Development of a Primer System to Study Abundance and Diversity of the Gene Coding for Alanine Aminopeptidase Pepn Gene in Gram-Negative Soil Bacteria
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Journal of Microbiological Methods 91 (2012) 14–21 Contents lists available at SciVerse ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth Development of a primer system to study abundance and diversity of the gene coding for alanine aminopeptidase pepN gene in Gram-negative soil bacteria Esther Enowashu a, Ellen Kandeler a, Michael Schloter b,⁎, Frank Rasche c, Marion Engel b a Institute of Soil Science and Land Evaluation, Soil Biology Section, University of Hohenheim, Emil-Wolf Straße 27, Stuttgart, D-70593, Germany b Research Unit for Environmental Genomics, Helmholtz Zentrum München, National Research Centre for Environmental Health, Ingolstädter Landstraße1, Neuherberg, D-85764, Germany c Department of Plant Production in the Tropics and Subtropics, University of Hohenheim, Garbenstraße 13, Stuttgart, D-70593, Germany article info abstract Article history: A new set of primers was developed allowing the specific detection of the pepN gene (coding for alanine Received 11 June 2012 aminopeptidase) from Gram-negative bacteria. The primers were designed in silico by sequence alignments Accepted 3 July 2012 based on available DNA sequence data. The PCR assay was validated using DNA from selected pure cultures. Available online 14 July 2012 The analysis of gene libraries from extracted DNA from different soil samples revealed a high diversity of pepN related sequences mainly related to α-Proteobacteria. Most sequences obtained from clone libraries Keywords: were closely related to already published sequences (b80% homology on amino acid level), which may be Alanine aminopeptidase fi pepN gene related to the conserved character of the ampli ed region of pepN. By linking the diversity data obtained Environmental samples by the clone library studies to potential enzymatic activities of alanine aminopeptidase, lowest diversity of PCR primer pepN was found in those soil samples which displayed lowest activity levels, which confirms the importance Proteobacteria of diversity for the ecosystem function mainly when transformation processes of complex molecules are studied. © 2012 Elsevier B.V. All rights reserved. 1. Introduction genes for neutral proteases and subtilisin was proven. (Watanabe and Hayano, 1993a, b, 1994; Mrkonjic Fuka et al., 2008). However data on Up to 90% of the total nitrogen (N) in soils is stored in the organic drivers for peptide degrading microbes in nature is still missing. In N fraction (Miltner and Zech, 1999). Compared to the turnover of in- the present study, we describe a new set of primers to amplify the organic N forms, like ammonia or nitrate, the cycling of organic N oc- gene encoding alanine aminopeptidase (EC 3.4.11.12; PepN) from soils curs often slowly over many years and thus it is steady mineralization and other ecosystems to investigate abundance and diversity of the which provides a sustainable source of N for above and below-ground corresponding functional groups of microbes. PepN has been firstly processes that contribute to plant nutrition (Kirk et al., 2004) mainly described as the major membrane bound aminopeptidase for Escherichia in non-fertilized soils. The most abundant organic N-containing com- coli in the 70s of the last century (Lazdunski et al., 1975a, 1975b), but pounds in soils are proteins, peptides, amino acids and amino sugars turned out to be well distributed among a wide range of Gram- positive (Stevenson, 1994; Schulten and Schnitzer, 1998). Whereas organisms and Gram-negative bacteria. Therefore not surprisingly enzymatic essays of different trophic levels including several plant species, animals focussing on the detection of potential activities of pepN in soil revealed and microbes are able to uptake amino acids (Senwo and Tabatabai, higher activities compared to other peptidases (e.g. leucine aminopepti- 1998), which are either utilized as energy source or as matrix for bio- dase) mainly in forest soils (Enowashu et al., 2009). The primers were mass formation, proteins and peptidases are exclusively transformed constructed in silico and their suitability was tested using selected by microbes (Kaye and Hart, 1997) Such microbes harbor the genetic bacterial strains. Finally, gene libraries from three different soil samples potential to express of extracellular hydrolytic enzymes, including were constructed and a diversity pattern for the alanine aminopepti- proteases and peptidases. dase was described to prove the suitability of the developed primer Only very few studies in the past investigated factors that drive the systems. abundance and the diversity of microbes being able to catalyse protein and the peptide degradation. These rare examples include several stud- 2. Materials and methods ies, where the influence of the soil type and the agricultural manage- ment on abundance, diversity and activity of microbes carrying the 2.1. Bacterial strains and culture conditions Strains, which are known to carry the pepN gene covering