Microbial Ecology

Metabolic Profiling of Burkholderia cenocepacia, Burkholderia ambifaria, and Burkholderia pyrrocinia Isolates from Maize Rhizosphere
Chiara Alisi1, Giovanna Jona Lasinio2, Claudia Dalmastri3, AnnaRosa Sprocati1, Silvia Tabacchioni3, Annamaria Bevivino3 and Luigi Chiarini3
` Protezione dellAmbiente, Ente Nazionale per le Nuove Tecnologie, lEnergia e lAmbiente (ENEA) C. R. Casaccia, Rome, Italy (1) Unita ` di Statistica, Universita ` BLa Sapienza^, Rome, Italy (2) Facolta ` Biotecnologie e Protezione della Salute e degli Ecosistemi, ENEA C. R. Casaccia, Via Anguillarese 301, 00060 S. Maria di Galeria, Rome, Italy (3) Unita Received: 26 October 2004 / Accepted: 27 January 2005 / Online publication: 25 November 2005

Abstract

Introduction

Burkholderia cenocepacia, Burkholderia ambifaria, and Burkholderia pyrrocinia are the Burkholderia cepacia complex (Bcc) species most frequently associated with roots of crop plants. To investigate the ecophysiological diversity of these species, metabolic proling of maize rhizosphere isolates was carried out by means of the Biolog system, using GN2 and SFN2 plates and different parameters related to optical density (OD). The metabolic proles produced by the SFN2 and GN2 plates were identical, but the SFN2s narrower range of OD values and signicantly longer reaction times made these plates less suitable for differentiation of isolates. Principal component analysis of maximum OD (ODM) and maximum substrate oxidation rate (mM) data generated by GN2 plates allowed the selection of a reduced number of carbon sources. Statistical analysis of ODM values highlighted marked differences between the metabolic proles of B. cenocepacia and B. ambifaria, whereas metabolic proles of B. pyrrocinia clustered very often with those of B. cenocepacia. Analysis of the mM parameter resulted in a slightly lower differentiation among the three Bcc species and a higher metabolic diversity within the single species, in particular within B. cenocepacia. Finally, B. cenocepacia and B. pyrrocinia showed generally higher oxidation rates than B. ambifaria on those GN2 substrates that commonly occur in maize root exudates.

Correspondence to: Luigi Chiarini; E-mail: luigi.chiarini@casaccia. enea.it

DOI: 10.1007/s00248-005-0223-y

The Burkholderia cepacia complex (Bcc) has attracted considerable interest because of its great biotechnological potential as a biopesticide for protecting crops against fungal diseases and as a bioremediation agent for breaking down recalcitrant herbicides and pesticides [3, 12, 16]. At the same time, Bcc has emerged also as an opportunistic human pathogen responsible for various nosocomial infections, in particular among patients with cystic brosis [22, 26]. Advances in the taxonomy of Bcc revealed that it comprises at least nine genetically closely related species, i.e., B. cepacia genomovar I, B. multivorans, B. cenocepacia, B. stabilis, B. vietnamiensis, B. dolosa, B. ambifaria, B. anthina, and B. pyrrocinia, which can be differentiated on the basis of molecular and biochemical tests [5, 6, 2730]. Over the past two decades, most studies investigating the relationships among Bcc isolates have focused mainly on their genetic diversity by means of a number of genetic ngerprinting methods, including ribotyping, randomly amplied polymorphic DNA typing (RAPD), pulsed-eld gel electrophoresis, multilocus restriction typing, and agellin typing [4, 7, 10, 32, 33]. In contrast, little is known about the metabolic diversity among and within the different Bcc species, reecting the scarce knowledge about the ecophysiological differences of the various Bcc species. Indeed, it is becoming increasingly accepted that the extent of physiological diversity among phylogenetically closely related bacterial species may be much larger than has been previously assumed [18]. Assessment of the metabolic proles of natural populations of Bcc species is essential to evaluate their ecological properties in natural habitats and the extent to which their ecological niches overlap.
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In our laboratory, we started studying the metabolic proles of Bcc species colonizing the rhizosphere of maize by means of the Biolog system [9]. Recent works indicate that B. cepacia genomovar I, B. cenocepacia, B. ambifaria, and B. pyrrocinia are the Bcc species most frequently associated with roots of crop plants [1, 9, 11]. In the present study, we carried out the metabolic proling of a subset of a rhizosphere Bcc population originally isolated from a single maize eld in Northern Italy; the subset includes isolates belonging to the species B. cenocepacia, B. ambifaria, and B. pyrrocinia [8, 9]. The only B. cepacia genomovar I isolate found in the whole rhizosphere Bcc population was not included in this study. The aims of the present work were to (i) evaluate the suitability of Biolog GN2 and SFN2 microplates and of different OD-related parameters for studying Bcc metabolic diversity, (ii) analyze the relative importance of the different substrates present in the Biolog system in shaping the metabolic proles of the various bacterial isolates, and (iii) evaluate maize root exudate-related substrates used by the different Bcc species.
Materials and Methods

