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Paenibacillus granivorans sp. nov., a new Paenibacillus Species which Degrades Native Potato Starch Granules
System. Appl. Microbiol. 23, 344-348 (2000) © Urban & Fischer Verlag _ht-,-tp_://w_w_w_ .ur_ba_nf_is_ch_er_.de--'./jo_u_rn_als_/s_am ____________
SYSTEIV14TIC AND APPLIED MICROBIOLOGY
Paenibacillus granivorans sp. nov., a new Paenibacillus Species which Degrades Native Potato Starch Granules M. J. E. C. VAN DER MAAREL 1,2, A. VEEN2, and D. J. WIJBENGA2 Centre for Carbohydrate Bioengineering TNO-RUG, Haren, The Netherlands Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands 2 Carbohydrate Technology Department, TNO Voeding, Groningen, The Netherlands 1
Received July 19,2000
Summary From a native potato starch-degrading enrichment culture, strain A30 had been isolated and had tentatively been identified as a member of the Bacillus firmusllentus group (WIJBENGA et al. Appl. Microbiol. Biotechnol. 35, 180-184, 1991). In this paper the isolate A30 is further characterized using phylogentic analysis of the 16S rDNA and determination of a number of additional phenotypic characteristics. These data are compared to those of Paenibacillus amylolyticus, P. chibensis, and P. thiaminolyticus. It is concluded that strain A30 is a new Paenibacillus species, for which the name Paenibacillus granivorans is suggested. Key words: native potato starch - 16S rDNA phylogeny - phenotypic characterization - Paenibacillus
Introduction Starch is one of the most abundantly available carbohydrates on earth. It is present in leafs, stems, tubers, seeds, and roots of plants as a storage material and serves as an important food and feed stock for humans and animals (ROBYT 1998). The main storage organelle is the amyloplast, in which the starch is present as waterinsoluble granules. Starch is composed of the two glucose-based polymers: (I) amylose containing only a,1-4 glucosidic bonds and having an avarage number of glucose residues between 250 and 5,000 and (II) amylopectin which has between 10,000 and 100,000 glucose residues with a,l-4 glucoside bonds in the backbone and side chains connected via a,1-6 glucosidic bonds (ROBYT 1998). Usually, starch contains 15-30% amylose and 85-70% amylopectin. Starch itself is, due to the presence of amylose, insoluble in water. Industrial use of starch starts with the solubilization and gelatinization by heating a water-starch mixture at high temperatures in a jet cooker (FOGARTY 1983). This process has two major disadvantages: (I) it requires large amounts of energy and (II) it can suffer from viscosity problems. During a study on the breakdown of native starch at moderate temperatures by extracellular bacterial enzyme complexes, WIJBENGA et al. (1991) isolated a Gram-positive, spore forming and motile bacterial strain that grew 0723-2020/00/23/03-344 $ 15.00/0
rapidly on native potato starch granules at moderate temperatures. They identified the isolate as a strain of the genus Bacillus. Based on a number of phenotypic characteristics the closest relatives were Bacillus firm us and Bacillus lentus. In this paper the identification of the isolate is further elaborated using phylogenetic analysis of the 16S rDNA and determination of a number of additional phenotypic characteristics (mol% G+C, fatty acid composition, fatty acid composition, substrates utilized). Based on the new data it is concluded that the isolate is not related to B. firmus or B. lentus but is a new species of the genus Paenibacillus. We propose the name Paenibacillus granivorans sp. nov., which means the Paenibacillus eating (starch) granules.
Materials and Methods Bacterial strain and cultivation The isolate, strain A30 was cultivated in mineral medium as described by WIJBENGA et aI., 1991 under aerobic conditions in 250 ml erlemeyer flask at 200 rpm and 37°C during 16 h. The medium contained the following (per liter distilled water): tryptone (Difco), 5.0 g; (NH 4JzS04' 2.0 g; KH 2 P0 4, 2.5 g; K2 HP0 4, 2.5 g; KCl, 0.5 g; MgS04 . 4H 2 0, 0.2 g; FeCl 3 6H 2 0, 0.02 g;
Paenibacillus granivorans sp. nov.
