Functions of the mismatch repair gene mutS from Acinetobacter sp. strain ADP1

Abstract

The genus Acinetobacter encompasses a heterogeneous group of bacteria that are ubiquitous in the natural environment due in part to their ability to adapt genetically to novel challenges. Acinetobacter sp. strain ADP1 (also known as strain BD413) is naturally transformable and takes up DNA from any source. Donor DNA can be integrated into the chromosome by recombination provided it possesses sufficient levels of nucleotide sequence identity to the recipient's DNA. In other bacteria, the requirement for sequence identity during recombination is partly due to the actions of the mismatch repair system, a key component of which, MutS, recognizes mismatched bases in heteroduplex DNA and, along with MutL, blocks strand exchange. We have cloned mutS from strain ADP1 and examined its roles in preventing recombination between divergent DNA and in the repair of spontaneous replication errors. Inactivation of mutS resulted in 3- to 17-fold increases in transformation efficiencies with donor sequences that were 8 to 20% divergent relative to the strain ADP1. Strains lacking MutS exhibited increased spontaneous mutation frequencies, and reversion assays demonstrated that MutS preferentially recognized transition mismatches while having little effect on the repair of transversion mismatches. Inactivation of mutS also abolished the marker-specific variations in transforming efficiency seen in mutS(+) recipients where transition and frameshift alleles transformed at eightfold lower frequencies than transversions or large deletions. Comparison of the MutS homologs from five individual Acinetobacter strains with those of other gram-negative bacteria revealed that a number of unique indels are conserved among the Acinetobacter amino acid sequences.

FIG. 1

Cloning of mutS and flanking DNA from the chromosome of Acinetobacter sp. strain ADP1. Small arrows indicate the degenerate primers MUTSF2 (F) and MUTSR3 (R) used for PCR amplification of a 1.9-kb segment of mutS. Shaded regions indicate this portion of mutS throughout the figure. Strain ADP7003 was formed by integration of pZR7000 into the chromosome of strain ADP1. pGEM-3Zf(+) does not replicate in strain ADP1, so selection for ampicillin resistance (ap^r), encoded on the vector, demanded strain ADP7003. Digestion of chromosomal DNA from strain ADP7003 with EcoRI yielded a fragment containing pGEM-3Zf(+) fused to a segment of upstream DNA that included the 5′ end of mutS, and chromosomal DNA extending to the first EcoRI site upstream of the gene. Circularization of the restriction fragments by ligation followed by transformation into E. coli DH5α and selection for Ap^r resulted in pZR7009. The 3′ end of mutS and downstream DNA were cloned in the same manner except that ADP7003 DNA was digested with BamHI rather than EcoRI, and the resulting plasmid was designated pZR1010.

FIG. 2

Genetic organization of the mutS region of the Acinetobacter sp. strain ADP1 chromosome. The annealing sites of primers MUTSF2, MUTSR3, and MUTC are indicated as horizontal arrows. The symbol  represents putative transcription terminators downstream from mutS (5′-ATAAGTAGCCATCGTGCTACTTAT-3′) and downstream from fdxA (5′-AAAAGATCAGCATTAGCTGATCTTTT-3′). Horizontal lines indicate inserts in plasmids containing overlapping portions of mutS.

FIG. 3

Conserved and divergent amino acid sequences in Acinetobacter MutS. Horizontal arrows indicate degenerate primers originally used to amplify a portion of mutS from the chromosome of strain ADP1. Boxes surround amino acid residues conserved in the Acinetobacter MutS and in the E. coli MutS for which the crystal structure has been determined (29). A vertical arrow indicates a conserved phenylalanine residue that has been shown to be required for mismatch binding in other MutS homologs. Numbers in parentheses indicate positions in the E. coli MutS sequence corresponding to the starts of indels that distinguish the primary sequences of the two MutS proteins.

FIG. 4

Indels that distinguish Acinetobacter MutS homologs from those of other gram-negative bacteria. Portions of a multiple sequence alignment depicting Acinetobacter MutS indels relative to other gram-negative homologs. Dashes indicate gaps in the aligned sequences. Numerals indicate positions corresponding to amino acids in the E. coli MutS primary sequence (29) that are located immediately prior to the start of the indel. Bold type indicates amino acids that are identical to those in Acinetobacter sp. strain ADP1.

FIG. 5

Phylogenetic tree based on alignments of MutS from Acinetobacter with those from nine other gram-negative bacteria. The tree was generated as described in Materials and Methods. Bootstrap values are indicated on tree branches.