Mucosal and systemic antibody responses to a C4/V3 construct following DNA vaccination of rabbits via the Peyer's patch.

A plasmid encoding T1-SP10MN(A), a peptide derived from immunodominant regions of human immunodeficiency virus type 1 gp120, was delivered to rabbit Peyer's patches using a helium-driven gene gun. Six weeks thereafter, 2 of 5 animals were given an intradermal booster immunization. Blood, feces, and vaginal washes were collected weekly and assayed by ELISA. High titer T1-SP10MN(A)-specific fecal and vaginal secretory IgA responses were observed, and the response appeared to be augmented following dermal booster immunizations. Specific serum IgG was also detected within 1 week of immunization and remained elevated through week 20 in the 2 animals receiving dermal boosts (titers > or = 6400). This study establishes the Peyer's patch as a promising target tissue for DNA vaccination and demonstrates the efficacy of gene gun-mediated delivery of foreign DNA to a mucosal tissue for the induction of an immune response.

A cryptic miniplasmid from the hyperthermophilic bacterium Thermotoga sp. strain RQ7.

An 846-bp cryptic plasmid has been discovered in the hyperthermophilic bacterium Thermotoga sp. strain RQ7. This is the first plasmid described for an organism from this ancient bacterial lineage and the smallest plasmid described to date for any organism. Nucleotide sequencing revealed a single open reading frame possibly encoding a 25,460-Da basic protein (212 amino acids). Upstream of the putative promoter lie five 11-bp direct repeats, each separated by 1 to 4 bp, while between the promoter and the open reading frame lies an 11-bp palindromic sequence. Its mode of replication is unknown, but its sequence bears similarities to those of plasmids which replicate by a rolling-circle mechanism.

DNA sequence analysis of artificially evolved ebg enzyme and ebg repressor genes.

The ebg system has been used as a model to study the artificial selection of new catalytic functions of enzymes and of inducer specificities of repressors. A series of mutant enzymes with altered catalytic specificities were previously characterized biochemically as were the changes in inducer specificities of mutant, but fully functional, repressors. The wild type ebg operon has been sequenced, and the sequence differences of the mutant enzymes and repressors have been determined. We now report that, contrary to our previous understanding, ebg enzyme contains 180-kD alpha-subunits and 20-kD beta-subunits, both of which are required for full activity. Mutations that dramatically affect substrate specificity and catalytic efficiency lie in two distinct regions, both well outside of the active site region. Mutations that affect inducer specificity of the ebg repressor lie within predicted sugar binding domains. Comparisons of the ebg beta-galactosidase and repressor with homologous proteins of the Escherichia coli and Klebsiella pneumoniae lac operons, and with the galactose operon repressor, suggest that the ebg and lac operons diverged prior to the divergence of E. coli from Klebsiella. One case of a triple substitution as the consequence of a single event is reported, and the implications of that observation for mechanisms of spontaneous mutagenesis are discussed.

IS103, a new insertion element in Escherichia coli: characterization and distribution in natural populations.

IS103 is a previously unknown insertion sequence found in Escherichia coli K12. We have sequenced IS103 and find that it is a 1441-bp element that consists of a 1395-bp core flanked by imperfect 23-bp inverted repeats. IS103 causes a 6-bp duplication of the target sequence into which it inserts. There is a single copy of IS103 present in wild-type E. coli K12 strain HfrC. In strain X342 and its descendents there are two additional copies, one of which is located within the bglF gene. IS103 is capable of excising from within bglF and restoring function of that gene. IS103 exhibits 44% sequence identity with IS3, suggesting that the two insertion sequences are probably derived from a common ancestor. We have examined the distribution of IS103 in the chromosomes and plasmids of the ECOR collection of natural isolates of E. coli. IS103 is found in 36 of the 71 strains examined, and it strongly tends to inhabit plasmids rather than chromosomes. Comparison of the observed distribution of IS103 with distributions predicted by nine different models for the regulation of transposition according to copy number and of the effects of copy number on fitness suggest that transposition of IS103 is strongly regulated and that it has only minor effects on fitness. The strong clustering of IS103 within one phylogenetic subgroup of the E. coli population despite its presence on plasmids suggests that plasmids tend to remain within closely related strains and that transfer to distantly related strains is inhibited.

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.

Cryptic genes for cellobiose utilization in natural isolates of Escherichia coli.

The ECOR collection of natural Escherichia coli isolates was screened to determine the proportion of strains that carried functional, cryptic and nonfunctional genes for utilization of the three beta-glucoside sugars, arbutin, salicin and cellobiose. None of the 71 natural isolates utilized any of the beta-glucosides. Each strain was subjected to selection for utilization of each of the sugars. Only five of the isolates were incapable of yielding spontaneous beta-glucoside-utilizing mutants. Forty-five strains yielded cellobiose+ mutants, 62 yielded arbutin+ mutants, and 58 strains yielded salicin+ mutants. A subset of the mutants was screen by mRNA hybridization to determine whether they were expressing either the cel or the bgl beta-glucoside utilization operons of E. coli K12. Two cellobiose+ and two arbutin+-salicin+ strains failed to express either of these known operons. It is concluded that there are at least four gene clusters specifying beta-glucoside utilization functions in E. coli populations, and that all of these are normally cryptic. It is estimated that in any random isolate the probability of any particular cluster having been irreversibly inactivated by the accumulation of random mutations is about 0.5.

Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state.

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta-glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta-glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.

Sequence of the ebgA gene of Escherichia coli: comparison with the lacZ gene.

We have sequenced the ebgA (evolved beta-galactosidase) gene of Escherichia coli K12. The sequence shows 50% nucleotide identity with the E. coli lacZ gene, demonstrating that the two genes are related by descent from a common ancestral gene. Comparison of the two sequences suggests that the ebgA gene has recently been under selection. A significant excess of identical, rather than synonymous, codons used to encode identical amino acids at the same positions in the aligned sequences implies that some form of selection is operating directly at the DNA level. This selection is independent of, and in addition to, selection based on codon usage or on function of the gene products.