trace element solution 10 ml; vitamin solution, 10 ml. Before autoclaving NaOH (1M) was added to adjust the pH to 9. The trace element solution had the following composition (per liter): MnCl 2 . 4H20, 400 mg; Na2B407 . 10H2 0, 225 mg; ZnS0 4 . 7H 2 0, 20 mg; CuCl 2 . 2H 2 0, 5 mg; Na2Mo04 . 2H20, 3 mg; VOS0 4 . 5H20, 2 mg; CoS0 4 . 7HP, 1 mg; H 3B01, 20 mg; 0.5M H 2S0 4 , 50 ml. The vitamin solution was composed of the following components per liter: 100 mg thiamine-HCl, 50 mg biotin, 20 mg pyridoxin-HCl, 50 mg riboflavin, 50 mg paminobenzoic acid, 50 mg nicotinic acid, 25 mg D-Ca-pantho then ate; 5 mg folic acid, and 0.5 mg cyanocobalamin. The vitamin solution was sterelized by passing it over an 0.2 ).1m filter. Soluble potato starch (Sigma) was autoclaved seperately. Phenotypic characterization Additional to the standard phenotypic characterization as had been performed by the Deutsche Sammlung fiir Mikroorganismen und Zellkulturen GmbH (WIJBENGA et aI., 1991), it was tested whether a number of substrates not included in the standard analysis of the DSMZ supported growth of strain A30. These substrates were: raffinose, glycerol, alginate, succinate, tween 80, acetate, ureum, fumarate, and malate. This was done by inoculating 1 % of a preculture on glucose to 15 ml of medium with substrate (1 %) in a 100 ml erlemeyer flask and incubating this at 200 rpm and 37°C for 1-2 days. Growth was measured by following the increase in the optical density at 600 nm. Cells for determination of cellular fatty acids and mol % G+C were cultivated for 24 h on 1 % (w/v) soluble potato starch (Sigma) and harvested by centrifugation (15 min 12,000 x g). The cell pellets were send to the DSMZ for determination of mol% G+C by high-performance liquid chromatography (n = 3) and cellular fatty acid composition by gas-chromatography. In the latter case the cells were suspended in isopropanol. Sequencing of the 16S rRNA gene and phylogenetic analysis The 16S rRNA gene sequence was amplified with the S-DBact-0008-a-S-19 and S-D-Bact-1492-a-A-22 primers (notation according to WHEELER ALM et aI., 1996) from one colony which
345
was picked from an agartube and transferred to the PCR reaction mixture without the extraction of genomic DNA. The PCR mixture (50 ).II) contained 50 ).1M of each deoxynucleoside triphosphate, 0.2 ).1M of primer (each) and 5 ).II of 10 x Taq DNA polymerase buffer. After heating the colony with the PCR reaction mixture at 95°C for 5 min 2U Taq polymerase were added and the amplification was started. The PCR conditions were as follows: 30 cycles of 1.5 min denaturation, 1.5 min annealing at 51°C, and extension at 72 °C for 1.5 min. The final step of the PCR protocol consisted of 5 min at 72 0c. PCR was performed on an Eppendorf thermal cycler. The PCR products were analyzed by electrophoresis in 1 % (wt/vol) agarose gels. The nucleotide sequence of the amplified PCR product was determined using the big dye-terminator cycle sequencing kit of Perkin Elmer and an ABI 310 automated DNA sequencer according to the manufacturer's guidelines. The following six primers were used for the cycle sequencing reactions: 1. S-DBact-0050-a-S-19; 2. S-"-Univ-0519-a-S-18; 3. S-"-Univ.-0519a-A-18; 4. S-':--Univ-0909-a-S-20; 5. V--Univ-0909-a-A-20; 6. S-" -Univ-1392-a-A-15. The 16S rRNA gene sequence of strain A30 was sent to the National Center for Biotechnology Information for a BLAST analysis. A number of sequences from the top of the BLAST similarity-ranking list were chosen for a more detailed phylogenetic analysis. Sequences, which were retrieved from the National Centre for Biotechnology Information database, were aligned manually using the Dedicated Comparative Sequence Editor software programme of DE RIJK and DE WACHTER (1993). They had the following accession numbers: P. burgondia, AJ011687; P. azotofixans, D78318; P. polymyxa, AJ223988; P. lautus, D78473; P. chibensis, D85395; P. amylolyticus, X60606; P. thiaminolyticus, D78475; P. lentimorbus, AF071861; P. popilliae, AF071859; B. benzoevorans, Y14693; B.lentus, D78315; B. firmus, D78314; B. subtilus, AB018486. Phylogenetic trees were generated using the TREE CON software package (VAN DE PEER and DE WACHTER, 1994) by the algorithm described by KIMURA (1980) and the neighbor joining method. Calculation of bootstrap values was also done with the TREECON software package.
Paenibacillus burgondia Paenibacillus azotofixans Paenibacillus polymyxa 75 ....---- Paenibacillus lautus Paenibacillus chibensis 71 Paenibacillus amylolyticus 100 ' - - - - - - - - Paenibacillus granivorans Paenibacillus thiaminolyticus 100 59 Paenibacillus lentimorbus Paenibacillus popilliae 100 Fig. 1. Phylogenetic relationship of '----- Bacillus benzoevorans Paenibacillus granivorans to members 98 . . . - - - - Bacillus lentus of the genus Paenibacillus and a few 2% ' - - - - - Bacillus firmus Bacillus species. The phylogenetic tree is based on a comparison of the 16S 1--___ Bacillus subtilis rDNA gene sequence (position 1-1435) and is constructed using the neighbor-joining method. 16S rDNA sequences were retrieved from the Antwerpen small subunit rRNA and the National Centre for Biotechnology Information database. The values indicate bootstraps per 100. The bar represents 2 % nucleotide difference. The sequences were retrieved from the National Center for Biotechnology Information database and have the following accession numbers: P. burgondia, AJ011687; P. azotofixans, D78318; P. polymyxa, AJ223988; P. lautus, D79473; P. chibensis, D85395; P. amylolyticus, X60606; P. thiaminolyticus, D78475; P. lentimorbus, AF071861; P. popilliae, AF071859; B. benzoevorans, Y14693; B.lentus, D78315; B. firmus, D78314; B. subtilus, AB018486.
