Role of pfkA and general carbohydrate catabolism in seed colonization by Enterobacter cloacae

Enterobacter cloacae A-11 is a transposon mutant of strain 501R3 that was d eficient in cucumber spermosphere colonization and in the utilization of ce rtain carbohydrates (D. P. Roberts, C. J. Sheets, and J. S. Hartung, Can. J . Microbiol. 38:1128-1134, 1992). In vitro growth of strain A-11 was reduce d or deficient on most carbohydrates that supported growth of strain 501R3 but was unaffected on fructose, glycerol, and all amino acids and organic a cids tested. Colonization by strain A-11 was significantly reduced (P less than or equal to 0.05) for cucumber and radish seeds compared to that of st rain 501R3, but colonization of pea, soybean, sunflower, and sweet corn see ds was not reduced. Pea seeds released several orders of magnitude more tot al carbohydrates and amino acids than cucumber and radish seeds and approxi mately 4,000-fold more fructose. Fructose was the only carbohydrate detecte d in the seed exudates which supported wild-type levels of in vitro growth of strain A-11. Soybean, sunflower, and sweet corn seeds also released sign ificantly greater amounts of fructose and total carbohydrates and amino aci ds than cucumber or radish seeds. The exogenous addition of fructose to cuc umber and radish seeds at quantities similar to the total quantity of carbo hydrates released from pea seeds over 96 h increased the populations of str ain A-11 to levels comparable to those of strain 501R3 in sterile sand. Mol ecular characterization of strain A-11 indicated that the mini-Tn5 kanamyci n transposon was inserted in a region of the genome with significant homolo gy to pfkA, which encodes phosphofructo kinase. A comparison of strain A-11 with Escherichia coli DF456, a known pfkA mutant, indicated that the nutri tional loss phenotypes were identical. Furthermore, the pfkA homolog cloned from E. cloacae 501R3 complemented the nutritional loss phenotypes of both E. coli DF456 and E. cloacae A-11 and restored colonization by strain A-11 to near wild-type levels. These genetic and biochemical restoration experi ments provide strong evidence that the quantities of reduced carbon sources found in seed exudates and the ability of microbes to use these compounds play important roles in the colonization of the spermosphere.