.05).During the stationary phase, cultures growing with excess glucose concentration exhibited a reduced viability count by one log unit and therefore the effect of excess glucose concentration on the mutation rate could not be estimated for this sample.
To test if the mutation rates for spontaneous mutations are increased or decreased by different glucose concentrations, fluctuation analysis was carried out by isolating mutants of nalidixic acid resistant (Nal R ) phenotype, which is due to point mutations in the A subunit of DNA gyrase.Results obtained showed that the mutation rates for the Nal R phenotype for bacteria growing with different glucose concentrations were fairly similar (not shown), suggesting that the varied mutation rates for Bgl + mutants were likely due to altered physiology under these growth condition.
The proportion of IS1 transposition varied with the glucose concentration in the M9 medium (Table 2).The reference value for comparisons was 24% in the standard (0.4%) glucose grown ZK126 during exponential growth.The proportion of IS1 insertions into the bglR region increased up to 36% with limited glucose (0.1%), and it increased to 83% with excess glucose (4%) concentration.A parametric Chi-square analysis on IS1 versus non IS1 mutations indicated that the increase of IS1 transposition observed with limiting and excess glucose concen-   Lea & Coulson (1949).Analysis of the Z-test on the data indicated mutation rates were significantly increased at extreme glucose (P < 0.05) whereas the differences of mutation rate in ideal conditions but in two genetic backgrounds (wt and rpsL150) were not statistically significant (P < 0.05).
IS1 transposition at limiting and excess glucose concentrations trations was statistically significant (P < 0.05).Compared to that of the standard glucose concentration, the mutant frequency with limiting glucose was not statistically significant (shown in Table 1, Z-test with P < 0.05).These observations suggest that the glucose concentration has a strong impact on IS1 transposition.
During the stationary phase, the proportion of IS1 transpositions appeared to be unchanged with limiting and standard glucose concentrations, 18% and 22%, respectively, on the other hand it dropped from 22% to 7% for cultures grown with excess glucose concentration.

IS1 transposition induced by translation errors
Since at extreme glucose concentrations (limiting or excess) IS1 transposition was enhanced, we sought to test if this was a result of enhanced translation errors under these physiological conditions.To test this hypothesis streptomycin was provided in growth medium at concentrations that were sub- A Chi-square analysis of mutants with insertion versus non insertion of the ZK126 strain during the exponential phase indicated that the increase of IS1 transposition seen in excess glucose is highly significant at P < 0.05.

Table 3. distribution of IS1 transposition at sub-inhibitory concentrations of streptomycin.
Both ZK126 (wt) and GBE 150 (rpsL150) strains were grown in M9 and 0.4% glucose unless described in parentheses.a 0.156 and 1.250 µg/ml are respectively 1:32 and 1:4 of MIC.Sm, streptomycin.A Chi-square analysis on the data of ZK126 strain grown in standard glucose containing sub-MIC concentrations of streptomycin indicated that the increase of IS1 transposition frequency is highly significant at P < 0.05.

Strain
MIC (i.e., below the minimum inhibitory concentration).At concentrations above the MIC streptomycin blocks protein synthesis, whereas at sub-MIC concentrations the effect is only to reduce translation accuracy without affecting viability (Gorini, 1971;Ruusala & Kurland, 1984).The streptomycin MIC for ZK126 was 5 µg/ml (obtained by double dilution method), and concentrations of 0.156 and 1.25 µg/ml (corresponding to 32 and 4 times lower than the MIC) were used in the fluctuation test analysis.
Growth at these streptomycin concentrations had no effect on either culture doubling times or the number of viable cells in the exponential or stationary phase (not shown).
We compared the effect of sub-MIC streptomycin on the transposition of IS1 into the bglR region by growing bacteria with standard glucose concentrations and supplemented with streptomycin (0.156 µg/ml and 1.25 µg/ml) or by growing with limiting or excess glucose concentrations supplemented with 0.156 µg/ml streptomycin (Table 3).When strain ZK126 was grown in a medium with standard glucose without streptomycin the frequency of IS1 transpositions during exponential growth and stationary phase were comparable (24% and 22%), but transposition was enhanced during exponential growth depending on streptomycin amounts in growth medium, 62% and 79% for 0.156 and 1.25 µg/ml, respectively.A Chi-square analysis on these data indicated that the increase of IS1 transpositions was highly significant at P < 0.05.However, the effect of streptomycin was not significant during stationary phase.This may be due to the fact that IS1 transposition at the bglR region was lower (4% with 0.156 µg/ml streptomycin and 21% with 1.25 µg/ml).A Chi-square analysis of the data indicated that IS1 transpositions were not significantly increased.These results show that IS1 transposition is greatly increased by streptomycin only during exponential growth, i.e. when the ribosomes are more active, and that limiting or excess glucose concentration could lead to increased translation errors, which in turn is likely to result in an increase of IS1 transpositions under these physiological conditions.

