Introduction

Psychrobacter, a genus within the family Moraxellaceae of the class Gammaproteobacteria, was proposed by Juni and Heym in 1986. This group includes various strains found in diverse environments like animals, sea ice, nonhost habitats, and food products (Juni and Heym 1986). Members of the group Psychrobacter are Gram-stain negative, osmotolerant, psychrophilic or psychrotolerant, aerobic and grow at temperatures between − 10 and 42 °C (Kim et al. 2012). A few were isolated from humans and cause illnesses, including endocarditis and peritonitis (Bowman 2006). As of January 2022, 43 species of Psychrobacter have been taxonomically characterized and their names validly published (Parte et al. 2020). With the availability of whole-genome sequencing, comparison of the taxon-specific, conserved gene-contents is a valuable tool for examining taxonomic assignments. In this study, we performed phylogenomic analyses on the 42 available genomes of Psychrobacter spp., excluding P. pocilloporae S6-60T, for which the genome sequence was not available.

Analyses based on phylogenomics and overall genome relatedness indices indicated that P salsus is a later heterotypic synonym of P. submarinus, and the union retains the name P. submarinus. P. oceani is a later heterotypic synonym of P. pacificensis. Lastly, P. marincola and P. adeliensis are later heterotypic synonyms of P. proteolyticus.

Materials and methods

The phylogenetic comparison of strains (n = 43) was performed based on 16S rRNA gene sequences using Geopsychrobacter electrodiphilus A1T as an outgroup to root the tree. The evolutionary distances were computed using the Jukes–Cantor method (Jukes and Cantor 1969) and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated, leaving 914 positions in the final dataset. Evolutionary analyses were conducted in Mega11 (Tamura et al. 2021). The results were comparable when trees were drawn using neighbor-joining (Saitou and Nei 1987) and maximum parsimony approaches (Fitch 1977).

The genomic information for the type strains of the 42 species for which data was available was obtained from the NCBI genome database (Supplementary excel file 1). ANI was calculated using the BLAST (ANIb) in pyani with default parameters (Pritchard et al. 2019). The available genomes (Table 1) were subjected to the calculation of the overall genome-related index, which included average nucleotide identity (ANI), digital DNA–DNA hybridization (dDDH) calculated using the Genome-to-Genome Distance Calculator (GGDC) with blast + and formula 2 (Goris et al. 2007). In addition, the AAI values were calculated using the Kostas web server (Konstantinidis and Tiedje 2005a).

Table 1 General genomic attributes of the whole-genome sequence assemblies of Psychrobacter marincola, Psychrobacter adeliensis, Psychrobacter proteolyticus; Psychrobacter salsus, Psychrobacter submarinus; and Psychrobacter oceani and Psychrobacter pacificensis
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For the core genome phylogeny, ORFs were predicted using Prodigal (Hyatt et al. 2010). All ortholog single-copy proteins were identified using OrthoMCL pipeline implemented in Get_homologues (Contreras-Moreira and Vinuesa 2013). The proteins were identified as a core only if present in all studied genomes and sharing a minimum 75% sequence identity. The proteins were aligned using MAFFT (Katoh et al. 2002), and multiple sequence alignments of all core amino acids were concatenated. Phylogeny of the concatenated core proteome was reconstructed using a maximum-likelihood approach using IQ tree v2.0.3 (Minh et al. 2020) with default parameters and based on LG + F + I + G4 (Kalyaanamoorthy et al. 2017) identified as the best-fit model. The robustness of tree topology was assessed using 1000 bootstrap replicates, and the resulting tree was visualized in iTOL (Letunic and Bork 2007). OrthoVenn2 at default parameters (Ling et al. 2019) was used for determining the orthologs and paralogs cluster in similar strains.

Results and discussion

In the 16S rRNA tree, several species were closely related, with gene sequence similarities > 98%. P. salsus DD48T and P. submarinus KMM 225T formed one clade, while P. oceani 4K5T and P. pacificensis NIBH-P2K6T formed another, having 16S rRNA gene sequence similarity values of 98.72 and 98.64%, respectively. It is important to highlight that P. pocilloporae S6-60T falls within the established group of P. pacificensis species in the 16S rRNA gene tree. However, due to the unavailability of its genome sequence, a more in-depth investigation of this relationship could not be conducted. In addition, P. adeliensis SJ14T and P. marincola KMM 277T share 99.51 and 99.77% similarities with P. proteolyticus 116T (Fig. 1).

