MURDOCH RESEARCH REPOSITORY http://researchrepository.murdoch.edu.au This is the author's final version of the work, as accepted for publication following peer review but without the publisher's layout or pagination. Ardley, J.K. , O'Hara, G.W. , Reeve, W.G. , Yates, R.J. , Dilworth, M.J. , Tiwari, R.P. and Howieson, J.G. (2009) Root nodule bacteria isolated from South African Lotononis bainesii, L. listii and L. solitudinis are species of Methylobacterium that are unable to utilize methanol. Archives of Microbiology, 191 (4). pp. 311-318. http://researchrepository.murdoch.edu.au/2046 Copyright © 2009 Springer-Verlag It is posted here for your personal use. No further distribution is permitted. Root nodule bacteria isolated from South African Lotononis bainesii, L. listii and L. solitudinis are species of Methylobacterium that are unable to utilize methanol Julie. K. Ardley1,+, Graham W. O’Hara1, Wayne G. Reeve1, Ron J. Yates1,2, Michael J Dilworth1, Ravi P. Tiwari1 and John G. Howieson1,2. 1 Centre for Rhizobium Studies, Murdoch University, Perth W.A. 6150, Australia 2 Department of Agriculture Western Australia, Baron-Hay Court, South Perth, WA 6151, Australia. + Corresponding author. Telephone: + 61 8 9360 2372 Fax: + 61 8 9360 6303 E-mail address: J.Ardley@murdoch.edu.au Correspondence address for proofs: Centre for Rhizobium Studies School of Biological Sciences and Biotechnology Murdoch University Murdoch WA 6150 Australia 1 1 Abstract 2 The South African legumes Lotononis bainesii, L. listii and L. solitudinis are 3 specifically nodulated by highly effective, pink-pigmented bacteria that are most 4 closely related to Methylobacterium nodulans on the basis of 16S rRNA gene 5 homology. Methylobacterium spp. are characterized by their ability to utilize 6 methanol and other C 1 compounds, but eleven Lotononis isolates neither grew on 7 methanol as a sole carbon source nor were able to metabolize it. No product was 8 obtained for PCR amplification of mxaF, the gene encoding the large subunit of 9 methanol dehydrogenase. Searches for methylotrophy genes in the sequenced genome 10 of Methylobacterium sp. 4-46, isolated from L. bainesii, indicate that the inability to 11 utilize methanol may be due to the absence of the mxa operon. While methylotrophy 12 appears to contribute to the effectiveness of the Crotalaria/M. nodulans symbiosis, 13 our results indicate that the ability to utilize methanol is not a factor in the 14 Lotononis/Methylobacterium symbiosis. 15 16 Keywords: Methylobacterium, Lotononis, Methylotrophy, Root nodule bacteria. 17 2 18 1. Introduction 19 Leguminous plants in the genus Lotononis and their associated root nodule bacteria 20 are being studied because of their potential as well-adapted pasture legumes able to 21 combat dryland salinity in southern Australian agricultural systems (Yates et al., 22 2007). The genus Lotononis is of mainly southern African origin, comprising some 23 150 species of herbs and small shrubs (Van Wyk, 1991). Species in the Listia section 24 are of particular interest, as they are perennial, stoloniferous and lack the poisonous 25 metabolites found in some other species of Lotononis (Van Wyk & Verdoorn, 1990). 26 The Listia section includes L. angolensis, L. bainesii, L. listii, L. macrocarpa, L. 27 marlothii, L. minima, L. solitudinis and L. subulata. Nodulation has been described 28 for L. angolensis, L. bainesii and L. listii, which characteristically form collar nodules 29 (Norris, 1958; Yates et al., 2007). 30 31 The root nodule bacteria from L. bainesii were first described by Norris (1958), who 32 reported that isolates from L. bainesii were red- or pink-pigmented and that the 33 symbiosis was highly specific. These pigmented bacteria were subsequently 34 characterized and identified as a species of Methylobacterium (Jaftha et al., 2002). 