Characterization of heterotrophic nitrifying bacteria with respiratory ammonification and denitrification activity – Description of Paenibacillus uliginis sp. nov., an inhabitant of fen peat soil and Paenibacillus purispatii sp. nov., isolated from a spacecraft assembly clean room

Systematic and Applied Microbiology 33 (2010) 328–336

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Characterization of heterotrophic nitrifying bacteria with respiratory ammonification and denitrification activity – Description of Paenibacillus uliginis sp. nov., an inhabitant of fen peat soil and Paenibacillus purispatii sp. nov., isolated from a spacecraft assembly clean room夽 Undine Behrendt a,∗ , Peter Schumann b , Michaela Stieglmeier c , Rüdiger Pukall b , Jürgen Augustin a , Cathrin Spröer b , Petra Schwendner c , Christine Moissl-Eichinger c , Andreas Ulrich a a

Leibniz-Centre for Agricultural Landscape Research (ZALF), Institute of Landscape Matter Dynamics, Eberswalder Str. 84, D-15374 Müncheberg, Germany DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany c University of Regensburg, Institute for Microbiology and Archea Center, Universitätsstr. 31, D-93053 Regensburg, Germany b

a r t i c l e

i n f o

Article history: Received 30 March 2010 Keywords: Paenibacillus uliginis sp. nov. Paenibacillus purispatii sp. nov. Heterotrophic nitrification Denitrification Dissimilatory nitrate/nitrite reduction to ammonium Space craft associated clean rooms Planetary protection

a b s t r a c t In the course of studying the influence of N-fertilization on N2 and N2 O flux rates in relation to soil bacterial community composition of a long-term fertilization experiment in fen peat grassland, a strain group was isolated that was related to a strain isolated from a spacecraft assembly clean room during diversity studies of microorganisms, which withstood cleaning and bioburden reduction strategies. Both the fen soil isolates and the clean room strain revealed versatile physiological capacities in N-transformation processes by performing heterotrophic nitrification, respiratory ammonification and denitrification activity. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that the investigated isolates belonged to the genus Paenibacillus. Sequence similarities lower than 97% in comparison to established species indicated a separate species position. Except for the peptidoglycan type (A4alpha l-Lys–d-Asp), chemotaxonomic features of the isolates matched the genus description, but differences in several physiological characteristics separated them from related species and supported their novel species status. Despite a high 16S rRNA gene sequence similarity between the clean room isolate ES MS17T and the representative fen soil isolate N3/975T , DNA–DNA hybridization studies revealed genetic differences at the species level. These differences were substantiated by MALDI-TOF MS analysis, ribotyping and several distinct physiological characteristics. On the basis of these results, it was concluded that the fen soil isolates and the clean room isolate ES MS17T represented two novel species for which the names Paenibacillus uliginis sp. nov. (type strain N3/975T = DSM 21861T = LMG 24790T ) and Paenibacillus purispatii sp. nov. (type strain ES MS17T = DSM 22991T = CIP 110057T ) are proposed. © 2010 Elsevier GmbH. All rights reserved.

Introduction Production of nitrous oxide by microbial processes is significant due to its high global warming potential and involvement in the destruction of stratospheric ozone [28]. One important source is the microbial utilization of nitrate as a terminal electron acceptor in respiratory metabolism under oxygen limited conditions. Nitrate

夽 The EMBL accession numbers for the 16S rRNA gene sequences of the type strains Paenibacillus uliginosus N3/975T (DSM 21861T = LMG 24790T ) and Paenibacillus purispatii ES MS17T (DSM 22991T = CIP 110057T ) are FN556467 and EU888513, respectively. ∗ Corresponding author. Tel.: +49 0 33432 82460; fax: +49 0 33237 82344. E-mail address: [email protected] (U. Behrendt). 0723-2020/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2010.07.004

respiration occurs via two dissimilar pathways that utilize the same initial substrate and involve formation of the intermediate nitrite, although they end up in different products [48]. One of these pathways, nitrate ammonification, or dissimilatory nitrate reduction to ammonium (DNRA), is characterized by the reduction of nitrite to ammonia. During this reaction, toxic intermediate nitric oxide can be formed that is further reduced to nitrous oxide by detoxification enzymes [43]. The second pathway of nitrate respiration is denitrification, which differs in the reduction of nitrite to gaseous nitrogen oxides by a multi-step process with nitrogen gas as a principal end product [48]. Nitrous oxide, an obligate intermediate of denitrification or end product of incomplete denitrification, can be released in relation to environmental influences. A further important microbial source of nitrous oxide is nitrification. Oxidation of