Translation accuracy and IS1 transposition
Transposition of IS1 requires a programmed translational frameshift.The transposase of IS1 was produced as the InsA-B'-InsB fusion protein by a -1 frameshifting event (Sekine & Ohtsubo, 1989;Escoubas et al., 1991).Physical evidence for the synthesis of such InsAB', composed of domains derived from two consecutive reading frames: insA (phase 0) and insB' (phase -1) and demonstrated that the InsAB' protein stimulates IS1 transposition (Escoubas et al., 1994).To examine the role of translation fidelity in IS1 transposition, strain GBE150 was constructed by replacing the rpsL gene of ZK126 strain with the rpsL150 mutation from strain MC4100 conferring resistance to streptomycin (Materials and Methods).The nucleotide sequencing of the rpsL gene from MC4100 and GBE150 strains confirmed that both these strains carried the rpsL150 mutation as published in (Post & Nomura, 1980), but it differed from the rpsL gene of ZK126 by an AàG transition (Genbank accession number AF312716 and AF312717).This mutation resulted in a Lys42 to Arg change.An identical mutation described earlier as A60 or sm-5 was shown to result in enhanced translation accuracy (Breckenridge & Gorini, 1970;Funtasu et al., 1972).As expected, the rpsL150 mutation slightly increased the generation time of GBE150 (Levin et al., 2000).The mutation rates in GBE150 were reduced by a small fraction when compared to those of strain ZK126 grown under similar conditions.However, the reduction in the mutation rates observed in GBE150 was not statistically significant when examined by the Z-test (P < 0.05).The mutation rates reduced in rpsL150 strain are in agreement with the marginal drop of mutation rates for an rpsL999 strain documented by Boe (1992).
In contrast to the mutation rates, the presence of the rpsL150 allele caused a major reduction in IS1 transposition, it went down from 24% to 0% during the exponential phase for bacteria grown with standard glucose concentrations.This finding may suggest that in the rpsL150 mutant background translation fidelity is enhanced which in turn is likely to reduce the -1 programmed translation frameshift thus leading to a drop of IS1 transposition.However, this effect was confined only to exponentially growing bacteria and for unknown reason it disappeared during the stationary phase.
The effect of rpsL150 on IS1 transposition was also examined in the exponential phase with sub-MIC streptomycin concentrations, for which the cultures were grown with either limiting or excess glucose concentrations and supplemented with sub-MIC concentration of streptomycin (0.156 µg/ml) and fluctuation analysis was performed.Under these conditions the IS1 transpositions were restored from 0% (standard glucose) to 10% (0.1% limiting glucose) or 16% (4% excess glucose) (Table 3).These data suggest that the translation fidelity enhanced by rpsL150 may be insufficient to repress IS1 transposition in exponential phase bacteria that grew with limiting or excess glucose concentrations.

Transcription analysis of IS1 transposase
Having shown that IS1 transposition is induced at sub-MIC concentrations of streptomycin, and at extreme (limited or excess) glucose concentra-IS1 transposition at limiting and excess glucose concentrations tions, whereas it was repressed by the rpsL150 mutation, we proceeded to investigate the transcription of the IS1 transposase gene under these experimental conditions.The functional transposase represents a small subset of peptides that are generated by mistranslation and our aim was to discriminate between an increase in the amount of IS1 mRNA and an increase of translation infidelities (transcriptional vs. post-transcriptional regulation) as alternative mechanisms for the enhanced IS1 transposition that we observed under conditions of extreme glucose concentrations in the medium and/or at sub-MIC concentrations of streptomycin.Strain ZK126 was grown in M9 with limiting, standard, and excess glucose or in M9 containing standard concentration of glucose supplemented with sub-MIC streptomycin.cDNA was used as a template for conventional RT-PCR amplification and real-time PCR of insB and 16S rRNA.Results of conventional RT-PCR and real-time PCR were used to quantify the level of insB mRNA, which was expressed as the ratio of the insB to 16S-rRNA RT-PCR products (Fig. 3).Compared to strain ZK126 growing in standard glucose concentration without streptomycin, transcription of IS1 mRNA was lower in all experimental conditions, with the exception of excess glucose where the transcription was enhanced by at least 8-fold.Thus, in most cases, when less IS1-mRNA was measured, more IS1 insertions were observed, suggesting that the regulation of IS1 transposition occurs principally at a step subsequent to transcription.In the case of excess glucose, the IS1-16S ratio increased, suggesting that this condition enhances transcription of (at least) the IS1 transposase gene or lower 16S transcription.One may then envision that from this larger pool of IS1 mRNA, a larger amount of misread InsAB transposase may be translated.