346
M. J. E. C. VAN DER MAAREL et al.
Results and Discussion Phenotypic characterization A number of different phenotypic characteristics had already been reported by WIJBENGA et al. 1991. Based on their data it was concluded that strain A30 belonged to Bacillus firmus or Bacillus lentus. However, the exact affiliation of strain A30 could not be resolved based on the available phenotypic characteristics. We made a more detailed analysis of the phylogeny of strain A30, by including a number of additional phenotypic characteristics as well as the phylogeny based on the 16S rRNA gene sequence.
From the additional substrates that were tested, only raffinose and glycerol supported growth of strain A30 (Table 1). The avarage mol% G+C was 47.8 :!: 0.3. The majority of the phenotypic characteristics of strain A30 are indeed similar to those of Bacillus firmus or Bacillus lentus (SMITH, 1957; SLEPECKY and HEMPHILL, 1992). Small differences between strain A30 and B. firmus are the growth in 7% NaCI and the hydrolysis of casein of the latter and not by A30. B. lentus is able to hydrolyse casein (SMITH, 1957) but it is not able to grow at 5% NaCl. A major difference between A30, B. firmus, and B. lentus is the mol% G+C: that of strain A30 (47.8%) is
Table 1. Phenotypic characteristics of P. granivorans, P. chibensis, P. thiaminolyticus, and P. amylolyticus.
P. chibensis 2
P. thiaminolyticus 2
P. aminolyticus 3
NRRLB-142
NRRLB-1456
NRRL B-1456
Rod 0.4-0.6 1.5-4.0 Yes Positive Ellipsoid Yes No Positive Negative Positive Negative 5.8 No
Rod 0.5-0.8 3.0-5.0 Yes Positive Ellipsoid Yes No Positive Negative Positive Negative 4.6 No
Rod 0.5-1.0 2.0-3.0 Yes Positive Ellipsoid Yes Yes Positive Positive Positive Negative 4.9-5.5 Yes
Rod 0.7-0.9 3.5-5.0 Yes Positive Oval Yes Yes Positive Negative Positive Negative 5.2 No
No Yes No No No
No Yes No No No
Yes Yes NR Yes NR
NR4 Yes NR No No
No No No
No No No
Yes NR Yes
No No Yes
Yes No Yes Yes Yes Yes No No No 37 No No 47.8
Yes Yes Yes NR No Yes No No No 37 No Yes 52.8
Yes Variable Variable NR NR No No No Yes 28 No No 52-54
Yes Yes Yes Yes NR NR No No No 37 No Yes 48 .1
No No No Yes No
Yes No Yes Yes Yes
Yes Yes No Yes Yes
Yes No No Yes No
P. granivorans l
Cell morphology Width (pm) Length (pm) Motile Gram reaction Spore Swollen sporangia Anaerobic growth Catalase Oxidase Nitrate reduction Voges-Proskauer reaction pH Voges-Proskauer Decomposition of tyrosine Hydrolysis of Casein Starch Gelatin Production of indole Phenylalanine deaminase Use of Citrate Propionate Acetate Fermentation of Glucose Mannitol Arabinose Raffinose Glycerol Xylose Gas from glucose Lecithinase Growth in 5% NaCl Optimum growth temp. (OC) Growth at 50°C Growth at pH 5.7 Mol% G+C Major cellular fatty acids C16:0 Iso C15:0 Iso C16:0 Anteiso C15:0 Anteiso C17:0
IData partially from WlJBENGA et aI., 1991; 2Data from SHIDA et ai., 1997; 3Data from NAKAMURA, 1984; 4NR - not reported
Paenibacillus granivorans sp. nov.