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ammonia to nitrite by litho-autotrophic nitrifiers, as well as the oxidation of inorganic or organic nitrogen compounds to nitrite by chemo-organotrophic bacteria, called “heterotrophic nitrification”, can release nitrous oxide as a by-product [9]. Except for autotrophic nitrification, heterotrophic bacteria contribute considerably to the conversion of nitrogen species by these metabolic pathways [9,48]. Although most of the characterized bacteria belong to the Proteobacteria, Gram-positive bacteria are also represented [48]. It was assumed that a number of bacilli were denitrifying species [59], but detailed studies have shown that products of nitrate respiration in several species are compatible with the pathway of nitrate ammonification [43,49,58]. Heterotrophic nitrification is also described primarily for Gram-negative bacteria [37,39]. Nevertheless, the isolation of a nitrifying strain highly related to species of the genus Bacillus by Lin et al. [33] indicated that this ability is more widespread than previously assumed. There is limited knowledge about the occurrence of these pathways in the genus Paenibacillus. Although various Paenibacillus species are able to reduce nitrate to nitrite [42], nitrate respiration by ammonification has only been described for the species Paenibacillus macerans [46], which was formerly classified as a member of the genus Bacillus. Formation of nitrous oxide from nitrate or nitrite by strains of the genus Paenibacillus isolated by Horn et al. [26] and Heylen et al. [25] cannot be definitely related to denitrification or nitrate ammonification as no nitrogen gas was detected or determined and a possible formation of ammonium was not analysed. Therefore, it remains unknown whether denitrification is performed by members of the genus Paenibacillus. In the context of studying the influence of N-fertilization on N2 and N2 O flux rates, in relation to the soil bacterial community composition of a long-term fen peat grassland fertilization experiment [2], a group of bacteria was isolated which was related to the genus Paenibacillus by partial 16S rRNA gene sequence analysis. Investigation of metabolic characteristics showed a high versatility in formation and conversion of soluble nitrogen oxides. Furthermore, a strain related to the fen soil isolates was obtained in the course of biodiversity studies of the cultivable microbial community in European spacecraft-associated clean rooms harbouring the Herschel Space Observatory [51]. Comparative 16S rRNA gene studies revealed a clear demarcation of the isolates from recognized species of the genus Paenibacillus. As a consequence, an extensive phylogenetic and phenotypic investigation was performed to show their unambiguous taxonomic positions. Furthermore, a comprehensive metabolic characterization of the isolates was conducted to determine their potential capacities to participate in nitrogen transformation processes.

Materials and methods Sample preparation and strain isolation For measurement of gas flux rates and microbiological analysis, fen soil cores (250 cm3 ) to a depth of 10 cm [35] were obtained from plots differing in nitrogen fertilization levels of a long-term experiment located at Paulinenaue (Germany, 52◦ 41 N, 12◦ 41 E). For microbiological analysis, soil from a core was homogenized by sieving (2.5 mm mesh size) and 3 g thereof was extracted in 27 mL of 0.2 M Sörensen sodium phosphate buffer (pH 7) by shaking for 30 min (250 rpm). To isolate the denitrifying bacterial community, 1 mL was serially diluted and plated in duplicate on nutrient agar (SIFIN) supplemented with 0.5 g L−1 KNO3 . Additionally, the solid media G2M11 and G3M12 optimized for the isolation of denitrifying bacteria, as described by Heylen et al. [25], were inoculated in the same manner. The agar plates were incubated for 2 weeks at 21 ◦ C in an anaerobic chamber (gas composition: 10% CO2 , 5% H2 ,

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and 85% N2 ). From each growth medium, 40 isolates were randomly picked and purified to determine the abundance of bacterial taxa. Isolates were grouped on the basis of their morphology and physiological tests [4,7]. A representative number of strains for each group were investigated using partial sequence analysis of the 16S rRNA gene to determine their phylogenetic affiliation, as described by Ulrich et al. [53]. One group related to the genus Paenibacillus was subjected to further taxonomic investigation. Three strains, originally derived from different plots, corresponding in their partial 16S rRNA gene sequence, were selected for a detailed phenotypic and phylogenetic characterization. In the framework of the European Space Agency (ESA) biodiversity study, samples were taken from clean rooms during ATLO activities for the Herschel Space Observatory [51]. The strain ES MS17T was isolated from a sample obtained from the ISO 8 clean room (Hall Hydra, Fh) at the European Space Research and Technology Centre (ESTEC) in Noordwijk, The Netherlands, as described by Stieglmeier et al. [51]. Detailed information about the isolated and reference strains investigated in this study are shown in Supplementary Table S1. Phylogenetic analysis Phylogenetic analyses, on the basis of 16S rRNA gene sequences of strain N3/975T and strain ES MS17T , were performed as described by Behrendt et al. [5] and Stieglmeier et al. [51], respectively. A phylogenetic tree for established Paenibacillus species and the sequenced strains was constructed using the neighbour joining [44] and maximum likelihood [16] algorithms (phylip, version 3.6; [17]). DNA–DNA hybridization studies between strains N3/975T , ES MS17T and the type strain of Paenibacillus campinasensis DSM 21989T were performed in 2× SSC at 68 ◦ C, according to Martin et al. [36]. Determination of the DNA base composition of strain N3/975T and ribotyping of the investigated isolates in comparison to close phylogenetic neighbours were performed as described previously [6]. Phenotypic characterization For morphology studies by transmission electron microscopy (TEM), vegetative cells of strains ES MS17T and N3/975T were cultivated at 37 ◦ C in basal medium [51] supplemented with carbon-coated gold grids (Plano GmbH, Wetzlar, Germany). Samples were prepared as described by Näther et al. [38] and analyzed by TEM on a Philips CM12 (Fei Co., Eindhoven, NL) with a LaB6 cathode at 120 keV. For scanning electron microscopy (SEM), cells of both strains grown on nutrient agar (SIFIN) were fixed on glass slides and sputter-coated with gold–palladium. The samples were examined on an SEM JSM-6060 LV (JEOL Ltd., Tokyo, Japan) in high-vacuum mode at 14 kV. Standard tests for physiological characterization were performed as described by Behrendt et al. [7]. A growth experiment for pH tolerance, ranging from 4.0 to 11.0, was performed in nutrient broth with the buffer systems described by Lee et al. [32]. Additionally, the growth at pH 4.0 and 5.0 was tested in nutrient broth containing 100 mM citric acid/200 mM Na2 HPO4 . Cultures were incubated at 30 ◦ C and the growth was estimated by monitoring the optical density at 680 nm. Oxidation of carbon sources was tested using Biolog GP2 microplates (AES). By modification of the test conditions described by Elo et al. [15], the strains were grown on BIOLOG universal growth medium (BUG) supplemented with 1% (w/v) glucose. Oxidative acid production from carbohydrates was investigated using API 50CH strips (bioMérieux) inoculated with a bacterial suspension in CHB/E medium (bioMérieux). The API 20E (bioMérieux) test system was applied to determine additional physiological and biochemical features. Acid formation from carbohydrates in the API 20E

