MLSA of the five concatenated HKGs
Of all 223 Proteus strains collected in this study, the phylogenetic tree of the concatenated 5 genes divided them into eleven clusters (Fig. 1), representing thirteen species. Among the clusters, ten contained one type strain of each. However, cluster 5 was comprised of three type strains, i.e., Proteus genospecies ATCC51470T, P. cibarius JCM 30699T and P. terrae LMG 28659T.
As expected, among the 223 Proteus strains, P. mirabilis (cluster 1) is the largest cluster (n=157, 70.4%) distinctly separated from the others, and there are three subclusters within this cluster. Cluster 5 is the second largest among Proteus strains (n=33, 14.8%) followed by P. vulgaris (cluster 2) (n=14, 6.3%) and P. penneri (cluster 7) (n=6, 2.7%).
Identification of Proteus species by phylogenetic analysis of five individual genes
Phylogenetic trees based on five individual HKGs were also constructed (Fig. 2). Phylogenetic trees of the five HKGs (dnaJ, mdh, pyrC, recA and rpoD) can be divided into eleven clusters, representing eleven species and corresponding to that of the concatenated tree. Meanwhile, phylogenetic trees of four individual HKGs (dnaJ, mdh, pyrC and rpoD) were the same as that of the concatenated tree, both in numbers of species (cluster) and strain numbers within each species (cluster). There is one inconsistency between trees of recA and concatenated 5-gene: recA identified four strains as unclusters, whereas the four strains were identified by concatenated 5 genes, and the other four HKGs were identified as genospecies 6 (Fig. 2). The results showed that it is inaccurate to classify the species of Proteus by using a single housekeeping to reflect general gene phylogenetic tree and it only reflects the evolution by itself, which is caused by genetic recombination or specific selection. While the phylogenetic tree constructed by five concatenated HKGs can overcome the basis.
Inter- and intraspecies distances of HKGs
The inter- and intraspecies distances of HKGs were summarized in a boxplot of the concatenated 5 genes (Fig. 3). All interspecies distances were clearly different from intraspecies distances. Among the interspecies boxplots, two species, P. mirabilis, and P. hauseri, indicated compacted distance ranges (both standard deviations, SD=0.004), whereas the remaining nine species shared dispersive distance ranges (SD ranges from 0.024 to 0.065). On the other hand, among the intraspecies boxplots, P. hauseri possessed a compacted distance range (SD=0.000) compared to that of five species (SD range from 0.012 to 0.058). Meanwhile, boxplots of the five individual genes (Figure S1) indicated the same trends of intra- and interspecies distance as that of the concatenated 5 genes, although there were small parts overlapping in species 5 and 6 of pyrC. The detailed genetic distance and median values of individual genes and the concatenated 5 genes are summarized in Table S1.
Web-based DNA-DNA hybridizations among species
To confirm the correctness of strains among the eleven species, we used web-based DDH, such as dDDH and ANI, to detect their similarity values. Among the eleven species defined in this study, the dDDH and ANI values of the type/representative strains were 23.5-51.4% and 80.8-94.4% (Table 2), less than the proposed cutoff level for species delineation, i.e., 70% and 95%, respectively. Notably, among the three subclusters within cluster 5 (Fig. 1), either among the three published type strains (Proteus genospecies ATCC51470T, P. cibarius JCM30699T and P. terrae LMG28659T) or representative strain (CA142267) among the three subclusters, their dDDH and ANI values were more than the proposed cutoff level for species delineation. These results indicate that strains within cluster 5 actually belong to the same species.
Table 2. dDDH relatedness and ANI values among the eleven species.
Strains #
|
a
|
b
|
c
|
d
|
e
|
f
|
g
|
h
|
i
|
j
|
k
|
l
|
m
|
n
|
Species
|
P. mirabilis
|
P. penneri
|
P. vulgaris
|
P.hauseri
|
Proteus genospecies 4
|
Proteus genospecies 5
|
P. cibarius
|
P. terrae
|
CA142267
|
Proteus genospecies 6
|
P. columbae
|
P. alimentorum
|
P. faecis
|
P. cibi
|
DDH/ANI
|
DDH
|
ANI
|
DDH
|
ANI
|
DDH
|
ANI
|
DDH
|
ANI
|
DDH
|
ANI
|
DDH
|
ANI
|
DDH
|
ANI
|
DDH
|
ANI
|
DDH
|
ANI
|
DDH
|
ANI
|
DDH
|
ANI
|
DDH
|
ANI
|
DDH
|
ANI
|
DDH
|
ANI
|
Strains#
|
a
|
100.0
|
100.0
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
b
|
24.7
|
82.1
|
100.0
|
100.0
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
c
|
24.4
|
81.7
|
37.5
|
89.4
|
100.0
|
100.0
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
d
|
23.5
|
80.8
|
25.8
|
83.3
|
25.1
|
82.6
|
100.0
|
100.0