35 Yates et al. (2007) further found isolates from L. bainesii, L. listii and L. solitudinis to 36 be pink pigmented, highly effective, most closely related to Methylobacterium 37 nodulans (with > 97% similarity of the 16S rRNA gene sequence) and to form a 38 cross-inoculation group. The non-pigmented M. nodulans that specifically nodulates 39 Senegalese Crotalaria spp. (Sy et al., 2001) is the only other Methylobacterium 40 species so far reported to nodulate legumes. 41 42 Free-living methylobacteria are found in a variety of habitats, such as soil, dust, and 43 fresh water (Green, 1992). Methylobacteria are also ubiquitous in the plant 3 44 phyllosphere and rhizosphere (Trotsenko et al., 2001). They promote the germination 45 or growth of soybeans, rice and other plants, probably because of their ability to 46 synthesise auxins, cytokinins, vitamin B 12 and other plant growth-promoting 47 substances (Basile et al., 1985; Holland & Polacco; 1994, Ivanova et al., 2000; 48 Trotsenko et al., 2001; Madhaiyan et al., 2004; Abanda-Nkpwatt et al., 2006; Ryu et 49 al., 2006). The closeness of the association between plants and Methylobacterium 50 spp. varies; epiphytes (Omer et al., 2004;), endophytes (Van Aken et al., 2004) and 51 nitrogen-fixing symbionts (Sy et al., 2001; Jaftha et al., 2002; Yates et al., 2007), 52 have all been described. 53 54 Methylobacterium spp. are characterized by their ability to utilize methanol and other 55 C 1 compounds, as well as a variety of multicarbon substrates (Green, 1992; Lidstrom, 56 2006). Utilization of carbohydrates as a sole carbon source is variable and can be 57 used to differentiate the various species (Green, 1992). Methylotrophy in 58 Methylobacterium spp. involves over 100 genes constituting a set of metabolic 59 functional modules (Chistoserdova et al., 2003). In the model organism 60 Methylobacterium extorquens AM1, such modules involve the primary oxidation of 61 methanol or methylamine to formaldehyde, the oxidation of formaldehyde, and the 62 assimilation of C 1 products via the serine cycle (Chistoserdova et al., 2003; Lidstrom, 63 2006). Methanol is oxidized by methanol dehydrogenase (MDH), a protein with an 64 α 2 β 2 tetramer structure, a pyrroloquinoline quinone (PQQ) cofactor and a calcium 65 ion, essential for maintaining the PQQ in its active configuration, in the active site of 66 each α-subunit (Anthony, 1996; Goodwin & Anthony, 1998). The genes encoding the 67 MDH structural polypeptides, the specific cytochrome c electron acceptor, proteins 68 essential for the insertion of the calcium ion, a regulatory protein and several proteins 4 69 of unknown function are transcribed in a single operon, mxaFGIRSACKLDEHB 70 (Chistoserdova et al., 2003). 71 72 The M. nodulans isolates from Crotalaria, like all previously described 73 Methylobacterium species, can use methanol as a sole carbon source and contain a 74 copy of mxaF, the gene that codes for the large subunit of MDH (Sy et al., 2001). 75 Isolates from L. bainesii are also reported as able to grow in minimal media with 76 methanol as a substrate (Jaftha et al., 2002). In contrast, our isolates from L. bainesii, 77 L. listii and L. solitudinis appeared unable to grow on methanol in minimal media. 78 The aim of this work was thus to determine the ability of these isolates to grow on or 79 utilize a variety of C 1 and other carbon substrates. 80 81 The genomes of five Methylobacterium spp., including the L. bainesii symbiont 82 Methylobacterium sp 4-46, have recently been sequenced and are available on the 83 Integrated 84 (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi) in either draft or finished form. 85 Methylobacterium sp 4-46 is closely related to xct9 on the basis of 16S rRNA gene 86 homology (Darryl Fleischman, personal communication). We therefore performed 87 BLASTP searches for sequences required for methylotrophy in these genomes, to 88 determine the genetic basis for our isolates’ inability to grow on methanol. Microbial Genomes database of the Joint Genome Institute 89 90 2. Materials and Methods 91 2.1. Bacterial strains, origins and cultural conditions 92 The bacterial strains (11 Lotononis isolates and four reference strains) used and their 93 collection details are listed in Table 1. Root-nodule bacteria were isolated (Yates et 5 94 al., 2007) from L. bainesii, L. listii and L. solitudinis plants growing in eight sites 95 across north-eastern South Africa, between latitudes 24°S and 30°S and form part of 96 the Western Australian Soil Microbiology (WSM) collection, Murdoch University, 97 Western Australia. Strain xct9 was isolated from a South African L. bainesii nodule. 