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L Boe

Publications and source records attributed to L Boe.

At least 19 recordsLinked to original sources
PubMed2000

The frequency of mutators in populations of Escherichia coli.

Owing to occasional spontaneous mutations in genes encoding DNA repair, any population of a reasonable size is expected to harbor a sub-population of genetic mutators. Using a genetically modified strain of Escherichia coli K-12, we have estimated the frequency of mutators to be about 3x10(-5). By and large, this corresponds to a mutation rate from non-mutators to mutators of 5x10(-6) per bacterium per generation. Using a mutS∷Tn10 derivative as representative for mutators, we estimated the increase in mutation rates in mutators to be 19- to 82-fold, depending on the test-mutation under consideration. The load associated with this increase in mutation rate resulted in a growth inhibition of 1%. From these data, we estimated that the rate of detrimental mutations in the non-mutators to be 2x10(-4)-8x10(-4). The situations where adaptive mutations may result in an increase in the frequency of mutators are discussed.

Adenosine Triphosphatases↗
PubMed1998

Kinetics of conjugative transfer: a study of the plasmid pXO16 from Bacillus thuringiensis subsp. israelensis.

The aggregation-mediated conjugation system of Bacillus thuringiensis subsp. israelensis, encoded by the 200-kb plasmid pXO16, is highly potent in transferring itself and efficient in mobilizing other nonconjugative plasmids. The present study reveals some salient features of this conjugation system. Our observations can be summarized as follows: (i) The conjugative transfer takes about 3(1/2) to 4 min. For a 200-kb plasmid this corresponds to about 1 kb per second. (ii) The ability to transfer the plasmid seems to be evenly distributed among the donors. (iii) Functionally, the mating complex was found to consist of one donor and one recipient cell, even though aggregates comprising thousands of interconnected cells are formed. (iv) Having donated the plasmid, the donor needs a "period of recovery" of about 10 min before it can redonate the plasmid. (v) Secondary transfer, i.e., transfer from newly formed transconjugants, is delayed about 40 min. This maturation time exceeds the generation time, and it may indicate that to display donor activity, a surface protein (the aggregation substance) has to be uniformly incorporated into the cell wall. Lastly, we found that when the experiments were sufficiently short and when the recipient cells were in excess compared with the donors, the process of conjugation could be reasonably described by a kinetic model analogous to the Michaelis-Menten model for enzyme catalysis. This allowed us to estimate (vi) the maximal conjugation rate to be about 0.05 transconjugant per donor per minute, and (vii) the Km value, i.e., the concentration of recipient that results in half of the maximal conjugation rate, to be about 4 x 10(6) recipients/ml.

Bacillus thuringiensis↗
PubMed1998

Non-genetic population heterogeneity studied by in situ polymerase chain reaction.

Expression of a lac operon in Salmonella typhimurium single cells was monitored using lac mRNA targeting in situ reverse transcription-polymerase chain reaction (RT-PCR). It is demonstrated that suboptimal induction of the lac operon in a culture of S. typhimuriuml/F'lac+ cells generates a subpopulation in which transcription of the lac operon occurs and another subpopulation in which transcription of the lac operon is repressed, whereas suboptimal induction of the lac operon in a culture of S. typhimuriuml/F'lacY cells generates a population with uniform levels of lac mRNA. The outcome of the single-cell lac mRNA detection assay was compared with the outcome of a single-cell beta-galactosidase assay. In cultures grown under different suboptimal lac induction conditions, the fraction of cells in which transcription of the lac operon occurred was concurrent with the fraction of cells showing beta-galactosidase activity. Besides supporting the hypothesis that the lactose permease has a role in generating non-genetic heterogeneity in suboptimally induced cultures of Lac+ cells, these results demonstrate the usefulness of in situ RT-PCR for the study of non-genetic population heterogeneities.

Escherichia coli Proteins↗
PubMed1998

atp Mutants of Escherichia coli fail to grow on succinate due to a transport deficiency.

