Fundicoccus ignavus gen. nov., sp. nov., a novel genus of the family Aerococcaceae isolated from bulk tank milk

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This dataset contains the digitized treatments in Plazi based on the original journal article Siebert, Annemarie, Huptas, Christopher, Wenning, Mareike, Scherer, Siegfried, Doll, Etienne V. (2020): Fundicoccus ignavus gen. nov., sp. nov., a novel genus of the family Aerococcaceae isolated from bulk tank milk. International Journal of Systematic and Evolutionary Microbiology 70 (8): 4774-4781, DOI: 10.1099/ijsem.0.004344, URL: http://dx.doi.org/10.1099/ijsem.0.004344

Abstract

Three strains of a Gram-stain-positive, catalase-negative, facultative anaerobic, and coccoid species were isolated from German bulk tank milk. Phylogenetic analyses based on the 16S rRNA gene sequences indicated that the three strains (WS4937 T, WS4759 and WS5303) constitute an independent phylogenetic lineage within the family Aerococcaceae with Facklamia hominis CCUG 36813 T (93.7–94.1%) and Eremococcus coleocola M1831/95/2 T (93.5%) as most closely related type species. The unclassified strains demonstrated variable growth with 6.5% (w/v) NaCl and tolerated pH 6.5–9.5. Growth was observed from 12 to 39 °C. Their cell-wall peptidoglycan belongs to the A1α type (L-Lys-direct) consisting of alanine, glutamic acid and lysine. The predominant fatty acids were C 16:1 ω 9 c, C 16:0 and C 18:1 ω 9 c and in the polar lipids profile three glycolipids, a phospholipid, phosphatidylglycerol, phosphoglycolipid and diphosphatidylglycerol were found. The G+C content of strain WS4937 T was 37.4mol% with a genome size of ~3.0Mb. Based on phylogenetic, phylogenomic and biochemical characterizations, the isolates can be demarcated from all other genera of the family Aerococcaceae and, therefore, the novel genus Fundicoccus gen. nov. is proposed. The type species of the novel genus is Fundicoccus ignavus gen. nov., sp. nov. WS4937 T (= DSM 109652 T = LMG 31441 T).

The Gram-stain-positive family Aerococcaceae belongs to the order Lactobacillales and contains eight genera: Aerococcus [1], Globicatella [2], Abiotrophia [3], Facklamia [4], Ignavigranum [5], Eremococcus [6], Dolosicoccus [7], and Suicoccus [8]. The genus described most recently is Suicoccus [8] which is the only catalase-positive genus within the family. In a cultivation-dependent biodiversity study of German bulk tank raw milk, aerobic, mesophilic bacteria were isolated from samples collected from several farms. Among them, three hitherto unknown isolates, WS4937 T, WS4759 and WS5303, were found in milk samples from three different farms. According to their 16S rRNA gene sequences, they were most closely related to members of the family Aerococcaceae but could not be assigned to any validly named species or genus. In this study it is suggested, based on phylogenetic, genomic, phenotypic and chemotaxonomic characterizations, that these unknown bacteria belong to a single species of a novel genus.

Extraction of genomic DNA and amplification of almostcomplete 16S rRNA sequences from the three isolates were performed according to von Neubeck et al. [9]. Due to pairwise sequence similarities ranging from 99.6 to 99.7%, it is assumed that strains WS4937 T, WS4759 and WS5303 are members of a single species. Comparison against the EzBioCloud database [10] revealed that strain WS4937 T is most similar to Facklamia miroungae ATCC BAA-466 T (94.3% similarity) followed by Facklamia tabacinasalis CCUG 30090 T (94.1%) and Facklamia languida CCUG 37842 T (93.7%). The most closely related type species of the genera within the family Aerococcaceae are Facklamia hominis CCUG 36813 T and Eremococcus coleocola M1831/95/2 T, sharing a sequence similarity of 93.7 and 93.5%, respectively. As previously described [11 – 13], the demarcation threshold to separate bacterial genera corresponds to 94.5–95.0%. Thus, based on 16S rRNA sequence similarity, strain WS4937 T represents a novel genus within the family Aerococcaceae.

Using the CLUSTAL_W functionality of MEGA X version 10.0.4 [14, 15], a maximum-likelihood tree based on the almost-complete 16S rRNA gene sequences of the three novel isolates and all type strains within the family Aerococcaceae was calculated. In the resulting phylogeny the three isolates form a separate monophyletic clade with maximal bootstrap support (Fig. 1). Consistent with the observations made by pairwise 16S rRNA sequence comparison, Eremococcus coleocola M1831/95/2 T and Facklamia hominis CCUG 36813 T were most closely related. Therefore, it can be assumed that strains WS4937 T, WS4759 and WS5303 represent a species of a novel genus.