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test strips was tested by aerobic incubation and by covering the tubes with sterile paraffin oil to test fermentation abilities. Results of all test systems were read visually after 24 and 48 h incubation at 30 ◦ C. Spectral analysis by MALDI-TOF MS (matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) of the isolates and the type strain of P. campinasensis DSM 21989T was performed according to the method of Tóth et al. [52]. The peptidoglycan structure was analyzed as described by Halpern et al. [20]. Cellular fatty acid composition of the isolates and their close phylogenetic neighbours was determined for cells grown on tryptic soy agar (Becton–Dickinson) for 24 h at 28 ◦ C with the Sherlock Microbial Identification system (MIDI version 4.5), as described by Behrendt et al. [7]. The isoprenoid quinone composition was analysed for strains N3/975T , ES MS17T and P. campinasensis DSM 21989T , according to the method of Groth et al. [18]. Polar lipid patterns of strains N3/975T and ES MS17T were determined as described by Schumann et al. [47]. Tests for abilities in N-transformation processes Potential nitrogen fixation of the isolates was investigated by growth tests on semisolid diazotrophic medium RBA [34] and the nitrogen-free Azotobacter-medium modified by omission of mannitol (http://www.dsmz.de/microorganisms/ medium/pdf/DSMZ Medium3.pdf). The latter was also used as a basal medium to test the isolates’ abilities to grow aerobically or anaerobically with nitrate, nitrite or ammonium as the sole nitrogen source. The medium was supplemented separately or in addition to (NH4 )2 SO4 (1.0 g L−1 ) with KNO3 , (1.0 g L−1 ), KNO2 (0.255 g L−1 , filter sterilized) and glucose (5.0 g L−1 ) as a carbon source. Furthermore, growth was tested for comparison on the basal medium supplemented with 5.0 g L−1 meat peptone (Fluka). Heterotrophic nitrification ability was tested for the fen soil isolates and strain ES MS17T using a peptone-meat extract medium (PM), as described by Papen et al. [39], and the Verstraete and Alexander (VA) medium [55], which contained acetamide as the sole source of nitrogen and carbohydrate. Culture flasks (total volume 250 mL) containing 100 mL of medium were inoculated with cells in the exponential growth phase to a cell density of approximately 106 cells mL−1 and incubated at 28 ◦ C by shaking (120 rpm). To determine the content of nitrate, nitrite and ammonium during the course of cultivation, portions (5 mL) of the culture suspension were centrifuged (20 min; 4 ◦ C; 24,000 × g). The supernatants were frozen at −80 ◦ C and stored until analysis. Immediately after thawing, the nitrate (NO3 − –N), nitrite (NO2 − –N) and ammonium (NH4 + –N) of the supernatants were measured colorimetrically with a segmented flow analyzer (SFAS; Skalar Analytic GmbH). For investigation of nitrous oxide formation by heterotrophic nitrification, a 30 mL cell suspension of the late exponential growth phase was transferred aseptically into glass beakers (total volume 50 mL) and covered by a gauze compress that allowed gas exchange. Emission of nitrous oxide, dinitrogen and carbon dioxide was measured directly by a helium atmosphere incubation method which worked similarly to the principle described by ButterbachBahl et al. [11]. Culture jars containing the bacterial inoculums and a control without any inoculum were introduced into gas tight incubation vessels. Dinitrogen was removed using four evacuation/flushing cycles with an artificial gas mixture consisting of 21.3% O2 , 78.6% He, 330 ppm (v) CO2 , 330 ppb (v) N2 O, 1800 ppb (v) CH4 and approximately 2 ppm (v) N2 . For evacuation, a vacuum with a pressure up to 4.6 kPa was used. The evacuation/flushing cycles were followed by establishment of flow equilibrium by continuously scouring out the vessel headspaces with the appropriate gas mixture by 20 mL min−1 for 20 h. After this procedure, the gas concentrations in the air stream were measured three times with an hour interval by gas chromatography at the entrance and the exit

