1 TITLE: Candidimonas nitroreducens sp. nov. and Candidimonas humi sp. nov. , isolated

2 from sewage sludge compost

3 4 AUTHORS: Ivone Vaz-Moreira1,2, Vânia Figueira1, Ana R. Lopes2, Evie De Brandt 3, Peter 5 Vandamme3, Olga C. Nunes2, Célia M. Manaia1 6

7 1CBQF - Escola Superior de Biotecnologia, Universidade Católica Portuguesa, 4200-072 8 Porto, Portugal 9 2LEPAE – Departamento de Engenharia Química, Faculdade de Engenharia, Universidade 10 do Porto, 4200-465 Porto, Portugal 11 3 Laboratorium voor Microbiologie, Vakgroep Biochemie en Microbiologie, Universiteit 12 Gent, Gent, Belgium 13 14

15 AUTHOR FOR CORRESPONDENCE: 16 C.M. Manaia, PhD 17 Escola Superior de Biotecnologia 18 Universidade Católica Portuguesa 19 4200-072 Porto 20 Portugal 21 Tel: +351 22 5580059 22 Fax: +351 22 5090351 23 e-mail: [email protected] 24 25 RUNNING TITLE: Candidimonas gen. nov.

26 SUBJECT CATEGORY: NEW TAXA –

27 FOOTNOTE: The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene

28 sequence of the isolate SC-089T and SC-092T are FN556191 and FN556192, respectively.

29

1 30 SUMMARY

31 Two bacterial strains isolated from sewage sludge compost (SC-089T and SC-092T) were

32 characterized using a polyphasic approach. The isolates were Gram-negative short rods,

33 catalase- and oxidase-positive, with good growth at 30 ºC, pH 7 and 1 % (w/v) of NaCl.

34 Ubiquinone 8 was the major respiratory quinone and phosphatidylethanolamine,

35 phosphatidylglycerol and diphosphatidylglycerol were amongst the major polar lipids. On

36 basis of the 16S rRNA gene sequence analysis, the strains were observed to be members of

37 the family , but could not be identified as members of any validly named

38 genus. The low 16S rRNA gene sequence similarity with other recognized taxa and the

39 comparative analysis of phenotypic traits and cellular fatty acid composition supported the

40 proposal of a new genus within the family Alcaligenaceae for which the name

41 Candidimonas gen. nov. is proposed. Strains SC-089T and SC-092T, which shared 99 %

42 16S rRNA gene sequence similarity, could be differentiated at the phenotypic level and the

43 DNA-DNA hybridization supported their inclusion in distinct species. Hence, the species

44 names Candidimonas nitroreducens and Candidimonas humi are represented by the type

45 strains SC-089T (=LMG 24812T =CCUG 55806T) and SC-092T (=LMG 24813T =CCUG

46 55807T), respectively.

47

2 48 Two bacterial strains, designated SC-089T and SC-092T, were isolated from sewage sludge

49 compost during a study on the diversity of culturable from final composts (Vaz-

50 Moreira et al., 2008). As discussed below these isolates shared about 99 % 16S rRNA gene

51 sequence similarity and were observed to be members of the family Alcaligenaceae.

52 Among the closest neighbours of strains SC-089T and SC-092T were members of the genera

53 Parapusillimonas, , , , Pigmentiphaga and

54 , with Parapusillimonas granuli, Pigmentiphaga kullae and

55 representing nearest neighbours. In order to test the hypothesis that strains SC-089T and

56 SC-092T could be members of a new genus, these strains were compared with the type

57 strains of the type species of those genera, as well as, with the type strain of closest species

58 within each genus. The only exception was made for the type strain of the type species,

59 , which given its potential hazardous for handling and manipulation

60 was substituted by Bordetella bronchiseptica, with which strains under study shared

61 identical 16S rRNA gene sequences. Thus, strains SC-089T and SC-092T were compared

62 with the strains Parapusillimonas granuli LMG 24012T, Pigmentiphaga kullae CCUG

63 47266T, Bordetella petrii CCUG 43448T, Bordetella bronchiseptica LMG 1232T,

64 Achromobacter xylosoxidans LMG 1863T, Achromobacter denitrificans LMG 1231T,

65 Castellaniella defragrans LMG 18538T, Castellaniella denitrificans LMG 25533T and

66 Pusillimonas noertemannii DSM 10065T, which were supplied by the respective culture

67 collections. Unless otherwise stated, the organisms were cultivated on Plate Count Agar

68 (PCA, Pronadisa), incubated at 30 ºC and stored at -80 ºC in nutritive broth with 15 % (v/v)

69 glycerol. The colony and cell morphology, Gram-staining, cytochrome c oxidase and

70 catalase tests, and motility were analysed based on the methodologies of Murray et al.

71 (1994) and Smibert & Krieg (1994). Additional phenotypic characterization was based on

3 72 the methods described before (Vaz-Moreira et al., 2007). Biochemical and nutritional tests

73 were performed using the commercial kits API 20E, API 20NE, API ZYM and API ID

74 32GN (BioMérieux) following the manufacturer’s instructions. The Voges-Proskauer test

75 was assayed in Methyl-red and Voges-Proskauer medium (Oxoid), incubated at 30 ºC

76 during 48 h. Simmons citrate was tested on Simmons citrate agar (Pronadisa), incubated 7

77 days at 30 ºC. Urease activity was assayed as described by Smibert & Krieg (1994).

