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1 Institute of Bioengineering and Agroecology, National University of Ireland, Maynooth, Co. Kildare, Ireland
2 Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland
Correspondence
Michael R. Enright
michael.r.enright{at}may.ie
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence of strains NEM1aT, NEM1b, NEM2 and NEM3 are respectively AF480935, AF480937, AF480936 and AF480934.
A list of the accession numbers of reference sequences used in the phylogenetic analysis is available as supplementary material in IJSEM Online (http://ijs.sgmjournals.org).
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TOPABSTRACTMAIN TEXTREFERENCES |
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), as well as non-pathogens/facultative pathogens such as Paenibacillus apiarus (Nakamura, 1996) and Paenibacillus alvei (Krieg, 1981). Enright et al. (2001) found a Paenibacillus sp. associated with an insect-pathogenic nematode, Heterorhabditis megidis EU17. This nematode was isolated during a survey conducted in Estonia (Griffin et al., 1999).
Heterorhabditis spp. nematodes are lethal pathogens of insects. Infective juveniles (IJs), the only infective stage of these nematodes, carry in their intestine a symbiotic bacterium belonging to the genus Photorhabdus (Forst et al., 1997). Upon infection of a suitable host insect, the symbiont is released into the haemocoel and causes the death of the insect within 48 h (Poinar, 1990). The Photorhabdus sp. then completely colonizes the dead insect cadaver, assisting in the breakdown of the insect tissues by secretion of an array of enzymes (Forst & Nealson, 1996). It is upon this bacterium and the degraded tissues that the nematodes feed. The nematodes complete their development and reproduce within the dead host. Photorhabdus spp. also produce several antibiotics (Forst & Nealson, 1996). Together, these have a broad spectrum of activity (Akhurst, 1982) and help to ensure the dominance of Photorhabdus within the insect cadaver (Forst et al., 1997). Until its nutritive status declines, the insect cadaver supports several nematode generations. Thousands of the non-feeding IJs then emerge from the insect into the soil, in search of a new insect host (Poinar, 1990).
The association reported by Enright et al. (2001) involves spindle-shaped swollen sporangia of a Paenibacillus sp. adhering to the surface of H. megidis EU17 nematode IJs (Fig. 1). There is a level of specificity to this adherence; sporangia adhere to IJs of all Heterorhabditis spp. tested and of closely related parasites of the order Strongylida but not to any other soil nematodes tested (Enright et al., 2001; M. R. Enright and C. T. Griffin, unpublished). Sporangia of closely related Paenibacillus species (eight species tested) did not adhere to Heterorhabditis spp. IJs (Enright et al., 2001). The Paenibacillus sp. reproduces within the dead insect despite the Photorhabdus-produced antibiotics and emerging IJs can then carry large numbers of its sporangia out of the cadaver. IJs bearing sporangia then infect new insect hosts. Other Heterorhabditis spp. strains have also been found in similar association with endospore-forming bacteria. We noted the presence of bacteria in a Heterorhabditis indica strain from India contained in our collection and a Heterorhabditis sp. from Georgia, USA, was found associated with a bacterium initially identified as a Bacillus species (Marti & Timper, 1999). In both cases, the bacteria produced sporangia that were morphologically similar to those described by Enright et al. (2001). These bacteria–Heterorhabditis associations are recognizable by the tendency of the sporangia to ‘clump’ the nematodes in water (nematodes stick together in large cross-linked groups). In each of the cases mentioned, the nematodes were associated with the endospore-forming bacteria from the time that they where first isolated from soil (D. Marti and S. Easwaramoorthy, personal communication). In this paper, we have characterized four bacterial isolates that produce spindle-shaped sporangia and which were isolated from the H. megidis, H. indica and Heterorhabditis sp. strains mentioned above. A novel species, Paenibacillus nematophilus sp. nov., represented by two of these isolates, is proposed.