higher than that of B. firmus or B. lentus, being 41.4% and 36.3% respectively (SLEPECKY and H EMPHILL, 1992). Beacause of this considerable differences in mol% G+C, we also sequenced the 16S rRNA gene of strain A30 and compared this in a neighbor-joining analysis to that of members of the genus Bacillus. The phylogenetic analysis based on the 16S rRNA gene sequence showed that strain A30 is only distantly related to B. firmus or B. lentus, with 78.3% similarity to the two Bacillus species. Strain A30 groups within the Paenibacillus genus and is most closely related to P. amylolyticus, P. chibensis, and P. thiaminolyticus with respectively 94.2%, 93.7% and 93.0% similarity (Fig. 1). Strains with approximately 70% or greater DNA-DNA relatedness are considered to be members of the same species (WAYNE et al. 1987). STACKEBRANDT and GOEBEL (1994) concluded that the DNA of organisms that have less than 97.0% 16S rDNA or 16S rRNA similarities will not reassociate to more than 60%, irrespective of the hybridization method used. It is therefore very likely that strain A30 will have not have more than 70% DNA relatedness with other Paenibacillus species and thus can be considered as a new Paenibacillus species. The major cellular fatty acid of strain A30 is anteiso C15:0, which is present as a dominant cellular fatty acid in all members of the genus Paenibacillus (SHIDA et aI., 1997). Only small amounts of the fatty acids C16:0, iso C15:0, iso C16:0, and anteiso C17:0, which are major constituents of P. chibensis, P. amylolyticus, or P. thiaminolyticus were found in strain A30 (Table 1). Comparison of the cellular fatty acid composition of different strains has to be interpreted with caution because differences in the composition may arise because of different culture conditions. Therefore, we do not compare the exact percentages of the fatty acids but only take the major ones into account. Based on results of the phylogenetic analysis of the 16S rRNA gene and the phenotypic characterization, we propose that strain A30 is considered to be a representative of a new species within the Paenibacillus genus. We suggest the name Paenibacillus granivorans, meaning the Paenibacillus eating (starch) granules, for this new species.
Nucleotide sequence accession number The 16S rRNA gene sequence of strain A30 has been deposited in the GeneBank database of the National Center for Biotechnology Information under accession number AF237682.
Description of Paenibacillus granivorans sp. nov.
Paenibacillus granivorans sp. nov. (gra.ni.vo'rans. L. n. grani, granules; L. v. vorare devour to eat, granule-eating bacterium). Cells stain Gram positive, are rod-shaped (0.5-0.8 rm x 1.5-4.0 rm, width x length) forming ellipsoid spores with swelling of the sporangium. The cells are motile by means of flagella which are located peritrichously. Growth is strictly aerobic. Reduction of nitrate to nitrite was observed. Cells are catalase positive and oxi-
347
dase negative. No indole is formed. Phenylalanine deaminase is not present. Growth is possible at temperatures The pH range up to 45°C, with an optimum at 37 for growth is between 6 and 8.5 with an optimum at pH7. No growth was observed at 5% NaCI or higher. The Voges-Proskauer reaction is negative; the pH in Voges-Proskauer broth is 5.8. No decomposition of tyrosine or production of indole was observed. Strain A30 is able to hydrolyse starch but not casein or gelatin. It forms acid from glucose, L-arabinose, raffinose, glycerol, and xylose. No gas is formed from glucose. It does not use citrate, propionate, or acetate. The DNA base composition is 47.8% G+C as determined by HPLC. The major cellular fatty acid is anteiso C15:0. The cellular fatty acids C15:0, C16:0, iso C15:0, iso C16:0, and anteiso C17:0 are present but only in minor amounts. The closest relatives of strain A30 are P. amylolyticus, P. chibensis, and P. thiaminolyticus with respectively 94.2 % , 93.7%, and 93% similarity based on comparison of the 16S rRNA gene. Strain A30 was isolated using granular starch as a substrate from a laboratory reactor fed with waste water from a potato starch production plant. The type strain is strain A30 and is deposited at the Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands (CBS 229.89).
0c.
Acknowledgements
An anonymous reviewer is thanked for critical comments.
References DE RIJK, P., DE WACHTER, R.: DCSE v2.54, an interactive tool for sequence alignment and secondary structure research. Comput. App!. Biosci. 9, 735-740 (1993). FOGARTY, W. M.: Microbial amylases. In: Microbial enzymes and biotechnology, pp. 1-93. Edited by W. M. FOGARTY. London: Applied Science Publishers (1983). KIMURA, M.: A simple model for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mo!. Evo!. 16, 111-120 (1980). NAKAMURA, L. K.: Bacillus amylolyticus sp. nov., nom. rev., Bacillus lautus sp. nov., nom. rev., Bacillus pabuli sp. nov., nom. rev., and Bacillus validus sp. nov., nom. rev. Int. ]. Syst. Bacterio!' 34,224-226 (1984). ROBYT, ]. E: Essentials of carbohydrate chemistry. New York: Springer Verlag (1998). SHIDA, 0., TAKAGI, H., KADOWAKI, K., NAKAMURA, L. K., KOMAGATA, K.: Transfer of Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus to the genus Paenibacillus and emended description of the genus Paenibacillus. Int. J. Syst. Bacterio!' 47, 289-298 (1997). SHIDA, 0., TAKAGI, H., KADOWAKI, K., NAKAMURA, L. K., KOMAGATA, K. : Emended description of Paenibacillus amylolyticus and description of Paenibacillus illinoisensis sp. nov. and Paenibacillus chibensis sp. nov. Int. ]. Syst. Bacterio!' 47, 299-306 (1997). SLEPECKY, R. A., HEMPHILL, H. E.: The genus Bacillus-nonmedical, pp. 1663-1696. In: The Prokaryotes (A. BALOS, H. G.