98 It is synonymous with CB376, the current commercial inoculant for L. bainesii in 99 Australia (Ian Law, personal communication). These root nodule bacteria and the 100 reference strains Sinorhizobium medicae WSM419 and Bradyrhizobium japonicum 101 USDA6 were grown on half lupin agar (½ LA) medium (Howieson et al., 1988). All 102 strains were stored in ½ LA plus 15% (v/v) glycerol broths at -80°C. M. nodulans 103 ORS 2060 and the non-symbiont Methylobacterium organophilum DSM 760 (kindly 104 supplied by Dr Catherine Boivin-Masson, INRA) were streaked onto agar plates of 105 minimal mineral medium M72 (Belgian Co-ordinated Collection of Micro-organisms, 106 1998) supplemented with 1% (v/v) methanol. They were then grown in broths of M72 107 plus 1% (v/v) methanol and stored as detailed above. 108 109 2.2 Acidification or alkalinization of ½ LA medium 110 The ability of the isolates to acidify or alkalinize ½ LA medium was tested on 111 unbuffered ½ LA agar plates, adjusted to pH 7.0 and containing 5 ml l-1 of Universal 112 range pH indicator (Vogel, 1962). The isolates and reference strains were streaked 113 onto the ½ LA plus Universal indicator plates and colour change recorded after 7 d 114 incubation at 28ºC. Strains were scored as acidifying if the medium turned yellow 115 (pH 6.0), alkalinizing if the medium turned blue/green (pH 8) or strongly alkalinizing 116 if the medium turned blue (pH 9.0). 117 6 118 2.3. Growth on sole carbon substrates 119 2.3.1. General growth procedures 120 All isolates and reference strains were streaked from -80ºC stocks onto fresh ½ LA 121 plates, except for ORS2060, which was streaked onto M72 agar containing 1% (v/v) 122 methanol. All media were adjusted to pH 7.0. All plates were incubated at 28ºC for 7 123 d. Glassware used to grow cultures (McCartney bottles and conical flasks) was 124 soaked in a 10% (v/v) hydrochloric acid solution for at least 24 h prior to use and then 125 rinsed twice in reverse osmosis deionized water. All broth cultures were grown at 126 28ºC with shaking (200 rpm). Lids of McCartney bottles were wrapped with parafilm 127 prior to incubation to prevent contamination. Optical densities were read on a Hitachi 128 U-1100 spectrophotometer. 129 130 2.3.2. Growth on methanol and multicarbon substrates 131 Isolates were tested for growth on arabinose, glucose, galactose, mannitol, succinate, 132 glutamate and methanol as sole carbon sources. Cells were inoculated into 5 ml 133 broths of M72 medium, supplemented with sodium pyruvate (10 mM), yeast extract 134 (0.5 g l-1) and vitamins (thiamine HCl, 1.0 mg l-1; pantothenic acid, 1.0 mg l-1 and 135 biotin, 20 µg l-1) and grown for 40 h to an optical density at 600 nm (OD 600 ) of 136 between 0.6 and 0.9. The cultures were centrifuged (20 800 g for 30 s), washed twice 137 with 0.89% (w/v) saline, resuspended in M72 medium containing vitamins (M72v) 138 and devoid of carbon source, then added to duplicate 5 ml broths of M72v and one of 139 the carbon substrates to a final OD 600 of 0.05. The concentration of all carbon 140 substrates was 20 mM, except for methanol, where the concentration was 1% (v/v) 141 (approximately 260 mM). Several strains were also grown in broths with 50 mM 142 methanol. The methanol and the stock solutions of the other carbon substrates 7 143 (adjusted to pH 7.0 where necessary) were filter sterilized (0.2 μm filter) and added to 144 the autoclaved M72v medium prior to inoculation. Inoculated culture media were 145 incubated for 10 d before a visual assessment was made. Growth on the carbon 146 substrate was assessed as being no growth (OD 600 was the same as for the minus 147 carbon substrate control), poor (0.1 < OD 600 < 0.2), moderate (0.2 < OD 600 < 0.5) or 148 abundant (OD 600 > 1.0). Two negative controls were used - an uninoculated control 149 containing M72v medium and various carbon sources and a control of M72v devoid 150 of carbon substrate but containing bacterial inoculant. 