Fig. 1. Maximum-likelihood tree based on the almost-complete 16S rRNA gene sequences of strains WS4937 T, WS4759, WS5303 and type strains of all species of the family Aerococcaceae. Type strains of the type species are marked in bold. Sequences of the strains were retrieved from EzBioCloud.The tree was calculated using the Tamura–Nei model.Bootstrap values (>50%) based on 500 repetitions are shown at branch nodes. Analyses were performed using MEGA X. Lactococcus lactis subsp. lactis was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.

For more detailed insights into the taxonomic status of the novel genus, phylogenomic analyses were conducted [16]. To uncover their genomic sequences, the three unclassified strains were cultivated on trypticase soy agar (TSA; Oxoid) and DNA was isolated using the Qiagen QIAamp DNA Mini kit according to the manufacturer’s instructions. Preparation of sequencing libraries was conducted as described by Huptas et al. [17], using the Illumina TruSeq DNA PCR-Free Sample Preparation kit. Sequencing of the genomes was carried out on an Illumina MiSeq platform. Quality control and read filtering was done with FastQC version 0.10.1 (www.bioinformatics.babraham. ac.uk/projects/fastqc/) and the NGS QC Toolkit version 2.2.3 [18], respectively. Genomes were assembled to highquality draft level with SPAdes version 2.5.1 [19]. Detailed assembly statistics can be found in Table S1 (available in the online version of this article). Genome annotation was performed by the NCBI Prokaryotic Genome Annotation Pipeline [20, 21]. Genome reconstruction of strain WS4937 T revealed 64 contigs with a cumulative size of ~3.0 Mb and a G+C content of 37.4 mol%. Interestingly, strain WS4937 T has the largest genome of all those currently available from members of the family Aerococcaceae.

Phylogenomic analyses were performed by selecting the genome data of publicly available type species of the eight genera belonging to the family Aerococcaceae. After generating a multiple sequence alignment comprising 92 bacterial core genes via UBCG software version 3.0 [22], a phylogenomic tree was calculated using MEGA X software. Similar to the 16S rRNA single-gene phylogeny, isolates WS4937 T, WS4759 and WS5303 cluster together in a clearly separated monophyletic clade (Fig. 2). Within the phylogenomy, the three strains are most closely related to the genus Globicatella represented by Globicatella sanguinis NBRC 15551 T. The average amino acid identity (AAI) is a whole genome similarity measure that provides robust resolution to discriminate between more distantly related taxa [23, 24]. Using CompareM (https://github.com/dparks1134/ CompareM), pairwise AAI values were calculated for strain WS4937 T and all type strains of the family’s eight genera. In this analysis, the AAI values between validly published genera ranged from 53.5 to 61.4%. Comparing strain WS4937 T to type species of the Aerococcaceae, AAI values in the same range (56.2–62.1%) were obtained, confirming its status as a novel genus.

Fig. 2. Up-to-date bacterial core gene tree (UBCG) based on a multigene alignment with 92 housekeeping genes. Genome data of type strains of the type species from the family Aerococcaceae available on the EzBioCloud database were used. The three isolates of this study are marked in bold. The tree was calculated based on the Tamura–Nei model using MEGA X software. Two hundred repetitions were performed to reveal statistic support which is shown at branch nodes (bootstrap values>50 %). Lactococcus lactis subsp. lactis was used as an outgroup. Bar, 0.1 substitution per nucleotide position.