dISCUSSIon
The present study was aimed at examining the transposition of IS elements in response to manipulations of the host cell physiology by providing different amounts of glucose in the growth medium.Our major finding is that both limiting and excess glucose concentrations enhance transposition of IS1 into the bglR region.The results presented here also demonstrate that IS1 transposition depends on the translation fidelity of the host cells.This was achieved by provoking translation infidelity by addition of streptomycin at sub-MIC concentrations, and by enhancing translation accuracy by genetic means which in turn inhibited transposition of IS1 into the bglR region.To our knowledge, our findings represent the first experimental evidence demonstrating that limiting and excess glucose or carbon source concentrations enhance translation errors and as a result of this the transposition frequency of an insertion sequence -IS1 -is increased.
Since transposition of IS1 requires a programmed translational frameshift to produce a functional transposase InsAB'.Escoubas et al. (1994) in their construct that could produce InsAB constitutively without requiring translational frameshift enhanced IS1 transposition rates by four orders of magnitude confirming that regulation of IS1 transposition is balance between production of repressor InsA and transposase InsAB'.We assumed that IS1 transposition is directly controlled by the ability of the cell to promote -1 ribosomal frameshifting which in itself is likely to be dependent on the physiological status of the cells.Indeed, we observed that enhanced (by genetic means) or restricted (by physiology) translation accuracy reduced or increased IS1 transposition, respectively.Interestingly, the rpsL150 strain did not allow IS1 transposition except when glucose was limiting or in excess or when standard glucose cultures were supplied with sub-MIC concentrations of streptomycin.Also in the wild type strain grown at a standard glucose concentration and with streptomycin at sub-MIC, the IS1 transposition rate was increased in a streptomycin concentration dependent manner.A plausible explanation for these observations would be to suggest that they all depend on ribosomal accuracy, and thus glucose limitation affects IS1 transposition by increasing the amount of programmed translation frameshifting.In fact, this assumption was further tested by real-time RT-PCR analysis and the results represented here show that insB transcription was repressed in all conditions except excess glucose where it was enhanced by 8-fold.However, when compared to standard glucose all the other conditions led to elevated IS1 transposition into the bglR region, which is in agreement with our hypothesis that the regulation of IS1 transposition in the studied conditions is primarily at a step subsequent to transcription.
Finding increased IS1 transposition at extreme glucose is of special interest and may be due to different regulators being involved.Stressinduced accumulation of ppGpp (guanosine tetraphosphate) is known for E. coli.In E. coli (p)ppGpp synthesis is known to be catalyzed by two paralogous enzymes: synthetase I (relA gene product) and synthetase II (encoded by spoT), whose activities are regulated differently.Glucose starvation is one of the stressful conditions under which ppGpp is known to accumulate (Hernandez & Bremer, 1991;Xiao et al., 1991;Gentry & Cashel, 1996; for a recent review see: Cashel et al., 1996).The glucose starvation conditions described for ppGpp accumulation resemble to those of limiting glucose described in this paper and therefore accumulation of ppGpp in this condition is an interesting possibility to consider.Genetic analysis suggests that SpoT is a bifunctional protein having both ppGpp degradation and synthesis activities (Xiao et al., 1991;Hernandez & Bremer, 1991).Accumulation of ppGpp in response to energy source starvation occurs by blocking degradation of ppGpp, catalyzed by a ppGpp 3'-pyrophosphohydrolase encoded by the spoT gene.Murray and Bremer (1996) found that both ppGpp synthesis and degradation were decreased immediately after glucose had run out but the rate of degradation was affected more severely leading to accumulation of ppGpp.This observation is interesting and conditions are comparable to that of limiting glucose concentration presented in this article.Interestingly, Gentry and Cashel (1996) also found that the RelA protein activity accounts for the initial burst of ppGpp synthesis at the onset of glucose starvation and that it depends on the presence of amino acids during glucose starvation.Accumulation of ppGpp over a long time period is RelA-independent, and this observation is corroborated by studies in marine Vibrio sp.S14 (Flardh et al., 1994).In both organisms, E. coli and Vibrio sp.S14, relA mutants are known to exhibit an increase in ppGpp levels but the increase is gradual over a longer period of time.This delay is likely to be due to the time required for expression of some of the genes that are likely be critical for the temporal pattern of ppGpp accumulation suggesting that some carbon-starvation inducible alleles show a relA dependence.Since the ZK126 and GBE150 strains used in present study are deleted for relA, the accumulation of ppGpp through solitary spoT is very likely when grown in limiting glucose.On the other hand, enhanced IS1 transposition for cultures grown in excess glucose may include yet another regulator(s).Interestingly, in this condition the cfu dropped by an order of magnitude between expo-nential and stationary phase culture.This might suggest that certain deleterious conditions are generated from excess glucose utilization, e.g., accumulation of acids shifting the pH from neutral to acidic.Although both of these conditions exhibited enhanced IS1 insertions into the bglR region, transcription of insB was drastically different, reduced by 3-fold for limiting glucose culture while enhanced by 8-fold for excess glucose.
Ribosomal misreading is necessary in E. coli because a few essential genes require a programmed translational frameshift (Parker, 1989).Hyper-accurate ribosomes inhibit growth (Ruusala et al., 1984).In order to avoid the load of high translation infidelity, translation errors should be kept as low as possible.Indeed, it has been demonstrated that only a small portion from the mRNA pool is mistranslated, often as a result of a programmed translation frameshift sequence (Kurland, 1992).By using a βgalactosidase reporter construct that required a -1 translation frameshift Barak et al. (1996) showed that the enzyme activity was enhanced by two orders of magnitude near stationary phase when E. coli cells were grown with 0.2% glucose concentration.In an independent study Atkinson et al. (1997) showed that glucose-limited cultures exhibited enhanced frameshift frequency by an order of magnitude.The observation described in this paper that IS1 transposition was enhanced in limiting glucose cultures during exponential phase is in agreement with the above two studies and might represent a similar phenomenon.Also, our results suggest that the biochemical effect of streptomycin on the ribosome mimics conditions that may occur spontaneously in cells starved for amino acids (Atkinson et al., 1997).
Interestingly, in nature bacteria are known to grow with a reduced growth rate and with low translation accuracy compared to laboratory strains (Ruusala et al., 1984;Kurland, 1992;Mikkola & Kurland, 1992).During nutritional deprivation, IS1 transpositions are enhanced, and this may play an important role in the process of adaptability of natural populations requiring continuous genome rearrangements to cope with fluctuating environments (Chalmers & Blot, 1999).
We have now identified genetic and physiological tools that enable one to probe the regulation of IS1 mobility.Nevertheless, the molecular mechanism by means of which either amino acid starvation described in (Atkinson et al., 1997) or extreme carbon source studied by Barak et al. (1996) and presented in this article influence IS1 transposition needs to be studied further.In particular, one should focus on the possibility that the growth conditions directly affect ribosome accuracy, or alternatively, that the level of translation fidelity would be under a genetic control.

