Our goal in this work was to develop a strategy for identifying new targets for antibiotic potentiators. Using a straightforward replica-plating approach, we succeeded in identifying genes whose disruption renders A. baylyi hypersusceptible to several clinical antibiotics. The corresponding gene products could be considered putative targets for antibiotic potentiators. In these studies, we used A. baylyi ADP1 as a model organism. The very high degree of competence of A. baylyi greatly facilitates the transfer of the identified transposon insertion mutations into wild-type cells and the subsequent preparation of strains with chromosomal replacement of the identified genes. In general, however, our approach can be applied to any microorganism for which a gene-knockout library is available. The task of identifying the intrinsic antibiotic resistance genes was significantly aided by the use of a DNA microarray printer, which allowed the fast and easy replica plating of 10,000 individual strains on plates with 12 different antibiotics.
Eleven genes in A. baylyi
whose disruption leads to antibiotic hypersusceptibility were identified. Among these genes was the homolog of acrB
, which encodes a component of the multidrug resistance pump in E. coli
). Disruption of such pumps in other bacteria is known to confer multidrug resistance (12
). Some of the previous studies also indicated that mutations in the rec
genes may increase cell sensitivity to quinolones (18
). This result validated our approach and demonstrated that the colony-printing technique can be used effectively to detect mutants hypersusceptible to antibiotics. To the best of our knowledge, the other genes that we identified were not previously described as direct contributors to the intrinsic antibiotic resistance and thus were not previously considered targets for putative antibiotic potentiators.
Several of the genes identified provide intrinsic resistance to one or more β-lactam antibiotics (Table ). In three cases, ampD
, and pbpG
, this conclusion is in generally good agreement with the known functions of the corresponding gene products, since they are involved in biosynthesis or recycling of peptidoglycan and thus belong to the same biochemical pathway that is targeted by β-lactam antibiotics. AmpD is involved in the breakdown of the anhydromuropeptides produced upon degradation of old peptidoglycan (9
), whereas Mpl links the tripeptide l
-diaminopimelate, released by AmpD, to one of the main precursors of peptidoglycan synthesis, UDP-N
). Gene pbpG
encodes two low-molecular-weight penicillin-binding proteins (PBPs), PBPs 7 and 8, in E. coli
, which play a role in the remodeling of peptidoglycan (8
). However, beyond the general observation that the protein products identified are involved in the biochemical pathway affected by the antibiotic, it is hard to explain why disruptions of these particular genes and not of multiple others involved in the same pathway render cells hypersusceptible to the drugs. This uncertainty underscores the importance of experimental approaches, similar to the one described in this paper, for identification of putative targets for antibiotic potentiators.
Four other genes, argH
, and ACIAD0795, whose knockout increases the susceptibility of A. baylyi
to β-lactam antibiotics, encode proteins with functions seemingly unrelated to the biosynthesis of the bacterial cell wall. ArgH and HisF are involved in the biosynthesis of arginine and histidine, respectively, while gph
encodes the housekeeping enzyme 2-phosphoglycolate phosphatase, which is induced during oxidative stress (21
). The presumed function of Gph is to metabolize the 2-phosphoglycolate produced in the repair of DNA lesions (21
). A hypothetical 167-amino-acid protein is encoded by an A. baylyi
gene, ACIAD0795, that contains a domain that exhibits similarity to the conserved domain of the erfK
family of proteins in E. coli
, whose function is obscure. At the moment, it is unclear why disruption of any of these four genes in A. baylyi
causes hypersusceptibility to β-lactam antibiotics.
Finally, genetic knockouts of gshA
increased the sensitivity of A. baylyi
to metronidazole and ciprofloxacin, respectively. Metronidazole is a prodrug which, upon activation, forms highly active radical species that cause DNA damage (15
). Therefore, it makes sense that the disruption of gshA
, the gene whose product is involved in the biosynthesis of the reducing agent glutathione, increased the sensitivity of A. baylyi
to metronidazole. Similarly, it is not surprising that the genetic knockout of recD
, a component of the recBCD
complex that plays a major role in DNA repair and recombination (1), increases bacterial sensitivity to the DNA-damaging drug ciprofloxacin. Nevertheless, as mentioned earlier, without the experimental data obtained in this study, it would be difficult to “handpick” these particular genes as targets for antibiotics potentiators.
Although in our experiments a number of genes were found to contribute to the intrinsic antibiotic resistance of A. baylyi, we cannot accurately estimate how exhaustive our screening was and whether we identified all or even the majority of the A. baylyi intrinsic resistance genes. On the one hand, our screen revealed five independent insertions into recD. This result may indicate that either mutagenesis was close to saturation or that recD represents a transposon insertion hot spot. On the other hand, the random nature of colony picking and the testing of a fairly limited number of clones (which was of the same order as the number of genes in A. baylyi) could have left some mutants with the hypersusceptible phenotype untested.
One of the unexpected findings of this work was that disruption of many genes which contribute to the intrinsic resistance of A. baylyi to antibiotics had very little or no effect on the susceptibility of E. coli to these drugs. Although this result might be viewed as discouraging for the development of broad-range antibiotic potentiators, it opens the possibility of expansion of the spectrum of available drugs to specific classes of pathogens as well as the development of narrow-spectrum potentiators fine-tuned to combat particular infections.