Twenty-six bacterial isolates belonging to the species B. cenocepacia (11 isolates), B. ambifaria (11 isolates), and B. pyrrocinia (4 isolates) were obtained from the rhizosphere of Zea mays (cv. Airone, Agra) cultivated in a eld in Pieve dOlmi (Italy) [8, 9]. The identication of the different species was previously accomplished [9] by a combination of techniques including sodium dodecyl sulfatepolyacrylamide gel electrophoresis of whole-cell proteins and recA-based restriction fragment length polymorphism analyses. All isolates had unique RAPD ngerprints [9]. Bacterial isolates were cryopreserved at _80-C in 30% glycerol. When required for experimental use, cells were removed from the freezer and plated onto nutrient agar (Difco, Sparks, MD, USA).
Bacterial Strains. Metabolic Proling. Qualitative metabolic proles of bacterial isolates were generated by means of the Biolog Microstation System 4.2 (Biolog Inc., Hayward, CA, USA). Both GN2 and SFN2 plates were used. The GN2 plates contain 95 different substrates as the only carbon sources and tetrazolium redox dye, which is irreversibly reduced in an insoluble purple product when the cells are capable of using the carbon source present in the well and consequently causing an increase in the respiration process; the color formation is the result of an oxidation process. The SFN2 plates have the same composition in carbon sources as GN2 plates but lack the tetrazolium redox dye; any increase in the turbidity is due to the cell growth produced by substrate assimilation.

Cells grown for 24 h on Biolog universal growth (BUG) agar were collected and processed according to the manufacturers instructions with minor modications. Briey, the cell concentration was adjusted to 40% of transmittance and aliquots were added to the plates (150 ml/well for GN2 plates, 100 ml/well for SFN2 plates). The plates were incubated at 28-C in the dark. Three replica plates were prepared for each strain. Purple color formation for GN2 plates [change in optical density (OD) recorded as dual wavelength, OD590OD750] was measured after 4, 8, 12, 24, 48, 72, and 96 h of incubation, whereas turbidity increase for SFN2 plates (OD590) was measured every 12 h for up to a week, by the Biolog Multiwell Reader. Data were stored using Biolog software. All the replica plates showed very high reproducibility. Apart from the usual qualitative proles, quantitative metabolic proles of bacterial isolates were obtained by calculating the maximum OD (ODM) value reached by the different bacterial isolates for each carbon source considered. Those carbon sources that, for all the isolates considered, showed an ODM below an arbitrarily taken minimal threshold (0.2 for GN2, 0.1 for SFN2) were excluded. As an alternative data processing method, the OD increase in each well was plotted against the incubation time and a nonlinear curve tting was performed for each carbon source. The slope of the regression line of the nonlinear curve, i.e., the maximal rate of color development (mM), is expressed in absorbance units per hour. No mM was estimated for SFN2 data due to the low increase in the monitored OD and the low ODM. Data were arranged in an ODM and a mM data set, which were used to investigate metabolic differences between isolates.
Statistical Analyses. To reduce the dimension of the highly multivariate data sets, the nal ODM and mM data sets were separately analyzed by principal component analysis (PCA) with the carbon sources as variables. Since the 95 variables fairly exceeded the cases (i.e., isolate number), four data subsets were created according to the categories of organic compounds (i.e., amino acids, carbohydrates, organic acids, and miscellaneous) as shown in Table 1. Separate PCAs were performed on each subset, and PCA results were plotted. The rst four principal components (PCs) were retained because these account for about 50% of the total variability. Substrates showing the highest negative or positive loadings (cutoff T 0.25) on the chosen PCs were selected. Using the Ward method, we analyzed the ODM and mM values for the resulting carbon sources by cluster analysis. Since the reduced number of variables was still higher than the cases, data were organized into two random subsets (Table 2), each comprising representatives of all the categories of organic compounds. Cluster

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Table 1. Substrate utilization by rhizosphere isolates belonging to B. ambifaria, B. cenocepacia, and B. pyrrocinia

