Proposal of Henriciella barbarensis sp. nov. and Henriciella algicola sp. nov., stalked species of the genus and emendation of the genus Henriciella.

Item Type Article

Authors Abraham, Wolf-Rainer; de Carvalho, Maira Peres; da Costa Neves, Thais Souto Paula; Memoria, Marina Torquato; Tartuci, Iago Toledo; Vancanneyt, Marc; Smit, John; Rohde, M

Citation Proposal of Henriciella barbarensis sp. nov. and Henriciella algicola sp. nov., stalked species of the genus and emendation of the genus Henriciella. 2017, 67 (8):2804-2810 Int. J. Syst. Evol. Microbiol.

DOI 10.1099/ijsem.0.002024

Journal International journal of systematic and evolutionary microbiology

Download date 06/10/2021 03:29:09

Item License http://creativecommons.org/licenses/by-nc-sa/4.0/

Link to Item http://hdl.handle.net/10033/621103 1Proposal of Henriciella barbarensis sp. nov. and Henriciella algicola sp. nov., stalked 2species of the genus and emendation of the genus Henriciella 3 4Wolf-Rainer Abraham*1, Maira Peres de Carvalho1, Thaís Souto Paula da Costa Neves1, 5Marina Torquato Memória1, Iago Toledo Tartuci1, Marc Vancanneyt2, John Smit3, and 6Manfred Rohde4 7 81Helmholtz Centre for Infection Research, Chemical Microbiology, Inhoffenstrasse 7, 38124 9Braunschweig, Germany; 2BCCM/LMG Bacteria Collection, Universiteit Gent, K.L. 10Ledeganckstraat 35, Gent; Belgium; 3Dept. of Microbiology and Immunology, University of 11British Columbia, Vancouver, British Columbia, Canada; 4Central Facility for Microscopy, 12Helmholtz Centre for Infection Research 13 14 15Running title: Henricella barbarensis and H. algicola sp. nov. 16 17Subject category: New taxa; Subsection: Alphaproteobacteria 18 19Keywords: Henriciella, Hyphomonadaceae, lipids, marine bacteria 20 21 22*Corresponding author: 23Dr. Wolf-Rainer Abraham 24Helmholtz Centre for Infection Research, Chemical Microbiology 25Inhoffenstrasse 7, 38124 Braunschweig, Germany 26Tel. +49 531 6181 4300; Fax: +49 531 6181 4699 27e-mail: [email protected]

1 2 1 28Abstract 29Two Gram-negative, heterotrophic, aerobic, prosthecated, marine bacteria, designated strains 30MCS23T and MCS27T, were isolated from seawater samples. NaCl was required for growth. 31The major polar lipid detected in strain MCS27T was phosphatidylglycerol, whereas those 32detected in MCS23T were phosphatidylglycerol, sulfoquinovosyl diacylglycerol, and 1,2- 33diacyl-3-α-D-glucuronopyranosyl-sn-glycerol taurineamide. Most abundant cellular fatty

34acids were C18:1ω7 and C16:0, hydroxyl-fatty acids were 3-OH C12:0 in both strains and 3-OH

T T T 35C11:0 in MCS23 . Strains MCS23 and MCS27 had DNA G+C contents of 57.0 and 55.0 mol 36%, respectively. The two strains shared 99.3 % 16S rRNA gene sequence similarity; levels of 37similarity with the type strains of species of the genus were 99.4-97.8 % but DNA-DNA 38hybridizations were 53% or lower. Beside by their 16S rRNA gene sequences they can be 39differentiated by cell morphology, lipid and fatty acid patterns and enzyme activities from 40other Henriciella species. The data obtained led to the identification of two novel species, for 41which the names Henriciella barbarensis sp. nov. (type strain MCS23T =LMG 28705T = 42CCUG 66934T) and Henriciella algicola sp. nov. (type strain MCS27T =LMG 29152T = 43CCUG 67844T) are proposed. Because these two novel species are the first prosthecate 44species in this genus, the description of Henriciella was here emended as well. 45 46The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequences of strain 47MCS23T is AJ227807 and for MCS27T is KP722025