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M. ]. E. C. VAN OER MAAREL et al.
TRUPER, M. DWORKIN, W. HARDER, K. H. SCHLEIFER, eds.) 2nd ed., New York, Springer Verlag 1992. SMITH, N. R.: Bacillus, pp. 613-634. In: Bergey's manual of determinative bacteriology (R. S. REED, E. G. D. MURRAY, N. R. SMITH, eds.) 7nd ed., Baltimore, The Williams & Wilkins Company 1957. STACKEBRANOT, E., GOEBEL, B. M.: Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. ]. Syst. Bacteriol. 44, 846-849 (1994). VAN DE PEER, Y., DE WACHTER, R.: TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft environment. Comput. Appl. Biosci. 10,569-570 (1994). WAYNE, L. G., BRENNER, D. J., COLWELL, R. R. & 9 other authors: International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. ]. Syst. Bacteriol. 37, 463-464 (1987).
WHEELER ALM, E., OERTHER, D. B., LARSEN, N., STAHL, D. A., RASKIN, L.: The oligonucleotide probe database. Appl. Environm. Microbiol. 62, 3557-3559 (1996). WIjBENGA, D.-]., BELOMAN, G., VEEN, A., BINNEMA, D. J.: Production of native-starch-degrading enzymes by a Bacillus firmusllentus strain. Appl. Microbiol. Biotechnol. 35, 180-184 (1991).
Corresponding author: M. J. E. C. VAN OER MAAREL, Centre for Carbohydrate Bioengineering TNO-RUG, P. O. Box 14, 9750 AA Haren, The Netherlands Tel.: +31-50-3 63-78 32; Fax: +31-50-3 63-21 54; E-mail: [email protected]
SYSTEIV14TIC AND APPLIED MICROBIOLOGY
Paenibacillus granivorans sp. nov., a new Paenibacillus Species which Degrades Native Potato Starch Granules M. J. E. C. VAN DER MAAREL 1,2, A. VEEN2, and D. J. WIJBENGA2 Centre for Carbohydrate Bioengineering TNO-RUG, Haren, The Netherlands Microbial Physiology Research Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands 2 Carbohydrate Technology Department, TNO Voeding, Groningen, The Netherlands 1
Received July 19,2000
Summary From a native potato starch-degrading enrichment culture, strain A30 had been isolated and had tentatively been identified as a member of the Bacillus firmusllentus group (WIJBENGA et al. Appl. Microbiol. Biotechnol. 35, 180-184, 1991). In this paper the isolate A30 is further characterized using phylogentic analysis of the 16S rDNA and determination of a number of additional phenotypic characteristics. These data are compared to those of Paenibacillus amylolyticus, P. chibensis, and P. thiaminolyticus. It is concluded that strain A30 is a new Paenibacillus species, for which the name Paenibacillus granivorans is suggested. Key words: native potato starch - 16S rDNA phylogeny - phenotypic characterization - Paenibacillus
Introduction Starch is one of the most abundantly available carbohydrates on earth. It is present in leafs, stems, tubers, seeds, and roots of plants as a storage material and serves as an important food and feed stock for humans and animals (ROBYT 1998). The main storage organelle is the amyloplast, in which the starch is present as waterinsoluble granules. Starch is composed of the two glucose-based polymers: (I) amylose containing only a,1-4 glucosidic bonds and having an avarage number of glucose residues between 250 and 5,000 and (II) amylopectin which has between 10,000 and 100,000 glucose residues with a,l-4 glucoside bonds in the backbone and side chains connected via a,1-6 glucosidic bonds (ROBYT 1998). Usually, starch contains 15-30% amylose and 85-70% amylopectin. Starch itself is, due to the presence of amylose, insoluble in water. Industrial use of starch starts with the solubilization and gelatinization by heating a water-starch mixture at high temperatures in a jet cooker (FOGARTY 1983). This process has two major disadvantages: (I) it requires large amounts of energy and (II) it can suffer from viscosity problems. During a study on the breakdown of native starch at moderate temperatures by extracellular bacterial enzyme complexes, WIJBENGA et al. (1991) isolated a Gram-positive, spore forming and motile bacterial strain that grew 0723-2020/00/23/03-344 $ 15.00/0
rapidly on native potato starch granules at moderate temperatures. They identified the isolate as a strain of the genus Bacillus. Based on a number of phenotypic characteristics the closest relatives were Bacillus firm us and Bacillus lentus. In this paper the identification of the isolate is further elaborated using phylogenetic analysis of the 16S rDNA and determination of a number of additional phenotypic characteristics (mol% G+C, fatty acid composition, fatty acid composition, substrates utilized). Based on the new data it is concluded that the isolate is not related to B. firmus or B. lentus but is a new species of the genus Paenibacillus. We propose the name Paenibacillus granivorans sp. nov., which means the Paenibacillus eating (starch) granules.
