Editor: Kornelia Smalla
Phylogeny of the 1-aminocyclopropane-1-carboxylic acid deaminase-encoding gene acdS in phytobeneficial and pathogenic Proteobacteria and relation with strain biogeography
Article first published online: 31 JAN 2006
FEMS Microbiology Ecology
Volume 56, Issue 3, pages 455–470, June 2006
How to Cite
Blaha, D., Prigent-Combaret, C., Mirza, M. and Moënne-Loccoz, Y. (2006), Phylogeny of the 1-aminocyclopropane-1-carboxylic acid deaminase-encoding gene acdS in phytobeneficial and pathogenic Proteobacteria and relation with strain biogeography. FEMS Microbiology Ecology, 56: 455–470. doi: 10.1111/j.1574-6941.2006.00082.x
- Issue published online: 13 FEB 2006
- Article first published online: 31 JAN 2006
- Received 27 July 2005; revised 27 October 2005; accepted 30 October 2005.First published online 21 January 2006.
- ACC deaminase;
Deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is a key plant-beneficial trait found in plant growth-promoting rhizobacteria (PGPR) and phytosymbiotic bacteria, but the diversity of the corresponding gene (acdS) is poorly documented. Here, acdS sequences were obtained by screening putative ACC deaminase sequences listed in databases, based on phylogenetic properties and key residues. In addition, acdS was sought in 71 proteobacterial strains by PCR amplification and/or hybridization using colony dot blots. The presence of acdS was confirmed in established AcdS+ bacteria and evidenced noticeably in Azospirillum (previously reported as AcdS−), in 10 species of Burkholderia and six Burkholderia cepacia genomovars (which included PGPR, phytopathogens and opportunistic human pathogens), and in five Agrobacterium genomovars. The occurrence of acdS in true and opportunistic pathogens raises new questions concerning their ecology in plant-associated habitats. Many (but not all) acdS+ bacteria displayed ACC deaminase activity in vitro, including two Burkholderia clinical isolates. Phylogenetic analysis of partial acdS and deduced AcdS sequences evidenced three main phylogenetic clusters, each gathering pathogens and plant-beneficial strains of contrasting geographic and habitat origins. The acdS phylogenetic tree was only partly congruent with the rrs tree. Two clusters gathered both Betaprotobacteria and Gammaproteobacteria, suggesting extensive horizontal transfers of acdS, noticeably between plant-associated Proteobacteria.
In the associative symbiosis (cooperation) between plant growth-promoting rhizobacteria (PGPR) and the plant, the PGPR benefit the latter through a variety of indirect (e.g. competition or antagonism towards phytopathogens) and/or direct (by enhancing nutrient availability for roots, inducing disease resistance in the plant, and/or modifying plant hormonal balance) effects (Haas & Keel, 2003; Moënne-Loccoz & Défago, 2004; Bally & Elmerich, 2005). It is well documented that some of the direct phytobeneficial effects displayed by PGPR, for example nitrogen fixation, can also be implemented by bacteria such as rhizobia, in a context of obligate/mutualistic symbiosis with the plant (Sullivan et al., 2002; Ma et al., 2003a). Recently, the diminution of ethylene concentration via microbial deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) (Glick et al., 1998; Holguin & Glick, 2003) was identified as another phytobeneficial mode of action exhibited by both PGPR and nitrogen-fixing rhizobia (Ma et al., 2003a, b).
The ACC deaminase [EC 18.104.22.168] is a pyridoxal 5′-phosphate (PLP)-dependent enzyme (Yao et al., 2000) that catalyses the conversion of ACC to ammonia and α-ketobutyrate. In PGPR closely associated with roots, this enzyme is thought to lead to a diminution of the amount of plant ACC available for conversion into ethylene (Glick et al., 1998). Because less ethylene will be synthesized by the plant, the inhibitory effects of this phytohormone on root elongation will be reduced. In parallel, the PGPR may use reaction products of ACC hydrolysis as nutrients for growth. Experimental data indicate that inoculation of plants with ACC deaminase-positive PGPR did reduce ethylene levels in the root zone while resulting in enhanced root proliferation (Penrose & Glick, 2003). Inactivation of acdS (encoding ACC deaminase) in the PGPR Pseudomonas putida GR12-2 and Enterobacter cloacae UW4 abolished the ability of the bacteria to promote the elongation of canola roots (Glick et al., 1994; Li et al., 2000). In the bacterial symbiont Rhizobium leguminosarum, inactivation of acdS reduced the extent of nodulation (Ma et al., 2003a). ACC deaminase activity is also documented in the yeast Hansenula saturnus (Minami et al., 1998) and the fungus Penicillium citrinum (Jia et al., 2000), but its ecological significance in eukaryotic microorganisms is unknown.
As ACC deaminase activity in bacteria can benefit the plant, it would be of interest to improve our knowledge of the range of bacteria displaying this activity. On one hand, ACC deaminase activity (and sometimes acdS) has mostly been documented in PGPR belonging to the genera Pseudomonas, Enterobacter and Kluyvera, and in the symbiont R. leguminosarum (Glick et al., 1995; Burd et al., 1998; Wang et al., 2001; Penrose & Glick, 2003; Ma et al., 2003b). ACC deamination ability has been identified by phenotypic means so far (Penrose & Glick, 2003). PCR amplification of acdS has been attempted before (Babalola et al., 2003) but without subsequent verification by sequencing, and there is a need to develop genetic tools to identify acdS in bacteria. Therefore, a genetic approach based on PCR and Southern hybridization was developed in this work to search for acdS in a range of Proteobacteria. On the other hand, putative ACC deaminase sequences derived from the annotation of whole-genome sequences are also available, but these sequences have not been validated by phylogenetic means. Therefore, phylogenetic methods were used to study ACC deaminase-related sequences, with the objectives of confirming their identity (based also on key residue analysis) and gaining insight into the evolutive history of acdS.