48

49Cauloform bacteria are dimorphic, prosthecate bacteria, which reproduce by separation of two 50cells that are morphologically different from each other. One of these cells is nonmotile, 51sessile by means of a prosthecum and the other cell is flagellated and motile, bearing one 52polar flagellum (Staley, 1968). The mode of reproduction of the dimorphic prosthecate 53bacteria reflects their life in oligotrophic habitats by fostering the dispersion of the population 54at each generation minimizing competition for resources (Poindexter, 1981). It took more 55than 100 years after the report of the first isolation of a Caulobacter sp. (Loeffler, 1890) to 56realize, by analyzing the 16S rRNA genes, that these bacteria belonged to two different 57families, Caulobacteraceae and Hyphomonadaceae (Stahl et al., 1992, Abraham & Rohde, 582014). A polyphasic study of a large set of cauloform isolates revealed that these strains 3 4 2 59belonged to the genera Caulobacter, Brevundimonas, Phenylobacterium and Maricaulis 60(Abraham et al. 1999, Abraham et al., 2008). Some isolates, however, were so different from 61all known species that they could not be assigned to one of them. We report here the 62characterization of two marine isolates obtained from different habitats that have their closest 63relatives in the genus Henriciella but are prosthecated and sufficiently distinct from its 64accepted members that we propose here the new species Henriciella algicola sp. nov. and 65Henriciella barbarensis sp. nov. for them.

66The strains of this study were obtained from the German Collection of Microorganisms and 67Cell Cultures (DSMZ), Laboratory of Microbiology, Department of Biochemistry and 68Microbiology, Faculty of Sciences of Ghent University (LMG) and from John Smit of the 69University of British Columbia (MCS strains). All strains were grown in the

70marine-Caulobacter-medium SPYEM: 30 g sea salts (Sigma), 0.5 g NH4Cl, 1 l deionized 71water. After autoclaving and cooling 20 ml 50xPYE, 2 ml 50 % glucose (sterile) and 5 ml 72riboflavin (0.2 mg ml-1, sterile filtrated, were added. 50xPYE: 10% peptone and 5% yeast 73extract in deionized water (autoclaved). The strains were cultivated in 2 l flasks at 30° C, 74shaken at 100 r p m and the biomass was harvested in the late exponential phase after 72 h. 75Anaerobic growth was tested on SPYEM plates in an anaerobic chamber (GasPak, BBL 76Microbiology Systems) (Anast & Smit, 1988).

77Genomic DNA was isolated from two inoculating loops full of bacterial cells using the 78DNeasy Blood and Tissue kit for purification of total DNA (Qiagen) with the addition of 79RNase A (Sigma), according to the manufacturers’ instructions. DNA was enzymatically 80digested as described by Gehrke et al. (1984) and the mean G+C content was determined by 81HPLC (Tamaoka & Komagata, 1984). Calculations were carried out according to Mesbah et 82al., (1989), with non-methylated lambda-phage DNA (Sigma) as a standard. The G+C 83contents of MCS23T (57.0%) and MCS27T (55.0%) were well within the range described for 84the genus Henriciella (Lee et al., 2011).

85For 16S rDNA sequencing and analysis single colonies were picked from plates, suspended in 86100 μl TE-buffer and boiled for 5 min. The suspension was briefly centrifuged and 1 μl of the 87supernatant was used for PCR. A nearly complete 16S rRNA gene sequence was obtained as 88described previously (Abraham et al., 1999) and the final contig was assembled using the

5 6 3 89program SEQUENCHER Version 4.0.5 (Gene Codes Corporation, USA). The 16S rRNA 90gene sequences determined have been deposited in the EMBL Nucleotide Database. The 16S 91rRNA gene sequences of the type strains of species of the genus Henriciella were obtained 92from the EMBL database (Leinonen et al., 2010). The nucleotide sequences were aligned 93using the evolutionary conserved primary sequence and secondary structure (Gutell et al., 941985) as references. Evolutionary distances (Jukes & Cantor, 1969) were calculated using 95CLUSTAL Omega software (Sievers & Higgins, 2014) using only identical, unambiguously 96determined nucleotide positions. Tree topologies were calculated with MEGA 6.06 software 97(Tamura et al., 2013) applying the maximum-likelihood algorithm (Fig. 1), and the 98neighbour-joining algorithm with 500 bootstrap replications (Fig. S1, available in IJSEM 99Online).

