Phylogenetic analysis of 16S rRNA gene
Different isolates from the gut of the armored catfish Parotocinclus maculicauda were phylogenetically characterized based on 16S rRNA gene sequence analysis. One strain (denoted P121T) was identified as belonging to the genus Paenibacillus, and its closest relatives were P. borealis DSM 13188T, P. silagei DSM 101953T and P. rhizoplanae DSM 103993T, all considered nitrogen-fixing Paenibacillus species (https://lpsn.dsmz.de/genus/paenibacillus; Elo et al. 2001; Tohno et al. 2016; Kämpfer et al. 2017). The phylogenetic similarity indicated by the 16S rRNA gene sequence data was in agreement with the levels of 16S rRNA gene sequence similarity obtained with the novel strain and P. rhizoplanae DSM 103993T (98.9% similarity), P. silagei DSM 101953T (98.3% similarity) and P. borealis DSM 13188T (97.6% similarity).
16S rRNA gene-based phylogenetic reconstruction using the maximum-likelihood algorithm, including the sequences of the most related species obtained from the GenBank database, showed that P121T clustered and in a separate clade together with P. borealis DSM 13188T, P. rhizoplanae DSM 103993T, and P. silagei DSM 101953T but in an independent branch (Fig. 1).
Genome sequence analysis
The draft genome sequence of strain P121T was determined in this study, and the Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under accession number JAIEUI000000000. The version described in this paper is version JAIEUI010000000. Genome sequencing of strain P121T resulted in a chromosome consisting of 7,513,698 bp. The G + C content was 53.9 mol%. According to the annotation, 6,955 coding sequences, 82 RNAs and 444 contigs were found in the P121T genome. The RAST analysis revealed 317 subsystems (Fig. S1). In subsystem categories, carbohydrates had the highest feature counts (299), followed by amino acids and derivatives with 279 feature counts. As a nitrogen-fixing bacterium, genome analysis of P121T revealed the presence of the nifK and nifD genes encoding dinitrogenase α and β subunits (Fe-Mo protein) and the nifH gene encoding the nitrogenase iron protein. Moreover, the nifB gene involved in the biosynthesis of the iron-molybdenum cofactor (FeMo-co or M-cluster) found in the dinitrogenase enzyme was found in the P121T genome.
AntiSMASH analysis resulted in the identification of eight predicted secondary metabolite biosynthetic gene clusters (BGCs). One of the BGCs matched paeninodine, a bacteriocin from the lassopeptide class, with 100% similarity. Lassopeptides are a class of ribosomally synthesized and posttranslationally modified natural products with diverse bioactivities (Maksimov et al. 2012). Another BGC showed 25% similarity to clusters encoding the polyketide aurantinin b/c/d. Polyketides are a large family of structurally diverse natural products with varied biological and pharmacological activities, including antibacterial, antitumor, and immunosuppressant activities (Nivina et al. 2019). These secondary metabolites have already been described in different Paenibacillus species, such as P. polymyxa KF-1 (Li et al. 2016), P. dendritiformis C454 (Zhu et al. 2016) and P. alvei MP1 (Pajor et al. 2020).
The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values were determined between strain P121T and the other three genomes of the closest related members of the genus Paenibacillus (P. borealis DSM 13188T, P. rhizoplanae DSM 103993T and P. silagei DSM 101953T) and are shown in Table 1. The ANIb values between strain P121T and P. borealis DSM 13188T, P. rhizoplanae DSM 103993T and P. silagei DSM 101953T were 80.47%, 83.52% and 84.28%, respectively. The accepted threshold for species delimitation using ANIb is 95–96% (Richter and Rosselló-Móra 2009). The in silico DDH results were in all cases lower than 45%, a value lower than 70%, which is the cutoff value for species delineation (Goris et al. 2007). Both ANI and DDH results indicate that strain P121T is a new species of the genus Paenibacillus.
Table 1
dDDH and ANI values between the genome of Paenibacillus piscarius P121T as the query genome and that of closely related species. ANI values were calculated using JSpecies with the ANIb algorithm (average nucleotide identity based on BLAST). Numbers between parentheses after ANI values are percentages of conserved aligned DNA between two genomes; numbers between parentheses after dDDH values are the confidence intervals.