Fig. 1
figure 1

Phylogenetic tree reconstructed using 16S rRNA gene sequences of 43 validly published species described in the genus Psychrobacter using the maximum likelihood algorithm (Welter et al. 2021) and Jukes-Cantor model of substitutions (Jukes and Cantor 1969). An additional sequence for Geopsychrobacter electrodiphilus A1T was taken as an outgroup. The 16S rRNA genes for Psychrobacter adeliensis SJ14T (CAJGYR010000004.1) and Psychrobacter marincola KMM 277T (CAJHAI010000030.1) were obtained using RNAmmer (Lagesen et al. 2007). The percentages of replicate trees (n = 1000) in which the associated taxa clustered together in the bootstrap test (> 0.51) are shown next to the branches (Felsenstein 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree

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Because 16S rRNA sequence similarity is not a reliable indicator at the species level, the ANIb was calculated for all pairs of the 42 species for which genomes were available. The ANIb values were > 95% between some pairs of species, indicating that they could be reclassified in the same species (Richter and Rosselló-Móra 2009, Fig. 2). For example, ANIb values of 99.89% and 99.93% were found between P. adeliensis SJ14T (CAJGYR000000000) and P. marincola KMM 277T (CAJHAI000000000), respectively, when compared to P. proteolyticus 116T (CAJHAS000000000). Additionally, the ANIb value between P. adeliensis SJ14T and P. marincola KMM 277T was 99.88% (Table 2). This high level of relatedness was further confirmed by dDDH values exceeding 99.5%. Similarly, the AAI values between these three strains were greater than 99.9%.

Fig. 2
figure 2

Heatmap with a dual dendrogram based on the average nucleotide identity (ANI) values of 42 members of the genus Psychrobacter for which complete genomic information was available. The strains were compared using ANIb with default parameters (Supplementary excel file 2). The ANIb value matrix was then plotted as a dual dendrogram in R using pheatmap package (Kolde and Kolde 2015)

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Table 2 ANIb (%) of closely related type strains based on whole-genome sequences of the genus Psychrobacter
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Furthermore, the genomes of P. salsus DD48T (CAJHAW000000000) and P. submarinus KMM 225T (CAJHBQ000000000) were very similar and shared values of 97.18% for ANIb, 76.8% for dDDH, and 96.93% for AAI (Table 2). Also found to be closely related were, P. oceani 4K5T (CAJHAM000000000) and P. pacificensis NIBH-P2K6T (CAJHAO000000000), with values of 97.69% for ANIb, 80.2% for dDDH, and 97.90% for AAI. The genome-related measures used for species identification, such as ANI, dDDH, and AAI values, exceeded the commonly accepted thresholds for species differentiation, which are > 95% for ANI, > 70% for dDDH, and > 95% for AAI values (Kim et al. 2014; Richter and Rosselló-Móra 2009; Konstantinidis and Tiedje 2005b). This indicates that the three groups of species belong to the same species.

The high similarities of the genome-related indices were also consistent with the similarity in size and mol % G+C for the genomes of each group (Table 1). These very similar genomes also shared most of their gene clusters as determined by OrthoVenn2 at default parameters (Ling et al. 2019), where each cluster consists of orthologs or paralogs (Supplementary Fig. 1). P. salsus DD48T and P. submarinus KMM 225T share 2174 out of 2207 clusters; P. oceani 4K5T and P. pacificensis NIBH-P2K6T share 2332 out of 2373 clusters. P. adeliensis SJ14T, P. marincola KMM 277T and P. proteolyticus 116T share 2497 out of 2501 clusters.

The phylogenomic analysis based on the core proteome of the strains under study confirmed that P. salsus DD48T and P. submarinus KMM 225T; P. oceani 4K5T and P. pacificensis NIBH-P2K6T; as well as P. adeliensis SJ14T, P. marincola KMM 277T and P. proteolyticus 116T formed monophyletic clades, indicative of a high level of genome sequence identity between these strains (Fig. 3).