100 101Figure 1: Maximum likelihood tree based on 16S rRNA gene sequences, showing the 102phylogenetic relationships of strains MCS23T and MCS27T and related taxa. Bootstrap 103percentages (based on 150 replications) are shown where >50 %. Escherichia coli served as 104outgroup. Bar, 0.02 substitutions per nucleotide position. 7 8 4 105From the analysis of the 16S rRNA gene sequences species of the genus Henriciella were 106found to be the closest relatives of strains MCS23T and MCS27T. The similarity of the 16S 107rRNA gene sequences of MCS23T to H. marina DSM 19595T, H. aquamarina LMG 24711T 108and H. litoralis DSM 22014T were 99.4%, 98.1% and 97.8%, respectively. For MCS27T the 109similarities to these type strains were determined to be 98.9%, 98.5% and 98.3%, while the 110similarity between MCS23T and MCS27T was 99.3%. Therefore, to decide whether MCS23T 111and MCS27T belong to any of the accepted three Henriciella species or are distinct species, 112DNA-DNA-hybridizations between these five type strains were required.

113DNA-DNA hybridization was done by the service of DSMZ and chromosomal DNA of high- 114molecular weight was isolated according to the method of Cashion et al. (1977). Degrees of 115DNA-DNA binding, expressed as percentages, were determined spectrophotometrically using 116the initial renaturation methods of De Ley et al. (1970) and Huss et al. (1983). DNA-DNA- 117Hybridization between strains MCS 23T and MCS 27T gave 53% similarity and similarities 118between MCS23T or MCS27T and Henriciella marina DSM 19595T, H. marina DSM 19595T 119or H. aquimarina LMG 24711T were 50% or lower (Suppl. Table S1). Because these values 120are below 70% (Wayne et al., 1987) this proved that strains MCS 23T and MCS 27T are both 121novel species within the genus Henriciella.

122For whole cell fatty acid analysis cells were saponified (15% (w/v) NaOH, 30 min, 100° C), 123methylated to fatty acid methyl esters (FAMEs) (methanolic HCl, 10 min, 80° C) and 124extracted (hexane/methyl-tert-butyl ether (1:1, v/v)) as described in detail by Osterhout et al. 125(Osterhout et al. 1991). Fatty acid methyl esters were analyzed on a Hewlett-Packard (HP) 1265890A gas chromatograph. Separation of fatty acid methyl esters was achieved with a fused- 127silica capillary column (25 m by 0.2 mm) with cross-linked 5% phenyl methyl silicone (film 128thickness 0.33 μm; HP Ultra 2). The computer-controlled parameters were the same as those 129described by Osterhout. The instrument was equipped with a flame ionization detector and an

130autosampler (HP 7673). H2 was serving as carrier gas. While C18:1ω7 was the main fatty acid 131in Henriciella marina DSM 19595T it was less dominant in strain MCS23T and in strain

T T 132MCS27 it was even less than C16:0. The occurrence of C20:1ω9 in strain MCS23 is novel for

133species of the genus Henriciella. 3-OH C12:0 occurred in considerable amounts in strain

T T 134MCS27 but was accompanied by 3-OH C11:0 in strain MCS23 (Table 1). The detection of 3-

T T 135OH C12:0 in both MCS23 and MCS27 corroborated their placement within the genus 9 10 5 136Henriciella as this is the common hydroxyl-fatty acid of Henriciella.