The experimental work was carried out using a collection of Proteobacteria encompassing subdivisions Alpha, Beta and Gamma, with an emphasis on the genera Azospirillum, Agrobacterium, Burkholderia and Pseudomonas. Azospirillum and Pseudomonas were selected as key PGPR genera, in which ACC deaminase activity is thought to be respectively absent (Holguin & Glick, 2001, 2003) and prevalent (Wang et al., 2001). Therefore, the current work included an investigation of AcdS activity, especially in the case of Azospirillum. With the recent recognition that certain emblematic virulence properties, for example the type-three protein secretion system (Ochman & Moran, 2001; Mazurier et al., 2004; Rezzonico et al., 2004), are widespread also in plant-beneficial bacteria, it is conceivable that key phytobeneficial traits may in fact be present in pathogens as well. To assess this hypothesis in the case of acdS, Agrobacterium and Burkholderia were included in the study because many strains from these genera have been extensively characterized as true or opportunistic pathogens affecting plants or animals.
Materials and methods
Bacterial strains and culture conditions
The bacterial strains used in this study are listed in Table 1. The majority of strains were taken from culture collections, and the Azospirillum strains isolated in this work were obtained from the roots of field-grown plants in Pakistan, using N-free NFB medium (Nelson & Knowles, 1978). Azospirillum was grown first overnight with shaking (200 r.p.m.) in Tryptone Yeast extract (TY) (Beringer, 1974) medium, and then overnight on TY agar. Rhizobium and Agrobacterium were grown for 48 h on TY agar supplemented with 6 mM CaCl2 and on MG agar (Keane et al., 1970), respectively. Burkholderia was grown for 72 h on Pseudomonas capacia azelaic acid trytamine (PCAT) medium (Burbage & Sasser, 1982). Pseudomonas and Kluyvera were cultured overnight on King's medium B (King et al., 1954), and Enterobacter strains were cultured in Tryptic Soybean Broth (TSB; Serlabo, Bonneuil sur Marne, France). All strains were grown at 28°C.
|Species||Geographic origin||Source||Strains||Reference||acdS hybridization*||acdS PCR analysis||AcdS activity|
|PCR product†||acdS sequence‡|
|Azospirillum lipoferum||Bangladesh||Rice||MRB16||Rhaman (1987)||0||−||ND|
|France||Corn||CRT1||Fages & Mulard (1988)||+||+||–||+|
|France||Rice||4B||Bally et al. (1983)||++||+||+§||+|
|Japan||Rice||B52||Elbeltagy et al. (2001)||0||+||−||ND|
|Japan||Rice||B510||Elbeltagy et al. (2001)||++||ND||ND|
|Japan||Rice||B518||Elbeltagy et al. (2001)||++||+||−||ND|
|Pakistan||Cotton||CN1, N4||This work||+||+||+||+|
|Vietnam||Rice||TVV3||Tran Van et al. (1997)||++||+||+||+|
|Azospirillum brasilense||Brazil||Digitaria||Sp7||Tarrand et al. (1978)||++||+||−||−|
|Brazil||Wheat||Sp245||Penot et al. (1992)||++||+||−||ND|
|France||Sorghum||L4||Kabir et al. (1996)||++||+||−||−|
|Japan||Rice||B506||Elbeltagy et al. (2001)||+||ND||ND|
|Pakistan||Wheat||Wb1, Wb3, WN1, WS1||This work||0||–||–|
|USA||Cynodon dactilon||Cd||Eskew et al. (1977)||++||−||−|
|Azospirillum irakense||Iraq||Rice||KBC1||Khammas et al. (1989)||0||+||−||ND|
|Azospirillum sp.||Cuba||Rice soil||R5(6)||Lavire (1998)||++||+||−||ND|
|Cuba||Rice soil||R5(15)||Lavire (1998)||0||+||−||ND|
|Mali||Soil||NC9||Kabir et al. (1996)||0||+||−||ND|
|Rhizobium leguminosarum||Unknown||Pea||128C53K||Ma et al. (2003a)||0||+||+¶||+¶|
|Genomic group 1||USA||Crown gall||S56||Mougel et al. (2002)||++||−||ND|
|Genomic group 2||France||Human urine||CIP 43-76||Kiredjian (1979)||+||+||ND||ND|
|Genomic group 3||France||Human cephalo-rachidian liquid||CIP 111-78||Kiredjian (1979)||0||−||ND|
|Genomic group 4 (Agrobacterium tumefaciens)||USA||Apple seedling||ATCC 23308||Yanagi & Yamasato (1993)||++||−||ND|
|Genomic group 6||Israel||Dahlia||Zutra F/1||Mougel et al. (2002)||++||−||ND|
|Genomic group 7||UK||Flacourtia ramontchi||NCPPB 1641||Mougel et al. (2002)||+||−||ND|
|Genomic group 8||USA||Cherry tree gall||C58||Mougel et al. (2002)||++||+||−||ND|
|Genomic group 9||South Australia||Potting soil||LMG26||Mougel et al. (2002)||+||−||ND|
|Genomic group 10 (A. rhizogenes)||USA||Apple||CFBP 2408||Mougel et al. (2002)||0||−||ND|
|Genomic group 11 (A. rubi)||USA||Rubus ursinus||LMG17935||Mougel et al. (2002)||0||−||ND|
|Achromobacter xylosoxidans||Russia||Pea/leaf mustard stand (PGPR)∥||Cm4||Belimov et al. (2001)||++||+||+||+¶|
|Burkholderia caledonica||UK||Rhizosphere (PGPR)||LMG19076||Coenye et al. (2001a)||++||+||+||−|
|Burkholderia caribiensis||Martinique||Vertisol||LMG18531||Achouak et al. (1999)||0||−||ND|
