Phenotypic variability of beta-glucoside utilization and its correlation to pathogenesis process in a few enteric bacteria

Utilization of beta-glucosides is markedly variable in the members of the family Enterobacteriaceae. The results presented here provide molecular clues for evolutionary events that resulted in the phenotypic variability seen amongst the members of these species. The genomic hybridization of selected Enterobacteriaceae members with the Escherichia coli bgl and cel genes resulted in detection of a complete homolog of the bgl and cel operons in Shigella sonnei, a member that is evolutionarily closest to E. coli. However, the Salmonella group of organisms have been shown to carry only a homolog of bglR and bglG regions and the deletions of the bglF and bglB genes. Similarly, Proteus mirabilis, Enterobacter aerogenes and a non-enteric Gram-negative bacterium Pseudomonas aeruginosa have been shown to carry a homolog of the bglR and bglG regions and deletions of the bglF and bglB genes. The homolog of the cel operon could be identified in S. sonnei and Salmonella groups of organisms. Possible implications of these observations, in connection with the phenotypic variability seen in beta-glucoside utilization amongst these members, are discussed.     

Mutational and recombinational events in carcinogen-modified plasmid DNA. Influence of host-cell repair genes

A plasmid containing the STR operon has been modified in vitro (i) by irradiation with UV light, (ii) by reaction with ethyl methanesulphonate (EMS), (iii) by reaction with N-acetoxy-2-acetylaminofluorene (AcO-AAF), (iv) by reaction with (+/-)trans-benzo[a]pyrene-7, 8-dihydrodiol-9,10-epoxide (BPDE), and (v) by heating at 70 degrees C to produce apurinic sites. Suitably modified plasmid DNA was then used to transform both repair-proficient and repair-deficient strains of Escherichia coli, and the mutation frequency in the plasmid-encoded rspL+ gene measured. The influence of host mutations in the uvrB+, recA+, umuC+ and lexA+, genes on the mutation frequency have been investigated. Transformation into a uvrB strain significantly decreased survival and increased the level of mutations observed for UV- and AcO-AAF-modified plasmid DNA, while only a small increase in mutation frequency was seen with EMS-modified DNA and no increase in mutation frequency with plasmid DNA containing apurinic sites. Mutagenesis in UV- and BPDE-modified DNA (and probably also DNA containing apurinic sites) was totally dependent on he recA+ gene product, while EMS and AcO-AAF induced mutagenesis was only partially independent on the recA+ gene. Transformation of UV- or BPDE-modified DNA into a umuC or lexA strain, on the other hand, showed no change in mutation frequency from that observed with wild-type strain. Pre-irradiation of the wild-type host with UV light before transformation led to a significant increase in mutation frequency for UV- and BPDE-modified plasmid DNA. These results are discussed in terms of mutational or recombinational pathways which may be available to act on modified plasmid DNA, and suggest that the majority of the mutational events measured in this system are due to recombination between homologous regions on the plasmid and chromosomal DNA.     

Activation of a cryptic gene by excision of a DNA fragment.

The cryptic bgl operon in Escherichia coli K-12 strain 1011A contains a 1.4-kilobase-pair fragment of foreign DNA within the bglF structural gene. The active allele found in its descendant strain, MK1, required the precise excision of that insertion for its activation. Molecular and genetic approaches have shown that strain 1011A possessed an active (bglR+) rather than a silent wild-type (bglR0) allele of the regulatory region and that this change was caused by a point mutation. Our model for the retention of cryptic genes (B. G. Hall, S. Yokoyama, and D. H. Calhoun, Mol. Biol. Evol. 1:109-124, 1983) suggested that the insertion might have been selected to silence a disadvantageous bglR+ allele. We examined the genealogy of strain MK1 and found that the insertion of foreign DNA was not selected for that reason, since it preceded the change to bglR+. This means that the change to bglR+ was also not selected, since the presence of the insertion would not allow expression of the operon. We have calculated the probability of isolating a bglR+ mutation by chance alone as less than 10(-8). We suggest that mutation rates estimated under the usual conditions of exponential growth may be irrelevant to the frequencies of these events under natural conditions.     