B. ambifaria ODM Carbohydrates N-Acetyl-D-glucosamine Adonitol L-Arabinose D-Arabitol D-Fructose L-Fucose D-Galactose -D-Glucose m-Inositol a-D-Lactose Lactulose D-Mannitol D-Mannose D-Psicose D-Rafnose D-Sorbitol Sucrose D-Trehalose Xylitol Carboxylic acids Acetic acid cis-Aconitic acid Citric acid Formic acid D-Galactonic acid lactone D-Galacturonic acid D-Gluconic acid D-Glucosaminic acid D-Glucuronic acid -Hydroxybutyric acid b-Hydroxybutyric acid g-Hydroxybutyric acid p-Hydroxyphenylacetic acid Itaconic acid -Ketobutyric acid -Ketoglutaric acid -Ketovaleric acid D,L-lactic acid Malonic acid Propionic acid Pyruvic acid methyl ester Quinic acid D-Saccharic acid Sebacic acid Succinic acid Amino acids D-Alanine L-Alanine L-Alanyl-glycine g-Aminobutyric acid L-Asparagine L-Aspartic acid D,L-Carnitine L-Glutamic acid Glycyl-L-glutamic acid L-Histidine Hydroxy-L-proline L-Leucine L-Ornithine L-Phenylalanine 1.068 0.842 1.299 0.683 1.157 0.976 1.160 1.189 0.970 0.320 0.854 1.108 0.926 1.063 1.039 1.121 1.243 1.184 0.479 0.594 1.310 1.271 0.894 1.180 1.260 1.402 1.057 1.290 0.540 0.962 0.562 1.100 0.690 0.400 0.748 0.326 1.298 1.019 0.875 1.233 1.246 1.351 1.046 1.013 0.840 0.874 0.817 1.318 1.275 1.328 0.637 1.311 0.370 1.150 1.144 0.547 0.458 0.841 T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T 0.136 0.168 0.119 0.220 0.143 0.231 0.116 0.118 0.138 0.110 0.250 0.127 0.210 0.294 0.118 0.160 0.122 0.118 0.308 0.228 0.142 0.137 0.129 0.099 0.119 0.135 0.293 0.137 0.145 0.189 0.148 0.107 0.241 0.092 0.219 0.046 0.152 0.242 0.144 0.123 0.131 0.086 0.149 0.091 0.148 0.133 0.168 0.108 0.126 0.151 0.103 0.086 0.240 0.175 0.139 0.134 0.055 0.147 mM 28.6 T 4.9 32.4 T 12.5 31.1 T 4.0 33.4 T 4.0 25.4 T 4.6 25.8 T 7.4 32.3 T 5.9 27.0 T 4.7 30.1 T 5.3 1.5 T 1.7 4.7 T 8.2 33.2 T 11.9 24.1 T 5.5 19.8 T 4.5 9.6 T 10.3 33.1 T 5.4 32.5 T 6.0 24.7 T 3.9 3.6 T 7.3 23.5 32.9 31.2 36.3 33.8 49.1 25.5 13.4 45.8 23.2 26.2 18.0 17.8 4.7 13.2 8.5 12.9 34.8 20.1 23.8 27.1 33.4 45.8 24.6 32.4 T T T T T T T T T T T T T T T T T T T T T T T T T 8.7 7.7 8.0 4.8 4.8 5.2 3.4 9.3 8.3 3.7 10.8 8.5 5.2 6.8 3.0 7.4 3.8 6.5 3.3 7.0 2.8 5.2 6.8 5.4 4.7

B. cenocepacia ODM 1.291 1.190 1.321 1.074 1.208 1.096 1.142 1.202 0.933 0.244 0.205 1.172 1.111 0.288 0.892 1.222 1.283 1.208 0.764 0.702 1.345 1.221 1.030 1.080 1.324 1.331 1.032 1.270 0.877 1.042 0.582 1.173 0.615 0.627 0.638 0.454 1.272 0.810 0.993 1.126 1.423 1.255 1.120 1.118 0.957 0.821 0.745 1.400 1.322 1.079 0.597 1.316 0.326 1.207 1.214 0.500 0.404 0.777 T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T 0.154 0.162 0.100 0.176 0.128 0.192 0.149 0.114 0.485 0.164 0.251 0.238 0.220 0.054 0.381 0.157 0.082 0.110 0.479 0.219 0.077 0.130 0.180 0.159 0.082 0.048 0.252 0.183 0.278 0.110 0.188 0.104 0.522 0.248 0.349 0.146 0.144 0.171 0.169 0.100 0.108 0.126 0.179 0.109 0.199 0.219 0.217 0.119 0.154 0.123 0.158 0.100 0.234 0.159 0.179 0.145 0.125 0.236 mM 29.4 33.1 33.6 42.3 28.3 37.5 38.4 34.7 29.1 0.4 0.0 47.4 26.9 7.7 21.9 44.8 41.7 29.6 28.4 30.8 40.5 48.9 45.5 32.6 57.6 37.0 26.4 49.3 28.0 40.2 22.0 14.9 17.0 17.8 6.6 19.3 34.6 27.6 28.9 30.1 39.3 55.6 28.2 29.9 23.1 25.3 20.0 29.7 37.7 25.0 11.4 33.9 3.5 36.2 39.7 17.2 8.4 18.8 T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T 11.1 14.6 4.1 8.4 6.5 12.0 7.5 9.4 19.5 1.1 0.0 10.7 7.3 9.7 8.0 7.7 8.8 7.0 21.4 7.6 9.2 8.4 10.5 6.0 10.7 5.6 4.9 9.0 5.2 6.7 8.9 6.4 14.2 6.8 10.2 8.2 5.8 4.0 3.5 4.5 9.0 20.4 11.0 7.3 3.5 5.8 4.2 6.8 9.8 4.5 5.5 6.1 4.5 7.2 7.3 7.3 6.4 9.9