78 After 48 hours of incubation at 30 ºC on PCA strains SC-089T and SC-092T formed convex

79 white colonies. Growth was best at 30 ºC, pH 7 and 1 % (w/v) of NaCl and was supported

80 by different amino acids and organic acids. The nucleotide sequence of the 16S rRNA gene

81 was determined after PCR amplification of total DNA extracts as described before (Ferreira

82 da Silva et al., 2007). The 16S rRNA gene sequence was compared with others available in

83 the GenBank/EMBL/DDJB database using the FASTA package from EMBL-EBI. The

84 phylogenetic analysis was conducted using the BioNumerics software package (version 6.0,

85 Applied Math, Belgium). Sequence similarity was estimated based on the model of Jukes &

86 Cantor (1969) and the dendrogram was created using the neighbour-joining method. Other

87 methods, namely, maximum parsimony and maximum likelihood, were used to assess the

88 tree stability. Non-homologous and ambiguous nucleotide positions were excluded from the

89 calculations. Strains SC-089T and SC-092T shared 99.1 % 16S rRNA gene sequence

90 similarity and lower values with the type strains Parapusillimonas granuli LMG 24012T

91 (DQ466075) (97.8 and 97.4 %, respectively), Pigmentiphaga kullae CCUG 47266T

92 (AF282916) (97.1 and 97.3 %, respectively) and Bordetella petrii CCUG 43448T

93 (AJ249861) (96.9 %), which represented their nearest phylogenetic neighbours (Figure 1).

94 Either both strains SC-089T and SC-092T or one of them presented lower similarity 16S

4 95 rRNA gene sequence values with members of other related species – respectively 97.0 and

96 96.6 % with Achromobacter xylosoxidans LMG 1863T (Y14908), 96.9 and 96.8 % with

97 Achromobacter denitrificans LMG 1231T (AJ278451), 96.3 and 95.8 % with Bordetella

98 bronchiseptica LMG 1232T (U04948), 96.5 and 96.0 % with Castellaniella defragrans

99 LMG 18538T, 96.6 and 96.1 % with Castellaniella denitrificans LMG 25533T (U82826),

100 and 96.2 and 95.9 % with Pusillimonas noertemannii DSM 10065T (AY695828). The two

101 strains represented a distinct line of descent in the Alcaligenaceae family and their

102 phylogenetic position was supported by a high bootstrap value and by the different treeing

103 algorithms that were used.

104 DNA-DNA hybridizations were performed between strains SC-089T and SC-092T, and

105 towards P. kullae CCUG 47266T and Parapusillimonas granuli LMG 24012T. DNA was

106 extracted from 0.25–0.5 g (wet wt) cells as described by Pitcher et al. (1989). DNA–DNA

107 hybridizations were performed with photobiotin-labelled probes in microplate wells (Ezaki

108 et al., 1989), using an HTS7000 Bio Assay Reader (Perkin Elmer) for the fluorescence

109 measurements. The hybridization temperature was 47 °C. Reciprocal experiments were

110 performed for every pair of strains and differed between 1 and 7 %. The DNA–DNA

111 hybridization level between strains SC-089T and SC-092T was 41 %; the other DNA–DNA

112 hybridization values were 10 % or lower (data not shown).

113 The fatty acid methyl esters (FAMEs) were analysed after an incubation period of 24 h or

114 48 h (C. defragrans LMG 18538T and P. noertemanii DSM 10065T) at 28 °C. A loopful of

115 well-grown cells was harvested and fatty acid methyl esters were prepared as described

116 previously (Vandamme et al., 1992), and separated and identified using the Sherlock

5 117 Microbial Identification System (version 3.1, MIDI Inc.). The polar lipid composition was

118 determined as described before (Manaia et al., 2004) (Figure 2).

119 The determination of the mol% of G+C content in genomic DNA and the respiratory

120 quinones were analysed as described previously (Vaz-Moreira et al., 2007) using,

121 respectively, the methods of Mesbah et al. (1989) and Tindall (1989). The respiratory

122 quinone was ubiquinone 8. The mol% G+C of strain SC-089T was 64 mol% and of strain

123 SC-092T 65 mol%. These chemotaxonomic traits confirm the affiliation of strains SC-089T

124 and SC-092T to the family Alcaligenaceae (Vancanneyt et al., 1995; Blümel et al., 2001;

125 von Wintzingerode et al., 2001; Busse & Auling, 2005; Stolz et al., 2005). Nevertheless,

126 the unique phylogenetic position suggests that they are most appropriately allocated into a

127 novel genus.

128 The phenotypic diversity of the closest neighbour genera, Parapusillimonas,

129 Pigmentiphaga, Bordetella, Achromobacter, Castellaniella and Pusillimonas (Figure 1),

130 limits their generic differentiation on basis of phenotypic traits alone (Blümel et al., 2001;

131 Busse & Auling, 2005). In this respect, the analysis of chemotaxonomic markers as the

132 polar lipids may give additional insights to genera differentiation. In fact, this analysis

133 permitted to distinguish strains SC-089T and SC-092T from members of their closely related

134 genera (Figure 2). Although phosphatidylethanolamine (PE), phosphatidylglycerol (PG)

135 and diphosphatidylglycerol (DPG) were present in the members of the different genera,

136 their proportion along with the presence/absence of unknown phospholipids and

137 aminolipids were distinctive characteristics among the taxa under study. Pigmentiphaga

138 kullae CCUG 47266T presented the most differentiated polar lipid pattern, with low

139 proportion of DPG and the absence of most of the unknown phospholipds and

6 140 amino(phospho)lipids. The absence of the unknown aminolipid AL2 permitted to

141 distinguish between strains SC-089T and SC-092T from members of other related genera, as

142 it constitutes a major lipid in Pusillimonas noertemannii DSM 10065T,.and is present in

143 low proportions in Achromobacter xylosoxidans LMG 1863T Additionally, the exclusive

144 presence of the unknown aminophospholipids APL2, APL3 and APL4 in strains SC-089T

145 and SC-092T contributed to distinguish these organisms from the related genera.

