he objective of this study was to examine and quantify the ability of
environmental isolates of Acinetobacter spp. to restore salicylate hydroxylase
(SalA+) phenotypes to five Acinetobacter baylyi strain ADPl sa/£ mutants, via
natural transformation, and to investigate the organization and diversity of
salicylate gene homologues within Acinetobacter. The ADP] salA allele was
disrupted using overlap extension PCR mutagenesis (OEPM) so that small 4 bp
deletions were incorporated within different gene regions, creating five mutant
strains, ADPW257 - ADPW261 . Out of 34 Acinetobacter isolates examined,
three salA homologues that had 60% DNA identity with the ADPI salA, were
isolated from Acinetobacter sp. strains AD3-l , FSS0 and BS6. The salA genes
from Acinetobacter sp. strains AD3-l and FSS0, shared 98% identity. A DNA
fragment cloned from the genome of Acinetobacter sp. strain AD3-l contained
three open reading frames, identified from the nucleotide sequence as: salA
(encoding a salicylate l-hydroxyalse), salK (encoding a putative salicylate
transport protein with closest homology to the ADP] BenK benzoate transport
protein) and salR (encoding a LysR-type transcriptional regulator).
A quantitative assessment of transformation frequency was conducted using
homologous ADPl and heterologous AD3-I donor DNA: consisting of
plasmid-borne salicylate operons, cloned copies of truncated salA and total
genomic DNA digests. Transformation frequencies resulting from natu.ral
transformation and homologous recombination with homologous DNA were
affected by the allelic position of the mutation and the length of flanking DNA
homology. Recombination using heterologous donor DNA appeared to be
confined to small regions of micro-homology resulting in overall
transformation frequencies of ~ 10·1 . Significantly reduced transformation
frequencies (centrally located, indicating that either recombination events occurring in this
region proved lethal to protein activity or that possibly the central region of the
gene was resistant to homologous recombination.