Besides the genetic and phylogenetic analysis of the novel genus, morphological, physiological and biochemical characteristics of the strains were documented. Isolates were routinely cultivated aerobically on TSA plates at 30 °C for 96 h unless otherwise stated. The analysis of physiological features was performed in two independent replicates. In order to observe cell shape and motility, a phase-contrast microscope (BX51, Olympus) was used in combination with the software package F-View Soft Imaging System (Olympus). Cells were grown at 30 °C for 48 h on TSA and suspended in Ringer’s solution (1/4-strength). Additionally, motility was examined on semi-solid trypticase soy broth (TSB; Merck) supplied with 0.3% (w/v) agar. According to Gregersen [25], the Gram-type was determined with the non-staining KOH method and presence of catalase was tested using 3% (v/v) H 2 O 2. The oxidase activity was checked with Bactident Oxidase strips (Merck), according to the manufacturer’s instructions. Haemolysis was examined on Columbia agar with 5% sheep blood (Oxoid) at 30 °C for 72 h. To check for gelatin hydrolysis, tubes with 5 ml nutrient gelatin (3.0 g l−1 beef extract, 5.0 g l−1 peptone, 120 g l−1 gelatin) were incubated at 30 °C. The tubes were analysed for turbidity and liquefaction after 48 h and 14 days by incubation at 5 °C for 15 min. Starch hydrolysis was checked by growing cells on starch agar (3.0 g l−1 beef extract, 10.0 g l−1 soluble starch, 12.0 g l−1 agar, pH 7.5±0.2) for 7 days at 30 °C and subsequent flooding of the plates with 1 ml of Lugol’s iodine. Starch hydrolysis was indicated by clear lysis zones. Nitrate and nitrite reduction was determined using reagents NIT 1, NIT 2 (bioMérieux) and zinc powder. Cultures were incubated at 30 °C for 24 h under semi-anaerobic conditions in TSB supplemented with 0.1% (w/v) KNO 3 and 0.17% (w/v) agar. Production of H 2 S and indole was tested using a ready-to use semi-solid SIM agar (Merck) and Kovacs’ reagent (Merck) according to the manufacturer’s instructions. To test optimal growth conditions, TSB was inoculated 1:500 with an overnight-culture (30 °C, 150 r.p.m., 24 h) of the strains and growth curves were recorded using the Bioscreen C instrument, measuring the optical density (OD 600) every 20 min under continuous shaking. The upper and the lower growth limits for temperature, NaCl concentration and pH were studied in 5 ml tubes. Visible turbidity after 7 days indicated a positive result. The growth temperature was assessed at 10–40 °C. Tolerance to 0%−8 % (w/v) NaCl (with 1% increments) and growth at pH 5–10 (with 0.5 increments) were analysed at 30 °C. For the pH media, TSB was supplemented with the respective buffer system described by Xu et al. [26] (pH 4–5.5, 0.1 M citric acid/0.1 M sodium citrate; pH 6–8, 0.1 M KH 2 PO 4 /0.1 M NaOH; pH 8.5–10, 0.1 M NaHCO

3 /0.1 M Na

2 CO

3) and filter-sterilized (0.22 µm pore size) after autoclaving and pH adjustment. The ability to grow under anaerobic conditions was tested in an anaerobic jar containing Oxoid AnaeroGen 2.5 l (Thermo Scientific) for 7 days.

Further physiological and biochemical tests were conducted using the API Rapid ID32 STREP and the API 50 CHL system (bioMérieux). Cells were grown at 30 °C for 24 h in 50 ml TSB, washed and resuspended in Ringer’s solution (1/4-strength). McFarland Standard 2 was then used to inoculate the API systems according to the manufacturer’s instructions. The API ID32 STREP strips were incubated at 30 °C for 24 h and a positive result was indicated by a colour change. The analysis of acid production from carbohydrates (according to API 50 CH) was conducted in 96-well microtiter plates. Each carbohydrate was dissolved in water and filter-sterilized. The concentration used was twice as high as specified in the API 50 CH strips manual in mg per well per 100 µl. Each carbohydrate was mixed aseptically with 100 µl of the inoculated API 50 CHL medium. Microtiter plates were sealed with Breathe-Easy sealing membrane (Diversified Biotech), covered with a plastic-lid and incubated at 30°C for 7 days without shaking. Acid production was indicated by a colour change of the pH indicator from purple to yellow.

Morphologically, the cells of the tested isolates were spherical with an average diameter of 1.3 µm. Single cells, but also pairs or chains occurred (Fig. S1). They were nonspore-forming and did not exhibit motility. Physiological properties to differentiate the type strain WS4937 T of the novel genus from the hitherto described genera of the family Aerococcaceae are displayed in Table 1. The three strains (WS4937 T, WS4759 and WS5303) resembled each other and shared similar characteristics, therefore, only data gained from WS4937 T are shown. The growth optimum was observed at 29–33 °C. All three isolates were able to grow down to 12 °C and up to 37 °C (WS5303) and 39 °C (WS4937 T and WS4759). A lack of growth at 10 °C and 45 °C is consistent with the growth features reported for most of the genera within the family Aerococcaceae (Table 1). Tolerated pH conditions were comparable between the three strains (pH 6.5–9.5). Growth in up to 6.5% (w/v) NaCl was monitored for two of the three strains, WS4937 T and WS4759. Remarkably, bacterial strains failed to produce acid from 47 tested carbohydrates of the API 50 CH. Moreover, strain WS4937 T showed differences in the activity of several enzymes compared to the evolutionarily most closely related genera Eremococcus coleocola M1831/95/2 T and Globicatella sanguinis NBRC 15551 T, respectively (Table 1). These phenotypic characteristics additionally support a delineation of the isolates into a novel genus.