of the vessels using two chromatographs (electron capture detector and flame ionization detector), respectively. These measurements were repeated after 24 h of continuously flushing the incubation vessels. The minimal gas flux rates that could be analysed by this method were 40 ␮g N2 –N m−2 h−1 , 0.5 ␮g N2 O–N m−2 h−1 and 200 ␮g CO2 –Cm−2 h−1 . To confirm the purity of the investigated cultures during gas measurement, they were plated subsequently on nutrient agar (SIFIN). Furthermore, formation of dinitrogen and nitrous oxide under anaerobic culture conditions was measured by the helium atmosphere incubation method, as already described, but using a gas mixture that differed by the content of O2 (0.3%), He (99.6%) and CO2 (0%). Nutrient broth (SIFIN) supplemented with KNO3 (0.5 g L−1 ) or KNO2 (0.255 g L−1 ) was inoculated with bacterial cells in exponential growth to a cell density of ca. 106 cells mL−1 and transferred immediately into the gas tight vessels of the test equipment. A culture jar containing medium without bacterial inoculums was used as a control. Following the procedure of gas measurement, the culture suspensions were investigated for nitrate, nitrite and ammonium, as already described. Presence of functional genes coding for enzymes of the denitrification pathway was tested using the primer sets nirK1F + nirK5R [10], F1aCu + R3Cu [19] and nirK876 + nirK1040 [22] for the nirK gene, nirS1F + nirS6R [10] for the nirS gene, and nosZ2F + nosZ2R [23] for the nosZ gene, as described in the respective references.

Results and discussion The nearly complete 16S rRNA gene sequences determined for phylogenetic analysis of strain N3/975T , representative of the fen soil isolates and strain ES MS17T , were continuous stretches of 1510 and 1318 bp, respectively. The nucleotides of strain N3/975T were variable at four positions (185, 187, 193 and 195 according to Escherichia coli numbering). Sequencing from the 3 end as well as from the 5 end indicated that the nucleotides were present approximately in the same abundance at the respective positions. Sequence comparison between strain N3/975T and strain ES MS17T showed a high similarity of 99.3%, but a DNA-DNA reassociation value of 63.7% (replicate value 62.7%) indicated that both strains represented different species, according to the recommendation for species delineation of Wayne et al. [57]. As shown in Fig. 1, strain N3/975T and strain ES MS17T clustered together in the maximum likelihood and neighbour joining tree. Their distinct position was supported by a bootstrap value of 100%. Furthermore, they were part of a subcluster of the genus Paenibacillus additionally harbouring P. campinasensis, P. lautus, P. glucanolyticus and P. lactis. Formation of this branch was found by both treeing methods and was substantiated by a high bootstrap value of 97%. Species of this branch shared sequence similarities in the range of 95.3 and 96.3% with the isolates, suggesting a separate species position for the investigated strains [50]. However, more than 1% sequence divergence of strain N3/975T and strain ES MS17T , in comparison to the next related species P. campinasensis, was based on gaps, and thus, DNA–DNA hybridization studies were performed. DNA–DNA reassociation values of 15.4% (replicate value 6.8%) between P. campinasensis and strain N3/975T and 2.5% (replicate value 7.3%), in comparison to ES MS17T , clearly demonstrated that the isolates represented separate species. Since the phylogenetic analysis of the fen soil isolates was performed for the representative strain N3/975T , it was necessary to analyze its relationship with isolates N3/340 and N3/727. For this reason, fingerprinting by MALDI-TOF MS and automated RiboPrinting in comparison to related species was performed. As demonstrated in Supplementary Fig. S1 (available in SAM online), the fen soil isolates formed a tight cluster on the basis of nearly identical mass spectra, which supported the view that they belonged to

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Fig. 1. Section of the neighbour joining tree showing the relationships between strains N3/975T and ES MS17T in comparison to species of the genus Paenibacillus. Filled circles indicate branches of the tree that were also obtained using the Felsenstein maximum likelihood method. The sequence of Bacillus subtilis subsp. subtilis strain BCRC 10255T (GenBank accession no. EF423592) was used as an outgroup (not shown). Numbers at nodes indicate bootstrap percentages >70% (based on 1000 resampled datasets). Bar, 0.01 changes per nucleotide position.

the same species. A similar but not identical spectrum was found for the isolate ES MS17T , which fell together with the spectra of the fen soil isolates into a cluster separate from the lineage of the type strain of P. campinasensis. These findings reflected the phylogenetic relationship revealed by 16S rRNA gene sequence analysis. Similar results were obtained for comparison of RiboPrint patterns (Supplementary Fig. S2, available in SAM online). Strain N3/975T showed nearly identical patterns to strain N3/340. Despite some differences in the RiboPrint pattern of strain N3/727, the fen soil isolates formed a coherent cluster, which additionally supported their affiliation to the same species. In correspondence with the results of MALDI-TOF MS, the most similar RiboPrint pattern was found for the closely related strain ES MS17T . Furthermore, the four isolates clustered separately from the type strains of phylogenetically related species. The cellular fatty acid profiles of the isolated strains did not differ remarkably from those of related species (Supplementary Table S2, available in SAM online). In particular, the relatively high variance in the proportion of several fatty acids of the fen soil isolates did not allow a clear demarcation from the other investigated strains.