137Lipids were extracted using a modified Bligh-Dyer procedure (Blight & Dyer 1959) as 138described previously (Vancanneyt et al. 1996). Isoprenoid quinones were analyzed using an 139Agilent 6410 Triple Quadropole LS/MS as described by Ruiz-Jimenez et al. (2007). Polar 140lipids were analysed using Fast Atom Bombardment (FAB) mass spectrometry. FAB-MS in 141the positive and negative mode was performed on the first of two mass spectrometers of a

142tandem high-resolution instrument in a E1B1E2B2 configuration (JMS-HX/HX110A, JEOL 143Tokyo) at 10 kV accelerating voltage with the resolution set to 1:1000. The JEOL FAB gun 144was operated at 6 kV with xenon. 3-Nitrobenzyl alcohol was used as matrix in the positive 145mode and a mixture of triethanolamine and tetramethylurea (Japanese matrix) in the negative 146mode. Positive and negative daughter ion spectra were recorded using all four sectors of the 147tandem mass spectrometer. High energy collision-induced dissociation (CID) took place in 148the third field free region. Helium served as the collision gas at a pressure sufficient to reduce 149the precursor ion signal to 30 % of the original value. The collision cell was operated at 150ground potential in the positive and negative modes. The resolution of MS2 was set to 1511:1000. FAB-CID spectra (linked scans of MS2 at constant B/E ratio) were recorded with 300 152Hz filtering on a JEOL DA 7000 data system.

153The dominant quinone in MCS23T and MCS27T was ubiquinone-10 (99%) as has been 154reported for all species of the Hyphomonadaceae. The glycolipids of both strains MCS23T and 155MCS 27T were dominated by α-D-glucopyranosyl- and α-D-glucopyranuronosyl- 156diacylglycerols, also common in Caulobacter, Brevundimonas, Maricaulis and some other 157species of the Hyphomonadaceae (Table S2). Additionally to these glycolipids phospho- and 158sulfolipids could be identified. Using MS/MS the structures of several of these polar lipids 159could be identified as listed in Table S3. The (-)-FAB CID mass spectrum of the molecular 160ions showed the loss of fatty acids and the corresponding ketenes. The relative abundance of 161the carboxylate anions provides evidence for the relative positions of the two acyl functions 162where the loss at the sn-2-acyl position is favoured, thus yielding a more abundant 163carboxylate anion (Murphy & Harrison, 1994). MS data led to the identification of the main 164phospholipids as 1-octadecenoyl-2-hexadecanoyl-sn-glycero-3-phosphoryI-glycerol and 1- 165nonadecenoyl-2-hexadecanoyl-sn-glycero-3-phosphoryI-glycerol, also common in 166Hyphomonas but not in Maricaulis species (Abraham et al., 1997). Only in strain MCS23T 11 12 6 167but not in MCS27T sulfoquinovosyl diacylglycerol and 1,2-diacyl-3-α-D-glucuronopyranosyl- 168sn-glycerol taurineamide were found (Table S3).

169

170Figure 2: Scanning electron micrographs of MCS23T (left row) and MCS27T (right row) 171depicting typical growth patterns of prosthecate bacteria in aggregates and connection by 172stalks (lower images in each row). Arrow heads point to flagella. Bars represent 1 µm. 173

174Parallel samples for routine scanning electron microscopy were fixed with 2% glutaraldehyde 175and 5% formaldehyde in cacodylate buffer, placed onto poly-L-lysine coated 12 mm glass 176cover slips, fixed for 10 min with 1% glutaraldehyde in TE-buffer, dehydrated with a graded 13 14 7 177series of acetone, and critical-point dried with liquid CO2 (CPD 030, Bal-Tec). After sputter 178coating with gold-palladium (SCD 500, Bal-Tec) samples were examined in a Zeiss Merlin 179field emission scanning electron microscope at an acceleration voltage of 5 kV with the 180Everhart-Thornley HE-SE-detector and Inlens SE-detector in a 25:75 ratio (Fig. 2). Contrast 181and brightness were adjusted with Adobe Photoshop CS5.

