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1 University of Campinas, 13084-510 Campinas, SP Brazil
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
D. Fritze
dfr{at}dsmz.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Published online ahead of print on 13 January 2003 as DOI 10.1099/ijs.0.02420-0.
Present address: Mühlwang 9, 25899 Bosbüll, Germany. 
Present address: Chemnitzer Str. 3, 37085 Göttingen, Germany.
The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences determined in this study are AJ345017 (DSM 1352), AJ345018 (DSM 6358), AJ345019 (DSM 1482), AJ345020 (DSM 1327T), AJ345021 (DSM 1474), AJ345022 (DSM 34), AJ345023 (DSM 1355T) and AJ345024 (DSM 1481).
Micrographs of cells and spores of strains DSM 1327T and DSM 1355T are available as supplementary data in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). As these strains were lost (K. T. Wieringa, personal communication), this species was not included in the Approved Lists of Bacterial Names (Skerman et al., 1989) and therefore has no standing in nomenclature. A new attempt to reisolate Wieringa's agarolytic spore-forming strains led to the collection of a considerable number of organisms (Hunger & Claus, 1978). Some of these isolates (designated growth requirement group I) matched the original description of ‘Bacillus agar-exedens’. These organisms were characterized by inhibition of growth by peptone on neutral to slightly acid mineral/glucose medium, an effect that was neutralized by the addition of urea to the medium. A second group of agarolytic strains was characterized by lack of growth inhibition by peptone and a moderate to strong requirement for carbohydrates (designated growth requirement group II); these strains matched the description of ‘Bacillus palustris var. gelaticus’ (Sickles & Shaw, 1934). Within groups I and II, phenotypic properties varied and the G+C content of the DNA covered a broad range (Hunger, 1978; Hunger & Claus 1978, 1981). By extensive DNA–DNA hybridization studies, the genomic diversity of the isolates was confirmed and group I was divided into DNA subgroups 1, 2, 3, 4 and 5, and group II into DNA subgroups 6 and 7. Hunger & Claus (1981) concluded that each of these groups might represent a novel species, but did not validly publish names for them.
Based on the results of Hunger & Claus (1978, 1981), the agarolytic strains were subjected to a renewed taxonomic study, using classical methods and novel genomic approaches that did not exist 25 years ago.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Twenty strains had been isolated during 1970–1976 at the DSMZ. One strain had been obtained from the American Type Culture Collection (ATCC), Manassas, VA, USA, and one strain was donated in 1991 by D. P. Stahly, University of Iowa, IA, USA. Cultures were grown in nutrient broth medium supplemented with 0·1 % (w/v) urea. The pH after autoclaving was 7·3–7·5. Sterile glucose solution was added to this basal medium to a final concentration of 1 % (w/v). For solid media, 1·5 % (w/v) agar was added. Incubation was carried out at 30 °C.
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, with the modifications described by Hunger & Claus (1981). Other tests were done as described by Hunger & Claus (1981). Several additional tests and repetitions were performed in the present study: hydrolysis of starch [using Lugol's iodine solution as well as ethanol precipitation according to Gordon et al. (1973)], hydrolysis of urea and pectin (Wieringa, 1947) and production of dextranase (Mencier, 1972). Diaminopimelic acid (A2pm) was determined by the method described by Schleifer & Kandler (1972). For microphotographs, cultures were prepared as described by Claus & Berkeley (1986).
DNA–DNA hybridization and ribotyping.
DNA was isolated by chromatography on hydroxyapatite, as described by Cashion et al. (1977). DNA–DNA hybridizations were carried out according to De Ley et al. (1970). Renaturation rates were calculated using the computer program TRANSFER.BAS (Jahnke, 1992). Automated ribotyping of the isolates was accomplished by using the DuPont Qualicon RiboPrinter system (Bruce, 1996) and EcoRI as the standard restriction enzyme for cutting genomic DNA.
16S rDNA sequence determination and analysis.
Genomic DNA extraction, PCR-mediated amplification of 16S rDNA and purification of PCR products were carried out as described by Rainey et al. (1996). Purified PCR products were sequenced with Taq DyeDeoxy Terminator Cycle Sequencing kits (Applied Biosystems), as directed in the manufacturer's protocol. An Applied Biosystems 373A DNA sequencer was used for electrophoresis of the sequence reaction products. The ae2 alignment editor (Maidak et al., 1999) was used to align the 16S rDNA sequences determined in this study against those of representatives of the main bacterial lineages, available from public databases. Pairwise evolutionary distances were computed by using the correction of Jukes & Cantor (1969). The phylogenetic dendrogram was reconstructed from a distance matrix using the treeing algorithm of DeSoete (1983) and Felsenstein (1993). Strains and accession numbers are indicated in Fig. 2.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, was unique (Table 1
, Fig. 1).