However, anteiso-C15:0 was the predominating fatty acid of all isolates, which corresponded to the genus description of Paenibacillus [30]. A further chemotaxonomic feature important for genus affiliation was the isoprenoid quinone composition. The quinone system of strains N3/975T and ES MS17T consisted of the predominating menaquinone MK-7 and small amounts of MK-6 in ratios of 83:16 and 94:6, respectively. A similar ratio of 97:3 was obtained for the related species P. campinasensis (DSM 21989T ). These results supported the affiliation to the genus Paenibacillus where the presence of MK-7 as the major quinone is a typical characteristic of the genus [30]. The peptidoglycan hydrolysate (6 N HCl, 120 ◦ C, 16 hours) of strains N3/975T and ES MS17T contained the amino acids lysine, glutamic acid, aspartic acid and alanine in the approximate ratios of 1.1:1.0:0.9:1.3 and 0.9:1.0:0.9:1.2, respectively. Under milder conditions (4 N HCl, 100 ◦ C, 16 h), the relatively stable peptide lLys–d-Asp was detected. From these data the peptidoglycan type A4alpha l-Lys–d-Asp was concluded for both strains N3/975T and ES MS17T . These results differed from the genus description, where meso-diaminopimelic has been shown as the diagnostic diamino acid of the peptidoglycan [1,49]. However, recent studies have

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indicated that several species of the genus Paenibacillus are characterized by the absence of meso-diaminopimelic in their cell wall [8,31,40,54]. The species Paenibacillus riograndensis displayed the peptidoglycan type A3alpha l-Lys–l-Ala–l-Ala [8]. On the basis of these results, an emendation of the genus description is proposed to include lysine as a possible diamino acid of the peptidoglycan. Investigation of the polar lipid composition revealed identical patterns for strains N3/975T and ES MS17T , which mirrored the close relationship between both. As shown by the example of strain ES MS17T (Supplementary Fig. S3, available in SAM online), the polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three aminophospholipids, phospholipids and two glycolipids. This lipid composition differed from the pattern reported for the type species Paenibacillus polymyxa [30]. However, heterogeneity in polar lipid patterns was also demonstrated for other species of the genus Paenibacillus [12,31,45]. The investigation of strains ES MS17T and N3/975T by TEM and SEM analysis showed nearly identical morphological features for both strains, as demonstrated in Supplementary Fig. S4 (available in SAM online). The growth of cells on carbon-coated gold grids enabled the analysis of their appendages without the possibility of them being sheared off by standard protocols for TEM cell preparation. Micrographs in Fig. S4C show two different kinds of appendages located laterally on the cell surface, which can be assumed to be flagella (20 nm) and fimbriae (13–15 nm) due to their diameters [3]. Furthermore, several cells of strain ES MS17T possessed an extracellular matrix with a width of approximately 150 nm (Fig. S4A and B), a feature not observed for strain N3/975T . The ellipsoidal spores of both investigated strains were located terminally in swollen sporangia (Fig. S4D and F), and their surfaces were characterized by a curvilinear rib pattern connecting the poles of the spores (Fig. S4G). The isolated strains were also subjected to a multitude of physiological tests to determine the similarities between them and to differentiate them from phylogenetic neighbours. Details are given in the species description and Table 1. The fen soil isolates were not able to assimilate any of the provided carbohydrates by acid production in the API 20E test system; although for some of them (i.e. sucrose, melibiose and amygdalin) a positive reaction was achieved in the API 50CH system. In contrast, strain ES MS17T , isolated from the spacecraft assembly clean room, produced acid from glucose, sucrose, amygdalin and l-arabinose. Furthermore, none of the tested carbohydrates were fermented by the soil isolates and strain ES MS17T in the API 20E test system. However, a positive reaction for glucose fermentation using the method of Hugh and Leifson [27], indicated that the application of the API 20E test system to investigate assimilation and fermentation of carbohydrates was not advisable for this bacterial group. With respect to the multitude of phenotypic features tested, the fen soil isolates displayed a relatively high level of conformity between each other, which supported the conclusion from fingerprinting by MALDI-TOF MS and ribotyping that all three isolates represented one species. Nevertheless, few differences were found in the API 50CH and Biolog GP2 test results, as shown in detail in Table 1. Furthermore, in the API 20E test system, strain N3/727 was able to reduce nitrate to nitrite. However, comparison of soil isolates with strain ES MS17T revealed differences in several physiological characteristics, which exceeded the intraspecific diversity of the fen soil isolates (Table 1). Therefore, a demarcation of strain ES MS17T from the soil isolates at the species level was clearly supported by several physiological characteristics. As also shown in Table 1, a comparison with phylogenetically related species of the genus Paenibacillus revealed a multitude of physiological features that enabled the investigated isolates to be clearly differentiated. This demonstrated the separate species status of the isolates from the phenotypic point of view.