151 152 2.3.3. Growth on C 1 sole carbon substrates 153 Isolates were examined for growth on methanol (0.2%, v/v), methylamine (0.1%, 154 0.2% or 0.5%, v/v), formaldehyde (0.5 mM or 1.0 mM) and formate (30 mM) as sole 155 carbon sources in JMM medium (O'Hara et al., 1989) devoid of galactose and 156 arabinose and with NH 4 Cl (10 mM) replacing glutamate as a nitrogen source. JMM 157 medium with succinate (20 mM) as a sole carbon source served as a positive control. 158 Isolates were also examined for growth in medium containing succinate (20 mM) 159 plus methanol (1.0% v/v). Stock solutions of methylamine and other carbon sources 160 were adjusted to pH 7.0 if required. Inoculum was prepared and grown as described 161 above, but with JMM replacing M72v medium. 162 163 2.3.4. Growth of WSM2799 on formate 164 Cells were inoculated into broths of JMM medium containing succinate (20 mM) and 165 NH 4 Cl (10 mM) as carbon and nitrogen sources, and grown for 40 h to an OD 600 of 166 0.6. The cultures were centrifuged and washed as described, resuspended in JMM 167 medium (containing either formate or succinate) and added to duplicate 250 ml 168 conical flasks containing pre-warmed 50 ml JMM broths with either succinate 8 169 (20mM) or formate (30mM) as sole carbon source to give an initial OD 600 of 0.05. 170 Duplicate samples were taken from each flask at regular intervals for OD 600 readings. 171 172 2.4. Biochemical assays for utilization of methanol 173 Cells of xct9, USDA6 and ORS 2060 were inoculated into broths of M72 medium 174 supplemented with sodium pyruvate (10 mM), yeast extract (0.5 g l-1) and vitamins. 175 The cultures were grown for 40 h to an OD 600 of between 0.6 – 0.9, centrifuged and 176 washed as described and resuspended in M72v medium, then added to duplicate 100 177 ml conical flasks containing 20 ml of M72v medium and either 25 mM or 100 mM 178 methanol, to a final OD of 0.05. These and duplicate uninoculated controls were 179 incubated for 70 h. Aliquots (1 ml) were taken from each flask at 0, 22, 40 and 70 h, 180 growth measured as OD 600 and the supernatant stored at –20ºC after centrifugation 181 (20,800 g for 30 s). 182 183 Sulfuric acid (0.5M) was added to the supernatant samples (10 µl from the cultures in 184 100 mM methanol, 40 µl from the 25 mM methanol cultures), to make a total volume 185 of 1 ml. For ORS 2060, the 40 h and 70 h 100 mM methanol samples were also of 40 186 µl. The samples were oxidized with potassium permanganate, excess permanganate 187 removed with sodium arsenite (Wood & Siddiqui, 1971), and formaldehyde 188 concentration determined by measuring OD 412 after reaction with Nash's reagent 189 (Nash, 1953). 190 191 The concentration of methanol in the succinate (20 mM) plus methanol (1% v/v) 192 media 10 d after inoculation was determined in the same way. 193 9 194 2.5. PCR amplification of mxaF 195 PCR amplification of mxaF was performed using the primers f1003 – 5' -GCG GCA 196 CCA ACT GGG GCT GGT-3' and r1561 – 5' -GGG CAG CAT GAA GGG CTC 197 CC-3'. Primers were obtained from GeneWorks Pty Ltd. 198 199 Whole cell DNA templates were prepared from isolates and reference strains. Cells 200 were suspended in PCR-grade water to an OD 600 of approximately 10. An initial PCR 201 amplification to optimize the magnesium chloride concentration of the PCR reaction 202 mix resulted in subsequent reactions using 1.5 mM MgCl 2 . The total volume of the 203 PCR reaction mix was 20 µl, consisting of 4 µl of 5X PCR polymerization