Escherichia coli atp mutants, which lack a functional H+-ATPase complex, are capable of growth on glucose but not on succinate or other C4-dicarboxylates (Suc- phenotype). Suc+ revertants of an atp deletion strain were isolated which were capable of growth on succinate even though they lack the entire H+-ATPase complex. Complementation in trans with the yhiF gene suppressed the growth of the Suc+ mutants on succinate, which implicates the yhiF gene product in the regulation of C4-dicarboxylate metabolism. Indeed, when the E. coli C4-dicarboxylate transporter (encoded by the dctA gene) was expressed in trans, the Suc- phenotype of the atp deletion strain reverted to Suc+, which shows that the reason why the E. coli atp mutant is unable to grow aerobically on C4-dicarboxylates is insufficient transport capacity for these substrates.

Bacterial Proteins↗
PubMed1997

Plasmid stability: comments on the dimer catastrophe hypothesis.

Using a derivative of the plasmid pBR322 we have tested the dimer catastrophe hypothesis of plasmid instability. Most of the theory was confirmed by our observations, but our data suggest that some of the quantitative aspects need modification. In a recF strain of Escherichia coli we estimated the difference in loss rate between the plasmid in the monomeric and the dimeric state to be a factor of 13-14 and the difference in the loss rate between the plasmid in the monomeric and the trimeric state to be a factor of 14-50. We were able to confirm that plasmid oligomers were heterogeneously distributed within a rec+ population, but we were unable to detect any pronounced difference in the level of growth inhibition exerted by the plasmid when in the monomeric, dimeric, or trimeric state. This leaves open the question as to whether runaway plasmid multimerization was prevented (i) by a small correlation between the inhibition of growth and the 'multimeric status' of the plasmid, (ii) by intramolecular homologous recombination, or (iii) whether the process of runaway multimerization is too slow to be recognized within the duration of the experiments, i.e. 200 generations of growth.

Escherichia coli↗
PubMed1996

Suggestions as to quantitative measurements of plasmid loss.

Suggestions are made for the estimation of plasmid loss rates in bacterial populations that multiply by binary fission. The plasmid loss rate is defined as the probability of a division of a plasmid-carrying individual giving birth to one plasmid-free and one plasmid-carrying daughter cell. The unit of time is consequently the interdivision time for plasmid-carrying individuals. The cautions that have to be taken when the population is cultivated in serial transfer (the usual stability experiment) are discussed.

Mathematical Computing↗
PubMed1996

Estimation of plasmid loss rates in bacterial populations with a reference to the reproducibility of stability experiments.

Two methods for estimation of plasmid loss rates were tested on data obtained from traditional (serial transfer) stability experiments. The first method was based on the assumption that the plasmid does not inhibit the growth of its host, whereas the second method takes differences in the interdivision time of plasmid-free and plasmid-carrying cells into account. In the cases where the loss rate is high and the plasmid does not exert strong growth inhibition, the estimates appear very reliable. When the plasmid loss rate is small and the plasmid exerts inhibition of growth to its host, the experimental design becomes unreliable.

Gene Dosage↗
PubMed1994

Complete nucleotide sequence of the Bacillus thuringiensis subsp. israelensis plasmid pTX14-3 and its correlation with biological properties.

The complete nucleotide sequence of the plasmid pTX14-3 from Bacillus thuringiensis subsp. israelensis has been determined. The circular DNA molecule was 7649 bp and had a G + C content of 35.1%. Twenty-two open reading frames larger than 50 codons were identified. Ten of these open reading frames are suggested to be protein coding regions. The existence of the polypeptides encoded by the mob14-3 and rep14-3 genes were verified by maxi-cells analysis in Escherichia coli. Even though the rep14-3 gene was expressed in E. coli the plasmid pTX14-3 was unable to replicate in this bacterium. The minimal region of the plasmid pTX14-3 required for replication in B. thuringiensis was identified. Potential secondary structures upstream of the rep14-3 gene indicated regulation by antisense RNA and transcription attenuation. Extensive sequence homology with the B. thuringiensis subsp. thuringiensis plasmid pGI2 was found in the last part of the mob14-3 gene, downstream of the rep14-3 gene, and in the region containing the single-strand origin of replication (i.e., the minus origin) of pTX14-3. A sequence of 700 bp containing multiple direct repeats was found in an ORF encoding a glycine and proline rich protein of 35.9 kDa. 1.2 kbp upstream and 0.1 kbp downstream of this ORF was found a large direct repeat of 230 bp (87% identity). The region between this direct repeat was often spontaneously deleted from plasmid derivatives containing the entire pTX14-3.