The comprehensive phylogenetic and phenotypic analyses demonstrated that the fen soil isolates and strain ES MS17T , isolated from the spacecraft assembly clean room, represented two novel species of the genus Paenibacillus when emended in order to include strains with lysine in the peptidoglycan, for which the names Paenibacillus uliginis sp. nov. and Paenibacillus purispatii sp. nov. are proposed. To investigate the abilities of the fen soil isolates and strain ES MS17T in nitrogen transformation processes, potential fixation of dinitrogen was tested using two selective media. None of the isolates were able to grow, which indicated that they were not diazotrophic. The tests for utilization of nitrate or nitrite as a sole nitrogen source for growth were negative, showing that the investigated isolates are not able to assimilate nitrate or nitrite. In contrast, growth was observed in the presence of ammonium but, in comparison to the medium containing peptone, the growth was weak, which indicated a preference for assimilation of organic nitrogen sources. For the investigation of heterotrophic nitrification, formation of nitrate and nitrite was monitored during aerobic growth in PM and VA medium. As shown for the type strains N3/975T and ES MS17T , when they were grown in VA medium with acetamide as a sole source of nitrogen and carbohydrate (Supplementary Fig. S5, available in SAM online), only minor amounts of nitrate-N and nitrite-N in the range of 0 and 0.2 mg L−1 were found. At first glance, the isolates appeared to be unable to perform heterotrophic nitrification as neither nitrate nor nitrite was accumulated. However, estimation of ammonium in the growth medium revealed a very high probability of nitrification activity. As shown in Fig. S5, the ammonium concentration increased significantly in the stationary phase of growth, indicating respiratory ammonification of nitrate or nitrite. None of these soluble nitrogen oxides was added to the media, and thus, it is concluded that the nitrogen source for ammonification was formed by heterotrophic nitrification – a process requiring aerobic conditions. This assumption was substantiated by the absence of ammonium formation during anaerobic incubation. As already mentioned for the test results of the API 20E test strips, with the exception of strain N3/727, the isolates were not able to reduce nitrate to nitrite. For this reason, the end product of nitrification and the starting compound for ammonification was most likely nitrite for the isolates with a negative test result for nitrate reduction. A possible source of the minor nitrate amounts determined in the culture media (Fig. S5) was the oxidation of nitrite by a chemical process. A further substantial argument for heterotrophic nitrification activity of the isolates was revealed by the helium atmosphere incubation method. All isolates emitted substantial amounts of dinitrogen during growth in both media tested. Formation of nitrogen gas indicated that denitrification activity also occurred during the incubation of the isolates in the nitrification media. Nitrate and nitrite are also starting compounds for the denitrification pathway. However, from this experiment, it cannot be assumed undoubtedly that the isolates were able to perform all steps of denitrification from nitrate/nitrite to nitrous oxide, as the latter can also be produced during ammonification. Nevertheless, the emission of dinitrogen required at least nitrite as the starting compound, independently of the microbiological pathway by which nitrous oxide was formed. Therefore, the emission of dinitrogen supported the assumption that the isolates performed heterotrophic nitrification activity. To clarify whether the isolates were able to perform all steps of denitrification, their activities were investigated anaerobically by using the helium atmosphere incubation method. A clear nitrous oxide formation from nitrite was determined for all isolates and from nitrate for strain N3/727, although none of the isolates formed dinitrogen in detectable amounts. To exclude ammonification of nitrate or nitrite as the source of nitrous oxide, the ammonium con-

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Table 1 Physiological characteristics of P. purispatii ES MS17T and strains of P. uliginis in comparison to phylogenetically related species of the genus Paenibacillus. Substrate Oxidation (Biolog GP2) of: Glycogen n-Acetyl-d-glucosamine n-Acetyl-␤d-mannosamine Amygdalin l-Arabinose d-Arabitol Arbutin d-Cellobiose d-Fructose d-Galactose d-Gluconic acid ␣-d-Glucose ␣-d-Lactose Lactulose d-Mannitol d-Mannose d-Melezitose d-Melibiose Methyl-␤d-galactoside 3-Methylglucose Methyl-␤d-glucoside Methyl-␣d-mannoside Palatinose d-Psicose d-Raffinose l-Rhamnose d-Ribose Salicin d-Sorbitol Stachyrose Turanose d-Xylose Acetic acid ␣-Ketoglutaric acid l-Lactic acid l-Malic acid Methyl pyruvate Mono-methylsuccinate Pyruvic acid Succinamic acid Succinic acid Glycerol Adenosine 2 -Deoxyadenosine Inosine Thymidine Uridine Uridine-5 -monophosphate d-Fructose 6-phosphate d-Glucose 6-phosphate Acid formation (API 50CH) from: Glycerol d-Arabinose l-Arabinose d-Xylose Methyl-D-xyloside d-Galactose d-Glucose d-Fructose d-Mannose Rhamnose Mannitol Sorbitol ␣-Methyl d-mannoside ␣-Methyl d-glucoside n-Acetylglucosamine Amygdalin Arbutin d-Melezitose d-Raffinose d-Turanose d-Fucose

1

2

3

4

5

6

+ + − − − w + − − + − + − − − + − − − − − − + − − − + + − − + w w − − − + + + − − + + − + w + − w w

− + + d(−) − − + d(w) + d(w) − + − − − + − − − − − − + − d(−) − + + − − + − + d(−) − − + + + − − + + w w w + − − −

+ − − +* w − +* +* + +* − + +* +* − +* +* + − −* +* +* − w +* +* −* +* −* − w* + −* − − − − − −* − − − w* − w* − w* − −* −*

+ w − w − − + + + + w + + + + + + + w w + − + + + + + + + w + + + w + + − − + + + − − − − − − + − −

+ − − − w − − − − − − − w − − − − − − − w − − − − − − − − − − − − − − − − − − − − − w − − − − − − −

+ − − − − − w w w w − − w w w + − + w − w − − − + + w − − − w − − − − − − − − − − − w − w − w − − −

+ − + − + + + + + − − − + + + + + + + + −

− − − − d(−) − + − + − − − − + + + + − d(−) + −

− − + + w − − + − − + − w w w + + + + − −

− + + + + + + + + + + + − + + + + w + + −

− w + + w − + + + − + − w w + + + − + w −

+ + + + − + + + + − + − − w + w w − + + w

334

U. Behrendt et al. / Systematic and Applied Microbiology 33 (2010) 328–336

Table 1 (Continued ) Substrate

1

2

3

4

5

6

d-Arabitol Gluconate

− +

− d(−)