146 The cellular fatty acid composition allowed the distinction between strains SC-089T and

147 SC-092T and the type strains of the related species (Table 1). The predominant fatty acids

T T 148 common to strains SC-089 and SC-092 were summed feature 2 (C14:0 3OH or C16:1 iso I

149 or both), C16:0 and C18:1 w7c. Both organisms differed in the abundance of cyclo-C17:0 and

150 summed feature 3 (C16:1 w7c or C15:0 iso 2OH or both) and in the presence of the

T 151 hydroxylated fatty acid C17:0 iso 3OH. The pattern of fatty acids of strains SC-089 and SC-

152 092T was different from all the strains examined. The most similar patterns belonged to

153 strains SC-089T and P. kullae LMG 21665T, which, nevertheless, differed on the proportion

154 of the components comprised in summed feature 2 and C18:1 w7c. For all other organisms,

155 differences higher than 5 % were observed in the percentage of more than four fatty acids.

156 The presence of hydroxyl fatty acids C16:0 2OH and C16:1 2OH were also distinctive being

157 only present in strains SC-089T and SC-092T and in P. kullae LMG 21665T. However, in P.

T 158 kullae LMG 21665 was not detected the component C12:0 2OH observed in the strains

159 under characterization. To complete if differences are found for Parapusillimonas...

160 At the phenotypic level strains SC-089T and SC-092T could also be distinguished from the

161 type strains of the type species of related genera, as well as, from the type strains of the

162 most closely related species (Table 2). For example, strains SC-089T and SC-092T could be

7 163 distinguished from the members of the other genera examined in this study by the absence

164 of motility (present in the members of the genera Achromobacter, Pigmentiphaga,

165 Castellaniella, Pusillimonas and Parapusillimonas), inability to assimilate L-alanine

166 (observed in the members of the genera Achromobacter, Bordetella, Castellaniella and

167 Parapusillimonas), and L-proline (absent also in the members of the genera Pigmentiphaga

168 and Pusillimonas), and the ability to assimilate 3-hydroxybenzoic acid (absent in the

169 members of the genus Bordetella) and several other organic acids and sugars. The

170 phenotypic differentiating characteristics, along with their unique phylogenetic position,

171 suggest that strains SC-089T and SC-092T should be most appropriately allocated into a

172 novel genus. Nevertheless, the distinction of genera of the family Alcaligenaceae on basis

173 of phenotypic characteristics is not possible, as was demonstrated in previous studies,

174 namely on the description of the genera Pigmentiphaga, Pusillimonas and

175 Parapusillimonas (Blümel et al., 2001; Stolz et al., 2005; Kim et al., 2010), which do not

176 refer any distinctive phenotypic trait of the genus. In fact, a literature review on basis of

177 studies of phenotypic description of members of the genera Achromobacter, Bordetella,

178 Castellaniella, Pusillimonas, Pigmentiphaga and Parapusillimonas shows that among a

179 group of more than 50 tests of carbon source assimilation, enzyme production/activity and

180 other physiological traits, these characteristics are variable within each genus and/or not

181 differentiating among these genera (Kersters et. al, 1984; Foss et al., 1989; Vandamme et.

182 al, 1995; Weyant et. al, 1995; Vandamme et. al, 1996; Blümel et. al, 2001; von

183 Wintzingerode et. al, 2001; Coenye et. al., 2003; Busse & Auling, 2005; Stolz et. al, 2005;

184 Kampfer et. al, 2006; Yoon et. al, 2007; Liu et. al, 2008; Chen et. al, 2009; Kim et. al,

185 2009; Kim et. al, 2010; Lee et. al, 2010; Srinivasan et. al, 2010). In this respect, the

186 comparison of the type strains of the type species and of the closely related species, as we

8 187 did in the current study and Kim et al. (2010) did in the description of the genus

188 Parapusillimonas, allows an objective comparison of related taxa, useful for future

189 identification or comparative assays. Due to the limitations mentioned above, some authors

190 have applied additional methods useful to allow the distinction of the genera of this family,

191 for example 16S rRNA gene sequence signatures or qualitative comparisons of polar lipid

192 patterns. For this reason, strains SC-089T and SC-092T were also compared with the type

193 strains of closest species and type strains of type species of related genera on basis of such

194 criteria (Tables 3 and 4, Figure 2). Both approaches allowed the distinction of these strains

195 from the related genera, supporting the proposal of a new genus.

196 Strains SC-089T and SC-092T were observed to be phenotypically distinct, namely in the

197 ability to reduce nitrate, to grow anaerobically, to oxidise glucose, to assimilate some

198 carbon sources, namely potassium 2-ketogluconate, 4-hydroxybenzoic acid, trisodium

199 citrate, malate and potassium gluconate, and in the mol% G+C content. These differences

200 associated with a value of 99.1 % of 16S rRNA gene sequence, the low DNA-DNA

201 hybridization values between these two strains further demonstrated that both organisms

202 are best classified within two novel species for which we propose the names Candidimonas

203 nitroreducens and Candidimonas humi, represented by the type strains SC-089T and SC-

204 092T, respectively.