|Burkholderia caryophylli||USA||Dianthus caryophyllus (P)||LMG2155||Yabuuchi et al. (1992)||+||−||ND|
|Burkholderia cepacia (I)**||USA||Allium cepa (P)||LMG1222||Yabuuchi et al. (1992)||++||+||+||+|
|Burkholderia cepacia (II)||Belgium||Clinical isolate||LMG13010||Vandamme et al. (1997)||++||−||ND|
|Burkholderia cepacia (III)||UK||Clinical isolate (P)||LMG16656||Vandamme et al. (1997)||+||+||+||−|
|Burkholderia cepacia (IV)||Belgium||Clinical isolate||LMG14294||Vandamme et al. (2000)||++||+||+||−|
|Burkholderia cepacia (V)||Vietnam||Rice (PGPR)||LMG10929||Gillis et al. (1995)||++||−||ND|
|Burkholderia cepacia (VI)||USA||Clinical isolate||LMG18941||Coenye et al. (2001b)||++||+||+||+|
|Burkholderia gladioli||USA||Gladiolus sp. (P)||LMG2216||Yabuuchi et al. (1992)||++||+||F||ND|
|Burkholderia glathei||Germany||Soil||LMG14190||Viallard et al. (1998)||0||−||ND|
|Burkholderia glumae||Japan||Rice (P)||LMG2196||Urakami et al. (1994)||0||+||−||ND|
|Burkholderia graminis||France||Corn||LMG18924||Viallard et al. (1998)||++||+||+||−|
|Burkholderia kururiensis||Japan||Aquifer||LMG19447||Zhang et al. (2000)||+||−||ND|
|Burkholderia phenazinium||Unknown||Soil||LMG2247||Viallard et al. (1998)||0||+||+||+|
|Burkholderia plantarii||Japan||Rice (P)||LMG9035||Urakami et al. (1994)||ND||−||ND|
|Burkholderia pyrrocinia||Unknown||Soil||LMG14191||Viallard et al. (1998)||++||+||F||ND|
|Burkholderia tropicalis||Mexico||Corn||BM16||Estrada et al. (2002)||+||+||−||ND|
|Mexico||Corn (PGPR)||BM273||Estrada et al. (2002)||++||+||+||+|
|Ralstonia solanacearum||Guyane||Tomato||GMI1000||Boucher et al. (1985)||ND||+||+||+|
|Pseudomonas fluorescens||Bhutan||Tomato||P97.26||Wang et al. (2001)||++||−||+¶|
|Czech Republic||Cucumber||K94.31||Wang et al. (2001)||++||+||+||+¶|
|Czech Republic||Wheat||P97.30||Wang et al. (2001)||++||+||+||+¶|
|Ireland||Sugar beet||F113||Fenton et al. (1992)||++||+||+||+¶|
|Italy||Tomato||PILH1||Keel et al. (1996)||0||+||F||+¶|
|Italy||Wheat||PITR2||Keel et al. (1996)||++||+||+||+¶|
|Switzerland||Cucumber||P97.38||Wang et al. (2001)||ND||+||F||+¶|
|Switzerland||Cucumber||CM1'A2||Fuchs & Défago (1991)||++||+||+||+¶|
|Switzerland||Tobacco||CHA0||Stutz et al. (1986)||0||−||−¶|
|Switzerland||Tomato||TM1A3||Fuchs & Défago (1991)||++||+||F||+¶|
|Enterobacter cloacae||Canada||Soil (PGPR)||UW4||Glick et al. (1995)||+||+||+¶||+¶|
|USA||Soil (PGPR)||CAL2||Glick et al. (1995)||++||+||+¶||+¶|
|Kluyvera ascorbata||Russia||Soil (PGPR)||SUD 165||Burd et al. (1998)||0||ND||+¶|
PCR amplification and sequencing
PCR amplifications were performed in 100 μL volumes containing 1 × PCR buffer, 1.5 mM MgCl2, 0.5 μM of each primer, 10 μM of each dNTP, 0.5 U Taq DNA polymerase (Gibco BRL/Life technologies, Cergy Pontoise, France), and cells from one colony as DNA source. Primers for amplification of acdS were defined according to nucleotide alignment of sequences available in Genbank (Pseudomonas sp. 65G, ACP and 17, E. cloacae UW4 and CAL2), and they are listed in Table 2. PCR cycles were as follow: (i) 95°C for 5 min, (ii) 95°C for 30 s, 50°C for 30 s, 72°C for 30 s (35 cycles), and (iii) 72°C for 7 min. Universal eubacterial primers PA and PH were used to amplify 1.5 kb of the 16S rRNA gene rrs (Bruce et al., 1992). PCR cycles for rrs were as follows: (i) 95°C for 5 min, (ii) 95°C for 30 s, 65°C for 30 s, 72°C for 45 s (35 cycles), and (iii) 72°C for 7 min.
|Target genes and primers||Sequence||acdS nucleotidic position in Pseudomonas fluorescens ACP*||Corresponding AcdS domain||Reference|
|F1936 (forward)||5′ GH GAM GAC TGC AAY WSY GGC 3′||38–44||EDCNSG||This work|
|F1937 (forward)||5′MGV AAG CTC GAA TAY MTB RT 3′||52–59||RKLEYLIP||This work|
|F1938 (reverse)||5′AT CAT VCC VTG CAT BGA YTT 3′||296–302||KSMHGM||This work|
|F1939 (reverse)||5′ GA RGC RTC GAY VCC RAT CAC 3′||218–224||VIGIDA||This work|
|PA||5′ AGA GTT TGA TCC TGG CTC AG 3′||Bruce et al. (1992)|
|PH||5′ AAG GAG GTG ATC CAG CCG CA 3′||Bruce et al. (1992)|
DNA bands were extracted from electrophoretic gels using the QIAquick Gel Extraction Kit (Qiagen, Courtaboeuf, France) and cloned into pGEM-T vector (Promega, Charbonnière-les-Bains, France) according to supplier's instructions. The presence of an insert (acdS and rrs amplicons) was checked by restriction and PCR using vector-annealing primers SP6 and T7. DNA sequencing (Genome Express, Grenoble, France) was performed on both strands on an ABI377 sequencer FS (Perkin-Elmer, Courtaboeuf, France) using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase.