+ +

− +

− −

− −

Data for all strains from this study: 1, P. purispatii ES MS17T ; 2, P. uliginis (N3/975T ; N3/340; N3/727); 3, P. campinasensis DSM 21959T ; 4, P. glucanolyticus DSM 5162T ; 5, P. lactis DSM 15596T ; 6, P. lautus DSM 3035T . +, Positive; −, negative; w, weak reaction; d, different reaction between strains; (), reaction of the type strain. All strains utilized the following substrates in the Biolog GP2 test system: ␣- and ␤-cyclodextrin*, dextrin, maltose*, maltotriose*, sucrose* and d-trehalose*, whereas inulin, Tween 40 and 80, l-fucose, d-galacturonic acid, gentiobiose, m-inositol, mannan, methyl-␣d-galactoside, methyl-␣d-glucoside, propionic acid*, sedoheptulosan, d-tagatose*, xylitol, ␣-hydroxybutyric acid, ␤-hydroxybutyric acid, ␥-hydroxybutyric acid, p-hydroxyphenylacetic acid, ␣-ketovaleric acid, lactamide, d-lactic acid methyl ester, d-malic acid, n-acetyl-l-glutamic acid, l-alaninamide, d-alanine, l-alanine, l-alanyl glycine, l-asparagine, l-glutamic acid, glycyl l-glutamic acid, l-pyroglutamic acid, l-serine, putrescine, 2,3-butanediol, adenosine 5 -monophosphate, thymidine 5 -monophosphate, ␣d-glucose 1-phosphate, dl-␣-glycerol phosphate were not utilized. In the API50CH test system, acid was produced from d-cellobiose, maltose, lactose, d-melibiose, salicin, sucrose, d-trehalose, ribose, starch, glycogen, gentiobiose, but no reaction was obtained for d-adonitol, erythritol, l-fucose, inulin, l-xylose, l-sorbose, dulcitol, m-inositol, xylitol, d-lyxose, d-tagatose, l-arabitol, 2-keto-d-gluconate and 5-keto-dgluconate. Hydrolysis of aesculin was positive for all strains. *Reaction differed from the result of P. campinasensis, as reported by Yoon et al. [58].

centration was determined in the test medium subsequent to the gas measurement. As the concentrations in the inoculated medium decreased in comparison to the control medium, this indicated that ammonium was assimilated and respiratory ammonification had not started at the time of measurement. Therefore, denitrification was the source of nitrous oxide formation under these test conditions. An alternative for characterization of isolates, as well as communities, is the study of functional genes coding for reductases involved in denitrification [14,24,41,56]. However, no (or no specific) amplicons for the nitrite reductase genes nirK or nirS, and for the nitrous oxide reductase gene nosZ were obtained for the isolates by the application of different primer sets commonly used in environmental studies. Tests of the specificity of nir and nos primer sets on bacterial strains by Heylen et al. [24] and Henry et al. [23] demonstrated restrictions of the presently available primers in their claim to amplify these functional genes from the majority of the denitrifiers. Thus, studies of the denitrifying bacterial community by culture-independent methods would not incorporate these fen soil isolates representative for a bacterial group, which formed a high proportion of the denitrifiers found by cultivation (data not shown). In conclusion, the investigations demonstrated that the isolates were able to perform three processes of N-transformation important in formation of nitrous oxide – heterotrophic nitrification, denitrification and respiratory ammonification. It is known that in several bacteria, heterotrophic nitrification is linked to denitrification activity [21], but it has not been reported that denitrification and respiratory ammonification occur in the same bacterial species [29]. This combination of abilities potentially enables the isolates to contribute to the nitrogen transformation processes of their habitats under aerobic and anaerobic conditions. High versatility in metabolic nitrogen conversion processes is an interesting and very helpful feature for the possibility of settling in novel habitats. In the scientific field of planetary protection [13], primary producers, resistant microbes and microorganisms that can deal with special nutrient conditions are the main focus, since the possible transportation of microbes to other planets could affect the integrity of not only the extraterrestrial environment, but also the life detection instruments [51]. For this reason, the presence of spore forming paenibacilli in spacecraft assembly clean rooms, revealing an extraordinary capability to deal with different nitrogen compounds, definitely poses a threat to planetary protection considerations. Strains of both proposed species, although derived from completely different habitats, have revealed novel insights into the capabilities of the genus Paenibacillus, whose representatives are widespread in most diverse natural and artificial habitats.