buffer 204 (Fisher Biotec), 9.8 µl of PCR-grade water, 4 µl of 7.5 mM MgCl 2 , 0.5 µl of each 50 205 µM primer, 0.2 µl of Taq DNA polymerase (5 U µl-1) (Invitrogen) and 1 µl of DNA 206 template. The PCR conditions for the thermocycler were an initial 4 min at 94ºC; 31 207 cycles of 94ºC for 1 min, 55ºC for 1 min and 72ºC for 1 min; then 1 cycle of 94ºC for 208 1 min, 55ºC for 1 min and 72ºC for 5 min. The samples were then subjected to gel 209 electrophoresis, or storage at -20ºC. 210 211 The PCR amplification product was visualized after gel electrophoresis using a 1% 212 (w/v) agarose in TAE gel submerged in TAE running buffer (40 mM Tris acetate, 1 213 mM EDTA, pH 8.0) run at 80V for about 2 h. with loading dye added to each sample 214 prior to electrophoresis. A 1 kb DNA ladder (Promega) was used as a marker. The 215 gels were stained in 0.5 µg ml-1 ethidium bromide for 40 min, destained in deionized 216 water and visualized under UV light. Images of the gels were captured using the Gel 217 Doc 2000 (BioRad) system. 218 10 219 2.6. Comparative genomics of Methylobacterium spp. 220 Homologs of genes required for methylotrophy were identified using BLASTP 221 searches 222 Methylobacterium extorquens AM1 (Chistoserdova et al., 2003) against the five 223 sequenced Methylobacterium spp. (M. chloromethanicum CM4, M. extorquens PA1, 224 M. nodulans ORS 2060, M. populi BJ001 and Methylobacterium sp. (Lotononis) 4- 225 46) and the S. medicae WSM419 genomes deposited in the Integrated Microbial 226 Genomes database of the Joint Genome Institute. 227 3. Results 228 3.1. Acidification or alkalinization of ½ LA media 229 The Lotononis isolates all alkalinized or strongly alkalinized the media, consistent 230 with organic acid utilization. USDA6 and ORS 2060 also alkalinized the media, 231 while WSM419 caused acidification. of sequences from the well-studied facultative methylotroph 232 233 3.2. Growth on sole carbon substrates 234 Isolates from L. bainesii, L. listii and L. solitudinis were highly selective in their 235 utilization of carbon sources (Table 2). None was able to utilize arabinose, galactose, 236 glucose or mannitol as sole carbon sources. All the isolates from Lotononis spp. grew 237 well on succinate and glutamate but none grew on methanol as a sole carbon source. 238 239 3.2.2. Growth on C 1 sole carbon substrates 240 Neither the Lotononis isolates nor WSM419 grew on methanol, methylamine or 241 formaldehyde at any of the concentrations supplied in the media (Table 3). ORS 2060 242 grew on methanol, but not on methylamine or formaldehyde. Growth of the 11 243 Lotononis isolates on formate was variable. WSM2603, WSM2666, WSM2678, 244 WSM2693, WSM3032, WSM3034 and WSM3035 did not grow, while WSM2598, 245 WSM2660 and xct9 grew poorly and WSM2799 grew moderately well. WSM419 246 grew poorly and ORS 2060 grew moderately well on formate. To determine whether 247 methanol had an inhibitory effect on growth, all strains were also grown in broths 248 containing both succinate and methanol as carbon sources (Table 3). All of the 249 Lotononis isolates and WSM419, as well as ORS 2060, grew abundantly in the 250 succinate plus methanol medium. 251 252 3.2.3. Growth of WSM2799 on formate 253 The growth rate of logarithmic phase cells of WSM2799 growing on formate was 254 compared with that of succinate-grown cells by measuring OD 600 . The mean 255 generation time (MGT) for WSM2799 grown on formate was 24 h, compared with 256 5.5 h MGT when grown on succinate. 257 258 3.3. Biochemical assays for utilization of methanol 259 3.3.1. Utilization by xct9, ORS2060 and USDA6 over 70 h 260 Three strains (xct9, ORS 2060 and USDA6) were chosen for determination of 261 methanol utilization at two different methanol concentrations (25 mM and 100 mM). 