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is a modification of a table shown by Hunger & Claus (1981), supplemented with new data. Intragroup DNA similarities were >80 %, while intergroup similarities were <35 %. Riboprint analysis assigned the phenotypically similar strains DSM 1486 and DSM 1355T to ribogroup 2; in contrast, DNA–DNA hybridization of these two strains (Hunger & Claus, 1981) revealed only 63 % similarity. Repetition of the DNA reassociation experiment gave a value of 98 %, indicating that these strains are indeed genomically closely related. Separation of ribogroups 1 and 7 was obvious from phenotypic results, but had not been verified by DNA–DNA hybridization. DSM 34 (ribogroup 7) shared only 20 % DNA similarity with DSM 1327T (ribogroup 1), thus supporting the results obtained by analysis of the rrn operon.
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. Binary similarity values are indicated in Table 2. Levels of 16S rDNA similarity range from 98·7 % (DSM 1355T versus DSM 6358) to 91·4 % (DSM 1352 versus DSM 34). All strains are members of the genus Paenibacillus and each representative occupies a distinct phylogenetic position. Similarity values obtained for each of the representative strains and all type strains of Paenibacillus species varied between 90·5 and 97·3 %, indicating that none of the agarolytic isolates is a member of a described species. These results suggest that all agarolytic strains in the present study should be renamed as Paenibacillus species. Interestingly, several of the known Paenibacillus species have been described as being capable of degrading unusual polysaccharides and are named accordingly, e.g. Paenibacillus alginolyticus, Paenibacillus chondroitinus, Paenibacillus curdlanolyticus and Paenibacillus glucanolyticus.
Cell-wall analysis
Whole-cell lysates were examined for the presence or absence of A2pm. A number of strains were found not to contain A2pm, but results were not consistent within subgroups. Repetition of the analysis using purified cell walls did improve the picture, but still not satisfactorily. Inconsistent detection of A2pm may be due to a very thin peptidoglycan layer in these organisms, as suggested by uneven or even Gram-negative staining.
Phenotypic characterization
Some of the key phenotypic traits that distinguish members of individual subgroups of growth requirement groups I and II (Hunger, 1978), were repeated in the present study. A complete listing of reactions is given by Hunger (1978) and Hunger & Claus (1981). While the results for pectin hydrolysis and dextranase activity were confirmed, those for starch hydrolysis only partially matched previous findings. Urea hydrolysis could not be confirmed at all. Different findings for starch hydrolysis were probably due to different methodologies: the method described by Gordon et al. (1973) resulted in a positive reaction for all strains. However, when starch hydrolysis was visualized by flooding the agar with Lugol's iodine solution, the results of Hunger & Claus (1981) were confirmed, i.e. subgroups 1, 5, 6 and 7 were positive, while subgroups 2, 3 and 4 were negative. The rationale for Hunger & Claus (1981) not to describe novel species for the emerging groups was the physiological inactivity and phenotypic similarity of the strains enclosed. Only a few properties were found that were coherent for members of a DNA subgroup, but discriminatory between the DNA subgroups (Table 3). By and large, re-examination of a selection of those properties of Hunger & Claus (1981) in the present work generally confirmed their findings.
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. Later, this name was accepted as being synonymous with Bacillus circulans and thus strain DSM 34 was included in this species by Gordon et al. (1973). However, the high G+C content of its DNA (51 mol%) and the high 16S rDNA sequence similarity to Paenibacillus species clearly exclude a close relationship to B. circulans sensu stricto, as defined by Nakamura & Swezey (1983a, b). The type strain of B. circulans, DSM 11T, has a DNA G+C content of 36 mol% and is an authentic member of the genus Bacillus.
The properties described by Wieringa (1941) are difficult to interpret today and can hardly be compared with modern results. According to findings obtained by Wieringa (1941), growth of all strains of ‘Bacillus agar-exedens’ is inhibited by peptone and the effect is reversed by the addition of urea. Members of DNA subgroups 1–5 do indeed react this way. Wieringa further described the strains as being strong starch hydrolysers, which is not true for members of DNA subgroups 2–4. This leaves members of DNA subgroups 1 and 5 as candidates to match the original description of ‘Bacillus agar-exedens’. As the single strain of DNA subgroup 5, DSM 1352, forms a yellowish pigment on mineral/glucose/yeast medium and Wieringa did not mention any pigmentation of the isolates studied, it is concluded that the six strains of DNA subgroup 1 most closely resemble Wieringa's original description.
16S rDNA and DNA–DNA similarities determined in the present study clearly point towards the presence of eight novel genospecies of the genus Paenibacillus. Additional ribotyping experiments on all genospecies confirm their distinctiveness and show, for those taxa that contain more than a single strain, that they are genomically homogeneous. Agarolytic activity, the salient phenotypic characteristic of the strains, has not been described so far for any of the Paenibacillus species with validly published names, and thus makes the novel species easily distinguishable from all of them. However, we refrain from describing as species those of our genospecies that contain three or fewer strains. It is suggested that additional strains in groups 3–8 should be isolated, to enable broadened investigation on physiological properties in order to better separate these putative species, and to search for intraspecies variation. For an overview of phenotypic traits of species of the genus Paenibacillus, see Heyndricks et al. (1996), Shida et al. (1997) and Petterson et al. (1999).