Materials and Methods Bacterial strain and cultivation The isolate, strain A30 was cultivated in mineral medium as described by WIJBENGA et aI., 1991 under aerobic conditions in 250 ml erlemeyer flask at 200 rpm and 37°C during 16 h. The medium contained the following (per liter distilled water): tryptone (Difco), 5.0 g; (NH 4JzS04' 2.0 g; KH 2 P0 4, 2.5 g; K2 HP0 4, 2.5 g; KCl, 0.5 g; MgS04 . 4H 2 0, 0.2 g; FeCl 3 6H 2 0, 0.02 g;
Paenibacillus granivorans sp. nov.
trace element solution 10 ml; vitamin solution, 10 ml. Before autoclaving NaOH (1M) was added to adjust the pH to 9. The trace element solution had the following composition (per liter): MnCl 2 . 4H20, 400 mg; Na2B407 . 10H2 0, 225 mg; ZnS0 4 . 7H 2 0, 20 mg; CuCl 2 . 2H 2 0, 5 mg; Na2Mo04 . 2H20, 3 mg; VOS0 4 . 5H20, 2 mg; CoS0 4 . 7HP, 1 mg; H 3B01, 20 mg; 0.5M H 2S0 4 , 50 ml. The vitamin solution was composed of the following components per liter: 100 mg thiamine-HCl, 50 mg biotin, 20 mg pyridoxin-HCl, 50 mg riboflavin, 50 mg paminobenzoic acid, 50 mg nicotinic acid, 25 mg D-Ca-pantho then ate; 5 mg folic acid, and 0.5 mg cyanocobalamin. The vitamin solution was sterelized by passing it over an 0.2 ).1m filter. Soluble potato starch (Sigma) was autoclaved seperately. Phenotypic characterization Additional to the standard phenotypic characterization as had been performed by the Deutsche Sammlung fiir Mikroorganismen und Zellkulturen GmbH (WIJBENGA et aI., 1991), it was tested whether a number of substrates not included in the standard analysis of the DSMZ supported growth of strain A30. These substrates were: raffinose, glycerol, alginate, succinate, tween 80, acetate, ureum, fumarate, and malate. This was done by inoculating 1 % of a preculture on glucose to 15 ml of medium with substrate (1 %) in a 100 ml erlemeyer flask and incubating this at 200 rpm and 37°C for 1-2 days. Growth was measured by following the increase in the optical density at 600 nm. Cells for determination of cellular fatty acids and mol % G+C were cultivated for 24 h on 1 % (w/v) soluble potato starch (Sigma) and harvested by centrifugation (15 min 12,000 x g). The cell pellets were send to the DSMZ for determination of mol% G+C by high-performance liquid chromatography (n = 3) and cellular fatty acid composition by gas-chromatography. In the latter case the cells were suspended in isopropanol. Sequencing of the 16S rRNA gene and phylogenetic analysis The 16S rRNA gene sequence was amplified with the S-DBact-0008-a-S-19 and S-D-Bact-1492-a-A-22 primers (notation according to WHEELER ALM et aI., 1996) from one colony which
345
was picked from an agartube and transferred to the PCR reaction mixture without the extraction of genomic DNA. The PCR mixture (50 ).II) contained 50 ).1M of each deoxynucleoside triphosphate, 0.2 ).1M of primer (each) and 5 ).II of 10 x Taq DNA polymerase buffer. After heating the colony with the PCR reaction mixture at 95°C for 5 min 2U Taq polymerase were added and the amplification was started. The PCR conditions were as follows: 30 cycles of 1.5 min denaturation, 1.5 min annealing at 51°C, and extension at 72 °C for 1.5 min. The final step of the PCR protocol consisted of 5 min at 72 0c. PCR was performed on an Eppendorf thermal cycler. The PCR products were analyzed by electrophoresis in 1 % (wt/vol) agarose gels. The nucleotide sequence of the amplified PCR product was determined using the big dye-terminator cycle sequencing kit of Perkin Elmer and an ABI 310 automated DNA sequencer according to the manufacturer's guidelines. The following six primers were used for the cycle sequencing reactions: 1. S-DBact-0050-a-S-19; 2. S-"-Univ-0519-a-S-18; 3. S-"-Univ.-0519a-A-18; 4. S-':--Univ-0909-a-S-20; 5. V--Univ-0909-a-A-20; 6. S-" -Univ-1392-a-A-15. The 16S rRNA gene sequence of strain A30 was sent to the National Center for Biotechnology Information for a BLAST analysis. A number of sequences from the top of the BLAST similarity-ranking list were chosen for a more detailed phylogenetic analysis. Sequences, which were retrieved from the National Centre for Biotechnology Information database, were aligned manually using the Dedicated Comparative Sequence Editor software programme of DE RIJK and DE WACHTER (1993). They had the following accession numbers: P. burgondia, AJ011687; P. azotofixans, D78318; P. polymyxa, AJ223988; P. lautus, D78473; P. chibensis, D85395; P. amylolyticus, X60606; P. thiaminolyticus, D78475; P. lentimorbus, AF071861; P. popilliae, AF071859; B. benzoevorans, Y14693; B.lentus, D78315; B. firmus, D78314; B. subtilus, AB018486. Phylogenetic trees were generated using the TREE CON software package (VAN DE PEER and DE WACHTER, 1994) by the algorithm described by KIMURA (1980) and the neighbor joining method. Calculation of bootstrap values was also done with the TREECON software package.