A plasmid vector for an extreme thermophile, Thermus thermophilus

The host-vector system for an extreme thermophile, Thermus thermophilus HB27, was developed. The host strain has a mutation in tryptophan synthetase gene (trpB), and the mutation was determined to be a missense mutation by DNA sequence analysis. A Thermus-E. coli shuttle vector pYK109 was constructed. pYK109 consists of Thermus cryptic plasmid pTT8, tryptophan synthetase gene (trpB) of Thermus T2 and E. coli plasmid vector pUC13. pYK109 transformed T. thermophilus HB27 trpB5 to Trp+ at a frequency of 10(6) transformants per microgram DNA.     

Adaptive Evolution That Requires Multiple Spontaneous Mutations. I. Mutations Involving an Insertion Sequence

Escherichia coli K12 strain chi 342LD requires two mutations in the bgl (beta-glucosidase) operon, bglR0----bglR+ and excision of IS103 from within bglF, in order to utilize salicin. In growing cells the two mutations occur at rates of 4 x 10(-8) per cell division and less than 2 x 10(-12) per cell division, respectively. In 2-3-week-old colonies on MacConkey salicin plates the double mutants occur at frequencies of 10(-8) per cell, yet the rate of an unselected mutation, resistance to valine, is unaffected. The two mutations occur sequentially. Colonies that are 8-12 days old contain from 1% to about 10% IS103 excision mutants, from which the Sal+ secondary bglR0----bglR+ mutants arise. It is shown that the excision mutants are not advantageous within colonies; thus, they must result from a burst of independent excisions late in the life of the colony. Excision of IS103 occurs only on medium containing salicin, despite the fact that the excision itself confers no detectable selective advantage and serves only to create the potential for a secondary selectively advantageous mutation.     

Activation of the bgl operon by adaptive mutation

In growing Escherichia coli K12 cells, the cryptic bgl operon is activated 98% of the time by insertions of IS1 or IS5 into the control region, designated bglR. The activated bgl operon permits utilization of the beta-glucoside sugar arbutin as a sole carbon and energy source. The bgl operon is also activated by late-occurring mutations during prolonged selection on arbutin. The late-occurring mutations that occurred during prolonged carbon starvation in the presence of arbutin were "adaptive mutations" because they were specific to the presence of arbutin, and they did not occur during prolonged starvation in the absence of arbutin. The spectrum of late-arising mutations differed from that of early-arising, growth-dependent mutations in that 20% of the late-arising mutants resulted from mutations at the hns locus. This provides the first direct evidence for adaptive mutagenesis mediated by the insertion of IS elements. Because no special genetic background is required to select Bgl+ mutants, this affords the opportunity to study IS-element-mediated adaptive mutagenesis in a variety of genetic backgrounds, including the backgrounds of natural isolates of E. coli.      

Widespread distribution of deletions of the bgl operon in natural isolates of Escherichia coli

A deletion that includes the bgl (beta-glucoside utilization) operon of Escherichia coli was originally detected in several rarely occurring natural isolates that utilize cellobiose. Here I show that bgl deletions are present in 95% of the Cel+ isolates obtained from diverse sources. They are also present in 29% of the Cel- strains in two different collections of natural isolates of E. coli. At least three versions of bgl deletions are present in E. coli populations. In the most common version approximately 8 kb of DNA around the bgl region of E. coli K12 is replaced by a specific 6.5-kb DNA fragment. In another version a deletion of similar length is not replaced by the same sequence. A third version involves deletion of approximately 14 kb without the replacement fragment being present. The distribution of these deletions suggests that the version 1 deletion occurred very early in the history of E coli. It also appears likely that there is selection for bgl deletions in Cel+ strains of E. coli. The presence of the version 1 deletion within distantly related phylogenetic groups of E. coli provides evidence for recombination within natural populations of E coli.      

Formation of the heterozygous tandem duplication in the process of conjugation recombination in Escherichia coli: study of the effect of mutations for the recQ, uvrD, and recJ genes]

Stable tandem duplications were shown to originate from conjugational recombination between Escherichia coli HfrH strains carrying mutations for the deo operon. The duplications deoC deoD/deoA deoB::Tn5 usually constitute approximately 5% of the Deo+ offspring. The effect of mutations for the recQ, uvrD, and recJ genes on the frequency of duplications was studied. The CM1563 strain carrying the recQ mutation was shown to give, as a recipient, 20% of duplications in the Deo+ offspring. However, this property of CM1563 seems to depend on the presence of a spontaneous mutation of unknown nature, which also increased UV sensitivity of bacteria. The recQ mutation itself increased the frequency of duplications by less than 50%. The recJ mutation did not virtually affect the frequency of duplications. The uvrD mutation possessing the recombinogenic effect was shown to increase the frequency of deo+ recombinants and simultaneously decrease the frequency of duplications. Tandem duplications are assumed to be normal intermediates of multi-stage conjugational recombination initiated by the integration of the proximal region of the Hfr chromosome into different nonhomologous regions of the recipient chromosome.     