205

206 Description of Candidimonas gen. nov.

207 Candidimonas (Can.di.di.mo'nas. L. adj. candidus -a -um, white; L. fem. n. monas, a unit,

208 monad; N.L. fem. n. Candidimonas, a unit (rod) that produces white colonies).

209

9 210 Cells are non spore-forming, Gram-negative, non-motile, curved short rods. Catalase- and

211 cytochrome c oxidase-positive. Mesophilic. Chemoorganotrophic with aerobic respiratory

212 metabolism. Amino acids and organic acids are used as single carbon sources. Anaerobic

213 growth, nitrate reduction, glucose oxidation and the assimilation of some organic acids as

214 sole carbon sources are variable characteristics of the genus. The respiratory quinone is

215 ubiquinone 8 and the DNA G+C content is 64-65 mol%. The fatty acids summed feature 2

216 (C14:0 3OH or C16:1 iso I or both), C16:0 and C18:1 w7c are among the major cellular fatty

217 acids. The polar lipids present are phosphatidylethanolamine, phosphatidylglycerol,

218 diphosphatidylglycerol and four unidentified aminophospholipids. Phylogenetically

219 belongs to the family Alcaligenaceae. The type species is Candidimonas nitroreducens.

220

221 Description of Candidimonas nitroreducens sp. nov.

222 Candidimonas nitroreducens (ni.tro.re'du.cens. N.L. n. nitras -atis, nitrate; N.L. pref. nitro-,

223 pertaining to nitrate; L. part. adj. reducens, leading back, bringing back

224 and, in chemistry, converting to a different oxidation state; N.L. part.

225 adj. nitroreducens, reducing nitrate).

226 Colonies are white, circular (~1 mm diameter) and convex on PCA after 48 h of incubation.

227 Cells are Gram-negative, non-motile, coccobacilli (0.53±0.11 µm). Catalase- and oxidase-

228 positive. Growth occurs between 15-40 ºC, pH 5-8 and up to 3 % NaCl, with optima at

229 about 30 ºC, pH 7 and 1 % NaCl, respectively. Under anaerobiosis growth is slow and

230 weak and nitrate is reduced to nitrite. Simmons citrate is not utilized. H2S, indole and

231 acetoin are not produced. Esculin is not hydrolysed. Glucose is oxidised (API 20E) but D-

232 mannitol, inositol, D-sorbitol, L-rhamnose, D-saccharose, D-melibiose, amygdalin and L-

10 233 arabinose are not. Glucose is not fermented. The enzymes acid- and alkaline phosphatase,

234 esterase C4, esterase lipase C8, leucine and valine arylamidase, naphtol-AS-BI-

235 phosphohydrolase are produced. The enzymes arginine dihydrolase, lysine and ornithine

236 decarboxylase, tryptophane deaminase, gelatinase, urease, lipase (C14), cystine

237 arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-

238 glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase

239 are not produced. The following sole carbon sources are assimilated: adipate, phenyl-

240 acetate, itaconic acid, sodium acetate, lactic acid, 3-hydroxibenzoic acid, propionic acid,

241 valeric acid, 3-hydroxibutyric acid and 4-hydroxibenzoic acid. The assimilation of malate

242 and potassium gluconate support weak growth. The following sole carbon sources are not

243 assimilated: D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, D-

244 maltose, citrate, L-rhamnose, D-ribose, inositol, D-saccharose, suberic acid, sodium

245 malonate, L-alanine, potassium 5-ketogluconate, glycogen, L-serine, salicin, D-melibiose,

246 L-fucose, D-sorbitol, capric acid, trisodium citrate, L-histidine, potassium 2-ketogluconate

247 and L-proline.

248 The predominant fatty acids were C16:0, summed feature 2 (C14:0 3OH or C16:1 iso I or both),

T T 249 cyclo-C17:0 and C18:1 w7c. DNA G+C content is 64 mol%. SC-089 (= CCUG 55806

250 =LMG 24812T) is the type strain, isolated from sewage sludge compost.

251

252 Description of Candidimonas humi sp. nov.

253 Candidimonas humi (hu'mi. L. n. humus, earth, soil and, in earth sciences or agriculture,

254 humus; L. gen. n. humi, of the soil, of the humus).

11 255 Colonies are white, circular (~1 mm diameter), convex and rough on PCA after 48 h of

256 incubation. Cells are Gram-negative, non-motile, coccobacilli (0.50±0.09 µm). Catalase-

257 and oxidase- positive. Growth occurs between 15-40 ºC, pH 5-8 and up to 3 % NaCl, with

258 optima at about 30 ºC, pH 7 and 1 % NaCl, respectively. Unable to grow under

259 anaerobiosis and to reduce nitrate. Simmons citrate is not utilized. H2S, indole and acetoin

260 are not produced. Esculin is not hydrolysed. Glucose is not fermented. The enzymes acid-

261 and alkaline phosphatase, esterase C4, esterase lipase C8, leucine and valine arylamidase,

262 naphtol-AS-BI-phosphohydrolase and α-chymotrypsin are produced. The enzymes arginine