Emended description of the genus Paenibacillus Ash, Priest, and Collins 1993 Paenibacillus (L. adv. paene, almost; L. masc. n. bacillus, a rod and also a bacterial genus name (Bacillus); N.L. masc. n. Paenibacillus, almost a Bacillus). The description is as given by Ash et al. [1] and Shida et al. [49] with the following amendment. The diamino acid of the peptidoglycan is either meso-diaminopimelic acid or lysine. Description of Paenibacillus uliginis sp. nov Paenibacillus uliginis (u.li.gi’nis. L. n. uligo -inis, moisture, marshy quality of the earth; L. gen. n. uliginis, of moisture). Vegetative cells (on average 4.0 × 0.8 ␮m) are motile by lateral flagellation. Cells produce ellipsoidal spores, located terminally in swollen sporangia. Spores are on average 1.8 × 1.1 ␮m with a curvilinear rib pattern on the surface connecting the poles of the spores. Young colonies are translucent, glistening, low convex and regular with an entire margin. With age the colonies become opaque with a white to cream colouring and adhere to the agar surface. The colonies are flat and sometimes spread on the agar surface forming a translucent morphotype. Cells are Gram-variable. Translucent colonies show a Gramnegative reaction by the KOH test but with age the reaction is Gram-positive. The Gram stain shows a Gram-positive reaction but the cells decolour easily. The optimal growth temperature is 30 ◦ C, and no growth is found at 4 or 41 ◦ C. Oxidase, catalase and ␤-galactosidase are present. Reactions for arginine dihydrolase, lysine and ornithine decarboxylase, tryptophan deaminase, urease, indole production and Voges Proskauer test are negative. Starch and aesculin are hydrolysed but gelatine and casein are not. Reduction of nitrate to nitrite is strain dependent. Produces acid from glucose anaerobically without formation of gas. Citrate utilization is negative. Acid formation in the API 20E test system is negative by assimilation and fermentation for d-glucose, d-mannitol, inositol, d-sorbitol, l-rhamnose, d-sucrose, d-melibiose, amygdalin and l-arabinose. Further physiological characteristics are as given in Table 1. The peptidoglycan type is A4alpha l-Lys–d-Asp and the major menaquinone is MK-7. Minor amounts of MK-6 are present. Predominant polar lipids are diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Additionally, two unidentified glycolipids, three unidentified phospholipids and aminophospholipids are present. The main fatty acids (average >5%) are anteiso-C15:0 (44.37 ± 5.04), C16:0 (17.51 ± 8.30), C16:1␻11c (9.40 ± 1.09), isoC15:0 (6.97 ± 0.50) and iso-C16:0 (5.62 ± 1.64). The DNA G + C content of the type strain is 45.2 mol% (HPLC). The type strain, N3/975T (=DSM 21861T = LMG 24790T ), was isolated from fen peat

U. Behrendt et al. / Systematic and Applied Microbiology 33 (2010) 328–336

soil of a nitrogen fertilization long-term experiment in Paulinenaue (Germany). Description of Paenibacillus purispatii sp. nov. Paenibacillus purispatii (pu.ri.spa’ti.i. L. adj. purus -a -um, clean, L. n. spatium, room; N.L. gen. n. purispatii, of a clean room). Vegetative cells (on average 4.0 × 0.8 ␮m) are motile by lateral flagellation. Cells produce ellipsoidal spores, located terminally in swollen sporangia. Spores are on average 1.8 × 1.1 ␮m with a curvilinear rib pattern on the surface connecting the poles of the spores. Young colonies are translucent, glistening, low convex and regular with an entire margin. With age the colonies become opaque with a white to cream colouring, flat and adhere to the agar surface. Cells are Gram-variable. Translucent colonies show a Gram-negative reaction by the KOH test but with age the reaction is Gram-positive. The Gram stain shows a Gram-positive reaction but the cells decolour easily. Growth is facultatively anaerobic in a temperature range of 10–39 ◦ C (optimum 32 ◦ C). The pH range of growth is 5.0–10.0 with an optimum at 7.0. Tolerates NaCl between 0.05 and 5% (w/v). Oxidase, catalase and ␤-galactosidase are present. Produces H2 S from sodium thiosulfate. Reactions for arginine dihydrolase, lysine and ornithine decarboxylase, tryptophan deaminase, urease, indole production and Voges Proskauer test are negative. Starch and aesculin are hydrolysed but gelatine and casein are not. Produces acid from glucose anaerobically without formation of gas. Citrate utilization is negative. Nitrate is not reduced to nitrite. Aerobic acid production in the API 20E system from glucose, sucrose, amygdalin, l-arabinose is positive, whereas the tests for d-mannitol, inositol, d-sorbitol, l-rhamnose and d-melibiose are negative. Acid production from fermentation is negative for all carbohydrates of the test system. Further physiological characteristics are as given in Table 1. The peptidoglycan type is A4alpha l-Lys–d-Asp and the major menaquinone is MK-7. Predominant polar lipids are diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Additionally, two unidentified glycolipids, three unidentified phospholipids and aminophospholipids are present. The main fatty acids (≥5%) are anteiso-C15:0 (45.1), C16:0 (17.4), anteiso-C17:0 (9.8), iso-C15:0 (6.3), iso-C16:0 (6.2) C16:1␻11c (6.0) and iso-C17:0 (5.0). The type strain, ES MS17T (=DSM 22991T = CIP 110057T ), was isolated from a spacecraft assembly clean room at the European Space Research and Technology Centre (ESTEC) in Noordwijk, The Netherlands. Acknowledgements Funding by the NitroEurope IP and the European Space Agency (ESA) is gratefully acknowledged. We wish to thank Mrs. B. Selch, Mrs. S. Weinert, Mr. B. Grossmann, Mrs. J. Busse, Mrs. D. Schulz (ZALF-Müncheberg), Mrs. A. Wasner, Mrs. B. Sträubler and Mrs. G. Pötter (DSMZ-Braunschweig) for their excellent technical assistance. Furthermore, we would like to acknowledge Prof. Dr. H.G. Trüper and Prof. Dr. J.P. Euzeby for their help with the Latin construction of the species names.

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