Isolation and Characterization of Agar-Degrading Paenibacillus Spp
Total Page：16
File Type：pdf, Size：1020Kb
Biosci. Biotechnol. Biochem., 67 (5), 1048–1055, 2003 Isolation and Characterization of Agar-degrading Paenibacillus spp. Associated with the Rhizosphere of Spinach Akifumi HOSODA,1 Masao SAKAI,2,† and Shinjiro KANAZAWA2 1Laboratory of Soil Microbiology, Department of Plant Resources, Graduate School of Bioresource and Bioenvironmental Sciences, and 2Laboratory of Soil Microbiology, Department of Plant Resources, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan Received November 8, 2002; Accepted January 11, 2003 Agar-degrading bacteria in spinach plant roots culti- which is an alternating polymer of D-galactose and vated in ˆve soils were screened, and four strains of 3,6-anhydro-L-galactose linked by alternating b-(1,4) Paenibacillus sp. were isolated from roots cultivated in and a-(1,3) bonds.12) Two types of agar-degradation three soils. The agar-degrading bacteria accounted for processes have been studied on several agar-degrad- 1.3z to 2.5z of the total bacteria on the roots. In con- ing bacteria. The ˆrst type was identiˆed in studies trast, no agar-degrading colony was detected in any soil on Pseudoalteromonas atlantica ATCC 19292.9,13) (non-rhizosphere soil samples) by the plate dilution P. atlantica hydrolyzes agarose by extracellular method, and thus these agar-degrading bacteria may b-agarase. This enzyme cleaves the b-(1,4) linkage speciˆcally inhabit plant roots. All isolates produced between D-galactopyranose and 3,6-anhydro-L-galac- extracellular agarase, and could grow using agar in the tose to give a series of neoagaro-oligosaccharides. culture medium as the sole carbon source. Zymogram Then, neoagarotetraose, one of the major end analyses of agarase showed that all four isolates products, is cleaved at the central b-linkage by extracellularly secreted multiple agarases (75–160 kDa). neoagarotetraose hydrolase to yield neoagarobiose. In addition, the isolates degraded not only agar but also Finally, neoagarobiose is degraded by periplasmic various plant polysaccharides, i.e., cellulose, pectin, a-neoagarobiose hydrolase to the D-galactose and starch, and xylan. 3,6-anhydro-L-galactose, which are metabolized by intracellular enzymes. The other type of agar-degra- Key words: rhizosphere; agarase; Paenibacillus; dation process involves the cleavage of a-(1,3) link- zymogram age in agarose by extracellular a-agarase,3) yielding a series of agaro-oligosaccharides. The agar-degrada- Several types of agarase-producing bacteria tion process of Alteromonas agarlyticus strain GJIB that degrade and utilize agar have been isolated. involves two enzymes: an a-agarase that cleaves the We previously found agar-degrading bacteria in a-(1,3) linkages and a b-galactosidase speciˆc for the rhizobacteria from a vegetable cropping soil, but we cleavage of the 3,6-anhydro-L-galactose units at the could not obtain the pure culture of the bacteria.1) reducing end. Agarotriose was the smallest product Generally, most previously reported agar-degrading detected in this system. bacteria were isolated from marine environments. A A few non-marine agar-degrading bacteria have wide range of agar-degrading bacteria, including been reported, including Cytophaga sp. and Altero- Alteromonas sp.,2,3) Bacillus cereus,4) Cytophaga monas sp.14) isolated from fresh water, and Bacillus sp.,5) Pseudoalteromonas sp.,6,7) Pseudomonas sp.8–10) sp.,15) Cytophaga sp.,16,17) and Streptomyces coe- and Vibrio sp.,11) have been isolated from marine licolor18) isolated from soils. From sewage, a Gram- environments. Since agar is a polysaccharide pro- negative bacterium (unidentiˆed) has been isolated.19) duced by marine red algae, it is natural that most In addition, we have recently reported the presence agar-degrading bacteria are inhabitants of marine of agar-degrading bacteria in plant rhizospheres. habitats. Agar-degrading bacteria are considered to There are various polysaccharides in plant utilize agar as a carbon and energy source to inhabit rhizospheres. Studies on such agar-degrading marine environments. rhizobacteria may provide a clue to the ecology of Agar is composed of two fractions, agarose and non-marine agar-degrading bacteria. agaropectin. Agarose, the main constituent, is a In this study, we isolated agar-degrading bacteria neutral polysaccharide that forms a linear chain from rhizospheres of spinach plants cultivated in structure consisting of repeating units of agarobiose, several soil samples, measured the activity of agarase † To whom correspondence should be addressed. TelWFax: ＋81-92-642-2863; E-mail: msakai＠agr.kyushu-u.ac.jp Isolation of Agar-degrading Paenibacillus spp. from Rhizosphere 1049 produced by these bacteria, and did zymogram tion and degradation of polysaccharides, agar analysis by activity staining. (Wako), CM-cellulose (Sigma Chemical Co., St. Louis, Mo, USA), chitin (NACALAI TESQUE, Materials and Methods INC., Kyoto, Japan), pectin (Sigma), starch (Sigma), and xylan (NACALAI). Agar (2 gWl), CM-cellulose Screening and isolation of agar-degrading bacter- (5 gWl), chitin (4 gWl), pectin (2 gWl), starch (2 gWl), ia. Soil was collected from ˆve vegetable cropping and xylan (5 gWl) were added to a basal medium (0.6 g ˆelds located in southwest Japan. These samples yeast extract, 1.0 g (NH4)2SO4,1.1gMgSO4･7H2O, consisted of a sandy loam (Futsukaichi soil; pH 6.9; 7.0 g K2HPO4,2.0gKH2PO4, and 1.0 l distilled 1.5z organic matter) located in Fukuoka Prefecture; water (pH 7.0)), and the resulting solution was used a silty clay loam located in Kagoshima Prefecture for the polysaccharide test. In the polysaccharide (Kagoshima soil; pH 6.7; 2.6z organic matter); a utilization test, isolates were inoculated in the liquid sandy clay located in Kumamoto Prefecture medium and cultured at 289C for one week in Erlen- (Minamata soil; pH 5.9; 2.0z organic matter); a meryer ‰asks, and the turbidity was measured at light clay (Ashikita soil; pH 6.2; 1.4z organic OD600 to evaluate the growth of the isolates. matter) located in Kumamoto Prefecture; and a loam In the polysaccharide degradation test, the above (Isahaya soil; pH 6.0; 1.2z organic matter) located basal medium supplemented with the polysaccharides in Nagasaki Prefecture. All ˆve soils had been was solidiˆed using gellan gum (15 gWl) and the solid cropped with vegetables. Five replicate soil cores media were used. Isolates were inoculated on the were obtained from each sampling site, pooled, media and cultured at 289C for one week. Degrada- homogenized, and stored without drying at 49C tion of agar and starch was detected by staining with before being used for enrichment and isolation of Lugol's solution;18,20) degradation of CM-cellulose, agar-degrading bacteria. Agar (Wako Pure Chemical chitin, and xylan was detected by staining with Industries, Ltd., Osaka, Japan) dissolved in distilled Congo Red solution;21) and degradation of pectin was water and sterilized was added to each soil sample detected by CTAB staining.22) andadjustedto0.1z (wWw), and the soil water con- Other phenotypic characterizations of the isolates tent was adjusted to 50z (wWw, waterWdry soil). were done as described by Shintani23) and Berge These agar-supplemented soil samples were packed in et al.24) pots and incubated at 289C for seven days before plant cultivation. Phylogenetic analysis of isolates based on 16S Spinach plants (Spinacea oleracea L. ``Atlas'') rDNA sequencing. We did 16S rDNA sequence anal- were cultivated in pots containing the soils in a ysis on four agar-degrading isolates from spinach growth chamber with a relative humidity of 70z at roots, strains M-2b, O-3b, O-4c, and St-4. Bacteria 209C. The daily light schedule consisted of 16 h of for DNA extraction were grown for 3 days at 289C light and 8 h of darkness, and the light intensity was on 0.1 TSA medium. A single colony of each isolate 250 mmol m－2 s－1. After growth for 21 days, plants was removed with a sterile toothpick, resuspended in were harvested and the roots were excised and shaken 20 ml of sterile distilled water, and heated at 959Cfor in a 10 mM phosphate buŠer solution (pH 7.0) to 10 min to lyse the cells. The lysate was then cooled on remove soil particles. The soil-free root samples were ice, brie‰y centrifuged with a microcentrifuge, and cut into fragments 3–5 mm long. The treated roots used for polymerase chain reaction (PCR) ampliˆca- (1.0 g) were then placed in tubes containing 5 ml of a tion. fresh buŠer solution, and glass beads 3 mm in The DNA coding for the 16S rRNA of each isolate diameter (2.5 g) were added. The samples were was ampliˆed with primers 8–27f (5?-AGAGTTTG- shaken on a Vortex mixer (5 min, full speed). The ATCCTGGCTCAG-3?) and 1543–1525r (5?-AAAG- root suspension was serially diluted, and 0.1-ml dilut- GAGGTGATCCAGCC-3?). These primers were ed portions were spread on 0.1 TSA agar medium designed on the basis of the conserved bacterial that contained (per liter) 1.7 g of peptone, 0.3 g of sequences at the 5? and 3? ends of the 16S rRNA soyton,0.25gofglucose,0.25gofK2HPO4,0.5gof gene, which allowed ampliˆcation of almost the NaCl, and 15 g of agar. For the preparation of non- entire gene.25) Ampliˆcation was done as follows: rhizosphere soil samples, the soils were maintained each mixture contained 2 ml of lysed cell suspension under the same conditions and serial dilutions were in 50 mlofTaq buŠer containing 2 mM Mg2＋,1.2mM plated onto 0.1 TSA agar. The plates were incubated of each primer, each deoxynucleoside triphosphate at at 289C for 10 days. Colonies in the cultured plates a concentration of 0.2 mM,and1.25UofTaq DNA were counted, and agar-degrading bacteria in colo- polymerase (TaKaRa Bio,Inc., Shiga, Japan).