Analysis of acdS and deduced AcdS sequences
BLASTN and BLASTP searches (Devereux et al., 1984; Altschul et al., 1990) were performed to detect similar DNA and deduced protein sequences (obtained using OrfFinder; NCBI web site), respectively. DNA sequences and deduced protein sequences were aligned using the multiple alignment software CLUSTAL X and CLUSTAL W version 1.74, respectively (Thompson et al., 1994). AcdS comparisons were computed in SeqVU (J. Gardner, Garvan Institute of Medical Research, Sydney, Australia). The deduced AcdS sequences from a selection of three strains (E. cloacae UW4, Pseudomonas sp. ACP and R. leguminosarum 128C53 K) were analysed by comparison with the well-studied AcdS from H. saturnus (Yao et al., 2000).
Phylogenetic analyses of acdS and deduced AcdS sequences were conducted using MEGA version 2.1 (Kumar et al., 2001). Distances between sequence pairs, the deduced phylogenetic tree, and bootstrap values were all computed using the global gap removal option. For acdS sequences, the distance-based tree-making methods used included the neighbour-joining method (NJ) (Saitou & Nei, 1987), using both the Kimura 2 parameter (K2P) and Jukes-Cantor (JC; with gamma parameter), and the maximum parsimony method (MP) (Berry & Gascuel, 1996). For deduced AcdS sequences, trees were obtained with the NJ method, using both Poisson correction and observed divergence (Galtier et al., 1996), and with the MP method. Nodal robustness of the trees was assessed using 1000 bootstrap replicates.
Analysis of acdS by hybridization
Bacterial colonies were transferred onto Gene Screen Plus nylon membranes, hybridized and washed (twice with saline-sodium citrate (SSC) 2 × for 5 min, and twice with SSC 2 × containing sodium dodecyl sulphate (SDS) 10% for 30 min) at 65°C according to the manufacturer's recommendations (NEN life science products, Le Blanc Mesnil, France). Autoradiography was performed according to Amersham-Pharmacia protocols (Orsay, France). To obtain probes, primers F1936 and F1938 were used to amplify acdS in Pseudomonas fluorescens PITR2, Burkholderia caledonica LMG19076 and Azospirillum lipoferum CN1. PCR fragments were radioactively labelled with 32P-dCTP according to the Random Primed DNA Labelling Kit (Boehringer Mannheim, Mannheim, Germany) and used as probes in DNA blot analyses. Hybridization results were confirmed when the procedure was repeated.
ACC deaminase activity in selected Proteobacteria
ACC deaminase activity in 24 representative proteobacterial strains was assessed by quantifying the reaction product α-ketobutyrate, using the procedure described by Penrose and Glick (2003). Briefly, a tube containing 7.5 mL of TY medium was inoculated with A. lipoferum (strain 4B, RSWT1, CRT1, TVV3, N4 or CN1), A. brasilense (strain L4, Sp7, Cd, WN1, WB1 or WB3), Azospirillum irakense (strain RSB1), B. caledonica (strain LMG19076), Burkholderia tropicalis (strain BM273), Burkholderia cepacia genomovar I (strain LMG1222), III (strain LMG16656), IV (strain LMG14294) or VI (strain LMG18941), Burkholderia graminis (strain LMG18924), Burkholderia phenazinium (strain LMG2247), Ralstonia solanacearum (strain GMI1000) or P. fluorescens (strains PITR2 and CHA0 as AcdS-positive and AcdS-negative controls, respectively). The tubes were incubated overnight at 28°C with shaking. Bacterial biomass was harvested by centrifugation (8000 g) for 10 min at 4°C. The cells were washed twice in 5 mL of DF salt minimal medium (Penrose & Glick, 2003) and resuspended in 7.5 mL of DF salt minimal medium supplemented with 3 mM ACC to induce acdS transcription. The bacterial cells were incubated for 24 h with shaking at 28°C and harvested by centrifugation, as described above. The pellets were washed twice in 5 mL 0.1 M Tris HCl, pH 7.6. The cells were lysed using 30 μL of toluene, and the procedure of Penrose & Glick (2003) was completed by vigourous shaking of the tubes for 1 min in a MM200 Retsch mixer mill (Bioblock, Vaulx Milieu, France) to ensure complete cell lysis. The amount of α-ketobutyrate produced was determined with a colorimetric method (absorbance at 540 nm), by comparison with a standard curve, according to Penrose & Glick (2003). Total protein content in the assay was measured using the BCA Protein Assay Kit (Pierce, Rockford, IL). ACC deaminase activity was assessed at least twice for each strain. Data were log-transformed before means and standard deviations were computed, and analysis of variance was performed followed by Fisher's LSD tests (P<0.05) (S-PLUS 6 for Windows; Insightful Corporation, Seattle, WA).
