Rahnella perminowiae sp. nov., Rahnella bonaserana sp. nov., Rahnella rivi sp.

ABSTRACT

bioremediation properties were assigned to Rahnella based on partial housekeeping gene sequences.
The taxonomic status of these 21 strains was investigated using multilocus sequence analysis, whole genome analyses, phenotypic assays and fatty acid analysis.Phylogenetic and phylogenomic analyses separated the strains into five clusters, one of which corresponded to Rahnella aceris.The remaining four clusters could be differentiated both genotypically and phenotypically from each other and existing Rahnella species.Based on these results, we propose the description of four novel species: Rahnella is a genus of environmentally-linked species in the family Yersiniaceae [1].For many years Rahnella aquatilis was the only validly described species in the genus Rahnella [2], although two genomospecies were proposed containing strains that could not be phenotypically differentiated from R. aquatilis [3].R. aquatilis has long been acknowledged as a truly ubiquitous bacterium and has been isolated from a diverse range of sources, both environmental and clinical [4].The genus Rahnella has expanded exponentially in recent years with the description of six novel species from a range of ecological niches and the elevation of the two genomospecies to validly described species [5][6][7].These eight species contributed to the existing diversity of Rahnella with isolations of Rahnella victoriana, Rahnella variigena and Rahnella inusitata from bleeding cankers of oak; R. victoriana, R. variigena and R. woolbedingensis from asymptomatic alder and walnut; Rahnella bruchi from the gut of the Agrilus biguttatus beetle; Rahnella aceris and Rahnella laticis from sap of Acer pictum and Rahnella contaminans as a contaminant from MRSA agar plates [5][6][7].In addition to their isolation from the natural environment, Rahnella species have been linked to nitrogen-fixation [8], metal and radionuclide sequestration [9] and biological control [10]; and more recently as possible pathogens of oak [11], poplar [12] and onion [13].
A study by Asselin et al. [13] indicated the existence of several potential novel Rahnella species, isolated over a number of years from onion bulbs with signs of bacterial decay in the United States and Norway, and from creek water in the United States.Multilocus sequence analysis of a selection of onion isolates placed them in four separate clusters without reference strains in the Rahnella genus, suggesting they belong to four novel taxa [13].A further potential novel Rahnella taxon was identified in this study following gyrB gene sequencing of a strain previously isolated from a Quercus species displaying symptoms of Acute Oak Decline (AOD) in the Netherlands.The above mentioned strains were examined using a polyphasic approach based on genotypic, phenotypic, genomic and fatty acid assays to clarify their taxonomic position.Based on the results, we propose four novel Rahnella species: Rahnella perminowiae sp.nov., Rahnella bonaserana sp.nov., Rahnella rivi sp.nov.and Rahnella ecdela sp.nov.

Isolation and Ecology
Bacterial strains were previously isolated from onion bulbs in the United States (New York State and Oregon) and Norway (Vestfold, Østfold, Oppland and Hedmark) as described in Asselin et al. 2019 [13], either directly from onion tissue or following soaking and crushing in buffer or sterile water.Strain FC061912-K T was isolated from creek water following high-speed centrifugation and culturing.A Rahnella strain Y9602 able to sequester metals was isolated from a mixed-waste-contaminated subsurface in Tennessee, United States [14].FRB 231 T was isolated from the bleeding lesion on a symptomatic oak in the Netherlands displaying symptoms of Acute Oak Decline (AOD).A swab was taken from the lesion, suspended in sterile Ringers solution and the resulting suspension plated onto Luria-Bertani (LB) agar.All strains can be routinely cultured on LB agar or in LB broth incubated at 28 °C, and stored in 40 % glycerol at -80 °C.See Suppl.Table S1 for a list of strains investigated in this study.