263 dihydrolase, lysine and ornithine decarboxylase, tryptophane deaminase, gelatinase, urease,

264 lipase (C14), cystine arylamidase, trypsin, α-galactosidase, β-galactosidase, β-

265 glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase

266 and α-fucosidase are not produced. The following sole carbon sources are assimilated:

267 adipate, citrate, phenyl-acetate, itaconic acid, sodium acetate, lactic acid, 3-hydroxibenzoic

268 acid, propionic acid, valeric acid, trisodium citrate, potassium 2-ketogluconate and 3-

269 hydroxibutyric acid. The carbon sources D-glucose, L-arabinose, D-mannose, D-mannitol,

270 N-acetylglucosamine, D-maltose, malate, potassium gluconate, L-rhamnose, D-ribose,

271 inositol, D-saccharose, suberic acid, sodium malonate, L-alanine, potassium 5-

272 ketogluconate, glycogen, L-serine, salicin, D-melibiose, L-fucose, D-sorbitol, capric acid,

273 L-histidine, 4-hydroxybenzoic acid and L-proline are not assimilated.

274 The predominant fatty acids are C16:0, summed feature 2 (C14:0 3OH or C16:1 iso I or both),

275 summed feature 3 (C16:1 w7c or C15:0 iso 2OH or both) and C18:1 w7c. DNA G+C content is

276 65 mol%. SC-092T (= CCUG 55807T =LMG 24813T) is the type strain, isolated from

277 sewage sludge compost.

12 278

279 Acknowledgments:

280 The authors acknowledge the expertise of K. Molin and Professor Enevold Falsen (CCUG)

281 for their technical and scientific assistance.

282

283 References

284 Blümel, S., Mark, B., Busse, H.-J., Kämpfer, P. & Stolz, A. (2001). Pigmentiphaga

285 kullae gen. nov., a novel member of the family Alcaligenaceae with the ability to

286 decolorize azo dyes aerobically. Int J Syst Evol Microbiol 51, 1867-1871.

287 Busse, H.-J. & Auling, G. (2005). Family III. Alcaligenaceae De Ley, Segers, Kersters,

288 Mannheim and Lievens 1986, 412VP. In Bergey’s Manual of Systematic Bacteriology,

289 vol. 2, pp. 647-685. Edited by D. J. Brenner, N. R. Krieg & J. T. Stanley. Springer.

290 Chen, Y.-G., Zhang, Y.-Q., Huang, K., Tang, S.-K., Cao, Y., Shi, J.-X., Xiao, H.-D.,

291 Cui, X.-L. & Li, W.-J. (2009). Pigmentiphaga litoralis sp. nov., a facultatively anaerobic

292 bacterium isolated from a tidal flat sediment. Int J Syst Evol Microbiol 59, 521-525.

293 Coenye, T., Vancanneyt, M., Falsen, E., Swings, J. & Vandamme, P. (2003).

294 Achromobacter insolitus sp. nov. and sp. nov., from human

295 clinical samples. Int J Syst Evol Microbiol 53, 1819-1824.

296 Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid

297 deoxyribonucleic acid hybridization in microdilution wells as an alternative to

298 membrane-filter hybridization in which radioisotopes are used to determine genetic

299 relatedness among bacterial strains. Int J Syst Bacteriol 39, 224-229.

13 300 Ferreira da Silva, M., Vaz-Moreira, I., Gonzalez-Pajuelo, M., Nunes, O.C. & Manaia,

301 C.M. (2007). Antimicrobial resistance patterns in Enterobacteriaceae isolated from an

302 urban wastewater treatment plant. FEMS Microbiol Ecol 60, 166-176.

303 Foss, S., Heyen, U. & Harder, J. (1998). defragrans sp. nov., description of

304 four strains isolated on alkenoic monoterpenes ((+)-menthene, α-pinene, 2-carene, and α-

305 phellandrene) and nitrate. Syst Appl Microbiol 21, 237-244.

306 Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian

307 Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.

308 Kämpfer, P., Denger, K., Cook, A. M., Lee, S.-T., Jäckel, U., Denner. E. B. M. &

309 Busse, H.-J. (2006). Castellaniella gen. nov., to accommodate the phylogenetic lineage

310 of , and proposal of Castellaniella defragrans gen. nov., comb.

311 nov. and Castellaniella denitrificans sp. nov.. Int J Syst Evol Microbiol 56, 815-819.

312 Kim, M.K., Srinivasan, S., Kim, Y.J. & Yang, D.C. (2009). Castellaniella ginsengisoli

313 sp. nov., a β-glucosidase-producing bacterium. Int J Syst Evol Microbiol 59, 2191-2194.

314 Kim, Y.-J., Kim, M. K., Im, W.T., Srinivasan, S. & Yang, D.-C. (2010)

315 Parapusillimonas granuli gen. nov., sp. nov., isolated from granules from a wastewater-

316 treatment bioreactor. Int J Syst Evol Microbiol 60, 1401-1406.

317 Lee, M., Jung, H.M., Woo, S.G., Yoo, S.A. & Ten, L.N. (2010). Castellaniella

318 daejeonensis sp. nov., isolated from soil. Int J Syst Evol Microbiol 60, 2056-2060.