acdS and AcdS sequence data already available and included in this work were obtained from the NCBI database for E. cloacae UW4 (GenBank accession no. AF047710) and CAL2 (AF047840), Enterobacter aerogenes Cal3 (AY604544.1), Caulobacter crescentus CB15 (AE005876), Salmonella typhimurium LT2 (NC_003197), Salmonella enterica CT18 (NC_003198), Erwinia carotovora SCRI1043 (NC_004547), Yersinia pestis KIM (NP_669768) and CO92 (CAC90662), Yersinia pseudotuberculosis IP 32953 (BX936398), Bordetella parapertussis 12822 (BX640435), Desulfovibrio vulgaris Hildenborough (NC_002937), Pseudomonas sp. 6G5 (M80882), 17 (U37103) and ACP (M73488), Pseudomonas syringae DC3000 (AE016853), GR12-2 (AY604545.1) and B728a (CP000075), Pseudomonas marginalis (AY604542.1), Pseudomonas brassicacearum Am3 (AY604528.1), P. putida Bm3 (AY604533.1), P. fluorescens PfO-1(NZ_AAAT03000010), Ralstonia eutropha JMP134 (NZ_AADY01000003), R. solanacearum GMI1000 (AL646080), Serratia proteamaculans SUD (AY604543.1), Achromobacter sp. CM1 (AY604541.1), Achromobacter xylosoxidans A551 (AY604539.1) and BM1 (AY604540.1), Variovorax paradoxus 5C2 (AY604531.1), 2C1 (AY604530.1) and 3C3 (AY604532.1), Burkholderia xenovorans LB400 (NZ_AAAJ02000016), Burkholderia pseudomallei K96243 (NC_006350), Burkholderia mallei ATCC 23344 (CP000011), B. cepacia R18194 (NZ_AAEI01000001) and R1808 (NZ_AAEH01000001), Rhizobium sullae HCNT-1 (AY604534.1), R. leguminosarum 128C53 K (AF421376 and AY172673) and 99A1 (AY604535.1), Bradyrhizobium japonicum USDA110 (NC_004463), and Agrobacterium tumefaciens d3 (AF315580). Nucleotide and deduced protein sequence data obtained in this work are available at the NCBI database for A. lipoferum 4B (GenBank accession no. DQ125242), CN1 (DQ125253), N4 (DQ125255), and TVV3 (DQ125257), B. cepacia LMG16656 (DQ125243), LMG1222 (DQ125251), LMG14294 (DQ125259), and LMG18941 (DQ125258), B. caledonica LMG19076 (DQ125247), B. graminis LMG18924 (DQ125249), B. phenazinium LMG2247 (DQ125252), B. tropicalis BM273 (DQ125254), P. fluorescens PITR2 (DQ125244), TM1A3 (DQ125245), CM1′A2 (DQ125246), P97.30 (DQ125248), K94.31 (DQ125250), and F113 (DQ125256), and A. xylosoxidans Cm4 (DQ133504).
Analysis of acdS PCR amplicons in Proteobacteria
Two forward and two reverse degenerative primers for acdS amplification were designed based on three Pseudomonas and two Enterobacter cloacae sequences available in Genbank (Table 2). The four primer combinations were tested on nine AcdS-positive pseudomonads (strains CM1′A2, TM1A3, P97.38, F113, K94.31, P97.30, P97.26, PITR2, PILH1). One PCR band, which was of the expected size, was obtained in all strains, except that P. fluorescens P97.26 did not yield any PCR amplicon. Primers F1936 and F1938 were selected for further use because (i) they were the only ones that worked with Azospirillum (a key taxon targetted in this study), and (ii) they yielded the longest amplicon (about 800 bp).
Using primers F1936 and F1938, a PCR product of the expected size (along with other PCR bands in certain strains) was obtained with 42 of the 68 Proteobacteria tested, including three strains in which acdS had already been sequenced (Table 1). Sequencing was performed on the 39 other 800-bp PCR products. Sequencing failed in five cases (despite several attempts). BLASTN analysis confirmed the presence of acdS for 19 bacteria, whereas no significant hit with GenBank sequences was obtained for the other 15.
The acdS gene was found in all three proteobacterial subdivisions tested (Alpha, Beta and Gamma), including taxa for which (to our knowledge) acdS or ACC deaminase activity has not previously been published, for example Azospirillum. Indeed, we obtained an acdS sequence for A. lipoferum (in three of ten strains). In the genus Burkholderia, an acdS sequence was obtained for eight strains (including three clinical isolates), which encompassed the species caledonica, graminis, phenazinium, tropicalis and genomovars I, III, IV and VI within the cepacia complex (Table 1).
Phylogenetic validation of partial acdS sequences obtained from databases
In addition to sequences obtained in this work, all putative ACC deaminase sequences available in databases were considered. Among the acdS-related sequences available in GenBank, BLAST alignment and phylogenetic analysis of the deduced proteic sequence for the Alphaproteobacteria C. crescentus CB15 (AE005876), Betaproteobacteria Achromobacter sp. CM1 (AY604541.1), A. xylosoxidans BM1 (AY604540.1), B. parapertussis 12822 (BX640435), R. eutropha JMP134 (NZ_AADY01000003), B. pseudomallei K96243 (NC_006350), Deltaproteobacteria D. vulgaris Hildenborough (NC_002937) and the Gammaproteobacteria S. typhimurium LT2 (NC_003197), S. enterica CT18 (NC_003198), E. carotovora SCRI1043 (NC_004547), Y. pestis KIM (NP_669768) and CO92 (CAC90662), Y. pseudotuberculosis IP 32953 (BX936398), P. syringae GR12-2 (AY604545.1) and B728a (CP000075), P. fluorescens PfO-1(NZ_AAAT03000010), P. marginalis (AY604542.1), E. aerogenes Cal3 (AY604544.1) and S. proteamaculans SUD (AY604543.1) showed that these sequences compared poorly with established AcdS sequences, whereas significant homology was found with sequences of d-cystein desulfhydrases (which, like AcdS, are PLP-dependent enzymes) (Yao et al., 2000; Soutourina et al., 2001). This means that these sequences cannot be considered as acdS sequences.
In contrast, relevant acdS sequences in terms of phylogenic properties were recovered by screening databases in the case of R. sullae HCNT-1 (Hedysarum coronarium isolate from Italy), R. leguminosarum 99A1, B. japonicum USDA110 (soybean nodule isolate from the USA), A. tumefaciens d3 (i.e. genomic group 4; soil isolate from the USA), A. xylosoxidans A551, B. xenovorans LB400 (soil isolate from the USA), B. cepacia R18194 (genomovar unknown) and R1808 (genomovar V; soil isolate from the USA), B. mallei ATCC 23344 (human isolate from Burma), V. paradoxus 2C1, 3C3 and 5C2, P. brassicacearum Am3 (pea or Indian mustard isolate from Russia), Pseudomonas chlororaphis 6G5, P. putida Bm3 (pea or Indian mustard isolate from Russia), P. syringae DC3000 (tomato isolate from the UK), and Pseudomonas sp. strains 17 (soil isolate from South Africa) and ACP.