B. pyrrocinia ODM 1.099 0.817 1.142 0.694 0.956 1.052 1.086 1.167 0.895 0.132 0 1.044 0.922 0.255 1.088 1.094 0.990 1.186 0.422 0.608 1.135 1.036 1.029 0.455 1.298 1.079 0.961 1.105 0.683 1.011 0.087 0.708 0.302 0.553 0.519 0.350 1.232 0.752 1.078 1.133 1.134 1.166 0.871 1.015 1.135 1.138 0.922 1.277 1.232 1.059 0.578 1.292 0.093 1.170 0.998 0.424 0.534 0.525 T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T 0.214 0.201 0.260 0.150 0.161 0.216 0.159 0.184 0.284 0.093 0 0.182 0.214 0.051 0.119 0.207 0.287 0.181 0.140 0.134 0.138 0.154 0.143 0.125 0.125 0.299 0.173 0.163 0.096 0.149 0.099 0.088 0.075 0.191 0.203 0.098 0.141 0.133 0.252 0.120 0.150 0.088 0.187 0.099 0.184 0.043 0.232 0.169 0.147 0.034 0.152 0.141 0.053 0.168 0.276 0.126 0.172 0.127 mM 39.9 37.0 30.2 33.8 33.9 27.3 29.8 38.8 38.5 0.0 0.0 44.7 26.5 4.0 16.4 39.4 45.4 42.0 0.9 28.8 36.1 42.6 43.6 0.9 24.4 34.8 16.1 31.4 16.9 34.3 0.0 5.4 0.0 15.8 0.4 13.2 35.3 29.9 31.4 31.5 36.9 40.2 29.2 43.8 T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T 9.4 5.2 5.9 5.6 5.3 5.6 3.9 4.3 5.3 0.0 0.0 5.0 9.1 7.9 8.1 8.0 6.6 7.9 1.2 4.0 6.4 8.0 5.0 1.0 17.1 11.9 3.6 4.0 12.3 3.4 0.0 6.5 0.0 2.9 0.8 9.4 10.0 10.7 7.6 6.1 9.3 5.7 4.0 5.4

17.2 T 3.6 21.5 T 4.9 17.9 T 4.6 29.3 T 3.7 36.3 T 5.7 25.5 T 5.2 9.2 T 5.3 35.7 T 6.1 5.8 T 5.7 37.3 T 5.9 32.6 T 6.9 16.5 T 4.3 4.1 T 4.2 21.7 T 4.9

30.8 T 21.1 36.2 T 10.0 27.7 T 12.0 36.0 T 4.4 42.3 T 10.9 18.2 T 12.5 16.9 T 11.3 38.0 T 7.4 0.1 T 0.3 35.3 T 6.2 35.8 T 5.3 19.3 T 14.5 7.6 T 9.6 16.9 T 11.4

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Table 1. Continued

B. ambifaria ODM
L-Proline L-Pyroglutamic D-Serine L-Serine L-Threonine

B. cenocepacia mM 34.9 33.5 7.9 25.8 5.8 T T T T T 4.3 4.2 3.5 6.6 4.7 ODM 1.235 1.280 0.252 0.982 0.542 0.345 0.772 0.860 0.346 0.617 1.076 0.207 0.544 0.478 0.295 0.659 0.859 0.802 0.480 1.135 1.175 1.143 T T T T T T T T T T T T T T T T T T T T T T 0.134 0.166 0.067 0.218 0.196 0.130 0.195 0.199 0.052 0.225 0.119 0.097 0.215 0.159 0.048 0.168 0.274 0.140 0.230 0.186 0.115 0.189
_

B. pyrrocinia mM ODM 7.6 5.1 4.6 7.8 5.2 1.038 0.922 0.360 0.835 0.494 0.524 0.530 0.822 0.363 0.468 0.919 0.319 0.399 0.452 0.294 0.552 0.923 0.923 0.343 1.127 0.990 1.109 T T T T T T T T T T T T T T T T T T T T T T 0.262 0.344 0.052 0.206 0.186 0.242 0.258 0.057 0.064 0.114 0.162 0.098 0.117 0.175 0.045 0.151 0.070 0.112 0.096 0.212 0.042 0.258 32.3 42.6 11.4 24.2 17.7 mM T T T T T 10.9 8.8 10.1 3.7 7.8

acid

1.088 1.142 0.199 1.083 0.531 0.500 0.826 0.866 0.311 0.328 1.071 0.336 0.602 0.568 0.245 0.854 0.960 0.671 0 1.032 0.951 1.090

T T T T T T T T T T T T T T T T T T T T T T

0.133 0.112 0.064 0.164 0.093 0.128 0.150 0.168 0.065 0.097 0.073 0.079 0.099 0.111 0.056 0.165 0.217 0.101 0.0 0.210 0.100 0.165