Paenibacillus burgondia Paenibacillus azotofixans Paenibacillus polymyxa 75 ....---- Paenibacillus lautus Paenibacillus chibensis 71 Paenibacillus amylolyticus 100 ' - - - - - - - - Paenibacillus granivorans Paenibacillus thiaminolyticus 100 59 Paenibacillus lentimorbus Paenibacillus popilliae 100 Fig. 1. Phylogenetic relationship of '----- Bacillus benzoevorans Paenibacillus granivorans to members 98 . . . - - - - Bacillus lentus of the genus Paenibacillus and a few 2% ' - - - - - Bacillus firmus Bacillus species. The phylogenetic tree is based on a comparison of the 16S 1--___ Bacillus subtilis rDNA gene sequence (position 1-1435) and is constructed using the neighbor-joining method. 16S rDNA sequences were retrieved from the Antwerpen small subunit rRNA and the National Centre for Biotechnology Information database. The values indicate bootstraps per 100. The bar represents 2 % nucleotide difference. The sequences were retrieved from the National Center for Biotechnology Information database and have the following accession numbers: P. burgondia, AJ011687; P. azotofixans, D78318; P. polymyxa, AJ223988; P. lautus, D79473; P. chibensis, D85395; P. amylolyticus, X60606; P. thiaminolyticus, D78475; P. lentimorbus, AF071861; P. popilliae, AF071859; B. benzoevorans, Y14693; B.lentus, D78315; B. firmus, D78314; B. subtilus, AB018486.
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Results and Discussion Phenotypic characterization A number of different phenotypic characteristics had already been reported by WIJBENGA et al. 1991. Based on their data it was concluded that strain A30 belonged to Bacillus firmus or Bacillus lentus. However, the exact affiliation of strain A30 could not be resolved based on the available phenotypic characteristics. We made a more detailed analysis of the phylogeny of strain A30, by including a number of additional phenotypic characteristics as well as the phylogeny based on the 16S rRNA gene sequence.
From the additional substrates that were tested, only raffinose and glycerol supported growth of strain A30 (Table 1). The avarage mol% G+C was 47.8 :!: 0.3. The majority of the phenotypic characteristics of strain A30 are indeed similar to those of Bacillus firmus or Bacillus lentus (SMITH, 1957; SLEPECKY and HEMPHILL, 1992). Small differences between strain A30 and B. firmus are the growth in 7% NaCI and the hydrolysis of casein of the latter and not by A30. B. lentus is able to hydrolyse casein (SMITH, 1957) but it is not able to grow at 5% NaCl. A major difference between A30, B. firmus, and B. lentus is the mol% G+C: that of strain A30 (47.8%) is
Table 1. Phenotypic characteristics of P. granivorans, P. chibensis, P. thiaminolyticus, and P. amylolyticus.
P. chibensis 2
P. thiaminolyticus 2
P. aminolyticus 3
NRRLB-142
NRRLB-1456
NRRL B-1456
Rod 0.4-0.6 1.5-4.0 Yes Positive Ellipsoid Yes No Positive Negative Positive Negative 5.8 No
Rod 0.5-0.8 3.0-5.0 Yes Positive Ellipsoid Yes No Positive Negative Positive Negative 4.6 No
Rod 0.5-1.0 2.0-3.0 Yes Positive Ellipsoid Yes Yes Positive Positive Positive Negative 4.9-5.5 Yes
Rod 0.7-0.9 3.5-5.0 Yes Positive Oval Yes Yes Positive Negative Positive Negative 5.2 No
No Yes No No No
No Yes No No No
Yes Yes NR Yes NR
NR4 Yes NR No No
No No No
No No No
Yes NR Yes
No No Yes
Yes No Yes Yes Yes Yes No No No 37 No No 47.8
Yes Yes Yes NR No Yes No No No 37 No Yes 52.8
Yes Variable Variable NR NR No No No Yes 28 No No 52-54
Yes Yes Yes Yes NR NR No No No 37 No Yes 48 .1
No No No Yes No
Yes No Yes Yes Yes
Yes Yes No Yes Yes
Yes No No Yes No
P. granivorans l
Cell morphology Width (pm) Length (pm) Motile Gram reaction Spore Swollen sporangia Anaerobic growth Catalase Oxidase Nitrate reduction Voges-Proskauer reaction pH Voges-Proskauer Decomposition of tyrosine Hydrolysis of Casein Starch Gelatin Production of indole Phenylalanine deaminase Use of Citrate Propionate Acetate Fermentation of Glucose Mannitol Arabinose Raffinose Glycerol Xylose Gas from glucose Lecithinase Growth in 5% NaCl Optimum growth temp. (OC) Growth at 50°C Growth at pH 5.7 Mol% G+C Major cellular fatty acids C16:0 Iso C15:0 Iso C16:0 Anteiso C15:0 Anteiso C17:0
IData partially from WlJBENGA et aI., 1991; 2Data from SHIDA et ai., 1997; 3Data from NAKAMURA, 1984; 4NR - not reported
Paenibacillus granivorans sp. nov.