Utilization of amino acids as growth substrates was tested using API ID32 STREP strips. Only the reaction for arginine-dihydrolase was positive, alanyl-phenylalanylproline-arylamidase, glycyl-tryptophan-arylamidase and pyroglutamic acid arylamidase tested negative. To complement those phenotypic data, we interpreted the amino acid metabolism of type strain WS4937 T at a genomic level using the KEGG database [27]. Protein-coding genes were predicted in Prodigal [28]. The protein sequences were annotated by BlastKOALA [29] and assigned to orthologues in the KEGGKEGG molecular network.

Of the 2661 protein-coding genes present in the genotype of WS4937 T, a total of 1611 (60.5%) could be annotated, 95 of which were assigned to the amino acid metabolism. The genome-based analysis by reconstruction of pathways using the KEGG Mapper [30] largely supported the enzyme activities determined by the API Rapid ID32 STREP system. It uncovered the presence of enzymes involved in the degradation of arginine, aspartate, glutamate, serine and cysteine, producing substrates of the citrate cycle and indicating their potential role in the energy metabolism. Pathways for proline and tryptophan were incomplete or not present, as well as pathways for all other amino acids. In addition, an orthologue of the alkaline phosphatase was annotated, which was not active under the conditions tested in the API Rapid ID32 STREP system.

Chemotaxonomic analyses of peptidoglycan structure, cellular fatty acid composition and polar lipids were carried out by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). To obtain biomass for the cell-wall peptidoglycan and polar lipids analyses, TSB was inoculated with the type strain WS4937 T at a dilution of 1:500 using a pre-culture of cells grown at 30 °C for 24 h. The inoculated medium was incubated at 30 °C for 48 h and the biomass was freeze-dried for both analyses. Disruption of the cells for peptidoglycan analysis was performed according to Schleifer [31]. Cells were then hydrolysed (4 N HCl, 100 °C, 16 h; 6 N HCl, 120 °C, 16 h and 4 N, 100 °C, 45 min) and the peptidoglycan contents were investigated by TLC as described by Schleifer and Kandler [32]. The approximate molar ratio of amino acids was determined according to Schumann [33]. The peptidoglycan profile contained the amino acids alanine, glutamic acid and lysine in molar ratio of 1.3 Ala:1.3 Glu:1.0 Lys and indicates the peptidoglycan type A1α L-Lys-direct for the type strain WS4937 T. This is in accordance with the peptidoglycan type mainly occurring in the family Aerococcaceae in which only the genus Facklamia (type A4 α) [4] contains a different murein type. Extraction and analysis of the polar lipids were performed as previously described [34 – 36]. Three glycolipids, phospholipid, phosphatidylglycerol, phosphoglycolipid and diphosphatidylglycerol were found as polar lipids (Fig. S2). Besides phosphatidylglycerol, none of the other polar lipids were found for any strain within the Aerococcaceae when analysed by Li et al. [8], not even in the most closely related type species Facklamia hominis CCUG 36813 T [4] and Eremococcus coleocola M1831/95/2 T [6].

Determination of the cellular fatty acids was conducted with the proposed type strain WS4937 T and Globicatella sanguinis NBRC 15551 T. The strains were cultivated on Columbia agar supplemented with 5 % (v/v) horse serum (Biorad) for 24 h at 37 °C, in air plus 5% CO 2 as described by Li et al. [8]. Fatty acid methyl esters of the cell material were obtained using minor modifications of the methods of Miller [37] and Kuykendall et al. [38]. Analyses revealed that the fatty acid profile of type strain WS4937 T was mainly dominated by C 16:1 ω9 c, having a fraction of 27.6% (Table 2). This was followed by C 16:0 with 23.5% and C 18:1 ω9 c (17.6%). The fatty acid composition of strain WS4937 T thereby enables its demarcation from all other type species of the Aerococcaceae [8], in particular from the closely related genera Facklamia hominis CCUG 36813 T, Eremococcus coleocola M1832/95/2 T and Globicatella sanguinis NBRC 15551 T. The major fatty acid of WS4937 T, C 16:1 ω9 c, was detected only in sparse amounts in Globicatella sanguinis NBRC 15551 T (4.3%) and was not found in Facklamia hominis CCUG 36813 T and Eremococcus coleocola M1832/95/2 T at all. While summed feature 5 (C 18:2 ω6,9c/C 18:0 ante) is a major fatty acid (>10%) of the three closest relatives, it was only detected with 5.6 % in WS4937 T.

