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 – PROTEOBACTERIA
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 Alcaligenaceae, 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 bacteria 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, Achromobacter, Bordetella, Castellaniella, Pigmentiphaga and
54 Pusillimonas, with Parapusillimonas granuli, Pigmentiphaga kullae and Bordetella petrii
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 Bordetella pertussis, 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
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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 Achromobacter ruhlandii ATCC15749T (AB010840) 100 83 426 Achromobacter denitrificans DSM 30026T (AJ278451) 58 Achromobacter insolitus LMG6003T (AY170847) 427 50 Achromobacter piechaudii ATCC43552T (AB010841) 99 98 428 Achromobacter spanius LMG5911T (AY170848) Bordetella petrii DSM12804T (AJ249861) 429 Bordetella trematum DSM11334 T (AJ277798) 54 59 430 Bordetella avium 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 Bordetella parapertussis 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