182To test salt tolerance strains were grown in 20 ml medium PYEM (2 g peptone, 2 g yeast

183extract, 0.5 g NH4Cl, 1 l Milli Q water). After autoclaving and cooling 5 ml riboflavin (0.2

-1 184mg ml ) sterile filtered, 2 ml 50 % glucose (sterile), 1 ml 20 % MgSO4 (sterile) and 1 ml 10

-1 185% CaCl2 (sterile) were added and amended with 0, 5, 10, 20, 30, 40, 60, 80 or 100 g l NaCl.

186OD600 of the cell suspension was determined at the beginning of the experiment and after 2 187days. The differences between these two measurements were used to determine salt 188tolerances. The growth was tested at different temperatures or different pH in SPYEM 189medium with the same OD protocol and the results are given in the species descriptions.

190Enzyme activity tests with the use of API ZYM test strips and substrate specificity tests with 191the use of API 20NE and API100 test strips (bioMerieux), respectively, were conducted 192according to the protocol supplied by the manufacturer. The test strips were incubated at 30° 193C for 7 days and monitored after 1, 2 and 7 days. A test was considered positive when the 194interface between sample well and air was visibly turbid due to bacterial growth after a 7 day 195incubation period. The results are given in the species descriptions. Contrary to all other 196species of Henriciella strain MCS27T was negative for cystine arylamidase. It should be noted 197here that together with strains MCS23T and MCS27T all species of Henriciella are positive for 198α-glucosidase, although the activity in H. litoralis SD10T is only weak. This discerns 199Henriciella from all other genera of the family Hyphomonadaceae. 200 201The characteristics of strains MCS23T and MCS27T are sufficiently different from each other 202and from the accepted Henriciella species (Table 2) that they are proposed here as 203Henriciella barbarensis MCS23T species nova and Henriciella algicola MCS27T species 204nova. Furthermore, the description of the genus Henriciella is emended.

205Proposal of new species and amendment of the genus Henriciella Quan et al. 2009

206Emended description of the genus Henriciella Quan et al. 2009 15 16 8 207The description of the genus Henriciella is as given by Quan et al. (2009), amended by Lee et 208al. (2011) with the following amendments:

209Cells of some species possess a stalk, ca. 0.1-0.2 μm in diameter, 0.5-0.7 μm long, varying in 210length depending on the species and environmental conditions, extending from one pole as a 211continuation of the long axis of the cell. Adhesive material is present at the distal end of the 212stalk. Occur singly. Multiplication by binary fission. Colonies circular, convex, colorless. 213Chemoorganotrophic, aerobes, can grow anaerobically probably using amino acids as 214fermentable carbon sources (Anast & Smit 1988). Cells can store carbon as poly-ß- 215hydroxybutyrate and show activity of α-glucosidase. Species have complex growth 216requirements and grow on peptone-yeast extract media with 30 g l-1 NaCl, with optimal 217growth between 10-50 g l-1 NaCl. No growth occurs with salt concentrations at or below 5 g l- 2181. Temperature range is 10-40° C, 20-35° C optimal. Polar lipids are α-D-glucopyranosyl 219diacylglycerol, α-D-glucopyranuronosyl diacylglycerol, phosphatidyl diacylglycerol and α-D- 220glucuronopyranosyl diacylglycerol taurine amide.