262 The L. bainesii isolate xct9 was included as Jaftha et al. (2002) reported that it grew 263 on M72 medium containing methanol as a sole carbon source. M. nodulans ORS 264 2060 and B. japonicum USDA6 served as positive and negative controls, 265 respectively. Over a period of 70 h and at both 25 mM and 100 mM of methanol, 266 ORS 2060 was the only strain for which the concentration of methanol in the medium 267 notably decreased (Fig. 1). The methanol concentrations in the cultures of xct9 and 12 268 USDA6 showed slight decreases equal to those in uninoculated media. Over the same 269 period of time, and at both 25 mM and 100 mM methanol, the OD 600 of the ORS 270 2060 culture increased, whereas the OD 600 of the xct9 and USDA6 cultures remained 271 unchanged. 272 273 3.3.2. Utilization of methanol in succinate plus methanol media 274 The concentration of methanol in inoculated medium containing both succinate and 275 methanol (Table 3) was determined for all strains after 10d incubation. In media 276 inoculated with the Lotononis isolates or with WSM419, the methanol concentration 277 was similar to that of the uninoculated control, but decreased by 50% in media 278 inoculated with ORS 2060 (results not shown). 279 280 3.4. PCR amplification of mxaF 281 To evaluate the presence of methanol dehydrogenase genes in the Lotononis isolates, 282 PCR amplification of mxaF, the gene that codes for the large subunit of methanol 283 dehydrogenase, was performed on Lotononis isolates WSM2603, WSM2660, 284 WSM2666, WSM2678, WSM2693, WSM2799, WSM3035 and xct9 and the 285 methylotrophic strains ORS 2060 and DSM 760. The primers used have previously 286 been shown to specifically amplify methylotrophic DNA (McDonald et al., 1995). 287 The correct size 555-bp PCR product was obtained only for OR S2060 and DSM 760. 288 No amplification was obtained for any of the Lotononis isolates, or for the reaction 289 devoid of template DNA. 290 13 291 3.5. Comparative genomics of Methylobacterium spp. 292 Several different homologs of the M. extorquens AM1 α-subunit of MDH, coded for 293 by mxaF, were found in each of the genomes of the five sequenced methylobacteria 294 and of S. medicae WSM419. However, only the genomes of the methylotrophic M. 295 chloromethanicum CM4, M. extorquens PA1 and M. populi BJ001 had a mxaF 296 homolog present in a mxaFGIRSACKLDEHB operon. The M. nodulans ORS2060 297 mxaF appeared to be in a separate section of the genome, but this may be due to its 298 genome sequence being still in a draft state. The remaining ORS2060 mxa genes were 299 present in an orthologous operon. These four mxaF homologs had a high similarity 300 (>88% over the length of the protein). In contrast, mxaF homologs found in 301 Methylobacterium sp. 4-46 and S. medicae WSM419 were not in an orthologous 302 operon and had a similarity of only 50% or less. No homologs of more than 50% 303 similarity could be found in Methylobacterium sp. 4-46 for the MDH β-subunit 304 (mxaI), the specific cytochrome c electron acceptor (mxaG), proteins essential for 305 Ca2+ insertion (mxaACKL) or for the protein products of mxaSH. Similarly, no 306 homologs were found in WSM419 for the products of mxaGIACKLD. 307 308 4. Discussion 309 Methylobacterium species are described as able to grow on methanol, formaldehyde 310 and formate (Green, 1992; Lidstrom, 2006). Sy et al. (2001) demonstrated that M. 311 nodulans, isolated from root nodules of Crotalaria, was able both to fix nitrogen 312 symbiotically and to utilize methanol as a sole carbon source. They also confirmed 313 the presence in M. nodulans of mxaF, the gene that codes for the alpha subunit of 314 methanol dehydrogenase. In contrast, our results from the carbon substrate utilization 315 tests, the methanol assay and the mxaF PCR indicate that our isolates from L. 14 316 bainesii, L. listii and L. solitudinis differ from other described Methylobacterium 317 species (Green, 1992; Gallego et al., 2006; Kato et al., 2008) in being unable to 318 utilize or oxidize methanol. Kato et al. (2008) did, however, report that two of their 319 Methylobacterium strains, isolated from freshwater, only grew weakly on methanol as 320 a sole carbon