Amino Acid Sequence↗
PubMed1994

Observations on the formation of deletions on monomeric and dimeric plasmids in Escherichia coli.

We have studied the formation of spontaneous mutations on plasmids present in the monomeric and dimeric states in a recF strain of Escherichia coli. Two test systems were employed: (i) the precise excision of Tn5 from the tetA gene of the plasmid pBR322 and (ii) operator constitutive (Oc) mutations on the pBR322-derived plasmid pPY97. The rate of Oc mutations was increased by a factor of three when this plasmid was present in the dimeric state compared to the monomeric state and the Oc phenotype was caused by small deletions in the operator sequence. No apparent mutational hot-spot was found. The rate of Tn5 excision was increased on dimeric compared to monomeric plasmids. Excision from a dimeric plasmid usually resulted in two types of mutant plasmids; a dimeric plasmid, where the Tn5 had excised from one of the plasmid units, and a monomeric parental pBR322. A mechanisms to account for this is suggested. Complementation tests revealed that the increased mutation rate on dimeric plasmids is the result of dimers being mutaphilic per se, rather than the result of a general, trans-acting increase in mutation rates of the host, induced by the presence of the dimeric plasmid. Furthermore, it was found that the rate of Tn5 excision from plasmids in the monomeric state was increased when the region carrying the inserted Tn5 was duplicated.

Antiporters↗
PubMed1994

Fluctuation analysis of mutations to nalidixic acid resistance in Escherichia coli.

Mutations of Escherichia coli from sensitivity to nalidixic acid resistance were studied by fluctuation analysis. The mutant distributions in replicate cultures were not significantly affected either by the age of the carbon-starved preculture used for inocula or by the inoculum size. The data from 23 fluctuation tests (48 cultures each) were pooled. The mean number of mutations per culture was estimated to be 0.71 from the fraction of cultures without mutants or 0.74 and 0.77 by maximum-likelihood estimation based on the two models under consideration. When the pooled data were compared with the theoretical expectations, the fits were unsatisfactory (P < 0.005). The lack of fit was caused mainly by too high a frequency of cultures with between 17 and 32 mutants and too high a frequency of cultures with more than 128 mutants. Possible reasons for the lack of fit and its implications with respect to estimation of mutation rates from fluctuation tests are discussed.

Chi-Square Distribution↗
PubMed1994

A statistical analysis of the formation of plasmid-free cells in populations of Escherichia coli.

By methods analogous to those used in the classical statistical analysis of bacterial mutation, we have analyzed the formation of plasmid-free cells in populations of Escherichia coli harboring pBR322-derived plasmids. Application of fluctuation tests and papilla analysis suggested that there is a high variance in the probability that a plasmid-containing cell will produce a plasmid-free daughter cell. Apparently a subpopulation of plasmid-containing cells gives rise to progeny that produces plasmid-free cells with a high and unpredictable rate. This finding raises the question of whether plasmid maintenance can be adequately described by the conventional mathematical models.

Colony Count, Microbial↗
PubMed1993

Fine mapping and DNA sequence of replication functions of Bacillus thuringiensis plasmid pTX14-3.

pTX14-3 is a 7.5-kb cryptic plasmid isolated from a Bacillus thuringiensis subspecies israelensis strain. Like many other small plasmids in gram-positive bacteria, pTX14-3 replicates via a single-stranded DNA intermediate. The nucleotide sequence of the replication region was determined and an open reading frame of 636 base pairs encoding a protein necessary for plasmid replication was identified by deletion analysis. No significant homology was found between this open reading frame and those encoding replication proteins identified on other plasmids isolated from gram-positive bacteria, nor could we find any homology to plus origins from other single-stranded DNA plasmids. Consequently, it seems that the replicon of pTX14-3 belongs to a new family of replicons in the group of single-stranded DNA plasmids. The sequence of the single-strand origin (i.e., the minus origin) responsible for the conversion of single-stranded plasmid DNA to double-stranded plasmid DNA was also determined. A partial homology between the minus origin of pTX14-3 and the Bacillus subtilis plasmid pBAA1 was identified. A previously identified locus that suppresses formation of high molecular weight multimers was also minimized and sequenced.