37.1 33.2 7.0 33.9 11.7

T T T T T

Miscellaneous L-Alaninamide 2-Aminoethanol Bromosuccinic acid 2,3-Butanediol a-D-Glucose-1-phosphate D-Glucose 6-phosphate Glucuronamide Glycerol D,L-a-Glycerol phosphate Glycogen Phenylethylamine Succinamic acid Succinate monomethyl ester Thymidine Tween 40 Tween 80 Urocanic acid

3.4 T 5.1 26.9 T 5.8 26.6 T 4.4 12.7 T 14.8 8.0 T 6.7 36.6 T 4.0 5.2 T 11.9 16.8 T 4.2 20.0 T 4.1 2.4 T 3.0 26.1 T 5.7 3.3 T 6.8 36.9 T 2.6 0.0 T 0.0 30.8 T 4.5 30.6 T 2.4 24.5 T 6.2

4.4 T 3.1 31.3 T 7.1 25.9 T 9.5 33.6 T 5.9 23.3 T 8.0 42.3 T 9.4 0.0 T 0.0 13.4 T 6.3 15.7 T 3.8 6.4 T 7.6 20.0 T 3.6 6.7 T 7.1 34.8 T 7.2 8.7 T 8.2 32.8 T 13.6 36.4 T 9.0 32.0 T 7.0

5.9 T 10.1 36.1 T 5.0 34.6 T 4.1 15.8 T 18.9 25.3 T 1.9 38.6 T 11.2 16.0 T 8.0 16.1 T 2.6 17.2 T 5.0 0.7 T 1.5 23.5 T 2.1 10.0 T 7.8 40.4 T 4.2 3.0 T 6.0 33.6 T 7.1 20.0 T 14.2 20.3 T 14.8

ODM T SD is expressed as absorbance units. mM T SD is expresssed as absorbance units (103) h 1. ODM and mM are the average of 11 isolates of B. ambifaria and B. cenocepacia and 4 isolates of B. pyrrocinia. The 15 substrates never utilized by all the isolates are listed in the Results section.

analysis was also performed on the different categories of metabolites taken separately, i.e., carbohydrates, carboxylic acids, amino acids, and miscellaneous substrates. Cluster analysis and one-way ANOVA were performed on the ODM and mM data produced by those substrates that commonly occur as components of maize root exudates, i.e., D-fructose, D-glucose, L-fucose, sucrose, citric acid, D,L-lactic acid, succinic acid, L-alanine, L-glutamic acid, and L-serine [2, 19, 25]. PCA and cluster analysis were performed by means of R software version 2.0.0 (The R Development Core Team, 2004, http://www.R-project.org). One-way ANOVA was performed by means of Prism (GraphPad Software Inc., CA, USA).
Results

The data obtained by Biolog GN2 microplates are summarized in Table 1, where the mean ODM and mM values obtained from the 11 B. cenocepacia, 11 B. ambifaria, and 4 B. pyrrocinia isolates are reported. Out of 95 carbon sources present in the microplates, 15 substrates (a-cyclodextrin, cellobiose, i-erythritol, gentiobiose, maltose, b-methyl-D-glucoside, melibiose, rhamnose, turanose, methylpyruvate, dextrin, inosine, uridine, putrescine, and glycyl-L-aspartic acid) were
Substrate Utilization.

never used by all the isolates of the three species. Based on the ODM values, B. cenocepacia, B. ambifaria, and B. pyrrocinia were able to oxidize 80, 79, and 73 of the 95 metabolites, respectively. B. cenocepacia showed low oxidation (G0.40 absorbance unit) of lactose, psicose, lactulose, D-serine, L-alaninamide, and glucuronamide. B. ambifaria showed a high oxidation capacity for lactulose and psicose, no reaction to thymidine, and low oxidation of lactose, a-ketovaleric acid and glycyl-Lglutamic acid. B. pyrrocinia showed no reaction with g-hydroxybutyric acid, glycyl-L-glutamic acid, lactulose, and lactose, and low oxidation of psicose. High intraspecies variability was observed for most of the substrates as shown by the high standard deviation values in Table 1. The_ mM values reached up to 0.057 absorbance units h 1, showing the highest values for some of the carboxylic acids (galacturonic acid, gluconic acid, and saccharic acid). In some cases, i.e., lactulose and glucuronamide for B. cenocepacia and itaconic acid for B. pyrrocinia, no mM was calculated due to a too slow increase in absorbance during the incubation time. Some of the substrates with the highest ODM values were also those most readily oxidized (arabinose, sorbitol, sucrose, saccharic acid, etc.), but some substrates, despite a quite low ODM, showed a high mM value, such as a-ketovaleric acid and 2,3-butanediol for

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Table 2. Substrates found to be discriminant (+ +) by PCA of ODM and mM data and included either in subset 1 or 2