Fig. 3
figure 3

Phylogenomic analysis of Psychrobacter species (n = 42) using core-genome alignments. The alignment was performed using MAFFT (Katoh et al. 2002) and maximum-likelihood phylogenetic tree was reconstructed using IQ tree v2.0.3 (Minh et al. 2020) with default parameters. The test of bootstrap was performed at 1000 re-samplings and resulting tree was visualized in iTOL (Letunic and Bork 2007)

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Given the high level of genetic similarity, it was not surprising that the physiological and biochemical features of the strains possessed a high degree of phenotypic coherence (Supplementary tables 1 and 2). The few differences observed could be because of intraspecies differences or differences in the laboratory conditions. Further, these observations are well-supported by Welter et al.’s recent study, which elucidates a link between ecological niches and Psychrobacter's evolutionary history (Welter et al. 2021). According to this study, P. salsus DD48T and P. submarinus KMM 225T, as well as P. adeliensis SJ14T, P. marincola KMM 277T, and P. proteolyticus 116T, all belong to the Restricted ecotype (RE), which can only grow between 4 and 25 °C. However, P. oceani 4K5T and P. pacificensis NIBH-P2K6T belong to the Flexible ecotype (FE), which can grow between 4 and 37 °C (Welter et al. 2021).

As per the priority of prokaryotic names governed by the International Code of Nomenclature of Prokaryotes (Parker et al. 2019), when species are united, the earliest validly published name is used for the union. P. submarinus was validly published in 2002 (Romanenko et al. 2002) and P. salsus was validly published in 2004 (Shivaji et al. 2004). Therefore, P. salsus is a later heterotypic synonym of P. submarinus, and the union retains the name P. submarinus. Similarly, P. pacificensis was validly published in 2000 (Maruyama et al. 2000) and P. oceani was validly published in 2015 (Matsuyama et al. 2015). Thus, P. oceani is a later heterotypic synonym of P. pacificensis. Lastly, P. proteolyticus was validly published in 2001 (Denner et al. 2001), whereas P. marincola and P. adeliensis were validly published in 2002 and 2004, respectively (Romanenko et al. 2002; Shivaji et al. 2004). Thus, P. marincola and P. adeliensis are later heterotypic synonyms of P. proteolyticus. To reflect these species unions, emended descriptions are provided.

Emended description of Psychrobacter submarinus

The description is as before (Romanenko et al. 2002) with the following modifications:

Psychrobacter salsus (Shivaji et al. 2004) is later heterotypic synonym of Psychrobacter submarinus Romanenko et al. (2002). The type strain is DSM 14161T (= KMM 225T), and strain DD 48 (= DSM 15338 = MTCC 4826) is an additional strain of Psychrobacter submarinus. The additional strain grows at the maximum temperature of 30 °C and optimum growth is observed at 22 °C, while growth of strain KMM 225T was observed at 4–35 °C. The whole-genome sequence accession number of Psychrobacter submarinus DSM 14161T is CAJHBQ000000000. The emended DNA G+C content (mol %) and the genome size (Mbp) of the species are 44.8 (WGS) and 2.89–3.01, respectively.

Emended description of Psychrobacter pacificensis

The description is as before (Maruyama et al. 2000) with the following modifications:

Psychrobacter oceani Matsuyama et al. (2015) is a later heterotypic synonym of Psychrobacter pacificensis Maruyama et al. (2000). The type strain is NIBH P2K6T (= DSM 23406T = IFO 16270T = NBRC 103191T), and strain 4k5 (= JCM 30235 = NCIMB 14948) is an additional strain of Psychrobacter pacificensis. The additional strain has optimum growth between 25 and 30 °C. NIBH P2K6T isolates have growth yields at 4 °C comparable to those at 20 °C. However, optimal growth occurs at about 25 °C, with the maximum growth temperature being 38 °C. The whole-genome sequence accession number of Psychrobacter pacificensis NIBH P2K6T is CAJHAO000000000. The emended DNA G+C content (mol %) and the genome size (Mbp) of the species are 44 (WGS) and 2.98–3.17, respectively.

Emended description of Psychrobacter proteolyticus

The description is as before (Romanenko et al. 2002; Shivaji et al. 2004) with the following modifications:

Psychrobacter marincola Romanenko et al. (2002) and Psychrobacter adeliensis Shivaji et al. (2004) are later heterotypic synonyms of Psychrobacter proteolyticus Denner et al. (2001). The type strain is 116T (= CIP 106830T = DSM 13887T), and strains KMM 277 (= DSM 14160) and SJ14 (= DSM 15333 = MTCC 4825) are additional strains of Psychrobacter proteolyticus. The additional strains grow to a maximum temperature of 37 °C, and the optimum range is 25–30 °C. Additionally, strain 116T is routinely cultivated at room temperature (22 °C ± 3 °C). The whole-genome sequence accession number of Psychrobacter proteolyticus 116T is CAJHAS000000000. The emended DNA G+C content (mol %) and the genome size (Mbp) of the species are 42.8–42.9 (WGS) and 3.01–3.09, respectively.