Genotypic characterisation
DNA for all PCR reactions was extracted using alkaline lysis [15] and stored at -20 °C.Multilocus sequence analysis (MLSA) was performed on strains which weren't included in the study by Asselin et al. [13], by amplification and sequencing of the gyrB, rpoB, infB and atpD housekeeping genes as previously described [16].The following modifications were used: annealing temperature of 46 °C for the gyrB PCR, alternative rpoB amplification and sequencing primers designed for Rahnella species [13] and an alternative atpD reverse sequencing primer atpD-08R 5' CCCAGAAGTGCGGACACTTC 3'.
Almost complete 16S rRNA gene sequencing was performed on a selection of strains (AR20, L31-1-12, C60, SL6 T , H11b T , FC061912-K T and FRB 231 T ) using the primers from Coenye et al. [17] and standard amplification cycles with an annealing temperature of 55 °C.Additional sequences for the closest phylogenetic relatives were downloaded from GenBank and added to the datasets which were aligned and trimmed in BioEdit v7.2.5 [18] to the following lengths: gyrB -741 bp, rpoB -636 bp, infB -615 bp, atpD -642 bp and 16S rRNA -1346 bp.Following concatenation of the four housekeeping genes, Smart Model Selection (SMS) [19] was performed on both the MLSA and 16S rRNA gene datasets before maximum likelihood phylogenetic analysis using PhyML 3.0 [20].Reliability of the generated clusters was assessed with 1000 bootstrap replicates.
In the maximum likelihood phylogenetic tree based on concatenated multilocus gene sequences (Fig. 1), the strains isolated from onion bulbs in the United States and Norway were separated into three clusters.The first cluster (Rahnella clade 1) comprised eight strains isolated from onion in the United States and Norway, strain Y9602 which can sequester heavy metals and the type strain of a recently described Rahnella species, R. aceris [6].As the cluster was strongly supported with a bootstrap value of 100 % and there was little sequence variation amongst the strains, it was concluded that strains in this cluster belonged to R. aceris.The remaining isolates from onion bulbs did not cluster with any reference or type strains.Rahnella clade 2, another large, well-supported group of nine strains from onion in the United States and Norway, clustered on the border of the type species, R. aquatilis, but was clearly separated and constituted a potential novel species.The remaining strain isolated from onion, H11b T (Rahnella clade 3) was situated on a separate branch between R. victoriana and R. variigena.Strains FRB 231 T , isolated from Quercus sp.(Rahnella clade 4), and FC061912-K T (Rahnella clade 5), isolated from creek water adjacent to an onion field, also had separate positions within the phylogenetic tree with no close association to a type strain indicating these three strains belonged to further novel Rahnella species.
The 16S rRNA gene sequence pairwise similarity for the selected strains was calculated using EZBioCloud [21], and was 99.3 % to R. aceris and R. aquatilis for strains from Rahnella clade 2; 99.2 -99.5 % similar to R. variigena, R. bruchi, R. wooldbedingensis and R. victoriana for H11b T , FRB 231 T and FC061912-K T (Rahnella clades 3, 4 and 5).As expected and as previously observed [5][6][7], due to the recognised high degree of homogeneity in the 16S rRNA gene of genera in the Enterobacterales, the taxonomic position of the potential novel species was not clearly or reliably represented by the 16S rRNA gene phylogenetic tree (Suppl.Fig. S1).The existing Rahnella species did not form a monophyletic clade and are interspersed by Rouxiella species and Ewingella americana.
BOX and ERIC PCR (repetitive element-based PCR) were performed on all isolates to examine their genetic diversity using the primers BOX-A1R and ERIC-2 and -1R, respectively [22].Included in the analyses were two representative strains from each existing Rahnella species.Amplicons were separated in 1.5 % agarose at 50 V for ~3 h.BOX PCR provided the best resolution for all strains tested and allowed the differentiation of the four potential novel species, not only from each other but also from existing Rahnella species (Suppl.Fig. S2).Although the fingerprint patterns for strains from Rahnella clade 2 had similar patterns, they were isolated from onion bulbs in different areas and countries and therefore cannot be clones.

Genome features
The whole genome sequences of nine strains isolated from symptomatic onion bulbs (Rahnella clade 1: AR20, F57b, L31-1-12, R92a; Rahnella clade 2: C60, L72c, L151-1A, SL6 T  Reads were adapter trimmed using Trimmomatic 0.30 with a sliding window quality cutoff of Q15 [23]. De novo assembly was performed using SPAdes version 3.11.1 [24] and the resulting contigs were annotated in Prokka 1.11 [25].Genome sizes from 5.40 to 5.75 Mbp and DNA G + C contents ranging from 51.4 to 53.2 mol % were observed for the sequenced strains.The genome sequences were submitted to Genbank under the BioProject number PRJNA706176); genome features and assembly accessions are listed in Suppl.Table S2.The 16S rRNA sequences derived by genome sequencing of the above strains were compared to those obtained with Sanger sequencing to ensure there was no contamination of the whole genome sequences.
To infer the phylogenomic position of the strains, pairwise comparisons between the genomes were conducted using Genome Blast Distance Phylogeny (GBDP) and accurate intergenomic distances inferred under the algorithm 'trimming' and distance formula d 5 [26] with 100 distance replicates each.The resulting intergenomic distances were used to construct a balanced minimum evolution tree including Subtree Pruning and Regrafting (SPR) post-processing using FASTME 2.1.6.1 [27].Branch support was inferred from 100 pseudo-bootstrap replicates and the tree was rooted at the midpoint [28].In the resulting phylogenomic tree (Fig. 2), all representative Rahnella strains from the present study formed a robust clade with existing Rahnella species with 100 % bootstrap support.The clustering of the strains agreed with that observed in the MLSA phylogenetic tree (Fig. 1), with five strains assigned to the recently validated species R. aceris and four strains isolated from onion forming a separate cluster representing a novel taxon.The remaining representative strains from various sources had unique positions within the Rahnella clade confirming their taxonomic status as three novel species.
Whole genome comparisons were performed between representative Rahnella strains from the present study and existing Rahnella species using average nucleotide identity with FastANI [29].
Strains from Rahnella clade 1 shared ANI values of 98.2 to 99.7 % with each other and 98.4 to 99.4 % with the type strain of R. aceris SAP-19 T (Suppl.Table S3).These values are above the suggested species limit of 95 % [30] confirming that the strains from onion and strain Y9602 belong to the same taxon, R. aceris.ANI values ranging from 99.1 -99.3 % were observed amongst strains from Rahnella clade 2 confirming they belong to a single taxon.The strains from the three single-strain species (Rahnella clades 3 -5) exhibited ANI values of less than 91.2 % to each other, and to strains from Rahnella clade 2. Furthermore, representative strains from Rahnella clades 2 -5 were less than 94 % related in terms of ANI to all existing Rahnella species (Table 1).The conclusions drawn from ANI analysis were confirmed by in silico DNA-DNA hybridisation (isDDH) using the genome-to-genome distance calculator (GGDC) [26] and are also presented in Table 1.