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, Parry et al. (1983), Smibert & Krieg (1994) and Claus & Berkeley (1986), using appropriate Paenibacillus spp. controls. API tests (bioMérieux) were carried out as in Logan & Berkeley (1984), except that, with API 50CHB tests, results were also recorded after 4 days. All tests were replicated at least twice. Growth under anaerobic conditions was tested using an anaerobic jar with an AnaeroGen sachet (Oxoid) according to the manufacturer's instructions. Fatty acid analysis was carried out at the DSMZ (Braunschweig, Germany) using high-resolution GC and the profile was analysed using the Microbial Identification System (MIDI; Microbial ID) according to the manufacturer's instructions. Both the G+C content and DNA–DNA hybridization studies were also conducted at DSMZ. For these, DNA was isolated and purified according to the method of Cashion et al. (1977). In the G+C content studies, DNA was hydrolysed according to the method of Mesbah et al. (1989) and HPLC was performed as described by Tamaoka & Komagata (1984). Calibrations were done as described by Mesbah et al. (1989). DNA–DNA hybridization studies were carried out as described by De Ley et al. (1970), with the modifications described by Huß et al. (1983) and Escara & Hutton (1980). Renaturation rates were computed with the program TRANSFER.BAS (Jahnke, 1992).
Standard methods were used to extract bacterial genomic DNA before PCR amplification of almost the full-length 16S rRNA gene. Conserved eubacterial primers EB-27F (5'-AGAGTTTGATCMTGGCTCAG- 3', M=A or C) and UN-1492R (5'-TACGGYTACCTTGTTACGACTT-3', Y=C or T) were used. PCR products were sequenced independently in both directions by Oswel Sequencing (Southampton, UK) using standard methods with an automated DNA sequencer and consensus sequences were determined. The 16S rRNA gene sequences of named Paenibacillus species and members of closely related genera were obtained from the GenBank database. All sequences were aligned using the program CLUSTAL X (Thompson et al., 1997). Accession numbers of reference sequences obtained from GenBank are available as supplementary material in IJSEM Online (http://ijs.sgmjournals.org). Short sequence stretches at both ends of the alignment were excluded because of large numbers of ambiguous positions before pairwise percentage similarities of the sequences were determined using the program PAUP* (Sinauer). The alignment was then manipulated manually, whereby positions of uncertain homology were excluded, using the program SEAVIEW (Galtier et al., 1996). Further phylogenetic analyses using distance, parsimony and maximum-likelihood criteria were carried out using the program PAUP*. Confidence in phylogenetic hypotheses was assessed using the bootstrap (n=1000) resampling method (Felsenstein, 1988) for distance and parsimony and quartet puzzling (n=1000) (Strimmer & Von Haeseler, 1996) for maximum-likelihood.
Selective isolations from the various sporangia-associated nematode strains yielded numerous slow-growing bacterial isolates, all with similar colony morphology and all of which met the selection criteria employed. Four isolates, with at least one from each nematode strain, were chosen for further study. Isolates NEM1aT and NEM1b were isolated from H. megidis EU17 (from Estonia), while NEM2 and NEM3 were respectively isolated from Heterorhabditis sp. ‘Line1’ (from Georgia, USA) and H. indica LN2 (from India). Vegetative cells of these isolates stained Gram-negative (Gram-variable in older cultures) and were motile and rod-shaped. All four isolates produced morphologically similar spindle-shaped sporangia, in which an ellipsoidal endospore occupied a central/paracentral position, clearly swelling the sporangium (Fig. 2). Endospores were retained within the sporangia. Colonies of all isolates were thin, non-pigmented, regular and smooth with an undulate edge. All four of these nematode-associated isolates were phenotypically very similar (Table 1).