different ⁎ Corresponding author. E-mail addresses: [email protected] (E. Enowashu), sub-groups of Proteobacteria (Labrenza aggregate (DMSZ 13394), [email protected] (M. Schloter). Pseudomonas putida (DMSZ 3226), Shinella zoogloeoides (DMSZ 287) 0167-7012/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2012.07.001 E. Enowashu et al. / Journal of Microbiological Methods 91 (2012) 14–21 15 Table 1 (AMC) derivative , as well as standards and buffer were obtained from Potential PepN activity measured in soil samples from different sites. Values in brackets Sigma-Aldrich (Germany). Briefly, soil suspensions were prepared indicate standard error (n=5). by adding 0.5 g soil into 50 ml of autoclaved water and dispersed by − Soil type Treatment Enzyme activity ultrasonication for 2 min with 50 J s 1 sonication energy. The suspen- −1 −1 (nmol AMC h g soil) sions were continuously stirred using a magnetic stir plate while 50 μl Forest FS-RN 985.21(±62.50) aliquots were dispensed into 96-well microplate (PP F black 96 well; Forest FS-C 522.29 (±18.81) Greiner Bio-one GmbH, Germany), followed by 50 μl of the buffer and Agricultural AS-ET 259.83 (±14.94) 100 μl of the substrate solution. Standard wells received 50 μl of soil Agricultural AS-C 72.73 (±0.64) fi Glacier GS-1953 189.71 (±5.76) suspension, standard solution buffer. All microplate wells had a nal Glacier GS-1900 295.27 (±18.68) volume of 200 μl as reaction medium. The plates were incubated at Glacier GS-1850 851.53 (±22.38) 30 °C. Fluorescence was measured at 360/460 nm wavelength in a microplate fluorescence reader (Bio-Tek Instruments Inc., FLX 800, Germany) after 0, 30, 60, 120 and 180 min. The enzyme activity and Rhizobium radiobacter (DSMZ 5172)) as well as Gram-positive bac- corresponded to an increase in fluorescence and was calculated in teria (Corynebacterium glutamicum (DSM 20300)) were purchased from nmol AMC AMC g−1 soil h−1. the German collection of microorganisms and cell cultures (DSMZ;, Germany). The strains were grown over night at 30 °C in peptone- 2.4. DNA extraction glucose broth (per l distilled water: 10 g peptone, 5 g glucose, 10 g of NaCl, 5 g yeast extract; pH 7.5) and cells were harvested by centri- Genomic DNA of pure cultures was obtained using the DNeasy® fugation at 5000×g for 10 min. Blood & Tissue Kit (Qiagen, Hilden, Germany) as specified by the manu- facturer. Total DNA of soils (300 mg) was extracted using the FastDNA® 2.2. Soils Spin Kit for soil (Qbiogene, Germany) as specified by the manufacturer. Isolated DNA was stored at −20 °C. We selected three different sites differing in their physico-chemical properties as well as in their vegetation cover for this study.. 2.5. In silico design of a PCR primer system for pepN Two forest soil (FS) samples were taken in October 2007 from the “clean rain” (reduced N) experiment in Solling, central Germany As the set of primers should be useful for qPCR as well as for diver- (51°31′N, 9°34′E, elevation ~500 m above sea level (a.s.l.)). The soils sity analysis of pepN an amplicon length of 400–450 bp has been con- were sampled from the Oa horizon (2 to 5 cm) with one sample being de- sidered as optimal, to obtain enough phylogenetic information for the rived from the treatment “clean rain” (FS-RN) and the other from the con- diversity analysis, as well as to reach satisfactory efficiencies for qPCR. trol (FS-C) plot. The “clean rain” treatment is characterized by a 65% lower For the design of the primers, nucleotide sequences of known bacterial al- N input than the control plot due to prior deionization of throughfall anine aminopeptidases were extracted from NCBI data base and aligned water. The mean annual precipitation and variation in temperature using the clustalW based alignment tool implemented in biology work- were approximately 1090 mm and 6.4 °C, respectively. The soil has bench (http://workbench.sdsc.edu/). The specificity of the obtained for- been characterized as a strongly acidic dystric cambisol. (pH 2.6-2.8; or- ward and reversed primer was tested using the Genomatix software ganic carbon 32.8–35 g 100 g−1 soil; total N 1.4 g 100 g−1soil). A de- package (http://genomatix.gsf.de) against the Bacteria Dataset (GenBank tailed description of the experimental site is given by Enowashu et al. release 154) in silco. The deduced sequences of the degenerated primers (2009). was as follows: pepN F5′-CARTGYGARBCISARG-3′ and pepN R5′-CYYTTR Soil under agricultural use (AS) was obtained from the Hohenheim TTYTCCATIGC-3′. Climate Change experimental field located at the Heidfeldhof experimen- tal field station (48°42′50′N, 9°11′26′E, 395 m a.s.l.) of the University of 2.6. PCR reaction Hohenheim. Samples were collected in June 2009 from both the elevated temperature treatment (AS-ET) where the surface soil was heated PCR reactions were done in 25 μl volume and contained 5 ng (pure (2.5 °C) by means of heating cables to a depth of 4 cm, and the control strains) respectively 10 ng (soils) of template DNA, 100 pmol of for- treatment (AS-C) (ambient temperature) from the upper 20 cm (Ap ward and reverse primer and 12.5 μl of a ready-to-use PCR mix (BioMix, horizont).