Experimental Studies On Escherichia Coli K12Mutants Induced By Ion Beam And High Voltage Electric Field

Accompanying the increasing worries about transgenic species the bioeffects of traditional physical factors, especially the mutational breeding of physical factors attract more and more attentions in the international circle of scientists. Electric field and low energy ion beam as two common mutagenic factors, after decades of research, have gained outstanding achievements; however, the biological mechanisms of such two mutagenic factors remain unclear.Can the bioeffects of electric field be hereditable? In other words, does electric field have mutagenic effects? There was a noncommittal answer on this question in scientists. This paper uses the model organism E.coli as a research object. Firstly, we studied the mutation rate of several plate electric field. Results indicated that both the high voltage DC, AC, and half-wave rectification electric fields all induced stimulative effects at low dosage, and with the dosage increasing these three plate electric fields induced inhibitory effects. Among three electric fields, the most evident effects were induced by DC plate electric field. At the field intensity of4.5kV/cm, the mutation rate reaches maximum.5.8×10-6, which is2.32times more than the controls. So, although these three plate electric field have mutagenic effects on E.coli, but the mutagenic effects seem not obvious. However, the case is different for prickle electrostatic field. Escherichia coli K12W3110was treated by high voltage prickle electrostatic field(HVPEF)10min with the field intensity of1,2,3and4kV/cm respectively. The mutant frequency reached31.2×10-6at field intensity of1kV/cm, which was about26-fold over the background (1.2×10-6) and12-fold over the controls (2.5×10-6). The result indicated that the HVPEF could act as an effective mutagen. It was observed that the base pair (bp) substitutions were the main type of HVPEF induced mutations. All types of base pair substitutions were observed,30%of the bp substitutions were G:Câ†'A:T transitions, and70%of the bp substitutions were A:Tâ†'T:A, G:Câ†'T:A, A:Tâ†'C:G, G:Câ†'C:G transversions. As compared with the spontaneous group, the base-pair substitutions, deletions and additions of the HVPEF exposure group were significantly increased. Although the deletion/addition of a5’-TGGC-3’sequence at hot spot was decreased from82%to26%, the remarkable increase in absolute Mutation Frequency (MF) of the deletion or the addition of a5’-TGGC-3’sequence at hot spot in the HVPEF exposure group suggested that HVPEF did induce the mutations of this type. The spectra of our HVPEF exposure group with increasing G:C to A:T transitions and A:T to T:A transversions show their similarity with SOS response and indicate that the HVPEF exposure may induce SOS response. On the other hand, from the occurrence of large deletion fragment of of280bp and the high base pair deletion/insertion ratio, we find that the bacterial genome evolution is biased to deletion.Low-energy ion beam biotechnology was developed in the1980s by Chinese scientists. Low-energy ion beam can cause strong mutagenesis in microbes and plants seeds and has been introduced as a useful tool for microbial and plant breeding. However the specificity and mechanism on the biological effect of low-energy ion beam remains unclear. Wild type Escherichia coli K12strain W3110was irradiated by10keV nitrogen ions. It was found that the multiple ion irradiations can increase genetic stability of mutants and generate a wider mutational spectrum. Through irradiation by N+ions six times and selection of lac constitutive mutants after each time a stable mutant S55was obtained. By sequencing the whole genome the fine map of S55was completed. Compared with reference sequences, a total of eighteen single nucleotide polymorphisms (SNPs), two insertions and deletions (Indels) and nine structural variations (SVs) were found in S55genome. Among18SNPs,11are transversional from A, T, or C to G, accounted for55.6%of point mutations, and7transitional accounted for44.4%. GCCA insertion occurs in the target gene lacI. Four SNPs (three SNPs in rlpB and one in ygbN) are connected with cell envelope and transport. The mutation of these genes possibly makes Escherichia coli genome more adapted to the etch and damage caused by ion irradiation. All nine structural variations of S55are of deletion type. Each of the deleted regions is longer than1000bp and contains insertion sequence (IS) elements. Six deleted SVs contain IS disrupted, nonfunctional pseudogenes and another one of length23252bp is the Rac prophage region. The result shows that the deletion bias observed in the Escherichia coli K12genome evolution is generally related to the deletion of some non-functional regions. Furthermore, since ISs are instable factors in a genome, the multiple ion irradiation that causes these fragments deleted in S55is beneficial to the genome stability.