Resistance of Dickeya solani strain IPO 2222 to lytic bacteriophage ΦD5 results in fitness tradeoffs for the bacterium during infection

Abstract

Resistance to bacteriophage infections protects bacteria in phage-replete environments, enabling them to survive and multiply in the presence of their viral predators. However, such resistance may confer costs for strains, reducing their ecological fitness as expressed as competitiveness for resources or virulence or both. There is limited knowledge about such costs paid by phage-resistant plant pathogenic bacteria in their natural habitats. This study analyzed the costs of phage resistance paid by the phytopathogenic pectinolytic bacterium Dickeya solani both in vitro and in potato (Solanum tuberosum L.) plants. Thirteen Tn5 mutants of D. solani IPO 2222 were identified that exhibited resistance to infection by lytic bacteriophage vB_Dsol_D5 (ΦD5). The genes disrupted in these mutants encoded proteins involved in the synthesis of bacterial envelope components (viz. LPS, EPS and capsule). Although phage resistance did not affect most of the phenotypes of ΦD5-resistant D. solani such as growth rate, production of effectors, swimming and swarming motility, use of various carbon and nitrogen sources and biofilm formation evaluated in vitro, all phage resistant mutants were significantly compromised in their ability to survive on leaf surfaces as well as to grow within and cause disease symptoms in potato plants.

Being spontaneous has its costs! Characterization of spontaneous phage □D5-resistant mutants ofDickeya solanistrain IPO 2222

“…In our earlier work, which analyzed the interaction of P. parmentieri with lytic phage ΦA38, we discovered that this phage required native LPS to infect its host 57 . Likewise, we also showed previously that mutations targeting the LPS cluster in D. solani 58 resulted, among other phenotypes, in resistance to bacteriophage infections 42 . It seems that modifications of LPS and capsule to prevent viral infections frequently occur in bacteria belonging to Soft Rot Pectobacteriaceae , as similar observations were made for other members of this group, including P. atrosepticum , P. carotovorum , and P. brasiliense 59–61 .…”

“…The total time of the assay was 20 min. Phage adsorption was calculated as described previously 42 : the percentage adsorption = (the average titer of unabsorbed phages per sample/average titer of phages in negative control) ×100. The averages and standard deviations (SD) of three independent repetitions per strain (WT or phage-resistant mutants) are shown.…”

“…The sequencing of the DsR34 and DsR207 genomes showed that spontaneous ΦD5 resistance was caused by mutations in two distinct loci not connected so far with phage resistance in SRP bacteria 42 viz . in the gene coding for secretion protein HlyD (mutant DsR34) and in the gene encoding an elongation factor Tu (EF-Tu) (mutant DsR207).…”

“…Bacteria surviving phage infections were purified into individual colonies by at least four passages on TSA agar plates 73 and collected for further studies. Verifying ΦD5 resistance of the obtained D. solani mutants was done as described previously 42 . D. solani IPO 2222 mutants with confirmed ΦD5 resistance were selected for further studies.…”

“…The virulence of spontaneous phage-resistant mutants was tested initially using a whole potato tuber assay 42 . Briefly, potato tubers (5 replicate tubers per bacterial strain) of cv.…”