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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%

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