Three proteobacterial partial sequences of acdS found in GenBank (P. brassicacearum Am3, P. putida Bm3 and V. paradoxus 3C3) were markedly different from all the others based on sequence alignment and phylogenetic analyses (data not shown), for example at distances of at least 1.73 base substitutions per site with the NJ method using K2P (data not shown). Therefore, they were removed from the dataset for acdS phylogenetic analysis (but were included in AcdS studies).
Phylogeny of acdS sequences
Significant polymorphism was found when comparing the acdS sequences obtained in this work or recovered from GenBank, as 227 of 415 sites were informative. Phylogenetic analyses of partial acdS sequences using the NJ method with K2P (Fig. 1a), the NJ method with Poisson correction or JC, or the MP method gave the same overall topology (data not shown). Three main acdS groups were arbitrarily defined for clusters that were highly robust and that contained a significant number of sequences (Fig. 1a). Variovorax paradoxus and R. solanacearum were not included within group I because their position varied somewhat according to the method, and R. sullae and B. japonicum were not included within group III because the distance was too long. The three acdS groups gathered bacteria displaying acdS pairwise distances up to 0.253 (group I), 0.257 (group II) and 0.253 (group III) base substitutions per site within each group.
Groups I and II both gathered sequences originating from the Beta- and Gammaproteobacterial subdivisions studied, whereas the seven sequences in group III corresponded to Alphaproteobacteria (three Rhizobiaceae and four A. lipoferum). The two E. cloacae strains clustered in acdS group I, whereas Burkholderia and Pseudomonas species clustered in groups I and II. Therefore, the phylogenetic relationship derived from acdS analysis was only partly congruent with the one based on 16S rRNA gene sequence analysis (data not shown). In addition, the GC content (%) of the acdS fragment for acdS group I (58.7±1.4; n=11) was statistically different (Kruskal–Wallis and Wilcoxon tests; P<0.005) from the one for acdS group II (65.3±1.8; n=13) or III (64.1±3.0; n=7).
Geographic distribution and habitat origin of acdS+Proteobacteria in relation to acdS groups
Each of the three acdS groups comprised strains originating (i) from several countries in different continents, and (ii) from different microbial habitats (Table 3). For instance, acdS group I included isolates from Europe (Ireland, UK, Switzerland, Italy, the Czech Republic), North America (Canada, USA) and South Africa, recovered from monocots, dicots or soil. Moreover, several geographic locations and host plants studied yielded isolates from more than one acdS group. However, all acdS+ clinical isolates analysed belonged to acdS group II.
|Irl and UK (n=5)†||W Europe (n=15)||C Europe (n=3)||Russia (n=3)||SE Asia (n=5)||Pakistan (n=9)||Africa (n=2)||N America (n=17)||C and S America (n=8)||Unknown (n=10)|
|B||Host plant‡||Bulk soil (n=15)||Clinical isolates (n=6)|
|Onion (n=1)||Corn (n=5)||Rice (n=12)||Wheat (n=7)||Cotton (n=2)||Cucumber (n=3)||Pea (n=2)||Sugarbeet (n=1)||Tomato (n=5)||Unknown (n=10)|
Phylogenetic analysis of deduced AcdS sequences
The phylogenetic tree derived from NJ analysis (using the number of differences) of deduced AcdS sequences is shown in Fig. 1b (61 of 137 sites were informative). Only minor topological differences were found when comparing with trees obtained using the NJ (with Poisson correction) or MP methods (data not shown). acdS and AcdS phylogenies were highly congruent when considering the Proteobacteria used in Fig. 1a, regardless of the method, and it appeared that the acdS-based groups I, II and III were also relevant with AcdS sequences. The three strains whose acdS sequence was very different from all the others (and therefore were not included in the acdS trees) clustered with acdS group I strains (i.e. for P. brassicacearum Am3), with the two V. paradoxus (i.e. for V. paradoxus 3C3), or with the two A. xylosoxidans (i.e. for P. putida Bm3) in the AcdS trees (see e.g. Fig. 1b).
Analysis of AcdS domains and residues based on deduced proteic sequences
Alignment of complete AcdS sequences (338 or 339 amino acids) for three strains encompassing acdS phylogenetic groups I (E. cloacae UW4), II (Pseudomonas sp. ACP) and III (R. leguminosarum 128C53K) showed a high level of conservation (Fig. 2). Identity levels ranged between 64 and 81%, and similarity levels between 79 and 87%. Structural analysis of the dimeric AcdS protein in H. saturnus (Yao et al., 2000) evidenced a PLP-binding domain (domain I) and an internal, smaller domain (domain II), which were readily identified in the bacterial sequences studied (Fig. 2).
Many residues considered to play a key role in protein conformation and functioning were highly conserved, such as the lysine residue K51. K51 is the PLP-binding site in H. saturnus (Yao et al., 2000) and is required for enzymatic activity in P. fluorescens ACP (Murakami et al., 1997), probably because it functions as the base for proton abstraction (Yao et al., 2000). Other residues (numbered as in Pseudomonas sp. ACP) important for conformation and functioning of AcdS in the yeast H. saturnus (Yao et al., 2000) and found in the three sequences included (i) four residue pairs implicated in monomer−monomer contact via hydrogen bonds, i.e. R23–A89, R38–G44, A89–E286 and R112–S332; (ii) four of 11 pairs of van der Waals contacts, i.e. F13–P17, L45–E286, A89–E286 and I121–F335; (iii) four aromatic (W102, Y110, Y268 and Y294) and three hydrophobic (V103, I137, and V293) residues forming two cavities involved in substrate/product transportation; (iv) E295 (specific of AcdS enzymes), which is within hydrogen bonding distance of the N1 nitrogen (pyridinium N) of the cofactor pyridine ring and stabilizes the PLP; (v) five residues (K54, V198, T199, G200 and T202) forming a pocket to which the phosphate group of PLP is tightly fixed; (vi) the tyrosine residue Y294, thought to contribute to hydrogen binding with the substrate (Yao et al., 2000); (vii) the serine residue S78, thought to act as nucleophile in the enzymatic attack (Yao et al., 2000); and (viii) T81, which is hydrogen-bonded to S78. The residue in position 162, which is important for enzymatic activity in Pseudomonas sp. ACP (Honma, 1985), is occupied by cysteine or alanine in PLP-dependent enzymes (e.g. C162 in AcdS from H. saturnus), which was the case for the three bacterial sequences studied. Therefore, it appears that functional traits of AcdS were highly conserved across the three acdS groups.