Mutations that activate the silent bgl operon of Escherichia coli confer a growth advantage in stationary phase

Wild-type strains of Escherichia coli are unable to utilize aromatic beta-glucosides such as arbutin and salicin because the major genetic system that encodes the functions for their catabolism, the bgl operon, is silent and uninducible. We show that strains that carry an activated bgl operon exhibit a growth advantage over the wild type in stationary phase in the presence of the rpoS819 allele that causes attenuated rpoS regulon expression. Our results indicate a possible evolutionary advantage in retaining the silent bgl operon by wild-type bacteria.     

Mobilization of phase I plasmid in Shigella sonnei and stability of its inheritance in rough strains of Shigella and Escherichia

The plasmid pSS120, determining the synthesis of species specific I phase antigen of Shigella sonnei is mobilized for genetic transfer into E. coli K12 recipient cells with the frequency 12-41%. The frequency depends on the type of mobilized plasmid and recipient strain. The I phase antigen is normally expressed in II phase recipient cells and in E. coli cells. During mobilization pSS120 forms cointegrates representing a recombinant of mobilizing and mobilized plasmids DNA. The study of pSS120 inheritance stability has shown the plasmid to be unstable during culturing of bacteria and to be partially lost from the parent Shigella sonnei strains as well as from the "hybrid" transconjugants obtained. The 60 Md plasmid present in the donor strains of Shigella sonnei is prone to structural fragmentation particularly expressed in Shigella sonnei/E. coli hybrids.     

New phenotypes associated with mucAB: alteration of a MucA sequence homologous to the LexA cleavage site.

Most mutagenesis by UV and many chemicals in Escherichia coli requires the products of the umuDC operon or an analogous plasmid-derived operon mucAB. Activated RecA protein is also required for, or enhances, this process. MucA and UmuD proteins share homology with the LexA protein, suggesting that they might interact with the RecA protein as LexA does. We used oligonucleotide-directed mutagenesis to alter a site in MucA homologous to the Ala-Gly cleavage site of LexA. The mutation, termed mucA101(Glu26), results in a change of Gly26 of MucA to Glu26. A lexA(Def) recA441 umuC122::Tn5 strain carrying a mucA101(Glu26)B+ plasmid did not exhibit the greatly increased frequency of spontaneous mutagenesis in response to RecA activation that a strain carrying a mucA+B+ plasmid did but retained a basal recA-dependent ability to confer increased spontaneous mutagenesis that was independent of the state of RecA activation. These results are consistent with a model in which RecA plays two distinct roles in mutagenesis apart from its role in the cleavage of LexA. A pBR322-derived plasmid carrying mucA+B+, but not one carrying mucA101(Glu26)B+, inhibited the UV induction of SOS genes, suggesting that MucA+ and MucA(Glu26) proteins may have different abilities to compete with LexA for activated RecA protein. The spectrum of UV-induced mutagenesis was also altered in strains carrying the mucA101(Glu26) mutation. These results are consistent with the hypothesis that activated RecA protein interacts with wild-type MucA protein, possibly promoting proteolytic cleavage, and that this interaction is responsible for facilitating certain mutagenic processes.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

Lethality of palindromic DNA and its use in selection of recombinant plasmids

A plasmid derived from ColE1 is constructed so that the removal of one restriction endonuclease HindIII fragment allows the ends of the remaining single fragment (the replicator) to be joined, generating a palindromic sequence 2394 bp in length. The circular species thus produced gives rise to transformants of E. coli at very low frequency. Since the palindromic sequence is effectively lethal to a plasmid containing it, the replicator will give rise to more transformants when the restriction fragment originally removed from it is replaced by another. This principle can be exploited to allow the efficient molecular cloning of unselected restriction fragments.