Physiology and Chemotaxonomy
Cell size, morphology and motility were determined using light microscopy and the microscopy imaging software CellSens version 1.11 (Olympus Life Science, Tokyo, Japan).Flagella arrangement for all proposed species, and existing Rahnella species (except R. contaminans and R. laticis) was observed by transmission electron microscopy (FEI Tecnai 12 120kV BioTwin Spirit TEM) following negative staining.Briefly, grids were floated on mid-log phase bacterial suspensions for 2 mins, washed 3 times in distilled water, stained with 3 % uranyl acetate for 30 sec and washed again 3 times before wicking away excess liquid and air drying.Colony morphology was examined following growth on tryptone soya agar (TSA, Sigma) incubated at 28 °C for 48 h, while the growth temperature range was determined on TSA incubated at 4, 10, 25, 28, 30, 37 and 41 °C in triplicate.Ranges for pH were tested in triplicate in tryptone soya broth (TSB, Oxoid) with the pH adjusted to 4 -10 (in increments of 1) with sodium acetate/acetic acid and carbonate/bicarbonate buffers.Salt tolerance was tested in saline-free nutrient broth (3 g l -1 beef extract, 5 g l -1 peptone) with the salt concentration adjusted to 1 -7 % (in increments of 1 % w/v) by supplemented NaCl.These were incubated overnight at 28 °C with shaking.Included in the temperature, pH and salt tolerance tests were the type strains for existing Rahnella species (except R. aceris, R. contaminans and R. laticis).Catalase and oxidase activity were determined by bubble production in 3 % v/v H 2 O 2 and staining with Kovács reagent (1 % tetramethyl-p-phenylenediamine dihydrochloride), respectively.
Cells from all strains are straight rods with an average size of 0.6 x 1.6 µm.They occur singly, or in pairs and are motile by peritrichous flagella (Fig. 3).Members of the order Enterobacterales are known for their motility by several peritrichous flagella, and a recent study confirmed that all examined Rahnella species possess the primary peritrichous flagella locus (flag-1), with most strains of R. variigena encoding an additional secondary predicted peritrichous locus (flag-3b) [31].However, the recently described R. aceris, R. contaminans and R. laticis are indicated to be motile by a single, polar flagellum [6,7] and it was suggested that as all Rahnella species have similar flagella gene profiles, they could all be motile by a polar flagellum [7].The original description of the genus Rahnella describes R. aquatilis as motile by peritrichous flagella, although electron microscopy images were not published [2,4].To clarify the flagella arrangement of the existing Rahnella species, strains of these were also imaged by TEM as described above (with the exception of R. contaminans and R. laticis).All species examined clearly displayed multiple flagella on the surface of the cells, not at the poles, providing evidence that the majority of Rahnella species are motile by peritrichous flagella (Suppl.Fig. S3).Additionally, the genomes of all existing Rahnella species and the proposed four novel species were screened for the presence of flag loci.All species were found to possess the primary peritrichous flagella locus (flag-1), while the additional secondary flag-3b locus was encoded in the genome of strain H11b T (Rahnella clade 3) along with the flag-1 locus (data not shown).
Following growth on TSA for 48 h, the resulting colonies are cream-coloured, round, slightly convex, smooth with entire margins and 2 -3 mm in diameter.All strains tested grew at 4, 10, 25, 28, 30 and 37 °C but not at 41 °C.Growth for all Rahnella strains included in this study was observed in the pH range of 5 to 8, with weak growth at pH 9. Strains from the four proposed novel species and existing Rahnella species grew well in nutrient broth supplemented with up to 6 % NaCl, while weak growth was observed at 7 % NaCl.All strains from the proposed novel species are oxidase negative and catalase positive.
The type strain of R. aquatilis, LMG 2794 T , was included as a positive control in the API 20E and 50 CH/B tests.Due to a lack of phenotypic data for existing Rahnella species based on the GEN III microplate system, the type strains and reference strains for these were included in the GEN III assays (with the exception of the type strains of R. aceris, R.contaminans and R. laticis).
Strains from the four proposed novel species were clearly differentiated from each other, and from the existing species in the genus Rahnella based on phenotypes.Even the three proposed single-strain species have clearly distinguishable phenotypic profiles.The most useful phenotypic characteristics for species differentiation are listed in Table 2.The full phenotypic profiles for each proposed species are described in the below protologues.It is acknowledged that the phenotypic profiles for the singlestrain species may change as further strains belonging to these taxa are isolated.Differing results for several phenotypic characteristics for existing Rahnella species were observed by Jeon et al. [7], such as citrate utilization, arginine dihydrolase, gelatinase and acetoin production.The phenotypic data generated in the present study and previous studies [5,6] were obtained following the manufacturer's instructions for incubation temperature and time, whereas the data presented by Jeon et al. was generated under different incubation conditions.This could account for the differences observed between the studies.Fatty acid methyl ester (FAME) analysis was performed on selected strains from Rahnella clade 1, now confirmed as belonging to R. aceris (AR20, L31-1-12, R92a and Y9602), clade 2 (C60, L72c, L151-1A and SL6 T ) and the three strains from clades 3 -5 (H11b T , FRB 231 T and FC061912-K T ) by Fera Science Ltd.
(York, UK).Strains were cultivated on TSA at 28 °C for 24 h and the protocol followed was based on the Sherlock Microbial Identification System Version 6.4 (MIDI Inc.).The results obtained were compared against the library RTSBA6 6.21.The fatty acid profiles obtained for all strains were similar in composition to those of existing Rahnella species [5,6].Complete fatty acid profiles for all Rahnella species are presented in Table 3.
In the past eight years, the genus Rahnella has evolved from a monotypic genus to a genus comprising species from a diverse range of hosts, sources and locations.The description of four novel Rahnella species contributes to an already extensive list of environmental niches and highlights a possible role for several species in bacterial decay of onion and AOD.Additionally, a large number of strains from onion bulb decay in the USA and Norway have been assigned to R. aceris, along with strain Y9602 that has the ability to sequester heavy metals, enhancing the description of this former single-strain species.