319 Liu, Q.M., Ten, L.N., Im, W.T. & Lee, S.T. (2008). Castellaniella caeni sp. nov., a

320 denitrifying bacterium isolated from sludge of a leachate treatment plant. Int J Syst Evol

321 Microbiol 58, 2141-2146.

322 Manaia, C. M., Nogales, B., Weiss, N. & Nunes, O. C. (2004). Gulosibacter

323 molinativorax gen. nov., sp. nov., a molinate-degrading bacterium, and classification of

14 324 ‘Brevibacterium helvolum’ DSM 20419 as Pseudoclavibacter helvolus gen. nov., sp. nov.

325 Int J Syst Evol Microbiol 54, 783-789.

326 Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the

327 G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J

328 Syst Bacteriol 39, 159-167.

329 Murray, R. G. E., Doetsch, R. N. & Robinow, F. (1994). Determinative and cytological

330 light microscopy. In Methods for general and molecular bacteriology, p. 21-41. Edited by

331 P. Gerhardt, R. G. E. Murray, W. A. Wood, & Krieg N. R. Washington, DC: American

332 Society for Microbiology.

333 Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial

334 genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151-156.

335 Sanden, G. N. & Weyant, R. S. (2005). Genus III. Bordetella Moreno-López 1952, 178AL.

336 In Bergey’s Manual of Systematic Bacteriology, vol. 2, pp. 662-671. Edited by D. J.

337 Brenner, N. R. Krieg & J. T. Stanley. Springer.

338 Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for

339 general and molecular bacteriology, p. 611-651. Edited by P. Gerhardt, R. G. E. Murray,

340 W. A. Wood, & Krieg N. R.. Washington, DC: American Society for Microbiology.

341 Srinivasan, S., Kim, M.K., Sathiyaraj, G., Kim, Y.J. & Yang D.C. (2010). Pusillimonas

342 ginsengisoli sp. nov., isolated from soil of a ginseng field. Int. Int J Syst Evol Microbiol

343 60, 1783-1787.

344 Stolz, A., Bürger, S., Kuhm, A., Kämpfer, P. & Busse, H.-J. (2005). Pusillimonas

345 noertemannii gen. nov., sp. nov., a new member of the family Alcaligenaceae that

346 degrades substituted salicylates. Int J Syst Evol Microbiol 55, 1077–1081.

15 347 Tindall, B. J. (1989). Fully saturated menaquinones in the archaebacterium Pyrobaculum

348 islandicum. FEMS Microbiol Lett 60, 251–254.

349 Vancanneyt, M., Vandamme, P. & Kersters, K. (1995). Differentiation of Bordetella

350 pertussis, B. parapertussis and B. bronchiseptica by Whole-Cell Protein Electrophoresis

351 and Fatty Acid Analysis. Int J Syst Bacteriol 45(4), 843-847.

352 Vandamme, P., Vancanneyt, M., Pot, B., Mels, L., Hoste, B., Dewettinck, D., Vlaes, L.,

353 Van Den Borre, C., Higgins, R., Hommez, J., Kersters, K., Butzler, J.-P. & Goossens

354 H. (1992). Polyphasic taxonomic study of the emended genus Arcobacter with

355 Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant

356 bacterium isolated from veterinary specimens. Int J Syst Bacteriol 42, 344-356.

357 Vandamme, P., Hommez, J. Vancanneyt, M., Monsieurs, M., Hoste, B., Cookson, B.,

358 Wirsing von König, C.H., Kersters, K. & Blackall, P.J. (1995). sp.

359 nov., isolated from poultry and humans. Int J Syst Bacteriol 45, 37-45.

360 Vandamme, P., Heyndrickx, M., Vancanneyt, M., Hoste, B., de Vos, P., Falsen, E.,

361 Kersters, K. and Hinz, K.H. (1996). sp. nov., isolated from

362 wounds and ear infections in humans, and reassessment of Alcaligenes denitrificans

363 Rüger and Tan 1983. Int J Syst Bacteriol 46, 849-858.

364 Vaz-Moreira, I., Nobre, M. F., Nunes, O. C. & Manaia, C. M. (2007). Gulbenkiania

365 mobilis gen. nov., sp. nov., isolated from treated municipal wastewater. Int J Syst Evol

366 Microbiol 57, 1108-1112.

367 Vaz-Moreira, I., Silva, E., Manaia, C. M. & Nunes, O. C. (2008). Diversity of bacterial

368 isolates from commercial and homemade composts. Microb Ecol 55, 714-722.

369 Weyant, R.S., Hollis, D.G., Weaver, R.E., Amin, M.F.M., Steigerwalt, A.G., O'connor,

370 S.P., Whitney, A.M., Daneshvar, M.I., Moss, C.W. & Brenner, D.J. (1995).

16 371 sp. nov., a new gram-negative species associated with septicemia. J

372 Clin Microbiol 33, 1-7.

373 von Wintzingerode, F., Schattke, A., Siddiqui, R. A., Rösick, U., Göbel, U. B. &

374 Gross, R. (2001). Bordetella petrii sp. nov., isolated from an anaerobic bioreactor, and

375 emended description of the genus Bordetella. Int J Syst Evol Microbiol 51, 1257-1265.

