Essentiality of the three carboxyl-terminal amino acids of the plasmid RK2 replication initiation protein TrfA for DNA binding and replication activity in gram-negative bacteria.

J L Cereghino,D R Helinski

doi:10.1016/s0021-9258(19)74553-0

J L Cereghino, D R Helinski

Open Access

https://doi.org/10.1016/s0021-9258(19)74553-0

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Abstract

In a previous study of mutations in trfA, the gene encoding the replication initiation protein of the broad host-range plasmid RK2, a carboxyl-terminal deletion of 3 amino acids of the TrfA protein was found to be completely nonfunctional for RK2 replication in Escherichia coli and other Gram-negative bacteria. In this work site-directed mutagenesis of the trfA gene was carried out to construct TrfA proteins altered in the three carboxyl-terminal positions. Specifically, TrfA proteins with deletions or substitutions of the terminal cysteine, lysine, and arginine (codons 380-382, respectively) were constructed and characterized for their ability to initiate replication from an RK2 origin in vivo in E. coli, Azotobacter vinelandii, Pseudomonas putida, and Agrobacterium tumefaciens and for binding activity to the iterons at the replication origin. Substitutions of the cysteine at position 380 with a glycine or an arginine resulted in a TrfA protein defective in binding to the RK2 origin and, therefore, defective in replication initiation activity in all four Gram-negative bacteria. Substitution of a serine at that position preserved limited function in replication and DNA binding. The lysine at position 381 could be changed to a glutamine without any obvious change in TrfA function. Deletion of the terminal arginine at position 382 did not affect the ability of TrfA to bind to origin iterons but caused a complete loss of replication activity in all four bacteria. Substitution of this terminal arginine with alanine, serine, or glutamic acid also produced replication-defective TrfA protein in all four bacterial hosts while not affecting iteron binding activity. However, substitution of this arginine with a lysine resulted in a loss of replication activity in E. coli and A. vinelandii but had no effect in P. putida and A. tumefaciens. These observations suggest that the terminal arginine plays an essential role in the activity of the TrfA protein, possibly interaction with host proteins, which can be separated from its iteron binding activity.

Highlights

Spectively)wereconstructedandcharacterizedfor their ability to initiate replication from RanK2 origin in vivo in E. coli, Azotobacter vinelandii, Pseudomonas putida, and Agrobacterium tumefaciens and for binding activity to the iteroants thereplication origin
The TrfA protein initiates repwith a lysine resulted in a loss of replication activity lication presumably by binding to the iterons at the RK2 in E. coli and A . vinelandii but had no effect in P. origin of replication and recruiting the appropriate host proputida and A. tumefaciens.These observations suggest teins, thereby allowing replication to proceed (Bramhill and that the terminal arginine plays an essential role Kionrntbheerg, 1988; Marians, 1992; Perri etal.,1991; Pinkney et activity of the TrfA protein,possibly interaction with al., 1987)
We have found that thehigh salt/ethidium bromide extraction method for cleaning up minipreparation plasmid DNA for sequencing does not work well if DNA is prepared from E. coli strain HB101

Summary

TABLEI Strains andplasmids used in this study

F- hsdS(rB- mB-) gal pol his rha F- leuB6proAB recAl3 thi-1 ara-14lacy galK2 xyl-5 mtl-1. DNase footprinting (Perri et al, 1991) and in vitro replication experiments (Kittell and Helinski, 1991; Lin and Helinski, 1992)were carried out as described previously using the purified wild-type and mutantTrfA proteins. Reactions were runon10%SDS-PAGE gels (Laemmli, 1970) and subjected to Western blotting(Durland and Helinski, 1990; Kyhse-Andersen, 1984; Lin and Helinski, 1992)to identify cross-linked products. Other techniques, including clone analysis (Birnboim and Doly, 1979; Holmes and Quigley, 1981),preparations of DNA for sequencing (Lin and Helinski, 1992; Stemmer, 19911, SDS-PAGE, Western blotting with a semidry horizontal blotting apparatus (Durland and Helinski, 1990; Kyhse-Andersen, 1984; Lin and Helinski, 1992), and DNA sequencing of mutations with Sequenase (Durland et al, 1990),were performed as described previously

RESULTS

TrfA in cisb

This study was undertaken as an extension of previous