Amino Acid Sequence↗
PubMed1993

Suicidal genetic elements and their use in biological containment of bacteria.

The potential risks of unintentional releases of genetically modified organisms, and the lack of predictable behavior of these in the environment, are the subject of considerable concern. This concern is accentuated in connection with the next phase of gene technology comprising deliberate releases. The possibilities of reducing such potential risks and increasing the predictability of the organisms are discussed for genetically engineered bacteria. Different approaches towards designing disabled strains without seriously reducing their beneficial effects are presented. Principally two types of strain design are discussed: actively contained bacteria based on the introduction of controlled suicide systems, and passively contained strains based on genetic interference with their survival under environmental-stress conditions.

Bacteria↗
PubMed1992

Translational errors as the cause of mutations in Escherichia coli.

The present work suggests that a significant proportion of spontaneous mutations in Escherichia coli are the result of translational errors. This idea is supported by the following observations: (i) Streptomycin can induce the formation of auxotrophic mutants in streptomycin-sensitive cells, but not in rpsL mutants resistant to streptomycin, and (ii) strains having hyperaccurate ribosomes (rpsL999 and rpsL1204 strains) show reduced mutation rates. The implications of these results are discussed with respect to the dogma of randomness of spontaneous mutations and the directed mutation hypothesis.

Alleles↗
PubMed1991

Cloning and characterization of two plasmids from Bacillus thuringiensis in Bacillus subtilis.

Bacillus thuringiensis subspecies israliensis plasmids pTX14-1 and pTX14-3 were cloned and analyzed by Southern blot hybridization for their replication mechanism in Bacillus subtilis. The cloning of pTX14-1 into the replicon deficient vector pBOE335 showed the usual characteristics of single-stranded DNA plasmids, i.e., it generated circular single-stranded DNA and high molecular weight (HMW) multimers. The other plasmid, pTX14-3, behaved differently; it generated neither single-stranded DNA nor HMW multimers. Treatment with rifampicin did not result in the accumulation of single-stranded DNA. However, deletion of an EcoRI-PstI fragment resulted in the accumulation of both single-stranded DNA and HMW multimers. From various deletion derivatives, we have mapped the minus origin and the locus responsible for suppression of HMW multimer formation. Full activity of the minus origin and of the locus suppressing HMW formation was only observed on the native replicon, indicating a coupling to the plus strand synthesis.

Bacillus subtilis↗
PubMed1991

Role of plasmid multimers in mutation to tetracycline resistance.

As an additional system for analysing mutations that appear to be specifically induced or directed, we have used a plasmid that contains the mnt repressor gene inserted as an operon fusion with the tet gene of the plasmid pBR322. Thus, the mnt gene product acts as a negative transcriptional regulator of tet gene expression. Mutations inactivating the Mnt repressor are recessive while those destroying operator recognition (Oc) are dominant in conferring tetracycline resistance on the host. When resistance mutations were isolated on plates with high levels of tetracycline they were preferentially mnt- and the plasmids were monomers. Pre-exposure to low concentrations increased the frequency of resistant mutants by 100- to 1000-fold, and the mutations were now mostly Oc, located on one unit of a plasmid multimer. Recessive repressor mutations on one unit would not have been selected. We suggest that the high frequency of mutation in tandem multimeric plasmids may be caused by the formation of single-stranded and hence highly mutable regions by homologous pairing out of register. The role of tetracycline in promoting mutations is discussed.

Escherichia coli↗
PubMed1990

Mechanism for induction of adaptive mutations in Escherichia coli.

When bacterial cells are subjected to a strong selective pressure it often induces specific mutations. Here a model is considered in which errors are introduced at random in one of the strands of the DNA molecule: a nick in one of the strands can initiate strand displacement rendering a region of the chromosome single-stranded. Upon conversion back to double-stranded DNA there is a certain probability of introducing errors creating a heteroduplex. If an error results in the production of an mRNA molecule encoding a product which provides a selective advantage, growth will be stimulated and the mutation can be immortalized by chromosomal replication. Otherwise, the error can be corrected by the DNA 'proof-reading' enzymes.

Adaptation, Physiological↗