376 Yoon, J.H., Kang, S.J., Kim, W. & Oh, T.K. (2007). Pigmentiphaga daeguensis sp. nov.,

377 isolated from wastewater of a dye works, and emended description of the genus

378 Pigmentiphaga. Int J Syst Evol Microbiol 57, 1188-1191.

17 379 Table 1: Average fatty acid composition of the strains SC-089T (1), SC-092T (2), and the 380 related type strains Pigmentiphaga kullae LMG 21665T (3), Bordetella bronchiseptica 381 LMG 1232T (4), Bordetella petrii CCUG 43448T (5), Achromobacter xylosoxidans LMG 382 1863T (6), Achromobacter denitrificans LMG 1231T (7), Castellaniella defragrans LMG 383 18538T (8), Castellaniella denitrificans LMG 25533T (9), Pusillimonas noertemannii DSM 384 10065T (10), Parapusillimonas granuli LMG 24012T (11). Fatty acids 1c 2 3d 4 5 6 7 8 9 10e 11

C10:0 2OH 1.7 T ND ND ND ND ND ND ND ND

C12:0 T T ND T T T T 4.8 6.5 8.2

C12:0 2OH 2.4 4.0 ND 2.7 2.5 3.9 3.4 ND ND 6.3

C14:0 ND ND ND 4.8 5.4 1.7 5.1 T T ND

C14:0 2OH ND ND 4.6 ND T 3.4 ND ND ND ND SF2a 20.4 16.7 6.8 7.6 8.4 10.6 10.7 6.4 8.6 22.7 SF3b 9.7 25.2 9.6 26.3 20.0 16.0 22.4 21.7 24.7 3.9

C16:0 25.9 25.1 28.4 34.1 32.2 33.8 30.2 30.4 31.6 10.3

C17:0 cyclo 17.9 6.2 17.1 10.6 16.0 19.6 15.2 15.8 13.0 18.9

C16:1 2OH 1.5 3.6 1.1 ND ND ND ND ND ND ND

C16:0 2OH 1.9 1.6 2.9 ND ND ND ND ND ND ND

C18:1 w7c 10.8 13.1 17.5 10.2 9.8 5.6 7.8 17.4 12.7 2.6

C18:0 2.4 3.6 1.1 2.4 2.6 1.5 1.5 T T 1.4

C19:0 cyclo w8c 1.3 ND 5.0 ND T T T T T 10.1 385 Those fatty acids for which the amount for all taxa was less than 1 % are not included. Therefore, the 386 percentages may not add up to 100 %. T, trace amount (less than 1 %); ND, not detected. a 387 SF 2, summed feature 2 (C14:0 3OH or C16:1 iso I or both). b 388 SF 3, summed feature 3 (C16:1 w7c or C15:0 iso 2OH or both). c T 389 strain SC-089 also contains 3.1 % C17:0 iso 3OH. d T 390 P. kullae LMG 21665 also contains 3.4 % C10:0 3OH , 2.4 % C18:1 2OH. e T 391 P. noertemanii DSM 10065 also contains 2.9 % C19:0 10 methyl, 4.2 % C16:0 3OH and 5.8 % C18:1 2OH

18 392 Table 2. Distinctive characteristics of strains SC-089T (1), SC-092T (2), and the related type strains Pigmentiphaga kullae CCUG 47266T 393 (3), Bordetella petrii CCUG 43448T (4), Bordetella bronchiseptica LMG 1232T (5), Achromobacter xylosoxidans LMG 1863T (6), 394 Achromobacter denitrificans LMG 1231T (7), Castellaniella defragrans LMG 18538T (8), Castellaniella denitrificans LMG 25533T (9), 395 Pusillimonas noertemannii DSM 10065T (10) and Parapusillimonas granuli LMG24012T (11) (tested in parallel in the present study). 1 2 3 4 5 6 7 8 9 10 11 Motility - - +1 -2 +3 +4 +4 +5 +5 +6 +8 Simmons Citrate - - - + - + - + - - - Urease - - - - + - - - - - + Assimilation Glucose - - + - - + - - - +w + D-Mannose - - + - - +w - - - - - L-Alanine - - - + + + + + + - + L-Proline - - - + - + + + + - + L-Serine - - - + - + - + - - + Adipate + + + + + + + - - - + Caprate - - - - +w + +w +w +w - - 3-Hydroxibenzoic acid + + + - - - + + + + + Itaconic acid + + +w + + + + - + - + Lactic acid + + - + + + + + + - + Phenyl acetate + + - - + + + +w - - + Propionic acid + + - + + + + + + + + Sodium acetate + + - + + + + + + + + Suberic acid - - - + - + + - - - - Valeric acid + + - + + + + + + - + G+C (mol%) 63.9 65.2 68.51 63.82 67-69*3 66.9- 63.9- 66.9*7 n.a. 61.8*6 67.98 69.8*4 68.9*4 Source of isolation of Sewage Sewage Soil Dechlorina Respirator Mostly Soil / Alkenoic Anaerobic Water Anaerobic the type strain sludge sludge ting y tract of clinical clinical monoterpe sludge sludge compost compost bioreactor animals isolates isolates nes digestor blanket / clinical reactor 396 Data from: 1Blümel et al., 2001; 2von Wintzingerode et al., 2001; 3Sanden & Weyant, 2005; 4Busse & Auling, 2005; 5Kämpfer et al., 2006; 6Stolz et al., 2005; 7Foss et al., 1998; 8Kim et al., 2010. 397 w weak reaction; n.a., not available; v, variable; *determined by Tm 398

