Temperature-responsive genetic loci in the plant pathogen Pseudomanas syringae pv. glycinea

Plant-pathogenic bacteria may sense variations in environmental factors, su ch as temperature, to adapt to plant-associated habitats during pathogenesi s or epiphytic growth. The bacterial blight pathogen of soybean, Pseudomona s syringae pv. glycinea PG4180, preferentially produces the phytotoxin coro natine at 18 degreesC and infects the host plant under conditions of low te mperature and high humidity. A miniTn5-based promoterless glucuronidase (ui dA) reporter gene was used to identify genetic loci of PG4180 preferentiall y expressed at 18 or 28 degreesC. Out of 7500 transposon mutants, 61 showed thermoregulated uidA expression as determined by a three-step screening pr ocedure. Two-thirds of these mutants showed an increased reporter gene expr ession at 18 degreesC whilst the remainder exhibited higher uidA expression at 28 degreesC. MiniTn5-uidA insertion loci from these mutants were subclo ned and their nucleotide sequences were determined. Several of the mutants induced at 18 degreesC contained the miniTn5-uidA insertion within the 32.8 kb coronatine biosynthetic gene cluster. Among the other mutants with incr eased uidA expression at 18 degreesC, insertions were found in genes encodi ng formaldehyde dehydrogenase, short-chain dehydrogenase and mannuronan C-5 -epimerase, in a plasmid-borne replication protein, and in the hrpT locus, involved in pathogenicity of P. syringae. Among the mutants induced at 28 d egreesC, insertions disrupted loci with similarities to a repressor of conj ugal plasmid transfer, UV resistance determinants, an isoflavanoid-degradin g enzyme, a HU-like DNA-binding protein, two additional regulatory proteins , a homologue of bacterial adhesins, transport proteins, LPS synthesis enzy mes and two proteases. Genetic loci from 13 mutants did not show significan t similarities to any database entries. Results of plant inoculations showe d that three of the mutants tested were inhibited in symptom development an d in plants multiplication rates. Temperature-shift experiments suggested t hat all of the identified loci showed a rather slow induction of expression upon change of temperature.