Analysis of acdS by colony hybridization in Proteobacteria
A total of 69 Proteobacteria were tested by acdS hybridization, performed using three different probes, for a more extensive assessment of the presence of the gene in the collection studied. Different hybridization results were obtained depending on the probe used. Briefly, when considering strains for which an acdS sequence is available, it appears that (i) the probes from P. fluorescens PITR2 and B. caledonica LMG19076 (acdS group I) hybridized with strains from acdS groups I and II; and (ii) the probe from A. lipoferum CN1 (acdS group III) hybridized with strains from acdS groups II and III.
Overall, a positive hybridization signal with at least one of the probes was obtained with 22 of 37 Alphaproteobacteria (i.e. 59%), 15 of 19 Betaproteobacteria (i.e. 79%) and 9 of 12 Gammaproteobacteria strains (i.e. 75%) (Table 1). Hybridization failed to identify acdS in a few strains in which an acdS sequence was obtained (i.e. R. leguminosarum 128C53 K and B. phenazinium LMG2247). More importantly, however, hybridization (especially with the P. fluorescens probe) evidenced acdS in additional strains (A. lipoferum CRT1 and B518, and A. brasilense L4, PH1, Sp245, Sp7, Cd and ZN1), genomovars (Agrobacterium genomic groups 1, 6, 7, 8 and 9, and B. cepacia genomovars II and V) and species (the Azospirillum species brasilense, and the Burkholderia species caryophylli, kururiensis, gladioli and pyrrocinia) when compared with acdS sequence results.
Analysis of ACC deaminase activity
In this work, 24 representative strains belonging to all proteobacterial taxa studied were selected to quantify ACC deaminase activity. Results indicated that (i) 11 of the 24 bacteria showed ACC deaminase activity in vitro; and (ii) levels of specific activity could differ statistically from one AcdS+ strain to the next, including within a particular species (Fig. 3). As expected, the positive control P. fluorescens PITR2 displayed ACC deaminase activity and the negative control P. fluorescens CHA0 did not. In A. lipoferum, five of six strains tested showed ACC deaminase activity, at levels ranging from 95.1±1.3 to 101.1±0.6 nmol α-ketobutyrate (mg protein)−1 h−1 (Fig. 3). Several acdS+A. brasilense were studied, but ACC deamination in vitro was not found. ACC deaminase activity was also evidenced in Burkholderia species and R. solanacearum. The prevalence of strains showing ACC deaminase activity in vitro was 80% in acdS phylogenic group I, 50% in group II and 100% in group III.
Understanding the genetic basis differentiating bacteria of contrasting ecology (e.g. pathogenic vs. beneficial bacteria) is a key issue. In recent years, features such as type-three protein secretion genes or genetic islands, thought to be specific to pathogens, have also been found in non-pathogens, including symbiotic bacteria (Ochman & Moran, 2001; Mazurier et al., 2004; Rezzonico et al., 2004). Similarly, certain genes often associated with beneficial interactions (e.g. nifU and nifS) may be important also for pathogens (Olson et al., 2000), but this possibility has received less attention in comparison.
In this context, we focused on ACC deaminase activity, a plant-beneficial property extensively surveyed by phenotypic means in several (but not all) key PGPR and phytosymbiotic bacteria (Glick et al., 1995; Wang et al., 2001; Penrose & Glick, 2003; Ma et al., 2003a, b), and whose assessment in other types of bacteria remained to be carried out. Our results indicate that ACC deaminase activity was not detected in vitro for several acdS+Proteobacteria, which means that these bacteria could not have been identified with the phenotype-based traditional methods used for screening strain collections (e.g. Wang et al., 2001). Therefore, the DNA approaches developed in the current work were useful to extend our knowledge on the distribution of acdS in Proteobacteria. PCR amplification of acdS was not as efficient as hybridization to detect acdS+Proteobacteria (Table 1). This is not surprising, and can be explained by variations in the short DNA sequences of the priming sites (data not shown). In addition, in a few cases, the PCR approach yielded nonspecific bands, or bands of the expected size but for which all sequencing attempts failed. Here, presence of the gene was established (i) in a range of pathogenic bacteria (i.e. certain Burkholderia species including several cepacia genomovars, and five Agrobacterium genomovars), and (ii) in a key PGPR genus believed so far to be devoid of ACC deaminase activity (i.e. Azospirillum) (Table 1).
First, acdS is present in several pathogenic Proteobacteria, and therefore the gene is not restricted to plant-beneficial taxa. acdS was evidenced in phytopathogens such as the Gladiolus pathogen B. gladioli, the onion pathogen B. cepacia genomovar I, the carnation pathogen B. caryophylli, and Agrobacterium strains (all of them harboring a pTi virulence plasmid) (Gelvin et al., 2000) belonging to genomovars 1, 4, and 6−9. In addition, the gene is also present in opportunistic human pathogens isolated from cystic fibrosis patients and belonging to B. cepacia genomovars II, III, IV and VI, such as the genomovar III strain LMG16656 implicated in the death of several patients (Govan et al., 1993), as well as in the true pathogen B. mallei (Nierman et al., 2004). ACC deaminase activity being a plant-beneficial trait, the occurrence of acdS (and sometimes ACC deaminase activity) in true or opportunistic pathogens is striking and raises the question of its contribution to pathogenesis, which may be especially relevant when dealing with phytopathogens. In addition, it questions the potential role of this trait for the survival of true and opportunistic human pathogens in terrestrial and/or non-terrestrial environments, because ACC deaminase activity may be important for nitrogen nutrition in microbial habitats containing ACC (Glick et al., 1998). For instance, strains belonging to B. cepacia genomovar III are found in the rhizosphere of corn (Balandreau et al., 2001), but it is not known whether the same strain can colonize both the rhizosphere and cystic fibrosis patients. The significance of acdS and/or ACC deaminase activity for survival in non-terrestrial environments is not obvious, because to our knowledge the occurrence of ACC is not documented in animal or human hosts.