19 399 400 Table 3. Pattern of 16S rRNA signature nucleotides that distinguish different genera within the family Alcaligenaceae, selected as proposed by Blümel 401 et al. (2001). 402 Positions given are relative to Escherichia coli numbering and this analysis included the type strains of the validly named species belonging to each of 403 the indicated genera. The accession numbers are as indicated in figure 1. 404 Positions Candidimonas Achromobacter Bordetella Castellaniella Pigmentiphaga Pusillimonas Parapusillimonas 43:399 G:C G:C G:C G:C C:G G:C G:C 82:87 C:G C:G C:G G:C C:G A:U C:G 83 U U U C U U6 U 85 C C C U C G6 C 123:238 U:A U:A U:A U:A C:G5 U:A U:A 200:217 G:C A:U G:C G:C G:C G:C G:C 207:212 U:A U:A C:G U:A U:A C:G U:A 208 G G U G G C6 G 209 C C U C C U6 C 210 A A C A A U6 A 211 A A G A A C6 A 412 U U U U A U U 624:616 C:G U:A1 C:G2 C:G2 C:G C:G6 C:G 673:717 A:U G:C1 G:C A:U G:C A:U A:U 1060:1197 U:A U:A U:A U:A C:G U:A U:A 1030 C C C C A C C 1137 A A A G A A A 1256 C U U3 U4 C U U 1283 C U C U C U6 C 1308 C C C C U U U 1329 G G G G A A A 405 1, in A. insolitus C:G and A:U pairs are observed in positions 624:616 and 673:717, respectively; 2, in B. avium, B. hinzii, C. defragrans a U:A pair is 406 observed; 3, C in B. petrii; 4, A in C. caeni; 5, in Pigmentiphaga litoralis a U:A pair is observed in this position; 6, in Pusillimonas gisengisoli is 407 observed G in position 83 and 208, U in position 87, C in position 209 and 1283, A in position 211 and 412 and the pair A:U in position 624:616. 408 409

20 410 Table 4. Qualitative comparison of the polar lipids profiles of different genera of the family Alcaligenaceae, according to criteria used by 411 Stolz et al. (2005) 412 Polar lipid* Candidimonas Achromobacter Bordetella Castellaniella Pigmentiphaga Pusillimonas Parapusillimonas PE + + + + + + + + + + + PG + + + + + + + + + + DPG + + + + + + ± + + + APL1 + - - - - - + APL2 + ------APL3 + ------APL4 + ------APL5 - - + - - - - APL6 - - + - - - - AL1 - + + + - + - AL2 - + - - - ++ - 413 * Details in Figure 2 414 PE, phosphatidylethanolamine; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; APL, unidentified aminophospholipid; AL 415 unidentified aminolipid.

21 416 0.01 Castellaniella defragransLMG18538T (AJ005447) T 417 100 Castellaniella caeni LMG23411 (AB166879) 97 Castellaniella ginsengisoli JCM15515T (EU873313) 418 90 Castellaniella daejeonensis MJ06T (GQ241321)) 95 T 419 Castellaniella denitrificans DSM11046 (AU82826) Pigmentiphaga litoralis JSM061001T (EU583723) 100 420 99 Pigmentiphaga kullae DSM13608T (AF282916) 100 T 421 Pigmentiphaga daeguensisJCM14330 (EF100696) SC‐092T (FN556192) 100 422 SC‐089T (FN556191) 74 T 423 Parapusilimonas granuli LMG 24012 (DQ466075) 87 Pusillimonas ginsengisoli DCY25T (EF672088)) 59 424 Pusillimonas noertemannii DSM10065T (AY695828) Achromobacter xylosoxidans DSM10346T (Y14908) 425 ATCC15749T (AB010840) 100 83 426 Achromobacter denitrificans DSM 30026T (AJ278451) 58 Achromobacter insolitus LMG6003T (AY170847) 427 50 ATCC43552T (AB010841) 99 98 428 Achromobacter spanius LMG5911T (AY170848) Bordetella petrii DSM12804T (AJ249861) 429 Bordetella trematum DSM11334 T (AJ277798) 54 59 430 ATCC35086T (AF177666) 64 Bordetella hinzii LMG13501T (AF177667) 431 T 60 Bordetella holmesii ATCC51541 (U04820) 79 Bordetella pertussis ATCC9797T (U04950) 432 99 Bordetella bronchiseptica ATCC19395T (U04948) 433 87 ATCC15311T (U04949) T 434 Zoogloea ramigera ATCC19324 (D14257) 435 Figure 1. Phylogenetic tree derived from 16S rRNA gene sequence analysis showing the 436 relationship of strains SC-089T and SC-092T with members of the family Alcaligenaceae. 437 The tree was generated by the neighbour-joining method and Zoogloea ramigera ATCC 438 19324T was used as outgroup. Bootstrap values, generated from 1000 re-samplings, at or 439 above 50 % are indicated at the branch points. Dark circles indicate branches recovered by 440 the maximum likelihood method. Bar, 1 substitution per 100 nt positions. 441

22 442

443

444

445 Figure 2. Polar lipid pattern of strains SC-089T, SC-092T, Pigmentiphaga kullae CCUG 446 47266T, Bordetella bronchiseptica LMG 1232T, Bordetella petrii CCUG 43448T, 447 Achromobacter xylosoxidans LMG 1863T, Castellaniella defragrans LMG 18538T, 448 Pusillimonas noertemannii DSM 10065T and Parapusillimonas granuli LMG 24012T. 449 PE, phosphatidylethanolamine; PG, phosphatidylglycerol; DPG, 450 diphosphatidylglycerol; PL, APL, GL and AL, respectively, unidentified phospholipid, 451 aminophospholipid, glicolipid and aminolipid.

23