Characteristic 1 2 3 4 5 6 7 8 9 Growth at 10°C − − − − − − − − + Growth at 45°C − − NR* − − − + − + Growth in 6.5% (w/v) NaCl + + +(W) + + − + − + Production of acid from: Glucose − − + + + + +(W) +(W) NR Glycogen − − − − − + − − − Lactose − − − + + − − +(W) − Mannitol − − − + + NR NR +(W) + Melibiose − − − + − − − − + Raffinose − − − + − + − − + Ribose − − − + +(W) − − − − Sucrose − − − + + + − − + Trehalose − − − + − + − − + Hydrolysis of: Aesculin − − − + + NR − − NR Hippurate + + + + +(W) − − − + Starch − + − + NR + − − NR Production of: Arginine dihydrolase + + + − − − + − − α -Galactosidase +(W) + − + − + − − + β -Galactosidase + + − + − + − − − β -Glucosidase + − − + − − − − − Pyroglutamic acid arylamidase − NR + + +(W) + + + + Urease − − − − − − + − − Catalase − − − − − − − − + Genome properties: DNA G+C content (mol%) 37.4 38.7 38.9 35.4 39.9 46.9 39.3 37.9 42.3 Size (Mb) 3.0 1.9 1.8 2.4 2.2 2.0 1.9 1.7 2.6 Murein type A1α A4α A1α A1α A1α A1α A1α NR A1α

Fatty acid 1 2 3 4 5 6 7 8 9 Saturated: C 10:0 0.7 NR NR 0.5 NR NR NR NR NR C 12:0 0.5 0.5 0.5 1.0 0.4 1.2 0.5 0.5 1.3 C 14:0 4.2 6.1 6.3 8.6 4.1 9.0 7.0 4.0 10.6 C15:0 anteiso – 0.4 4.2 – 0.3 – 1.0 0.5 0.2 C16:0 23.5 52.9 52.0 30.2 25.4 33.4 56.8 44.1 38.1 C 17:0 – NR NR 0.5 NR NR NR NR NR C 18:0 6.5 5.0 3.9 11.9 13.8 4.8 3.1 5.8 2.4 C 19:1 iso I 1.1 – – – – 1.1 – 1.8 0.2 Unsaturated: C: ω9 с 16 1 27.6 – – 4.3 15.3 18.1 – 7.9 18.4 C: iso ω5 с 17 1 0.5 0.9 0.6 0.7 0.3 2.2 0.8 3.4 0.5 C18:1ω9 с 17.6 11.8 9.3 17.0 9.3 12.1 10.0 10.1 12.7 C 20:1ω9 с 0.4 – – – 7.2 – – – – C 20:2 ω6,9 с 0.5 NR NR – NR NR NR NR NR C 20:4 ω6,9,12,15 с – NR NR 0.6 NR NR NR NR NR Hydroxy: C 13:0 2-OH – – – – – – – 0.6 – C15:0 2-OH – – – – – – – 0.9 – Summed features:* 3 6.0 2.9 2.5 4.5 1.4 5.6 2.8 3.1 2.1 5 5.6 15.9 12.2 16.4 1.6 5.6 13.3 9.4 9.6 7 – – – – 16.3 – – – – 8 4.1 1.2 1.1 2.9 1.7 3.8 1.2 1.9 0.8 9 1.2 – – 0.9 0.5 1.7 – – 1.0 *Summed feature 3, C16:1 ω 7с/C16:1 ω 6с; summed feature 5, C18:2 ω 6,9с/C18:0 ante; summed feature 7, unknown 18.85/C19:1 ω 6с; summed feature 8, C18:1 ω 7с/C18:1 ω 6с; summed feature 9, C17:1 iso ω 9с/C16:0 10-methyl.

In summary, phylogenetic reconstruction and genomic, phenotypic and chemotaxonomic data showed that strains WS4937 T, WS4759 and WS5303 form a robust and independent lineage that represents a novel genus within the family Aerococcaceae.

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