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). Using the same alignment, Paenibacillus macquariensis was the named species with highest sequence identity to isolate NEM1aT (94 %), followed by Paenibacillus amylolyticus and Paenibacillus azoreducens (93·6 %), Paenibacillus borealis and Paenibacillus durus (93·5 %) and Paenibacillus macerans (93·4 %). Most similar to isolate NEM2 was P. durus (93·3 %), while P. amylolyticus was most similar to isolate NEM3 (93·2 %). Across a shorter alignment (966 alignment positions), Paenibacillus illinoisensis and Paenibacillus chibensis were also among the species with higher sequence similarity to the nematode-associated isolates. The genomic heterogeneity of the nematode-associated isolates suggested by the 16S rRNA sequences was confirmed by the results of DNA–DNA hybridization studies. Isolate NEM1aT shared 90·3 % DNA–DNA similarity with NEM1b, but only 40·9 and 38·3 % with isolates NEM2 and NEM3. Isolate NEM2 shared 60·4 % DNA–DNA similarity with NEM3. In comparison, NEM1aT shared 25·8 % DNA–DNA similarity with P. azoreducens, one of the species with highest 16S rRNA sequence identity to that isolate. According to the recommendations of Wayne et al. (1987), isolates should have similarity values of 70 % or greater to be considered conspecific.
As is typical of Paenibacillus species (Ash et al., 1993), the fatty acids of the nematode-associated isolates were mainly of the straight-chain saturated, anteiso- and iso-branched types, with anteiso-C15 predominant, except in NEM2, where C16 : 0 was slightly higher. Analysis yielded two major fatty acid types, namely, anteiso-C15 and C16 : 0 (Table 2). Straight-chain C14 : 0 fatty acid was more common in the nematode-associated isolates than has been reported for most other Paenibacillus species (Shida et al., 1997a, b; Nakamura et al., 1996; Pettersson et al., 1999; Elo et al., 2001), an exception being P. borealis (Elo et al., 2001). While all the isolates shared a similar overall pattern, there were some differences between them in the actual levels of some fatty acids. For example, whereas NEM1aT and NEM1b had similar levels of anteiso-C17 (approx. 8 %), NEM2 and NEM3 had higher levels (13 and 16 %). Diagnostic amounts of C12 : 0 and 3-hydroxy fatty acids were found in all of the nematode-associated isolates. These have normally been undetected or unreported for Paenibacillus species (Shida et al., 1997a, b; Nakamura et al., 1996; Elo et al., 2001; Heyndrickx et al., 1996a; Meehan et al., 2001).
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). They could easily be distinguished from closely related species on the basis of numerous differential characteristics (Table 1
). For example, the endospore was always located in a central/paracentral position, while a terminal/subterminal position is the most common in their closest relatives. Strains of some related Paenibacillus can have both. For example, while all Paenibacillus polymyxa strains produce terminal/subterminal endospores, some also produce sporangia with central/paracentral endospores (Logan & Berkeley, 1984). Endospores of the nematode-associated isolates are retained within the sporangium, even following long periods of storage. This is not the case with the majority of other described Paenibacillus species (N. Logan, personal communication). All the nematode-associated isolates had distinct profiles in API tests, easily distinguishable from those of closely related species. In API 50CHB tests, NEM1aT utilized 16 carbohydrate substrates out of 49, while NEM1b utilized 15, NEM2 utilized 17 and NEM3 utilized 16, with 15 of these being common to all four isolates. These numbers are low compared with those found for other Paenibacillus species in both this and previous studies (Logan & Berkeley, 1984; Elo et al., 2001). Incubation of the API 50CHB tests for 2 days longer than described in Logan & Berkeley (1984) resulted in positive results for utilization of 5-keto-D-gluconate by the nematode-associated isolates. This was not the case with control Paenibacillus species.
Differentiation of the nematode-associated isolates from P. macquariensis, the species with highest 16S rRNA gene sequence identity to NEM1aT, is based on the position of the endospore within the sporangium, their inability to grow at 5 °C and their Voges–Proskauer reaction, as well as their inability to utilize a wide range of carbohydrates (Table 1).
The nematode-associated isolates differed somewhat in the conditions under which growth occurred. All four isolates grew at between 15 and 37 °C. However, only NEM1aT and NEM1b grew at 10 °C. This finding may be linked to climatic differences between the geographical origins of the different isolates. None of the isolates grew at 5 or 40 °C. Isolates NEM1aT, NEM1b and NEM3 grew at pH 6–11, whereas NEM2 grew at pH 6–10. Isolates NEM1aT and NEM1b grew in 2 % (w/v) NaCl, but not in 3 %, whereas NEM2 and NEM3 grew in 3 % (w/v) NaCl but not in 4 %.