source. Our findings also differ from those of Jaftha et al. (2002) who 321 reported that their L. bainesii isolates grew in the presence of methanol as a sole 322 carbon source. Consistent with our results, the L. bainesii isolate CB376 has 323 previously been described as unable to utilize methanol (O'Brien & Murphy, 1993), 324 but was identified as a species of Rhizobium at the time. The ability of the Lotononis 325 isolates to grow in media containing succinate and methanol (1% v/v) indicates that 326 their inability to grow on methanol as a sole carbon substrate is not due to any toxic 327 effects of methanol at this concentration. 328 329 In methylobacteria, methanol dehydrogenase is essential for the oxidation of 330 methanol to formaldehyde. The negative result for the PCR amplification of the mxaF 331 gene suggests that the Lotononis isolates in this study lack at least one of the genes 332 required to synthesize methanol dehydrogenase. Searches for methylotrophy genes in 333 the sequenced genome of Methylobacterium sp. 4-46, isolated from L. bainesii, 334 indicate that the inability to utilize methanol may be due to the absence of the mxa 335 operon, which is present in the other sequenced methylotrophic Methylobacterium 336 genomes, and the products of which are required for the primary oxidation of 337 methanol (Amaratunga et al., 1997; Chistoserdova et al., 2003). It is likely that the 338 mxaF homologs with similarity < 50% that are present in these genomes code for 339 PQQ-dependent alcohol or glucose dehydrogenases that are not specific for methanol 340 as a substrate. 341 15 342 Most species of Methylobacterium can only grow on a narrow range of carbohydrates 343 (Green, 1992). The growth of the L. bainesii, L. listii and L. solitudinis isolates on 344 other sole carbon sources is consistent with the substrate utilization patterns of other 345 Methylobacterium species and with results obtained from other L. bainesii isolates 346 (Jaftha et al., 2002) None of these isolates grew on the sugars or sugar alcohol 347 supplied. Their ability to grow on succinate and to alkalinize ½ LA + universal 348 indicator media also suggests that they preferentially utilize organic acids, including 349 dicarboxylic acids, which are the usual form of carbon supplied to nitrogen-fixing 350 bacteroids within legume nodules (Lodwig & Poole, 2003). M. nodulans ORS2060, 351 in contrast, appears to be able to use methanol within the Crotalaria podocarpa 352 nodule (Jourand et al., 2005). 353 354 Plants produce methanol as a by-product of pectin metabolism, with high pectin 355 methyl esterase activity correlating with areas of rapid growth, such as seen in 356 seedlings (Obendorf et al., 1990). Studies have suggested that the ability of 357 Methylobacterium spp to utilize this methanol confers an advantage in plant 358 colonization (Corpe & Rheem, 1989; Sy et al., 2005) and contributes to the 359 effectiveness of the specific C. podocarpa/ M. nodulans symbiosis (Jourand et al., 360 2005). Yates et al. (2007) have shown that the symbiosis between L. bainesii, L. listii 361 and L. solitudinis and their nodulating methylobacteria is a highly effective one. Our 362 results suggest that the inability to utilize methanol is not deleterious to effective 363 colonization or symbiosis in the association between these methylobacteria and 364 Lotononis and factors other than methylotrophy must be implicated in the specificity 365 of the symbiosis. 366 16 367 Acknowledgements 368 J.A. is the recipient of a Murdoch University Research Scholarship. 369 370 5. References 371 Abanda-Nkpwatt D, Musch M, Tschiersch J, Boettner M, Schwab W (2006) 372 Molecular interaction between Methylobacterium extorquens and seedlings: growth 373 promotion, methanol consumption, and localization of the methanol emission site. J 374 Exp Bot 57: 4025-4032. doi:10.1093/jxb/erl173 375 376 Amaratunga K, Goodwin PM, O’Connor CD, Anthony C (1997) The methanol 377 oxidation genes mxaFJGIR(S)ACKLD in Methylobacterium extorquens. FEMS 378 Microbiol Lett 146: 31-38. doi:10.1111/j.1574-6968.1997.tb10167.x 379 380 Anthony C. (1996) Quinoprotein-catalysed reactions. 