As already suggested by Hunger & Claus (1981), the specific epithet agar-exedens (ex Wieringa 1941) should be revived for a novel Paenibacillus species that embraces the six strains of DNA subgroup 1. We propose Paenibacillus agarexedens sp. nov., nom. rev., with the spelling corrected according to Rule 12a of the Bacteriological Code (1990 Revision) (Lapage et al., 1992). A detailed phenotypic description is given below.
We also propose Paenibacillus agaridevorans sp. nov. for the five members of DNA subgroup 2, which are physiologically distinguishable from all other agarolytic strains by the combination of characteristics shown in Table 3. A detailed phenotypic description is given below.
Description of Paenibacillus agarexedens (ex Wieringa 1941) sp. nov., nom. rev.
Paenibacillus agarexedens [a.gar.ex.e'dens. Malayan n. agar agar (algal polysaccharide); N.L. n. agarum agar; L. v. exedere to eat up, utilize; N.L. part. adj. agarexedens agar-utilizing].
Motile rods, width 0·5–1·4 µm, length 2–8 µm (type strain: 0·5–0·8 µm, 3–5 µm). Unstained cells are not granulated. Gram reaction in 12 h cultures is uneven (dappled); after 38 h, cells are Gram-negative. Spores are ellipsoidal and most (>50 %) of the sporangia are not swollen. Colonies on agar media sink into the agar within a few days (see Fig. 3); no liquefaction of agar occurs. Colonies on peptone/urea agar are whitish and round with entire margins; no pigmentation occurs on mineral/glucose/yeast extract medium. Chemo-organotrophic. Growth is inhibited by peptones; inhibition may be neutralized by urea. Catalase- and oxidase-positive. Mesophilic; maximum temperature for growth is 40 °C (type strain: 35 °C). Positive for hydrolysis of agar, starch, hippurate and aesculin. Acid is produced from agar and glucose. Shows weak aminopeptidase activity. Negative for anaerobic growth, growth at pH 5·7 and in 5 % NaCl, Voges–Proskauer test, urease, nitrate reduction, activities of egg-yolk lecithinase, dextranase, DNase and lysine decarboxylase, hydrolysis of poly-
-hydroxybutyric acid, casein, pectin, Tween 80 and chitin, production of indole, dihydroxyacetone and dextrin crystals, anaerobic gas production from nitrate, alkali or acid production in litmus milk, liquefaction of gelatin and resistance to lysozyme and sodium lauryl sulfate. Variable reactions are observed for deamination of phenylalanine, tyrosine degradation (type strain is positive) and methylene blue reduction (type strain is negative). The G+C content of the DNA is 47–49 mol% (type strain, 47 mol%), as determined by the thermal denaturation method.
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Description of Paenibacillus agaridevorans sp. nov.
Paenibacillus agaridevorans [a.ga.ri.de.vo'rans. Malayan n. agar agar (algal polysaccharide); N.L. n. agarum agar; L. part. adj. devorans consuming, devouring; N.L. part. adj. agaridevorans agar-devouring].
Motile rods, width 0·6–0·8 µm, length 2–5 µm. Unstained cells are not granulated. Gram-reaction in 12 h cultures is uneven (dappled); after 38 h, cells are Gram-negative. Spores are ellipsoidal and most of the sporangia (>50 %) are not swollen. Colonies on agar media sink into the agar within a few days; no liquefaction of agar occurs. Colonies on peptone/urea agar are whitish and round with entire margins; no pigmentation occurs on mineral/glucose/yeast extract medium. Chemo-organotrophic. Growth is inhibited by peptones; inhibition may be neutralized by urea. Catalase- and oxidase-positive. Mesophilic; maximum temperature for growth is 35 °C. Positive for hydrolysis of agar, dextran, hippurate and aesculin. Acid is produced from agar and glucose. Negative for aminopeptidase, anaerobic growth, growth at pH 5·7 and in 5 % NaCl, Voges–Proskauer test, nitrate reduction, urease, activities of egg-yolk lecithinase, DNase and lysine decarboxylase, hydrolysis of starch (ethanol precipitation, positive; flooding with Lugol's iodine solution, negative), poly--hydroxybutyric acid, casein, pectin, Tween 80 and chitin, tyrosine degradation, deamination of phenylalanine, production of indole, dihydroxyacetone and dextrin crystals, anaerobic gas production from nitrate, alkali or acid production in litmus milk, methylene blue reaction, liquefaction of gelatin and resistance to lysozyme and sodium lauryl sulfate. The G+C content of the DNA is 50–52 mol% (type strain, 51 mol%) as determined by the thermal denaturation method.
Type strain is 65T=DSM 1355T =CIP 107436T. Isolated from volcanic soil, Paricutin volcano, Mexico, in 1975.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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