221Henriciella barbarensis sp. nov. (=bar.ba.ren.sis. L. adj., barbarensis, derived from a site 222near to the town Santa Barbara, California). The description of Henriciella barbarensis is the 223same as that given for the genus, with the following additional characteristics. Cells 1.8 μm 224long, 0.3 μm thick; cells possess a stalk, 0.7 μm long, depending on growth conditions, with 225adhesive material at the distal end. Cells reproduce by binary fission and formation of a 226motile swarmer cells with single flagellum. The species is characterized by three major fatty 227acids, 16:0, 17:0 and 18:1ω7c, and minor amounts of 11:0 3-OH, 12:0 3-OH, 15:0, 17:1ω5, 22820:1ω9.The species can grow on peptone yeast extract media with 5-80 g l-1 NaCl with 229optimal growth between 20-80 g l-1 NaCl. Growth is observed between 15° C and 40° C and 230at pH 6-9 with the optimal temperature between 20°-40° C. Shows anaerobic growth on 231SPYEM-plates. Does not reduce nitrate, oxidize tryptophan to indole or hydrolyse arginine, 232urea, aesculin, gelatin or p-nitrophenyl-3-D-galactopyranoside. Does not use glucose, 233arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, 234malate, citrate, phenylacetate, aspartate, glutamate, proline, alanine, serine, malonate or 235propionate as carbon sources. Cells show activity for alkaline phosphatase, naphthol-ASBI- 236phosphohydrolase, leucine arylamidase, acid phosphatase, esterase (C4), esterase lipase (C8), 237α-glucosidase, cystine arylamidase, weak activity for trypsin but are negative for α- and β- 17 18 9 238galactosidase, α-glucuronidase, β-glucosidase, α-mannosidase, catalase and α-fucosidase. 239From filtered seawater from Santa Barbara marine laboratory, University of California. The 240G+C content is 57.0 %. The type strain is MCS 23T (=LMG 28705T, CCUG 66934T). 241 242Henriciella algicola sp. nov. (al.gi’co.la. L. fem. n., alga L. n. seaweed; L. n. cola inhabitant; 243N. L. n. algicola, inhabitant of algae). The description of Henriciella algicola is the same as 244that given for the genus, with the following additional characteristics. Cells 1.8 μm long, 0.4 245μm thick; cells possess a stalk, 0.5 μm long, depending on growth conditions, with adhesive 246material at the distal end. Cells reproduce by binary fission and formation of motile swarmer 247cells with single flagellum. The species is characterized by three major fatty acids, 16:0, 17:0 248and 18:1ω7c, and minor amounts of 12:0 3-OH, 15:0, 17:1ω8, 18:0, 18:1ω5t 11-Me.The 249species can grow on peptone yeast extract media with 5-100 g l-1 NaCl with optimal growth 250between 20-100 g l-1 NaCl. Growth is observed between 10° C and 40° C and at pH 6-9 with 251the optimal temperature between 20°-40° C. Does not reduce nitrate, oxidize tryptophan to 252indole or hydrolyse arginine, urea, aesculin, gelatin or p-nitrophenyl-3-D-galactopyranoside. 253The species does not use glucose, arabinose, mannose, mannitol, N-acetylglucosamine, 254maltose, gluconate, caprate, adipate, malate, citrate, phenylacetate, aspartate, glutamate, 255proline, alanine, serine, malonate or propionate as carbon sources. Cells show activities for 256alkaline phosphatase, naphthol-ASBI-phosphohydrolase, leucine arylamidase, acid 257phosphatase, esterase (C4), esterase lipase (C8), α-glucosidase, weak activity for trypsin but 258are negative for α- and β-galactosidase, α-glucuronidase, β-glucosidase, α-mannosidase, 259cystine arylamidase, catalase and α-fucosidase. Growing on Nannochloris sp. 260(Chlorophyceae), from Hull Bay, US Virgin Islands. The G+C content is 55.0 %. The type 261strain is MCS 27T (=LMG 29152T, CCUG 67844T).

262Acknowledgments

263We thank Dagmar Wenderoth and Peter Wolff for their excellent technical assistance. This 264work was supported by the European Union within the T-project “High Resolution 265Automated Microbial Identification and Application to Biotechnologically Relevant 266Ecosystems". T. S. P. da C. N., M. T. M., and I. T. T. acknowledge the support by stipends of 267the “Cienca sem Fronteiras” (Science without borders) program of Coordenação de 268Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil, and the Conselho Nacional 19 20 10 269de Desenvolvimento Científico e Tecnológico (CNPq) from Brazil and the German Academic 270Exchange Service (DAAD).

271Funding information – This work was supported by the European Union within the T- 272project “High Resolution Automated Microbial Identification and Application to 273Biotechnologically Relevant Ecosystems". Stipends of T. S. P. da C. N., M. T. M., and I. T. 274T. came from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), 275Brazil and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), 276Brazil.

277Conflicts of interest – There is no conflict of interest.

278Ethical statement – No experiments with humans or animals were done.

