Distinguishing Characteristics of Hyperrecombinogenic RecA Protein from Pseudomonas aeruginosa Acting in Escherichia coli

Abstract

In Escherichia coli, a relatively low frequency of recombination exchanges (FRE) is predetermined by the activity of RecA protein, as modulated by a complex regulatory program involving both autoregulation and other factors. The RecA protein of Pseudomonas aeruginosa (RecA Pa ) exhibits a more robust recombinase activity than its E. coli counterpart (RecA Ec ). Low-level expression of RecA Pa in E. coli cells results in hyperrecombination (an increase of FRE) even in the presence of RecA Ec . This genetic effect is supported by the biochemical finding that the RecA Pa protein is more efficient in filament formation than RecA K72R, a mutant protein with RecA Ec -like DNA-binding ability. Expression of RecA Pa also partially suppresses the effects of recF, recO, and recR mutations. In concordance with the latter, RecA Pa filaments initiate recombination equally from both the 5 and 3 ends. Besides, these filaments exhibit more resistance to disassembly from the 5 ends that makes the ends potentially appropriate for initiation of strand exchange. These comparative genetic and biochemical characteristics reveal that multiple levels are used by bacteria for a programmed regulation of their recombination activities.

Modulating cellular recombination potential through alterations in RecA structure and regulation

“…Measurement of FRE to assess elevated or depressed levels of recombination in vivo has been developed over the past 15 years (Namsaraev et al ., 1998; Bakhlanova et al ., 2001; Chervyakova et al ., 2001; Lanzov, 2002; Baitin et al ., 2003; 2006; 2008; Lanzov et al ., 2003). The method has the advantage that it can detect essentially all types of genetic exchanges that might occur during conjugation (Baitin et al ., 2008).…”

“…In this study, we are particularly interested in changes in FRE, using the wild‐type E. coli values as a benchmark called FRE 2 . Therefore, as described previously (Bakhlanova et al ., 2001; Lanzov, 2002; Baitin et al ., 2003; 2006; 2008; Lanzov et al ., 2003), our reported value of ΔFRE, or the ratio of FRE 1 /FRE 2 , indicates the measured FRE for the cross under investigation relative to FRE 2 . ΔFRE can also be calculated by use of the formula: ΔFRE = ln(2µ 1 − 1)/ln(2µ 2 − 1), where µ 1 is the linkage observed with either a modified RecA or the wild‐type RecA under particular conditions of HR, and µ 2 is the linkage observed with wild‐type EcRecA under standard conditions (Bakhlanova et al ., 2001).…”

“…It is also not clear to what extent recombination potential reflects RecA protein structure itself, the modulation of RecA protein function by other factors or the activities of entirely different recombination proteins. Therefore, to directly address the affects of these variables on the in vivo function of RecA, we took advantage of a quantitative genetic analysis of the linkage of donor markers following bacterial conjugation by determining the frequency of recombination exchanges per DNA unit length (FRE) (Namsaraev et al ., 1998; Bakhlanova et al ., 2001; Chervyakova et al ., 2001; Lanzov, 2002; Baitin et al ., 2003; 2006; 2008; Lanzov et al ., 2003). These methods have shown that RecA from E. coli (EcRecA) has a relatively moderate recombinase activity in vivo (Namsaraev et al ., 1998; Lanzov et al ., 2003; Baitin et al ., 2006; 2008), although the activity is presumably optimal for this bacterium.…”

“…Hyper‐recombination arises as a result of the induction of the SOS response, either transiently by treating normal cells with a DNA damaging agent (Sassanfar and Roberts, 1990) or constitutively as observed with certain RecA mutant variants such as RecA E38K ( recA 730) (Lavery and Kowalczykowski, 1992). Hyper‐recombination can also be SOS‐independent, as observed in a set of RecA proteins including the Pseudomonas aeruginosa RecA (PaRecA) (Namsaraev et al ., 1998; Baitin et al ., 2003; 2006) and different E. coli / P. aeruginosa chimeric RecAX proteins (Bakhlanova et al ., 2001; Baitin et al ., 2006; 2008) expressed in E. coli .…”

Strain Engineering by Genome Mass Transfer: Efficient Chromosomal Trait Transfer Method Utilizing Donor Genomic DNA and Recipient Recombineering Hosts

“…The RecA PA protein induces hyper recombination in E. coli, in the absence of SOS induction, and presence or absence of E. coli RecA protein [35].…”