Description of
The type strain H11b T (= LMG 32256 T = DSM 112610 T ) was isolated from onion in New York State, USA.Frequently isolated from fresh water and various environmental habitats including soils, the rhizosphere, woody tissues of oak, alder and walnut, tree sap and onion bulbs.Also found in the intestines of snails and insects such as beetles and moths.Can be isolated from foods or human clinical specimens, especially from immunocompromised patients.Major fatty acids include C 16:0 and C 17:0 cyclo.The presence of C 18:1 ω7c, summed feature 2 (iso-C 16:1 and/or C 14:0 3-OH) and summed feature 3 (C 16:1 ω7c and /or iso-C 15:0 2-OH) as major fatty acids is variable.

Description of
The G + C content ranges from 51.3 to 53.7 mol %.

FIGURES AND TABLES
T = type strain.as the average if more than one strain per species were investigated, with the standard deviation shown in parentheses.Data for 5, 7, 9, 11 -13 taken from [5], data for 8, 10 taken from [7] .n = number of strains.* Fatty acid analysis for these two species was performed by Jeon et al. [7] following growth on nutrient agar, whereas the remaining Rahnella species were cultured on TSA prior to analysis by Fera Science Ltd. (York, UK).
and Rahnella clade 3: H11b T ) and two single strains (Rahnella clades 4 and 5) from Quercus sp.(FRB 231 T ) and creek water (FC06191-K T ) were sequenced by Microbes NG (Birmingham, UK) on the Illumina HiSeq platform, following DNA extraction by cell lysis and DNA purification with SPRI (Solid Phase Reversible Immobilization) beads.

Figure 1 :Figure 2 :
Figure 1: Maximum likelihood tree based on concatenated partial gyrB, rpoB, atpD and infB gene sequences of proposed novel Rahnella species, existing Rahnella species and the closest phylogenetic neighbours.Bootstrap values after 1000 replicates are expressed as percentages (values > 50 % shown).Hafni aalvei (ATCC 13337 T ) is included as an outgroup.The scale bar indicates the fraction of substitutions per site.T = type strain

Table 3 :
Fatty acid composition (percentage of peak areas) of Rahnella species.