Discovery of a cutinase-producing Pseudomonas sp. cohabiting with an apparently nitrogen-fixing Corynebacterium sp. in the phyllosphere

Abstract

A phyllospheric bacterial culture, previously reported to partially replace nitrogen fertilizer (B. R. Patti and A. K. Chandra, Plant Soil 61:419-427, 1981) was found to contain a fluorescent pseudomonas which was identified as Pseudomonas putida and a Corynebacterium sp. The P. putida isolate was found to produce an extraceliular cutinase when grown in a medium containing cutin, the polyester structural component of plant cuticle. The Corynebacterium sp. grew on nitrogen-free medium but could not produce cutinase under any induction conditions tested, whereas P. putida could not grow on nitrogen-free medium. When cocultured with the nitrogen-fixing Corynebacterium sp., the P. putida isolate grew in a nitrogen-free medium, suggesting that the former provided fixed N2 for the latter. These results suggest that the two species coexist on the plant surface, with one providing carbon and the other providing reduced nitrogen for their growth. The presence of cutin in the medium induced cutinase production by P. putida. However, unlike the previously studied fungal systems, cutin hydrolysate did not induce cutinase. Thin-layer chromatographic analysis of the products released from labeled apple fruit cutin showed that the extracellular enzyme released all classes of cutin monomers. This enzyme also catalyzed hydrolysis of the model ester substrates, p-nitrophenyl esters of fatty acids, and optimal conditions were determined for a spectrophotometric assay with p-nitrophenyl butyrate as the substrate. It did not hydrolyze triacyl glycerols, indicating that the cutinase activity was not due to a nonspecific lipase. It showed a broad pH optimum between 8.0 and 10.5 with 3H-labeled apple cutin as the substrate. Diisopropylfluorophosphate severely inhibited the enzyme, whereas thiol-directed reagents did not inhibit it, suggesting that catalysis by this bacterial cutinase involves active serine.It has been suggested that a well-established and mixed population of microorganisms in the phyllosphere could make a substantial contribution to the nitrogen requirements of field vegetation (16). Some nitrogen-fixing organisms have been isolated from the leaf surface (8, 13, 18). These nitrogen fixers, when sprayed on the crop plants, increased crop yield when compared with crops which received no nitrogen fertilizers (8). The carbon source for these organisms on the leaf surface is not known. Proposed sources of nutrients include leaf exudates or organic deposits on the leaf surface. Whether these nitrogen fixers are capable of utilizing any of the leaf surface components has not been determined. If the microorganisms are to utilize the insoluble polymer cutin on the leaf surface, these organisms would have to produce an extracellular cutinase. Although cutinases have been purified and characterized from fungi and pollen (2, 3), little is known about bacterial cutinase. In this paper we report that a phyllospheric fluorescent Pseudomonas putida strain, coisolated with a nitrogen-fixing bacterium, may be induced to produce cutina...

Identification and Characterization of Bacterial Cutinase

“…There are a few reports of bacterial cutinases from Streptomyces scabies (19), Thermobifida fusca (20), and Pseudomonas putida (6,21); however, these studies were limited to screening of enzyme-producing strains and initial characterization on crude enzyme preparations. Most critically, no cutinase open reading frame has been identified in bacteria, thus it is impossible to obtain sufficient amounts of pure recombinant enzymes for in depth studies.…”

Three New Cutinases from the Yeast Arxula adeninivorans That Are Suitable for Biotechnological Applications

“…(21), Thermobifida fusca (7), and a Pseudomonas sp. (22), and in yeast, such as Cryptococcus spp. (4,23) and Pseudozyma antarctica (6,24).…”

Cutinase structure, function and biocatalytic applications

“…It was shown that inhibitors of cutinase could prevent fungal penetration of plants and thus prevent infection (Soliday and Kolattukudy, 1983). Since this enzyme appears to play an important role in virulence, several studies have been performed to elucidate its physiological and biochemical properties (Purdy and Kolattukudy, 1975;Lin and Kolattukudy, 1980;Dantzig et al, 1986;Murphy et al, 1996;Sebastian et al, 1987;Soliday and Kolattukudy, 1983). Cutinases are serine hydrolases specific for primary alcohol esters, the dominant linkage in cutin (Murphy et al, 1996).…”

“…Cutinases have been purified and characterized from several different sources, mainly fungi (Purdy and Kolattukudy, 1975 ;Lin and Kolattukudy, 1980;Dantzig et al, 1986;Murphy et al, 1996) and pollen Sebastian et al, 1987). Evidence of a bacterial cutinase produced by a phyllospheric fluorescent Pseudomonas putida strain, cohabiting with a nitrogen-fixing bacterium Corynebacterium sp., has also been demonstrated (Sebastian et al, 1987).…”