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27 28 14 362Table 1: Fatty acid content (mean percentage of total) of whole-cell hydrolysates of strains 1

363= Henriciella sp. MCS23T, 2 = Henriciella sp. MCS27T, 3 = Henriciella marina DSM 19595T,

3644 = Henriciella aquimarina LMG 24711T, and 5 = Henriciella litoralis DSM 22014T

Fatty acids 1 2 3 4 5

3-OH C11:0 2.1 - - - -

3-OH C12:0 1.1 8.6 1.1 4.1 -

C15:0 3.9 4.3 2.9 1.8 2.5

C16:0 13.4 32.8 7.8 21.0 21.2

C17:0 19.2 10.8 4.8 4.3 10.1

C17:1ω5 5.7 - 11.7 3.0 7.2

C17:1ω8 - 3.9 - 2.7 5.5

C18:0 1.8 3.7 - 3.4 3.3

C18:1ω7 29.3 30.0 53.5 38.4 41.2

11-Me-C18:1ω5t - 6.0 - 8.7 3.0

C20:1ω9 4.8 - - - 0.7 366

367

29 30 15 368Table 2. Comparison of selected characteristics of Henriciella species. Strains are 1 = H. sp. 369MCS23T, 2 = H. sp. MCS27T, 3 = H. aquimarina P38T, 4 = H. litoralis SD10T, 5 = H. marina 370Iso4T 371 Characteristic 1 2 3 4 5 Colony colour white white creamwhite yellow white Prosthecum + + - - - Growth at 40°C + + + + - Esculin hydrolysis - - - + - Enzyme activity β-glucosidase - - - + - Cystine arylamidase + - - - - Carbon utilization Tween 80 - - - - + L-arabinose - - - + + D-fructose - - - + - α-D-glucose - - + - - Raffinose - - - + - Sucrose - - + - +

3-OH C12:0 + + + - + DNA G+C content 57.0 55.0 61.0a 55.2a 56.2a 372 373adata taken from Lee et al., 2011

31 32 16 374Supplementary Material 375 376Proposal of Henriciella barbarensis sp. nov. and Henriciella algicola sp. nov., stalked 377species of the genus and emendation of the genus Henriciella 378 379Wolf-Rainer Abraham*1, Maira Peres de Carvalho1, Thaís Souto Paula da Costa Neves1, 380Marina Torquato Memória1, Iago Toledo Tartuci1, Marc Vancanneyt2, John Smit3, and 381Manfred Rohde4 382 3831Helmholtz Centre for Infection Research, Chemical Microbiology, Inhoffenstrasse 7, 38124 384Braunschweig, Germany 3852BCCM/LMG Bacteria Collection, Universiteit Gent, K.L. Ledeganckstraat 35, Gent; 386Belgium 3873Dept. of Microbiology and Immunology, University of British Columbia, Vancouver, British 388Columbia, Canada 3894Central Facility for Microscopy, Helmholtz Centre for Infection Research 390 391Running title: Henricella barbarensis and H. algicola sp. nov. 392Subject category: New taxa; Subsection: Alphaproteobacteria 393Keywords: Henriciella, Hyphomonadaceae, lipids, marine bacteria 394 395*Corresponding author: 396Dr. Wolf-Rainer Abraham 397Helmholtz Centre for Infection Research, Chemical Microbiology 398Inhoffenstrasse 7, 38124 Braunschweig, Germany 399Tel. +49 531 6181 4300; Fax: +49 531 6181 4699 400e-mail: [email protected]

33 34 17 401Supplementary Table S1: Results of DNA-DNA hybridizations between MCS23T and 402MCS27T and Henriciella type strains 403 MCS23T MCS27T MCS23T 53% MCS27T 53% H. aquimarina LMG 20% 18% 24711T H. litoralis DSM 22014T 15% 50% H. marina DSM 19595T 22% 12%

35 36 18 404Supplementary Table S2: Phospho- and sulfo-lipids as biomarkers in Henriciella and related 405genera (Abraham et al., 1999; Abraham et al., 2013) 406

