Physiological characteristics
Colonies of strain M2458T were cream-coloured, opaque, round and convex on YCFA plates. scanning electron microscopy analysis revealed coccoid or ovoid-shaped cells present singly or in short chains (Fig. 1). No spores and mycelium were observed and cells could not grow in a medium with 4% (w/v) NaCl. The tolerance to 4% NaCl clearly differentiated strain M2458T from Lactococcus formosensis NBRC 109475T. Besides, M2458T grew well at pH 5.0 but Lactococcus formosensis NBRC 109475T did not. M2458T could be differentiated from the reference strains by a combination of temperature and pH for growth, acid production from various substrates, and enzymatic activities. Detailed phenotypic and differential properties are presented in Table 1.
Table 1
Characteristics of strain M2458T and type strains of species and subspecies of the genus Lactococcus with validly published names. Strains: 1, M2458T; 2, Lactococcus formosensis 516T; 3, Lactococcus garvieae NBRC 100934T; 4, Lactococcus taiwanensis NBRC 109049T; 5, Lactococcus lactis subsp. lactis BCRC 12312T; 6, Lactococcus lactis subsp. cremoris BCRC 12586T; 7, Lactococcus lactis subsp. hordniae BCRC 80474T; 8, Lactococcus lactis subsp. tructae BCRC 80475T; 9, Lactococcus chungangensis KCTC 13185T; 10, Lactococcus piscium DSM 6634T; 11, Lactococcus plantarum DSM 20686T; 12, Lactococcus raffinolactis DSM 20443T. Data obtained from the present study except where indicated. +, Positive; -, negative; W, weakly positive; NA, not available.
Characteristic
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
8
|
9
|
10
|
11
|
12
|
Growth in 4% (w/v) NaCl
|
-
|
+
|
W
|
+c
|
+
|
-c
|
-c
|
+c
|
NA
|
-d
|
NA
|
-d
|
Growth in 6% (w/v) NaCl
|
-
|
+
|
-
|
+c
|
-c
|
-c
|
-c
|
-c
|
-
|
-d
|
-d
|
-d
|
Growth at 10 ℃
|
-
|
-
|
-
|
-c
|
-c
|
-c
|
-c
|
W
|
-
|
-d
|
-d
|
-d
|
Growth at pH 5.0
|
+
|
-
|
+
|
+c
|
Wc
|
-c
|
Wc
|
Wc
|
-
|
-d
|
-d
|
-d
|
Acid production from:
|
|
|
|
|
|
|
|
|
|
|
|
|
Ribose
|
+
|
+
|
+
|
+c
|
+c
|
-c
|
-c
|
+c
|
-d
|
་d
|
-b,d
|
-d
|
Mannitol
|
-
|
+
|
+
|
+c
|
-c
|
-c
|
-c
|
+c
|
་d
|
-d
|
་b,d
|
-d
|
Amygdalin
|
-
|
+
|
+
|
+c
|
+c
|
-c
|
-c
|
+c
|
+c
|
-d
|
་b,d
|
-d
|
Melezitose
|
-
|
-
|
-
|
-c
|
-c
|
-c
|
-c
|
-c
|
-c
|
་d
|
-b,d
|
་d
|
D-Xylose
|
-
|
-
|
-
|
-c
|
+c
|
-c
|
-c
|
-c
|
-c
|
་d
|
-b,d
|
་d
|
Enzymic activity
|
|
|
|
|
|
|
|
|
|
|
|
|
Leucine aminopeptidase
|
+
|
+
|
+
|
NA
|
Wd
|
-d
|
+d
|
NA
|
+d
|
+d
|
-d
|
+d
|
Acid phosphatase
|
+
|
+
|
+
|
NA
|
+d
|
-d
|
+d
|
NA
|
+d
|
+d
|
+d
|
+d
|
β-Glucuronidase
|
-
|
-
|
-
|
NA
|
+d
|
-d
|
-d
|
NA
|
+d
|
Wd
|
Wd
|
+d
|
*Data obtained from: a, the Japan Collection of Microorganisms, RIKEN BioResource Center; b, the study of Collins et al.(Collins et al. 1983); c, Chen et al. (Chen et al. 2013); d, Cai et al. (Cai et al. 2011). |
Phylogenetic tree analysis
The 16S rRNA gene sequence of strain M2458T has been deposited in the GenBank database with the accession number of OM926013. The EzBiocloud server showed that the 16S rRNA sequence of strain M2458T has less than 97.59% homology with only one related taxon of the genus Lactococcus. The novel isolate showed the highest sequence similarities to Lactococcus formosensis NBRC 109475T (97.59%) and Lactococcus garvieae NBRC 100934T (97.10%). The phylogenetic tree analysis indicated that strain M2458T had a separate branch within the genus Lactococcus and formed a group with Lactococcus formosensis NBRC 109475T (Fig. 2). Therefore, Lactococcus formosensis NBRC 109475T was selected as the closest reference strain for comparative analysis with strain M2458T.