Query genome
|
Reference genome
|
Bioproject NCBI number
|
Size (Mb)
|
G + C
(mol%)
|
dDDH (%)
|
ANI (%)
|
P. piscarius P121T
|
P. piscarius P121T
|
JAIEUI010000000
|
7.51
|
53.9
|
100
|
100
|
P. piscarius P121T
|
P. silagei DSM 101953
|
JAGGLV010000001
|
7.68
|
52.5
|
36.1 (33.7–38.6)
|
84.28 (72.69)
|
P. piscarius P121T
|
P. rhizoplanae DSM 103963
|
JAGGLX010000035.1
|
7.54
|
52.1
|
35.2 (32.8–37.7)
|
83.52 (70.12)
|
P. piscarius P121T
|
P. borealis DSM 13188
|
PRJNA258353
|
8.16
|
51.39
|
42.3 (39.8–44.8)
|
80.47 (60.36)
|
Finally, the comparative genome analysis for P. piscarius P121T, P. borealis DSM 13188T, P. silagei DSM 101953T and P. rhizoplanae DSM 103963T revealed that the strains formed 6874 clusters, 5015 orthologous clusters (at least containing two species) and 1859 single-copy gene clusters (Fig. 2). Paenibacillus piscarius P121T possesses 394 singletons, proteins not found in any cluster. Figure 3 shows a circular diagram illustrating the nucleotide similarity between P. piscarius P121T and other Paenibacillus genomes represented by concentric rings. The nif operon region in P121T is highlighted and shows more than 50% similarity with P. borealis DSM 13188T nif genes.
Multilocus sequence analysis (MLSA) using the housekeeping genes 16S rRNA, rpoB, gyrB and nifH
Multilocus sequence analysis (MLSA) was performed using the concatenated sequences of the 16S rRNA, rpoB, gyrB and nifH genes. Concatenation of the 16S rRNA gene and the three housekeeping genes (rpoB, gyrB and nifH) of the different nitrogen-fixing Paenibacillus species and the outgroup resulted in a phylogenetic tree (Fig. S2 a) showing the same distribution as that from the 16S rRNA gene reconstruction. Again, strain P121T grouped in a monophyletic group together with P. borealis DSM 13188T, P. rhizoplanae DSM 103993T and P. silagei DSM 101953T but formed an independent branch in the tree. Similar results were also found in phylogenetic analyses performed for each gene separately (Fig. S2 b, c, d). Furthermore, the use of neighbor-joining and maximum-parsimony methods for phylogenetic reconstructions, including the sequences of the P121T most related species, showed highly similar trees to those obtained using the maximum-likelihood algorithm (Fig. S3).
Phenotypic characteristics
Strain P121T was Gram-positive or Gram-variable, cells were rod-shaped measuring 0.63 ± 0.11 µm by 3.34 ± 0.45µm, motile with flagella (Fig. 4A). The spores were ellipsoidal, distending the sporangia and located in the central to subterminal position in the cell (Fig. 4B). The colonies were yellowish, circular, convex and mucoid, 10 to 15 mm in diameter on TSB agar.
Different phenotypic tests were used to characterize strain P121T based on the recommendations of Gordon et al. (1973) and Logan et al. (2009) and are described below in the species description. Strain P121T was also characterized by using API tests (API 50CH and API 20NE). It produced acid in API 50CH from L-arabinose, D-xylose, β-methyl-xyloside, glucose, D-fructose, D-mannose, mannitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, D-raffinose, starch, glycogen, β-gentibiose and turanose. A weak reaction was observed with methyl-D-mannoside, methyl-D-glucoside and xylitol. The novel strain was not able to produce acid with 22 of the other carbohydrates tested. Using API 20NE, strain P121T was able to reduce nitrate to nitrite, produce urease, β-galactosidase and arginine dehydrolase, and assimilate glucose, arabinose, mannitol and maltose. Phenotypic characteristics that differentiate the novel isolate from the three closely related species Paenibacillus borealis DSM 13188T (Elo et al. 2001), Paenibacillus rhizoplanae DSM 103993T (Kämpfer et al. 2017) and Paenibacillus silagei DSM 101953T (Tohno et al. 2016), also considered nitrogen-fixing Paenibacillus species, are presented in Table 2. When the phenotypic characteristics of the novel isolate were compared with those of the three closely related Paenibacillus species, it became clear that P121T could not be considered to represent typical members of any one of these previously established species (Table 2).
Table 2. Characteristics that differentiate strain P121T from the closest type strains of selected Paenibacillus species Species: 1, strain P121T; 2, P. rhizoplanae DSM 103963T; 3, P. silagei DSM 101953T; and 4, P. borealis DSM 13188T. +, positive reaction; -, negative reaction; nd, not determined.