ODM Carbohydrates N-Acetyl-D-glucosamine Adonitol L-Arabinose D-Arabitol D-Fructose L-Fucose D-Galactose a-D-Glucose m-Inositol a-D-Lactose Lactulose D-Mannitol D-Mannose D-Psicose D-Sorbitol Sucrose D-Trehalose Xylitol Carboxylic acids Acetic acid Formic acid D-Galactonic acid lactone D-Galacturonic acid D-Glucosaminic acid D-Glucuronic acid a-Hydroxybutyric acid g-Hydroxybutyric acid p-Hydroxyphenylacetic acid Itaconic acid a-Ketobutyric acid a-Ketoglutaric acid D,L-Lactic acid Malonic acid Propionic acid Quinic acid D-Saccharic acid Succinic acid + + + +

Subset 1 2 1 2

mM + + + + + + + +

Subset 2 1 1 2 1 2 1 2 Amino acids D-Alanine L-Alanine L-Alanyl-glycine g-Aminobutyric acid L-Asparagine L-Aspartic acid D,L-Carnitine L-Glutamic acid L-Histidine Hydroxy-L-proline L-Ornithine L-Phenylalanine L-Pyroglutamic acid D-Serine L-Threonine Miscellaneous L-Alaninamide Bromosuccinic acid 2,3-Butanediol a-D-Glucose-1-phosphate D-Glucose 6-phosphate Glucuronamide Glycerol D,L-a-Glycerol phosphate Glycogen Phenylethylamine Succinamic acid Succinate mono-methyl ester Thymidine Tween 40 Tween 80 Urocanic acid

ODM + + + + + + + + + + + + + + + + + + + + + + +

Subset 1 2 1 2 1 2 1 2 1 2 2

mM + + + + + + + + + + + +

Subset 2 1 2 1 1 1 2 1 2 1 2 2 1 2 1 2 1 2 2 1 1

+ + + + + + + + + + + +

1 2 1 2 1 2 1 2 1 2 1 2

+ + + + + + + + + + + + + + + +

1 2 1 2 1 1 2 1 2 1 2 1 2 1 2 1

2 1 1 1 2 1 1 2 2 2 1 1

+ + + + + + + + +

+ + + + + + + + + +

1 2 1 2 1 2 1 2 1 2

B. cenocepacia; arabitol, a-hydroxybutyric acid and succinate monomethyl ester for B. ambifaria; and leucine, 2-aminoethanol, and phenylethylamine for B. pyrrocinia. The results obtained for the SFN2 plates produced the same qualitative metabolic proles as the GN2 plates for each isolate (data not shown). Values of absorbance ranged from 0.1 to 0.5 absorbance units (average 0.25), and the growth rate was very low, taking up to 1 week to reach plateau value. Due to these characteristics (low ODM values and low OD increase) no mM values were calculated for the SFN2 data.
Statistical Analysis of Metabolic Proles

Principal Component Analysis. PCA was performed on the four subsets of substrates corresponding to the main categories of organic compounds (Table 1); data were

collected from the ODM and mM data sets generated by the GN2 plates. The rst four PCs accounted for about 50% of the total variance in our data. The rst two PCs for the different categories of organic compounds on the ODM data set are shown in Fig. 1. All the substrates received negative weights on PC1, with few exceptions, and only some substrates received weak positive weights on PC2. The isolates are scattered in the four quadrants (numbered counterclockwise starting from the upper right quadrant), B. pyrrocinia in particular. Whereas the amino acid and carboxylic acid data produce an evident overlap of B. cenocepacia and B. ambifaria isolates, carbohydrates scatter B. cenocepacia mainly in the second quadrant and B. ambifaria mainly in the fourth. Miscellaneous data produce a different scattering, with B. ambifaria distributed in the rst, second, and fourth quadrants, and B. cenocepacia mainly in the third.

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PCA of the mM data sets produced the results shown in Fig. 2. The substrates received mainly negative weights on PC1 except for the amino acids, which were all positive. Amino acids, carbohydrates, and carboxylic acids showed a low discriminant power, with the isolates randomly distributed in the four quadrants around the origin and very close to the axes, with few exceptions. Miscellaneous substrates showed a better discriminant power, scattering B. ambifaria mainly in the rst and fourth quadrants and B. cenocepacia in the rst and second. The substrates with the highest discriminant power (positive and negative loadings) in ODM and mM subsets on the rst four PCs (which represent about 50% of total

variance explained by those principal components) were pooled together and are listed in Table 2 (49 and 45 substrates for ODM and mM data, respectively). Discriminant substrates were those related to the principal components along which isolates were segregated (i.e., those responsible for the segregation along each principal component). The carbon sources shown in Table 2 were widely distributed across categories of metabolites, including 13 of the 26 starting carbohydrates for both ODM and mM data sets; 13 of the 24 carboxylic acids for the ODM data set and 11 for the mM data set; 10 of the 21 amino acids for the ODM data set and 11 for the mM data set; 13 of the 17 miscellaneous compounds for the ODM data set and 10 for the mM data set.

Figure 1. PCA of ODM values on amino acids (a), carbohydrates (b), carboxylic acids (c), and miscellaneous substrates (d). B. ambifaria

( ), B. cenocepacia (q), B. pyrrocinia ( ).