Second, in the PGPR taxa Azospirillum, acdS was found in two of three species tested, and several acdS+Azospirillum strains displayed ACC deaminase activity in vitro (Table 1). Holguin & Glick (2001) concluded that ACC deaminase activity was absent from A. brasilense Cd and Sp245, which is in accordance with the current phenotypic results obtained in vitro, but nevertheless acdS was discovered here in these and other A. brasilense strains (some of the latter actually displaying enzymatic activity). So far, the phytobeneficial effets of Azospirillum PGPR strains have been attributed to nitrogen fixation (Bally & Elmerich, 2005) and phytohormone production (Dobbelaere et al., 2001), and in certain cases biocontrol of phytopathogenic bacteria (Bashan & de-Bashan, 2002) or phytoparasitic plants (Michéet al., 2000). In light of the current results, this will need to be reassessed by including the contribution of ACC deaminase activity.
Phylogenetic analysis evidenced three main acdS phylogenetic groups. BLASTP comparison of deduced AcdS sequences within each of the groups failed to distinguish conserved domains from variable regions (data not shown). Indeed, polymorphism was observed throughout the 264-amino acid sequence, even though key residues, for example K51, S78, T81, C162/A162, Y294 and E295 (see Fig. 2), were conserved. Therefore, information is not available to suggest that AcdS sequence polymorphism has the potential to modulate enzymatic functioning in a group-dependent manner.
Membership of proteobacterial strains to a particular group did not correlate with a particular geographic or habitat origin (Table 3). For instance, strains from a given acdS group originated from different plant hosts, similarly to findings derived from the study of other genes implicated in plant−microbe interactions (Wang et al., 2001; Ramette et al., 2003; Rezzonico et al., 2004). All opportunistic human pathogens were found in a single Burkholderia cluster within acdS group II. This observation will need confirmation using a larger number of sequences, as here an acdS sequence was available for only three opportunistic human pathogens. When analysing the complete dataset, however, it appeared that there was no obvious relation with the ecological strategy of the bacteria, as each acdS group included both pathogenic and plant-beneficial bacteria. This means that acdS alleles will not be useful as molecular markers to distinguish pathogens from nonpathogenic bacteria.
Each of the acdS phylogenetic groups I and II gathered strains belonging to the Beta-and Gammaproteobacterial subdivisions, whereas group III was composed of Alphaproteobacteria (Fig. 1). Therefore, although the strain collection studied did not cover the entire proteobacterial division, it is clear that the relationship with the rrs phylogeny was not extensive in comparison with congruence levels often found when studying bacterial genes important for interaction with a eukaryotic host (Ramette et al., 2003; Lerat & Moran, 2004; Mazurier et al., 2004; Rezzonico et al., 2004). This is probably a result of the past occurrence of extensive horizontal gene transfers. This hypothesis is supported by the observations that acdS is plasmid-borne in R. leguminosarum (Ma et al., 2003a) and is located within a symbiotic island in R. loti (Sullivan et al., 2002), genetic islands being prone to such transfers.
Insight into the consequence of a horizontal gene transfer of acdS can be gained from experiments in which acdS from E. cloacae UW4 was intentionally introduced into A. brasilense Cd and Sp245. Indeed, the introduced acdS gene was readily expressed in the transconjugants and resulted in enhanced phytobeneficial effects (Holguin & Glick, 2001, 2003). Similarly, introduction of the gene into the acdS− indoleacetic acid-producing strain P. fluorescens CHA0 increased the ability of the latter to protect potato tubers from E. carotovora-mediated soft rot (in certain but not all experimental conditions) and cucumber from Pythium damping-off, but not tomato from Fusarium crown and root rot (Wang et al., 2000). In addition, it conferred direct plant-growth promotion ability on canola cultured in artificial soil. These experiments illustrate how transferred acdS can benefit the plant, and perhaps this may in turn benefit acdS+ root-colonizing bacteria via increased root exudation.
This work was supported in part by the Ministère de la Recherche, Paris, France. A postdoctoral fellowship awarded to M.S.M. by the Higher Education Commission, Pakistan is gratefully acknowledged. We are grateful to A. Menard, X. Nesme and P. Portier (UMR CNRS 5557 Ecologie Microbienne, Lyon 1) for Burkholderia and Agrobacterium strains; B. Glick (University of Waterloo, Waterloo, Ontario) for E. cloacae UW4 and CAL2, K. ascorbata SUD 65 and R. leguminosarum 128C53K; A. Belimov (University of Saint Petersburg, Saint Petersburg, Russia) for A. xylosoxidans Cm4; and G. Défago (ETH, Zürich, Switzerland) for Pseudomonas strains CHA0, TM1A3, CM1′A2, PITR2, P97.30, K94.31, F113 and P97.38. We thank V. Segealet, D. Mourier des Gayets, J. Morel and O. Crassard for technical assistance, L. Jocteur Monrozier and A. Dechesne (UMR CNRS 5557 Ecologie Microbienne, Lyon 1) for advice on statistical analysis, and J. Balandreau, X. Nesme and R. Bally (UMR CNRS 5557 Ecologie Microbienne, Lyon 1) and A. Ramette (Michigan State University, East Lansing, Michigan) for useful discussions.
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