Genus PG SQD TAU G

Henriciella +++ + +

Glycocaulis

Hyphomona +++ +++ s Hirschia +++

Maricaulis + +++ + 407 408PG=phosphatidyl-glycerol, SQDG=1,2-diacyl-3-O-sulfoquinovosylglycerol, TAU = 1,2- 409diacyl-3-α-D-glucuronopyranosyl-sn-glycerol taurineamide. 410+++ = present in all strains, + = present in some strains (<50 %), blank = absent

37 38 19 411Supplementary Table S3: Phospho- and sulfolipids identified by MS/MS in strains of 412Henriciella and related genera 413 Mass Type Fatty acids 1 2 3 4 5 6 7 8

R1COOH R2COOH 720 PG 18:1 14:0 + 734 PG 18:1 15:0 + 746 PG 18:1 16:1 + 748 PG 18:1 16:0 + + + + + 750 PG 19:0 15:0 + 760 PG 18:1 17:1 + + 762 PG 19:1 16:0 + + + + + 774 PG 18:1 18:1 + + + 788 PG 19:1 18:1 + + 820 SQDG 18:1 16:0 + + + + 834 SQDG 18:1 17:0 + + + 846 SQDG 18:1 18:1 + + + 848 SQDG 18:1 18:0 + + 850 SQDG 18:0 18:0 + 860 SQDG 18:1 19:1 + 862 SQDG 19:1 18:0 + 878 TAU 18:1 16:0 + 904 TAU 18:1 18:1 + + + 414 415Abbreviations: PG = phophatidyl glycerol, SQDG = sulfoquinovosyl diacylglycerol, Tau = 4161,2-diacyl-3-α-D-glucuronopyranosyl-sn-glycerol taurineamide; + = ion in negative FAB MS. 4171 = MCS23T; 2 = MCS27T; 3 = Hyphomonas jannaschiana ATCC 33883T; 4 = H. 418polymorpha DSM 2665T; 5 = Maricaulis indicus MCS26T; 6 = M. maris ATCC 15268T; 7 = 419M. salignorans MCS18T; 8 = M. virginensis VKM B-1513T.

39 40 20 420 Figure S1: Neighbour-joining tree based on 16S rRNA gene sequences, showing the 421phylogenetic relationships of strains MC23T and MCS27T and related taxa. Bootstrap 422percentages (based on 500 replications) are shown where >50 %. Escherichia coli was used as 423an outgroup. Bar, 0.01 substitutions per nucleotide position. 424 426425

T 100 Hyphomonas jannaschiana VP2 [AJ227814] 54 Hyphomonas adhaerens MHS-3T [AF082790] Hyphomonas atlantica LMG 27916T [KF863140] Hyphomonas oceanitis SCH89T [AF082797] 100 54 Hyphomonas johnsonii MSH-2T [AF082791] Hyphomonas polymorpha DSM 2665T [AJ227813] Hyphomonas hirschiana VP5T [AF082794] 100 93 T 94 Hyphomonas rosenbergii VP6 [AF082795] 90 Hyphomonas neptunium LE670T [AF082798] Ponticaulis koreensis GSW-23T [FM202497] Henriciella litoralis SD10T [FJ230835] 92 T 57 Henriciella aquimarina P38 [EU819081] 100 MCS27T [KP722025] 55 T 80 Henriciella marina Iso4 [EF660760] 73 [Caulobacter sp.] MCS23T [AJ227807] Robiginitomaculum antarcticum IMCC3195T [EF495229] Hellea balneolensis DSM19091T [AY576758] 100 Fretibacter rubidus JC2236T [JQ965646] 83 Litorimonas taeanensis G5T [FJ230838] Litorimonas cladophorae KMM 6395T [JX174422] 99 Algimonas arctica SM1216T [KJ144186] 60 T 83 Algimonas porphyrae 0C-2-2 [AB689189] 63 Algimonas ampicilliniresistens LMG 26421T [AB795010] Hirschia baltica IFAM1418T [AJ421782] 100 Hirschia litorea M-M23T [JQ995780]

0.01

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