Figure 1 .
Figure 1.Schematic representation of the bgl operon.A unique promoter controls three genes: bglG (positive regulator acting by anti-termination of Rho-independent structures), bglF (PEP-dependent phosphotransferase transporting aryl-β-glucosides and also negative regulator by phosphorylation of BglG in absence of β-glucosides) and bglB (phospho-β-glucosidase).The bglR region located upstream to the promoter is the regulatory element.The box designated as IS-TR indicates a 49 bp target region for the transposition of IS elements.The boxes -10 and -35 indicate location of the promoter.An arrow shows the transcription start site.Rho-independent terminators are depicted as a stem loop structure.

Figure 2 .
Figure 2. Generation time in M9 as a function of glucose concentration (0.04%, 0.1%, 0.2%, 0.4%, 0.8%, 1%, 2%, and 4%).Generation time was measured with ZK126 during the linear part of the exponential phase as the doubling time (three replicates).Growth in micro-titration plates is slower than that of the test tube culture because the agitation is restricted.

Figure 3 .
Figure 3.In vivo transcriptional analysis of IS1 by realtime PCr.Real-time PCR reactions were performed with 50 ng cDNA from cultures grown in M9 supplied with various concentrations of glucose with or without streptomycin.IS1 transcription was normalized by taking its ratio to 16S rRNA.The normalized ratios of IS1/16S rRNA for various conditions are expressed in relation to standard glucose concentration arbitrarily fixed at 1.

Table 1 . Mutation rates and mutation frequencies at different glucose concentrations for the wt (ZK126) and rpsL (GBe150) strains
a According to