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Figure 2. PCA of mM values on amino acids (a), carbohydrates (b), carboxylic acids (c), and miscellaneous substrates (d). B. ambifaria ( ), B. cenocepacia (q), B. pyrrocinia ( ).

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Cluster Analysis. The substrates selected by PCA exceeded the number of cases; therefore, two subsets of randomly chosen substrates (Table 2) were used to perform the cluster analysis. Clusters were dened on the basis of 30% dissimilarity. Using ODM data, cluster analysis of both substrate subsets (Fig. 3a, b) clearly separated the isolates into three or four different clusters. In both cases, all B. ambifaria strains, except strain A33, were grouped in one cluster. B. cenocepacia strains were grouped in two different clusters, which also comprised B. pyrrocinia strains in one case (Fig. 3a), whereas by analyzing the second substrate subset (Fig. 3b), all four B. pyrrocinia strains were grouped separately in a single cluster. Strain A33 clustered together with B. pyrrocinia and/or B. cenocepacia.

Cluster analysis of mM data of the two substrate subsets yielded a more detailed clustering. In one case, B. cenocepacia isolates were split into ve clusters that included the strain A33 of B. ambifaria, B. pyrrocinia isolates were split into two clusters, and B. ambifaria into three clusters (Fig. 3c). In another case, the clustering was similar, but for one including all the B. ambifaria plus two isolates of B. cenocepacia (Fig. 3d). To further evaluate the ability of the different categories of substrates to differentiate the three Bcc species, cluster analyses were separately performed on PCA-selected carbohydrates, carboxylic acids, amino acids, and miscellaneous substrates (Table 2). When the cluster analysis was performed using the ODM values reached by isolates on carbohydrates (Fig. 4b), the isolates were split into three clusters: the rst cluster

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Figure 3. Cluster analysis of ODM (a, b) and mM (c, d) data of substrates included in subsets 1 (a, c) and 2 (b, d). Bacterial isolates are designated by letters A (B. ambifaria), C (B. cenocepacia), and P (B. pyrrocinia) followed by numbers. Dashed lines represent the cutoff at 30% dissimilarity.

with all the B. ambifaria (except for A33), the second including the B. cenocepacia, and the third with the B. pyrrocinia. Cluster analyses performed on carboxylic acids (Fig. 4c) yielded a more detailed clustering, with two clusters for B. cenocepacia, one for B. ambifaria, one for B. pyrrocinia, and two with a mixture of B. ambifaria and B. cenocepacia. Cluster analyses performed on amino acids and miscellaneous (Fig. 4a, d) substrates produced seven and six clusters, respectively; all clusters turned out to be a mixture of isolates of two or three different species. The cluster analysis performed using the mM dataset on the four substrate categories (data not shown) yielded generally a slightly lower degree of separation among the three Bcc species than in the case of the ODM dataset; moreover, isolates, in particular those belonging to B. cenocepacia, were spread across a

high number of clusters (up to 11). Amino acids failed to group the three species into separate clusters, whereas carbohydrates, carboxylic acids, and miscellaneous substrates showed a better separation power.
Use of Root Exudate-Related Substrates. The use of Biolog substrates that commonly occur as components of maize root exudates (i.e., D-fructose, D-glucose, L-fucose, sucrose, citric acid, D,L-lactic acid, succinic acid, L-alanine, L-glutamic acid, and L-serine) was investigated by means of ANOVA and cluster analysis; both maximum and mM data were analyzed. No signicant differences in ODM values for the different substrates were observed among the three species (data not shown); in contrast, mM data were generally higher for B. cenocepacia and B. pyrrocinia than for B. ambifaria (Table 3). Cluster analysis

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Figure 4. Cluster analysis after PCA of ODM

data. (a) Amino acids, (b) carbohydrates, (c) carboxylic acids, (d) miscellaneous. Bacterial isolates are designated by letters A (B. ambifaria), C (B. cenocepacia), and P (B. pyrrocinia) followed by numbers. Dashed lines represent the cutoff at 30% dissimilarity.

of ODM data failed to separate the three species, as all major clusters included a mixture of isolates belonging to the different species, whereas by analyzing the mM data, a partial separation of only B. cenocepacia and B. ambifaria isolates was obtained (data not shown).

Discussion

We investigated in great detail the potential of the Biolog system as a tool for assessment of the metabolic diversity of Bcc species by analyzing 26 rhizosphere isolates belonging to the species most commonly found in the maize rhizosphere, i.e., B. cenocepacia, B. ambifaria, and B. pyrrocinia.

All 26 Bcc isolates were analyzed by means of SFN2 and GN2 microplates, which are commonly used for metabolic proling of pure and mixed bacterial cultures [14, 15, 23], to evaluate which microplate is best suited for metabolic proling of Bcc species. In terms of carbon sources used, the metabolic proles generated by the SFN2 and GN2 plates were identical. However, the narrower range of OD values and signicantly longer reaction time of the SFN2 plates made these plates less suitable for differentiation of isolates in terms of ODM and for accurate calculation of mM; therefore, GN2 plates were considered more suitable to perform statistical analysis on ODM and mM data and thus provide a more in-depth analysis of metabolic proles of the different Bcc isolates.