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
e
|
24.6
|
81.8
|
41.5
|
90.9
|
44.5
|
91.6
|
25.8
|
83.2
|
100.0
|
100.0
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
f
|
24.3
|
81.8
|
44.6
|
91.7
|
37.7
|
89.6
|
25.8
|
83.1
|
40.0
|
90.2
|
100.0
|
100.0
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
g
|
25.1
|
82.6
|
44.5
|
91.7
|
37.5
|
89.6
|
25.7
|
83.2
|
39.9
|
90.2
|
72.3
|
96.9
|
100.0
|
100.0
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
h
|
24.1
|
81.5
|
44.6
|
91.8
|
37.5
|
89.5
|
25.7
|
83.3
|
39.9
|
90.2
|
93.1
|
99.2
|
71.2
|
96.7
|
100.0
|
100.0
|
|
|
|
|
|
|
|
|
|
|
|
|
i
|
24.4
|
82.0
|
44.9
|
91.8
|
37.6
|
89.7
|
25.8
|
83.1
|
39.8
|
90.3
|
74.4
|
97.1
|
72.3
|
96.8
|
72.6
|
96.8
|
100.0
|
100.0
|
|
|
|
|
|
|
|
|
|
|
j
|
25.0
|
82.4
|
44.8
|
91.7
|
36.0
|
89.0
|
26.1
|
83.4
|
37.8
|
89.5
|
47.3
|
92.3
|
47.1
|
92.5
|
47.3
|
92.4
|
48.3
|
92.8
|
100.0
|
100.0
|
|
|
|
|
|
|
|
|
k
|
24.8
|
81.9
|
46.3
|
92.1
|
36.8
|
89.1
|
26.0
|
83.4
|
37.9
|
89.6
|
48.9
|
92.7
|
48.3
|
92.6
|
48.8
|
93.0
|
49.8
|
93.0
|
57.1
|
94.4
|
100
|
100
|
|
|
|
|
|
|
l
|
24.9
|
82.0
|
46.3
|
92.1
|
36.4
|
89.0
|
25.9
|
83.3
|
37.8
|
89.6
|
48.7
|
92.8
|
48.1
|
92.7
|
48.5
|
92.7
|
49.2
|
92.3
|
52.4
|
93.7
|
53.9
|
93.9
|
100
|
100
|
|
|
|
|
m
|
24.1
|
81.6
|
52.7
|
93.7
|
35.8
|
88.7
|
25.7
|
83.1
|
36.5
|
89.1
|
45.9
|
91.9
|
45.5
|
91.9
|
45.9
|
92.0
|
46.6
|
92.1
|
47.1
|
92.2
|
48.6
|
92.7
|
48.5
|
92.6
|
100
|
100
|
|
|
n
|
24.6
|
81.9
|
45.3
|
92.0
|
35.5
|
88.3
|
25.8
|
83.0
|
36.5
|
89.0
|
45.8
|
92.0
|
45.5
|
92.0
|
45.9
|
92.0
|
46.6
|
92.3
|
49.2
|
92.0
|
51.4
|
93.3
|
50.5
|
93
|
49.5
|
92.9
|
100
|
100
|
# Strain: a, P. mirabilis ATCC 29906T(GenBank accession no. ACLE00000000.1); b, P. penneri ATCC 33519T (PHFJ00000000); c, P. vulgaris KCTC 2579T (PHNN000000000); d, P. hauseri JCM 1668T (PGWU00000000); e, Proteus genospecies 4 ATCC 51469T (PENV00000000); f, Proteus genospecies 5 ATCC 51470T (PENU00000000); g, P. cibarius JCM 30699T (PGWT00000000); h, P. terrae LMG 28659T (PENS00000000); i, CA142267; j, Proteus genospecies 6 ATCC 51471T (PENT00000000); k, P. columbae 08MAS2615T (NGVR00000000); l, P. alimentorum 08MAS0041T (NBVR00000000); m, P. faecis TJ1636T (PENZ00000000); n, P. cibi FJ2001126-3T (PENW00000000).
Results were percentages based on Formula 2, calculate distances and DDH estimates with GGDC 2; ANI values were estimated using the web-based service ANI calculator (http://www.ezbiocloud.net/tools/ani). The gray shadow marked strains with dDDH>70% an23d ANI>95%, respectively, indicating they belong to the same species.
|
Reclassification of Proteus genospecies 5 and P. cibarius to P. terrae
Since either MLSA of the five concatenated HKGs or phylogenetic analysis of five individual genes indicated that three type strains, i.e., Proteus genospecies ATCC51470T, P. cibarius JCM 30699T and P. terrae LMG 28659T, fell into one cluster (cluster 5 in Fig. 1), further web-based DNA-DNA hybridizations, such as dDDH and ANI, confirmed that among the three subclusters within cluster 5, either among the three type strains or representative strain (CA142267) among the three subclusters, their dDDH and ANI values were higher than the proposed cutoff level for species delineation (70% for dDDH and 95% for ANI, Table 2). The genomic analysis provided evidence that strains within cluster 5 actually belonged to the same species.
Further phenotypic characteristics were detected among type strains of Proteus genospecies 5, P. cibarius and P. terrae, and slight distinctive properties were observed (Table 3). Only minor differences were obtained between the type strains of the three species, including growth at the optimum temperature, growth range in NaCl and pH, utilization of DNase, lipase and citric acid, and DNA G+C content. Combined with the genetic, genomic and phenotypic characteristics, three species, P. terrae reported by Behrendt et al. 2015, P. cibarius reported by Hyun et al. 2016 and Proteus genospecies 5 reported by O’Hara et al. 2000, should be regarded as the heterotypic synonyms of Proteus terrae reported by Behrendt et al. 2015.
Table 3. Distinctive phenotypic characteristics among the type strains P. terrae, P. cibarius and Proteus genospecies 5#.
Characteristic
|
P. terrae
|
P. cibarius
|
Proteus genomospecies 5
|
Growth in optimum temperature (°C)
|
37
|
35
|
37
|
Growth range in NaCl (%,w/v)
|
0-15
|
0-12
|
0-15
|
Growth range in pH
|
4-9
|
4-9
|
4-9
|
DNase (25°C) (3 days)
|
+
|
+
|
+
|
Lipase (olive oil) (7 days)
|
-
|
+
|
-
|
CIT
|
-
|
-
|
+
|
DNA G+C content (mol %)
|
37.9
|
37.8
|
37.8
|
#Species strain: P. terrae LMG 28659T; P. cibarius JCM 30699T; Proteus genospecies 5 ATCC 51470T.