higher than that of B. firmus or B. lentus, being 41.4% and 36.3% respectively (SLEPECKY and H EMPHILL, 1992). Beacause of this considerable differences in mol% G+C, we also sequenced the 16S rRNA gene of strain A30 and compared this in a neighbor-joining analysis to that of members of the genus Bacillus. The phylogenetic analysis based on the 16S rRNA gene sequence showed that strain A30 is only distantly related to B. firmus or B. lentus, with 78.3% similarity to the two Bacillus species. Strain A30 groups within the Paenibacillus genus and is most closely related to P. amylolyticus, P. chibensis, and P. thiaminolyticus with respectively 94.2%, 93.7% and 93.0% similarity (Fig. 1). Strains with approximately 70% or greater DNA-DNA relatedness are considered to be members of the same species (WAYNE et al. 1987). STACKEBRANDT and GOEBEL (1994) concluded that the DNA of organisms that have less than 97.0% 16S rDNA or 16S rRNA similarities will not reassociate to more than 60%, irrespective of the hybridization method used. It is therefore very likely that strain A30 will have not have more than 70% DNA relatedness with other Paenibacillus species and thus can be considered as a new Paenibacillus species. The major cellular fatty acid of strain A30 is anteiso C15:0, which is present as a dominant cellular fatty acid in all members of the genus Paenibacillus (SHIDA et aI., 1997). Only small amounts of the fatty acids C16:0, iso C15:0, iso C16:0, and anteiso C17:0, which are major constituents of P. chibensis, P. amylolyticus, or P. thiaminolyticus were found in strain A30 (Table 1). Comparison of the cellular fatty acid composition of different strains has to be interpreted with caution because differences in the composition may arise because of different culture conditions. Therefore, we do not compare the exact percentages of the fatty acids but only take the major ones into account. Based on results of the phylogenetic analysis of the 16S rRNA gene and the phenotypic characterization, we propose that strain A30 is considered to be a representative of a new species within the Paenibacillus genus. We suggest the name Paenibacillus granivorans, meaning the Paenibacillus eating (starch) granules, for this new species.
Nucleotide sequence accession number The 16S rRNA gene sequence of strain A30 has been deposited in the GeneBank database of the National Center for Biotechnology Information under accession number AF237682.
Description of Paenibacillus granivorans sp. nov.
Paenibacillus granivorans sp. nov. (gra.ni.vo'rans. L. n. grani, granules; L. v. vorare devour to eat, granule-eating bacterium). Cells stain Gram positive, are rod-shaped (0.5-0.8 rm x 1.5-4.0 rm, width x length) forming ellipsoid spores with swelling of the sporangium. The cells are motile by means of flagella which are located peritrichously. Growth is strictly aerobic. Reduction of nitrate to nitrite was observed. Cells are catalase positive and oxi-
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dase negative. No indole is formed. Phenylalanine deaminase is not present. Growth is possible at temperatures The pH range up to 45°C, with an optimum at 37 for growth is between 6 and 8.5 with an optimum at pH7. No growth was observed at 5% NaCI or higher. The Voges-Proskauer reaction is negative; the pH in Voges-Proskauer broth is 5.8. No decomposition of tyrosine or production of indole was observed. Strain A30 is able to hydrolyse starch but not casein or gelatin. It forms acid from glucose, L-arabinose, raffinose, glycerol, and xylose. No gas is formed from glucose. It does not use citrate, propionate, or acetate. The DNA base composition is 47.8% G+C as determined by HPLC. The major cellular fatty acid is anteiso C15:0. The cellular fatty acids C15:0, C16:0, iso C15:0, iso C16:0, and anteiso C17:0 are present but only in minor amounts. The closest relatives of strain A30 are P. amylolyticus, P. chibensis, and P. thiaminolyticus with respectively 94.2 % , 93.7%, and 93% similarity based on comparison of the 16S rRNA gene. Strain A30 was isolated using granular starch as a substrate from a laboratory reactor fed with waste water from a potato starch production plant. The type strain is strain A30 and is deposited at the Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands (CBS 229.89).
0c.
Acknowledgements
An anonymous reviewer is thanked for critical comments.
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Corresponding author: M. J. E. C. VAN OER MAAREL, Centre for Carbohydrate Bioengineering TNO-RUG, P. O. Box 14, 9750 AA Haren, The Netherlands Tel.: +31-50-3 63-78 32; Fax: +31-50-3 63-21 54; E-mail: [email protected]