Evolution of immunity and host chromosome integration site of P2-like coliphages

The amount and distribution of variation in the genomic region containing the genes in the lytic-lysogenic genetic switch and the sequence that determines the integration site into the host chromosome were analyzed for 38 P2-like phages from Escherichia coli. The genetic switch consists of two convergent mutually exclusive promoters, Pe and Pc, and two repressors, C and Cox. The immunity repressor C blocks the early Pe promoter, leading to the establishment of lysogeny. The Cox repressor blocks expression of Pc, allowing lytic growth. Phylogenetic analyses showed that the C and Cox proteins were distributed into seven distinct classes. The phylogenetic relationship differed between the two proteins, and we showed that homologous recombination plays a major role in creating alterations in the genetic switch, leading to new immunity classes. Analyses of the host integration site for these phages resulted in the discovery of a previously unknown site, and there were at least four regular integration sites. Interestingly, we found no case where phages of the same immunity class had different host attachment sites. The evolution of immunity and integration sites is complex, since it involves interactions both between the phages themselves and between phages and hosts, and often, both regulatory proteins and target DNA must change.     

Controlled expression of the transcriptional activator gene virG in Agrobacterium tumefaciens by using the Escherichia coli lac promoter

The Agrobacterium VirG protein is normally expressed from two promoters in response to multiple stimuli, including plant-released phenolics (at promoter P1) and acidic growth media (at promoter P2). To simplify the analysis of vir gene induction, we sought to create Agrobacterium strains in which virG could be expressed in a controllable fashion. To study the possibility of using the lac promoter and repressor, we constructed a plasmid containing the lac promoter fused to the lacZ structural gene. A derivative of this plasmid containing the lacIq gene was also constructed. The plasmid not containing lacIq expressed high levels of beta-galactosidase. The plasmid containing lacIq expressed beta-galactosidase at very low levels in the absence of o-nitrophenyl-beta-D-galactoside (IPTG) and at moderate levels in the presence of IPTG. We also fused the lac promoter to a virG::lacZ translational fusion and found that IPTG elevated expression of this translational fusion to moderate levels, though not to levels as high as from the stronger of the two native virG promoters. Finally, the lac promoter was used to express the native virG gene in strains containing a virB::lacZ translational fusion. virB expression in this strain depended on addition of IPTG as well as the vir gene inducer acetosyringone. In a similar strain lacking lacIq, virB expression was greater than in a strain in which virG was expressed from its native promoters. Expression of virG from the lac promoter did not alter the acidic pH optimum for vir gene induction, indicating that the previously observed requirement for acidic media was not due solely to the need to induce P2.     

A minor arginine tRNA mutant limits translation preferentially of a protein dependent on the cognate codon.

The Escherichia coli argU gene encodes a rare arginine tRNA (anticodon UCU) that translates the similarly rare AGA codon. The argU10(Ts) mutation is a transition that changes the first nucleotide of the mature tRNA from G to A, presumably destabilizing the acceptor stem. This mutation, when present in haploid condition in the chromosome, reduces the growth rate at 30 degrees C and results in cessation of growth after 60 to 90 min at 43 degrees C. The mutation also preferentially limits (compared with total protein synthesis) translation of an induced gene that depends on five AGA codons, i.e., the lambda cI repressor gene. Translation of another inducible protein, beta-galactosidase, which does not involve AGA codons, was inhibited to a much lesser extent. The chromosomal argU(Ts) mutation also confers the Pin phenotype, that is, loss of ability of the host, as a P2 lysogen, to inhibit growth of bacteriophage lambda, probably the result of reduced translation of the P2 old gene, which contains five AGA codons (E. Hagg?rd-Ljungquist, V. Barreiro, R. Calendar, D. M. Kurnit, and H. Cheng, Gene 85:25-33, 1989).     

Role of -35 sequence and its cooperativity with vir-box for the expression of virE gene

To elucidate the role of the -35 sequence and its cooperativity with vir box in the expression of the virE gene, various mutants were constructed by either site-directed mutation or deletional mutation of the virE promoter. The expression level of pHBAV, a mutant where its putative -35 sequences (CCGAGT) have been substituted with the consensus -35 sequences of the Escherichia coli promoter (TTGACA), was increased by 386%. pECHV, containing the conserved -35 sequence but lacking the vir box and the 5'-half of the imperfect dyad symmetry region (DSR) showed an increase of 286% in its promoter activity. pESHV, containing the conserved -35 sequence but lacking the complete 5'-upstream region from the mid-region of imperfect DSR, exhibited 244% of the native virE promoter activity. pHBCA, containing the conserved -35 sequence but destroying the vir box, was constructed by substitution of A, C, T at the positions -62, -63, and -65 on the vir-box to T, A, C, respectively. These mutations increased promoter activities by 319%. On the other hand, when the vir box was mutated from imperfect DSR to almost perfect DSR with T to A and G to T substitutions at -60 and -61 positions of the virE promoter containing the conserved -35 sequence (pHBNA), a higher activity of 671% was observed. These results demonstrate that when the putative -35 sequence of virE promoter is replaced with the consensus -35 sequence, the virE gene can be expressed independently without the binding of VirG protein to the vir-box and/or the induction of acetosyringone. Moreover, the presence of an almost perfect dyad symmetry of the vir-box can increase the expression of virE synergistically with the consensus -35 sequence.