Characteristics
|
1
|
2
|
3
|
4
|
Motility
|
+
|
-
|
-
|
+
|
Growth in the presence of
2% NaCl
oxidase
|
-
-
|
+
+
|
+
nd
|
+
-
|
Growth in pH 5.7
Growth at 5oC
|
-
-
|
-
-
|
nd
+
|
+
+
|
Nitrate reduction
|
+
|
nd
|
nd
|
-
|
Acid production from:
|
|
|
|
|
glycerol
L-arabinose
D-xylose
D-mannose
Rhamnose
Mannitol
Maltose
Lactose
Melezitose
D-arabitol
|
-
+
+
+
-
+
+
+
-
-
|
nd
-
-
-
-
-
-
-
nd
-
|
+
+
+
+
+
+
+
+
-
-
|
+
+
+
+
-
-
nd
+
+
+
|
Chemotaxonomic characteristic
In accordance with other species of the genus Paenibacillus, meso-diaminopimelic acid was detected. The quinone system was composed predominantly of menaquinones MK-7, which is also in line with other species of the genus.
The fatty acids comprised mainly iso- and anteiso-branched components, and the fatty acid profile was very similar to those of the most closely related Paenibacillus species. The major cellular fatty acids are anteiso-C15:0, iso-C16:0, iso-C15:0 and C14:0. The detailed cellular fatty acid profiles (%) of P. piscarius sp. nov. P121T and closely related Paenibacillus species is shown in Table S1.
Nitrogen fixation
The new isolate, together with the P. graminis and P. riograndensis type strains (RSA19T and SBR5T, respectively), effectively reduced acetylene, showing values varying from 2.08 to 4.73 nmol ethylene/mg protein/h (Table 3).
Table 3. Nitrogenase activity of strain P121T compared with two other nitrogen-fixing Paenibacillus species
The results are the means ± standard deviation of triplicates; (*) two independent experiments.
Strain
|
Acetylene reduction
(nmol ethylene per mg protein per h)
|
P121T
P. riograndensis SBR5T
|
2.43 ± 0.28/3.36 ± 0.23 *
4.73 ± 0.63
|
P. graminis RSA19T
|
2.08 ± 0.20
|
Description of Paenibacillus piscarius sp. nov.
Paenibacillus piscarius (pis.ca’ri.us. L. masc. adj. piscarius, of or pertaining to fish).
Cells are straight, motile rods (0.63 ± 0.11 µm in width, 3.34 ± 0.45 µm in length) with flagella. Spores are oval to ellipsoidal and predominantly central to subterminal and distend the sporangium. Young trypticase soy broth (TSB) cultures are Gram-positive or Gram-variable. On TSB agar, colonies are 10 to 15 mm in diameter, yellowish, circular, convex and mucoid. Do not grow at 10oC or 40oC; optimum is near 25oC. Growth was not observed at pH 5.7 (optimum pH 7.5-8) or in the presence of 2% NaCl. Resistant to 0.001% lysozyme. Facultatively anaerobic. Catalase and urease are produced. Voges-Proskauer negative. Nitrate is reduced to nitrite. Gelatin is not liquefied. Starch hydrolysis was negative, and esculin hydrolysis was positive. No crystalline dextrins are formed in rolled oat medium. Casein is weakly decomposed. Indole is not produced. Acid is produced from L-arabinose, D-xylose, β methyl-xyloside, glucose, D-fructose, D-mannose, mannitol, N acetyl glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, threalose, inulin, D-raffinose, starch, glycogen, gentibiose and turanose. Strain P121T does not produce acid from glycerol, erythritol, D-arabinose, ribose, L-xylose, adonitol, galactose, L-sorbose, rhamnose, dulcitol, inositol, sorbitol, melezitose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2 keto-gluconate or 5 keto-gluconate. A weak reaction was observed with xylitol, α-methyl-D-glucoside, and α-methyl-D-mannoside. Assimilation of maltose and mannitol was positive but negative for mannose and N-acetyl-glucosamine. Utilization of gluconate, caprate, adipate, malate, citrate and phenyl-acetate was not observed. Nitrogen fixation (acetylene reduction) was detected. The G¬C content of the type strain is 53.9 mol%. The major cellular fatty acids are anteiso-C15:0, iso-C16:0, iso-C15:0 and C14:0. Isolated from the gut of the armored catfish Parotocinclus maculicauda. The type strain is LFB-Fiocruz 1636, DSM 25072 (= P121T).