Despite the phenotypic and ecological similarities, genomic heterogeneity exists within this group of isolates, such that its members are likely to represent more than one species. Certain characteristics (growth temperatures, differential ability to tolerate NaCl during growth, ability to utilize D-ribose and features of their fatty acid profiles) separate one group, NEM1aT and NEM1b, from NEM2 and NEM3, but there is little to separate the latter two isolates. Strain NEM1aT grew in vivo (in infected insect hosts) with some, but not all Heterorhabditis spp. tested, while NEM2 and NEM3 grew with a similar spectrum of Heterorhabditis spp., but different from that of NEM1aT (M. R. Enright and C. T. Griffin, unpublished). Here, we propose isolates NEM1aT and NEM1b to represent a single species, Paenibacillus nematophilus sp. nov., with NEM1aT as the type strain. While NEM2 and NEM3 are phylogenetically very closely related to this proposed species, it is clear that they themselves are representative of at least one, but most likely two, novel species. To date, bacteria with these characteristics have been recovered only rarely fortuitously during the isolation of entomopathogenic nematodes from soil and the isolates studied here have been maintained in laboratory culture with nematodes for up to 10 years. Examination of further isolates from the wild (nematode-associated or otherwise) and of more phenotypic characters would be desirable so that the true extent of variation within these remaining potential species can be explored.
Description of Paenibacillus nematophilus sp. nov
Paenibacillus nematophilus (ne.ma.to'phi.lus. N.L. n. nematoda nematode; Gr. adj. philos loving or having affinity for; N.L. adj. nematophilus nematode-loving).
Cells are rod-shaped, motile, 3·5–7·0 µm long and 0·5–1·0 µm wide. Cells stain Gram-negative (Gram-variable in older cultures). Oval-shaped endospores are produced in swollen, spindle-shaped sporangia and lie in a central/paracentral position. Endospore is retained within the sporangium. Endospores measure on average 1·7x1·2 µm, while sporangia are 6·5–10·6 µm long. Relatively slow-growing on nutrient agar, with colonies measuring 0·5–4·0 mm in diameter. Colonies are thin, non-pigmented, regular, smooth and slightly umbonate with an undulate edge. Catalase-positive and oxidase-negative. Under anaerobic conditions, growth is weak and germination of endospores is poor compared with aerobic conditions. Grows in 2 % but not 3 % (w/v) NaCl. Grows optimally at 30 °C. Growth occurs at between 10 and 37 °C. No growth at 5 or 40 °C. Growth at pH 6–11; no growth at pH 5·6. As determined with API 20E strips, 
-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and urease are not produced. Citrate is not utilized. Hydrogen sulfide, indole and NO2 are not produced. Gelatin is not hydrolysed. The Voges–Proskauer reaction is positive. Starch is hydrolysed. Casein and Tween 80 are not hydrolysed. Isolates are negative for egg yolk lecithinase activity. No colour change in litmus milk. In API 50CHB galleries, when API CHB suspension medium is used, aesculin is hydrolysed and acid but no gas is produced from D-glucose, methyl 
-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, sucrose, trehalose, starch, glycogen and gentiobiose. Acid is produced at a slower rate from 5-keto-D-gluconate. Utilization of D-mannose is variable (type strain is positive). Glycerol, ribose, erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, adonitol, methyl -D-xyloside, galactose, fructose, sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl -D-mannoside, lactose, melibiose, inulin, melezitose, raffinose, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate and 2-ketogluconate can not be utilized as sole carbon sources. The major fatty acids are anteiso-C15 and C16 : 0 and the G+C content is approximately 44 mol% as determined by HPLC. The type strain, strain NEM1aT (=DSM 13559T=NCIMB 13845T), and strain NEM1b were found associated with the entomopathogenic nematode H. megidis EU17 isolated from Estonia.
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ACKNOWLEDGEMENTS |
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