Housekeeper genes are now commonly used to supplement 16S rRNA gene analysis to determine species levels. Comparing rpoA gene sequence with GenBank database sequence, strain M2458T has the highest similarity (96.56%) to Lactococcus formosensis NBRC 109475T and the second highest similarity (96.53%) to Lactococcus garvieae NBRC 100934T. Figure 3 shows the phylogenetic analysis of the rpoA gene. In addition, the rpoB gene was compared with that of other type strains of species of the genus Lactococcus. The results showed that strain M2458T has 93.57% sequence similarity to Lactococcus formosensis NBRC 109475T. The phylogenetic analysis of rpoB gene is shown in Fig. 4.
Chemotaxonomic analysis
The cellular fatty acids of strain M2458T and its closest reference strains are shown in Table 2. The major fatty acids of strain M2458T were C18:1ω9c, C16:0 and C18:1ω7c. Strain M2458T and its phylogenetically closest relatives exhibited some qualitative and quantitative differences in fatty acid content. PE, PG, and DPG were the major polar lipids, while 1 unidentified polar lipid was the minor polar lipid (Fig. 5).
Table 2
Fatty acid composition and content of the strain M2458T.
Fatty acid (%)
|
1
|
2
|
3
|
Saturated straight-chain
|
|
|
|
C14:0
|
ND
|
3.43
|
2.44
|
C16:0
|
13.26
|
28.78
|
27.37
|
C17:0
|
ND
|
ND
|
1.21
|
C18:0
|
3.19
|
2.75
|
4.08
|
C20:0
|
ND
|
1.63
|
ND
|
Branched fatty acid
|
|
|
|
C19:0 iso Ι
|
1.33
|
ND
|
ND
|
C19:0 iso
|
4.72
|
3.43
|
3.23
|
Unsaturated fatty acids
|
|
|
|
C16:1ω6c
|
ND
|
ND
|
1.04
|
C18:0ω6, 9c
|
ND
|
ND
|
2.51
|
C18:1ω7c
|
11.52
|
12.33
|
15.76
|
C18:1ω9c
|
39.68
|
18.44
|
15.54
|
C18:2ω6, 9c
|
9.61
|
4.12
|
ND
|
C19:1ω6c
|
4.72
|
9.59
|
ND
|
C20:1ω9c
|
ND
|
2.31
|
2.94
|
Hydroxy acids:
|
|
|
|
C17:0 2OH
|
10.19
|
5.57
|
2.49
|
Strains: 1, M2458T; 2, Lactococcus formosensis 516T; 3, Lactococcus garvieae NBRC 100934T. |
Draft genome sequencing
The genome sequence was submitted to the GenBank (www.ncbi.nlm.nih.gov/) database (JAKZEZ000000000). The genome size of strain M2458T was 4,171,867 bp and 3866 genes were annotated. Among them, 79 genes were tRNAs and 5 genes were rRNAs. Total DNA G + C content of strain M2458T was determined to be 38.29 mol%, which was within the G + C content range of the genus Lactococcus (35.5–47.5 mol%) (Chen et al. 2014; Chen et al. 2013). The ANI values of strain M2458T with the closely species Lactococcus formosensis NBRC 109475T, Lactococcus garvieae NBRC 100934T and Lactococcus petauri 159469T were 82.35, 82.45 and 82.46%, respectively, and the dDDH values estimated by the GGDC were 26.9, 26.8 and 26.4%, respectively (Table 3). In general, species boundaries for ANI and dDDH values are 95–96% and 70%, respectively (Chun et al. 2018).