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Table 3. Oxidation rates ( mM) of B. ambifaria, B. cenocepacia, and B. pyrrocinia isolates on the ten substrates that occur in maize root

exudates B. ambifaria
D-Fructose D-Glucose

B. cenocepacia 28.3 T 6.5 ab 34.7 T 9.4 b 41.7 T 8.8 b 48.9 T 8.4 b 34.6 T 5.8 a 29.9 T 7.3 a 25.3 T 5.8 a 33.9 T 6.1 a 33.9 T 7.8 b 37.3 T 12.0 b

B. pyrrocinia 33.9 38.8 45.4 42.6 35.3 43.8 36.2 38.0 24.2 27.3 T T T T T T T T T T 5.3 b 4.3 b 6.6 b 8.0 b 10.0 a 5.4 b 10.0 b 7.4 a 3.7 a 5.6 ab

Sucrose Citric acid D,L-Lactic acid Succinic acid L-Alanine L-Glutamic acid L-Serine L-Fucose

25.4 27.0 32.5 31.2 34.8 32.4 21.5 35.7 25.8 25.8

T T T T T T T T T T

4.6 4.7 6.0 8.0 6.5 4.7 4.9 6.1 6.6 7.4

a a a a a ab a a a a
_

mM values (TSD) are expressed as absorbance units (103) h 1. Within a row, values followed by the same letter are not statistically different (P 9 0.05) according to Tukey test.

Dalmastri et al. [9] observed that more than 90% of substrates used in GN2 plates were common to these three species. Qualitative metabolic proles based simply on the use of substrates hardly give valuable information on the ecophysiological differences of the Bcc species. Therefore, we focused our attention primarily on quantitative metabolic proles based on OD and mM, which may give more insight into the metabolic diversity of Bcc species. At the same time we tried to dene a reduced number of substrates able to differentiate Bcc species, which could ultimately represent the basis for tailormade systems intended for metabolic diversity studies of environmental Bcc isolates. By applying PCA to ODM and mM data, we were able to select 45 and 49 carbon sources, respectively, which proved to be highly discriminating by subsequent cluster analysis. Indeed, cluster analysis based on subsets of these substrates (around 25 carbon sources) generally grouped B. cenocepacia and B. ambifaria in separate clusters. In particular, it was observed that B. ambifaria isolates tended to group in just few, usually broader clusters, thus showing considerably less metabolic diversity than B. cenocepacia isolates, which were generally dispersed in a higher number of clusters. Furthermore, B. cenocepacia isolates were often intermingled with B. pyrrocinia, suggesting a higher metabolic similarity to this species than to B. ambifaria. The possibility to study Bcc metabolic diversity by the analysis of just about 25 carbon sources may imply a signicant cost and time reduction and hence the possibility to process a higher number of samples or replicates [17]. The use of two different parameters (ODM and mM) allowed a comparison between two different approaches to the data analysis. The ODM is a measure of the greatest extent of substrate oxidation and is a valid tool for metabolic characterization and differentiation of bacterial species, whereas mM represents the rate of substrate oxidation and, as a kinetic parameter, can be used as an

alternative way to process Biolog data and provide a greater analytical power for ecological studies than the ODM parameter [13, 31]. The analysis of ODM values highlighted marked differences among the metabolic proles of the three species, in particular between B. cenocepacia and B. ambifaria, as shown by the cluster analysis; at the same time, the analysis of the mM parameter, which is more relevant to the functional characterization of single bacterial isolates and/or species in natural habitats [13, 21], yielded a slightly lower differentiation among the three Bcc species and, on the other hand, a markedly higher metabolic diversity within a single species, in particular B. cenocepacia, in comparison with ODM analysis. GN2 microplates contain some substrates that commonly occur in maize root exudates, which represent a primary source of carbon (and possibly nitrogen) and energy and are likely to favor fast-growing microbes in the rhizosphere [2, 20, 24]. Thus, the ability to metabolize root exudates at high rates has been often related to the colonization of roots by bacteria [2]. Dalmastri et al. (unpublished data) observed that among the Bcc species commonly found in the rhizosphere of maize, B. cenocepacia and B. pyrrocinia have a signicantly higher root-colonizing ability than B. ambifaria. Accordingly, in this study we showed that, in general, B. cenocepacia and B. pyrrocinia have higher oxidation rates of root exudaterelated compounds than B. ambifaria. These data suggest that, among Bcc species, a high rhizosphere competence may be associated with the capacity to metabolize root exudates at high rates. In conclusion, our study demonstrates that the Biolog system may be a powerful tool for metabolic proling of Bcc species. B. cenocepacia and B. pyrrocinia proved to be metabolically more diverse than B. ambifaria. Furthermore, a reduced list of less than 50 substrates that can represent the basis for future Biolog microplates targeted toward Bcc species is provided.

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