Pseudomonas aeruginosa MutL protein functions in Escherichia coli

Abstract

Escherichia coli MutS, MutL and MutH proteins act sequentially in the MMRS (mismatch repair system). MutH directs the repair system to the newly synthesized strand due to its transient lack of Dam (DNA-adenine methylase) methylation. Although Pseudomonas aeruginosa does not have the corresponding E. coli MutH and Dam homologues, and consequently the MMRS seems to work differently, we show that the mutL gene from P. aeruginosa is capable of complementing a MutL-deficient strain of E. coli. MutL from P. aeruginosa has conserved 21 out of the 22 amino acids known to affect functioning of E. coli MutL. We showed, using protein affinity chromatography, that the C-terminal regions of P. aeruginosa and E. coli MutL are capable of specifically interacting with E. coli MutH and retaining the E. coli MutH. Although, the amino acid sequences of the C-terminal regions of these two proteins are only 18% identical, they are 88% identical in the predicted secondary structure. Finally, by analysing (E. coli-P. aeruginosa) chimaeric MutL proteins, we show that the N-terminal regions of E. coli and P. aeruginosa MutL proteins function similarly, in vivo and in vitro. These new findings support the hypothesis that a large surface, rather than a single amino acid, constitutes the MutL surface for interaction with MutH, and that the N- and C-terminal regions of MutL are involved in such interactions.

The small FNR regulon of Neisseria gonorrhoeae: comparison with the larger Escherichia coli FNR regulon and interaction with the NarQ-NarP regulon

“…However, as some sensor kinases have been shown to phosphorylate response regulators of a heterologous host [21,22], the ability of gonococcal NarQ to phosphorylate the E. coli NarP protein was assessed to investigate the ligand sensing and signal transduction characteristics of the gonococcal NarQ and NarP proteins. Strain JCB391 ( narXL narQ ) and JCB391 transformed with pBADgcQ, expressing gonococcal narQ , each co-transformed with pRNW15 carrying napF :: lacZ , were grown anaerobically in the presence or absence of nitrate and nitrite and their β-galactosidase activities determined (Table 2B).…”

Analysis of the Quaternary Structure of the MutL C-terminal Domain

“…Since some proteins e.g. UvrD helicase and MutH, can interact with both the NTD and the CTD of MutL, 5,11,13,44 prediction and identification of protein interaction sites in the CTD provides a platform for studying possible arrangements of the NTD and CTD of MutL.…”

“…We have shown before that the binding site of MutH in the NTD is located in a region close to residues 169, 251, 314 and 327 (Figure 9). 16 In addition, other data suggested that the CTD is also in direct physical contact to MutH and UvrD, 5,11,44 probably via the conserved surface patch. The model shown in Figure 9 offers the attractive opportunity that interaction partners such as MutH and UvrD could bind between the conserved patch of the CTD and the highly conserved NTD.…”

A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species

“…We suspect that pORTMAGE does not require high sequence conservation, as long as the functional role of MutL in the mismatch-repair system remains unchanged. Indeed, despite substantial differences in MutL sequences between Pseudomonas aeruginosa and E. coli, the P. aeruginosa copy is able to complement that of E. coli (38). In fact, even the human mismatchrepair protein MutL homolog 1 (hMLH1) functionally interacts with the E. coli MMR machinery and was able to induce a dominant mutator state in E. coli (39).…”