Table 3
Genomic homology analysis of strain M2458T and type strains
Query Genome
|
Reference genome
|
ANI (%)
|
dDDH (%)
|
M2458T
|
Lactococcus formosensis NBRC 109475T
|
82.35
|
26.9
|
M2458T
|
Lactococcus garvieae NBRC 100934T
|
82.45
|
26.8
|
M2458T
|
Lactococcus petauri 159469T
|
82.46
|
26.4
|
Taxonomic conclusion
In accordance with the data, strain M2458T displays numerous features that are common to members of the genus Lactococcus, e.g., PG and DPG as major lipids; and C18:1ω9c, C16:0 and C18:1ω7c as major fatty acids. However, chemotaxonomic and phenotypic differences differentiate the novel isolate from previously recognized species of the genus Lactococcus despite the phylogenetic tree of the 16S rRNA gene sequence is in the same group (Table 1). Therefore, strain M2458T should be assigned to the genus Lactococcus with the proposed name Lactococcus intestinalis sp. nov.
Description of Lactococcus intestinalis sp. nov.
Lactococcus intestinalis (intestinal’ is. L.gen. adj. intestinalis of intestinal, where the type strain M2458T was isolated).
Cells are Gram-stain-positive, catalase-negative, coccoid or ovoid-shaped, facultatively anaerobic and grow well anaerobically on YCFA agar at 37°C. Cells utilize d-glucose homofermentatively but do not produce gas. L-lactic acid is the main product. Growth occurs at 15–37°C but not at 10 or 45°C. The cells can grow at pH 5–7 and have a tolerance for NaCl from 0 to 3%. Optimum growth conditions are 37°C, pH 6–7 and 0–1% NaCl. The enzyme reaction is positive for leucine aminopeptidase, acid phosphatase, but is negative for β-Glucuronidase. Acid is produced from ribose, amygdalin, arbutin, D-glucose, D-fructose, D-mannose, salicin, cellobiose, maltose, trehalose, starch, gentiobiose, sucrose, D-tagatose, N-acetylglucosamine, aesculin, but is not produced from L-arabinose, D-galactose, D-mannitol, lactose, melibiose, D-xylose, D-adonitol, D-arabinose, D-arabitol, L-arabitol, dulcitol, erythritol, gluconate, glycerol, glycogen, L-fucose, D-fucose, inositol, inulin, D-lyxose, melezitose, L-rhamnose, L-sorbose, D-sorbitol, D-turanose, L-xylose, D-xylitol, methyl β-xyloside, methylα-D-mannoside, methyl α-D-glucoside, D-raffinose, 2-ketogluconate and 5-ketogluconate. The major fatty acids are C18:1ω9c, C16:0 and C18:1ω7c. The type strain is M2458T (= JCM 35706 = CGMCC 1.60066), isolated from intestinal contents of APPswe/PSΔE9 mouse model of Alzheimer’s disease. The DNA G + C content of the type strain is 38.29 mol%. The GenBank accession number for the 16S rRNA gene sequence of the type strain is OM926013 and the number for the whole genome sequence JAKZEZ000000000.