The novel gene(s) ARD of plasmid pKM101: alleviation of EcoK restriction

Summary The host-controlled K restriction of unmodified phage was 10-100-fold alleviated in the wild-type strain E. coli K12, carrying plasmid pKM101 of incompability group N. pKM101-mediated release of K restriction was also observed in lexA ^-, recA ^-, and recB ^- strains of E. coli K12. By restriction mapping Tn5 insertions in pKM101, which reduced pKM101-mediated alleviation of restriction, were shown to be located within the BglIIB fragment approximately 11 kb anticlockwise from the RI site of pKM101. We have termed the gene(s) promoting the alleviation of K restriction of phage ard (alleviation of restriction of DNA). It was shown (1) that ard function affected only the EcoK restriction system and not the EcoB, EcoRI, EcoRIII, or EcoPI system, (2) ard gene(s) did not mediate EcoK type modification of DNA and did not increase the modification activity of the EcoK system in a way similar to that observed with gene ral of bacteriophage .     

Role of “Antirestriction” Motif in Functional Activity of Antirestriction Protein ArdA pKM101 (IncN)

A number of mutant forms of the antirestriction protein ArdA encoded by theardA gene located in a transmissive IncN plasmid pKM101 have been constructed. Proteins belonging to the Ard family are specific inhibitors of type I restriction–modification enzymes. Single mutational substitutions of negatively charged amino acid residues located in the antirestriction motif with hydrophobic alanine, E134A, E137A, D144A, or a double substitution E134A, E137A do not affect the antirestriction activity (Ard) of ArdA but almost completely abolish the antimodification activity (Amd). Mutational substitutions F107D and A110D in the assumed interface ArdA, which determines contact between monomers in the active dimer (Ard)₂, cause an approximately 100-fold decrease in the antirestriction protein activity. It is hypothesized that the ArdA protein forms two complexes with the type I restriction–modification enzyme (R₂M₂S): (1) with a specific region in the S subunit involved in contact with the sK site in DNA; and (2) with a nonspecific region in the R subunit involved in DNA translocation and degradation by restriction endonucleases. The association of ArdA with the specific region inhibits restriction endonuclease and methyltransferase activities simultaneously, whereas the association of ArdA with a nonspecific region inhibits only restriction endonuclease activity of the R₂M₂S enzyme.     

Seroprevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.

     

The Effects of Regulatory Proteins RcsA and RcsB on the Expression of the Vibrio fischeri lux Operon in Escherichia coli

A study was made of the effect of RcsA and RcsB on the Vibrio fischeri lux expression in Escherichia coli. RcsA suppressed the LuxR activity and thereby inhibited expression of the lux genes coding for luciferase and reductase. In osmotic shock, RcsA–RcsB activated lux expression and, consequently, the bioluminescence of E. coli cells in the early log phase.     

Sequencing and Comparative Analysis of the lux Operon of Photorhabdus luminescens Strain Zm1: ERIC Elements as Putative Recombination Hot Spots

The EcoRI chromosomal fragment (6782 bp) containing the lux operon of Photorhabdus luminescens was cloned in pUC18 and completely sequenced. Enteric repetitive intergenic consensus (ERIC), an imperfect palindrome (125–127 bp) characteristic of Enterobacteriaceae genomes, was found in three sites. Strain Zm1 proved to differ in the ERIC number and location from strains Hb, Hm, and Hw. Nucleotide substitution analysis showed that luxC and luxB, which are more than 1 kb away from ERIC, are similar to the corresponding Hb genes, whereas luxD, luxA, and luxE, which are close to ERIC, are intermediate between their Hb and Hw counterparts. The Hb/Hw nucleotide substitution ratio was 1:1 in regions adjacent to ERIC. Hence ERIC can be thought to be recombination hot spots in the bacterial genome.     

DNA mimicry by proteins as effective mechanism for regulation of activity of DNA-dependent enzymes

Modern concepts on mechanisms of DNA-dependent enzyme regulation involving specific DNA-mimicking proteins are considered. There are proteins that share structural resemblance with DNA duplexes. These include inhibitors of type I restriction-modification enzymes (Ocr and ArdA), inhibitors of DNA gyrase MfpA and QnrABS, etc. We describe here structural features of these proteins and mechanisms responsible for their interaction with DNA-dependent enzymes and then discuss perspectives of use of DNA-mimicking proteins in analysis of replication, repair, recombination, mechanisms underlying resistance to antibiotics, and also fields of applied biotechnology.     

Antirestriction and antimodification activities of T7 Ocr: Effects of amino acid substitutions in the interface

The T7 antirestriction protein Ocr, encoded by 0.3 (ocr), specifically inhibits ATP-dependent type I restriction-modification systems. T7 0.3 (ocr) was cloned in pUC18. Ocr inhibited both restriction and modification activities of the type I restriction-modification system (EcoKI) in Escherichia coli K12. The Ocr F53D A57E mutant was obtained and proved to inhibit only restriction activity of EcoKI. The 0.3 (ocr) and Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P _ltetO-1 promoter, strongly controlled by the TetR repressor. The bioluminescence intensity and luciferase content varied up to 5000-fold in E. coli K12 MG1655Z1 tetR⁺ (pZE21-luxCDABE) cells, depending on the environmental concentration of the inductor anhydrotetracycline. The antirestriction activity of Ocr and Ocr F53D A57E was studied as a function of their concentration in the cell. The dissociation constant K _d, characterizing the binding with EcoKI, differed 1000-fold between Ocr and Ocr F53D A57E (10^?10 M versus 10^?7 M).