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List of strains used in this study Isolate /strain Host plant Geographical origin Collector(s)/Source Reference WSM2598 L. bainesii Sth Africa Yates, Real & Law (2002) This study; Yates et al., (2007) WSM2603 L. listii Sth Africa Yates, Real & Law (2002) This study WSM2660 L. listii Sth Africa Yates, Real & Law (2002) This study WSM2666 L. listii Sth Africa Yates, Real & Law (2002) This study WSM2678 L. listii Sth Africa Yates, Real & Law (2002) This study WSM2693 L. listii Sth Africa Yates, Real & Law (2002) This study; Yates et al., (2007) WSM2799 L. listii Sth Africa Yates, Real & Law (2002) This study; Yates et al., (2007) WSM3032 L. solitudinis Sth Africa Yates, Real & Law (2002 This study; Yates et al., (2007) WSM3034 L. solitudinis Sth Africa Yates, Real & Law (2002) This study WSM3035 L. bainesii Sth Africa Yates, Real & Law (2002) This study xct9 = L. bainesii Sth Africa Botha (1954); ARC Jaftha et al., (2002) WSM419 Medicago spp. Sardinia Howieson (1985) Howieson & Ewing (1986) USDA6 Glycine max Japan USDA ORS 2060 Crotalaria spp. Senegal INRA U.S.A. INRA a CB376 DSM 760* a b Jordan (1982) c Sy et al., (2001) c Patt et al., (1976) Agricultural Research Council-Plant Protection Research Institute, Pretoria, South Africa b U.S. Department of Agriculture, Beltsville, Maryland, U.S.A c National Institute for Agricultural Research, Montpellier Cedex 5, France 24 Table 2. Visual rating of growth of strains on various sole carbon sources after 10 days incubation. Growth was defined as 0 = no growth (OD < 0.1); x = poor growth (0.1 < OD < 0.2); xx = moderate growth (0.2 < OD < 0.5); xxx = abundant growth (OD > 1.0); ND = not determined. Carbon Source Isolate/ Strain L+ Arabinose D+ DDSuccinate Galactose Glucose Mannitol a Glutamate MeOH 266mM MeOH 50mM WSM2598 0 0 0 0 xxx xxx 0 ND WSM2603 0 0 0 0 xxx xxx 0 ND WSM2660 0 0 0 0 xxx xxx 0 ND WSM2666 0 0 0 0 xxx xxx 0 ND WSM2678 0 0 0 0 xxx xxx 0 ND WSM2693 0 0 0 0 xxx xxx 0 0 WSM2799 0 0 0 0 xxx xxx 0 0 WSM3032 0 0 0 0 xxx xxx 0 ND WSM3034 0 0 0 0 xxx xxx 0 ND WSM3035 0 0 0 0 xxx xxx 0 ND xct9 0 0 0 0 xxx xxx 0 0 Reference Strains a WSM419 xxx xxx xxx xxx xxx xxx 0 ND USDA6 xxx xx x xxx xxx xxx 0 ND ORS 2060 x 0 0 0 xxx xxx xxx xxx MeOH = methanol 25 Table 3. Visual rating of growth of strains on various sole C 1 carbon sources after 10 days incubation. Growth was defined as 0 = no growth (OD < 0.1); x = poor growth (0.1 < OD < 0.2); xx = moderate growth (0.2 < OD < 0.5); xxx = abundant growth (OD > 1.0) and was determined for the following concentrations of substrates: succinate (20mM); methanol [0.2% (v/v)]; formaldehyde (0.5mM or 1.0mM); methylamine [0.1%, 0.2% or 0.5% (w/v)]; formate (30mM); succinate plus methanol [20mM plus 1.0% (v/v) respectively]. Carbon Source Isolate/ Strain Succinate MeOH Methylamine Formaldehyde Formate Succinate + MeOH WSM2598 xxx 0 0 0 x xxx WSM2603 xxx 0 0 0 0 xxx WSM2660 xxx 0 0 0 x xxx WSM2666 xxx 0 0 0 0 xxx WSM2678 xxx 0 0 0 0 xxx WSM2693 xxx 0 0 0 0 xxx WSM2799 xxx 0 0 0 xx xxx WSM3032 xxx 0 0 0 0 xxx WSM3034 xxx 0 0 0 0 xxx WSM3035 xxx 0 0 0 0 xxx xct9 xxx 0 0 0 x xxx Reference Strains ORS 2060 xxx xxx 0 0 xx xxx WSM419 xxx 0 0 0 x xxx 26 30 1.6 1.4 25 1.2 1 15 0.8 OD 600 Methanol (mM) 20 0.6 10 0.4 5 0.2 0 0 0 10 20 30 40 50 60 70 80 Tim e (hours) Fig. 1. 27 Fig. 1. Non-utilization of methanol by the L. bainesii isolate xct9. Methylobacterium nodulans strain ORS 2060 was used as a positive control. An uninoculated control was also used. Shown is growth of xct9 (filled squares), growth of ORS 2060 (filled triangles) and OD 600 of the uninoculated control (filled circles). MeOH concentrations in supernatants of cultures of xct9 (empty squares) and ORS 2060 (empty triangles) and of the uninoculated control (empty circles) are also shown. 28