Expression of collagenase in Flavobacterium psychrophilum isolated from cold-water disease-affected ayu (Plecoglossus altivelis)

Figures & data

Fig. 1. The fpcol gene of F. psychrophilum.

Notes: (a) The gene map around fpcol gene in F. psychrophilum. The primers col1F and col1R were designed within the unknown gene and the tRNA-methyl transferase gene, which were neighboring the fpcol gene, respectively. (b) Comparison of the fpcol genes of the isolates WA-1, WA-2, and WB-1. (c) The fpcol gene of WA-1. The coding and noncoding nucleotide sequences are shown by uppercase and lowercase letters, respectively. Amino acids (one letter code) are shown beneath the ORF nucleotide sequence. The -35 Box, Pribnow Box, 6-nt repeat (caaaaa) region, and ribosomal protein S1-binding site are indicated by wavy, dashed, thick, and thin lines, respectively. The signal peptide, catalytic motif (HExxH), Por secretion tail, and translation terminator are indicated by a box, a double-lined box, dashed boxes, and an asterisk, respectively. Horizontal arrows denote the transcriptional terminator.

Table 1. Primers used in this study.

Primer	Target gene	Sequences (5′–3′)
8F	16S rRNA	AGAGTTTGATCCTGGCTCAG
357F	16S rRNA	CTCCTACGGGAGGCAGCAG
1492R	16S rRNA	GGTTACCTTGTTACGACTT
1541R	16S rRNA	AAGGAGGTGATCCAGCC
col1f	Collagenase	CCATCTAATTATGCACC
col2f*	Collagenase	ATGATGTACAAAAGACTCAGCG
col3F	Collagenase	TGACTTGAARAACCAYCGYYG
col4f	Collagenase	AGAGTTTGATCMTGGCTCAG
col5f	Collagenase	ATTACCGCGGCTGCTGG
col6f	Collagenase	TTAAAAATTGAAACTTATG
col7f	Collagenase	TACGGTTGGTTTAAAG
col8f	Collagenase	AACAATGGTACTTATAC
col9f	Collagenase	GAAACTTACGGTAAACG
col10f	Collagenase	TGGTGATGGTTATAC
col1r	Collagenase	CGAAACGCCACAATCTGCC
col2r	Collagenase	AATAAATTTCTCATTATCTACTG
col3r	Collagenase	CCATATTCTGTATTGTC
col4r	Collagenase	AAGGAGGTGATCCAGCC
col5r	Collagenase	CCTGGTTGTGTGTTGTAAGC
col6r	Collagenase	CATATCTACAAAAAATATACC
col7r	Collagenase	CAGCTCGTGTTGTGAAATGT
col8r*	Collagenase	TCTAGCTTGAAAAAAAGG
Fpp1F	Fpp1	ATGAAAAAAAAATTACTATT
Fpp1R	Fpp1	TTATTCTATAACAATACGTT
Fpp2F	Fpp2	ATGAAGAAAA ATACAAGTAA
Fpp2R	Fpp2	TTATTCTACAACAATACGTT
PPIC1388F*	PPIC	TTCATATCATCAATGTAACGGACAAGC
PPIC1593R*	PPIC	GTCAAGTGCTAATAGTTTAGCTGCTGGAGC

Note: Asterisks denote the primers used in RT-qPCR.

Fig. 2. The 5′ leader sequences and the start region of the fpcol genes.

Notes: The fpcol gene sequences of JIP02/86 (AM398681), WA-1, WA-2, and WB-1 were aligned using Clustal W.³⁵⁾ Asterisks denote identical nucleotides.

Fig. 3. Collagenolytic activity.

Notes: The collagenolytic activities of the culture supernatants of WA-1, WA-2, and WB-1 against FITC-labeled type I collagen were shown. The supernatant of WA-1 showed higher collagenolytic activity than that of WA-2. The supernatant of WB-1 showed no collagenolytic activity. One unit of collagenolytic activity = the amount of enzyme required to cleave 50 μg of collagen/min.

Table 2. Properties of the F. psychrophilum isolates used in this study.

Isolate	Gelatinolytic activity	fpcol gene	Proteolytic activity	fpp1 gene	fpp2 gene
WA-1	+	Active (960aa)	–	Frameshift (A^43 → deletion)^b	Insertion of transposon (2002th, 986 bp)^d
WA-2	+	Active (960aa)	+	Active (1138aa, >99%^c)	Nonsense mutation (G^466GA→TGA)^e
WB-1	–	Frameshift (G^499 → deletion)^a	+	Active (1138aa, >99%^c)	Insertion of transposon (279th, 1019 bp)^d

^a Figure in the parenthesis means the position of the nucleotide in the fpcol gene.

^b Figure in the parenthesis means the position of the nucleotide in the fpp1 gene.

^c Amino acid identity with that of the fpp1 gene.

^d Figures in the parentheses mean the insertion position and the length of the transposon in the fpp2 gene.

^e Figure in the parenthesis means the position of the nucleotide in the fpp2 gene.

Fig. 4. Proteolytic activity of the F. psychrophilum isolates.

Notes: The F. psychrophilum isolates were cultured on solid CY medium containing 1.5% agarose and 10% horse serum at 18 °C for 6 days. (a) WA-1 formed no clear zones. (b) WA-2 and (c) WB-1 formed clear zones around streaked colonies.

Fig. 5. The growth curves and the transcription levels of the fpcol gene.

Notes: The growth curves (OD₆₂₀) and the transcription levels of the fpcol genes (col ref⁻¹) were shown by filled circles and hollow squares, respectively. WA-1 was cultured in CY medium (a), CY medium + 0.1 mM calcium chloride (b), and CY medium + 0.1% gelatin (c) at 18 °C, and in CY medium at 10 °C (d). WA-2 was cultured in CY medium at 18 °C (e). In all conditions, the transcription of the fpcol gene was the highest in the death phase of each culture.

Fig. 6. Bacterial challenge.

Notes: The accumulated mortality of CWD-affected ayu injected with 10⁷ CFU of WA-1 (filled circles), WA-2 (hollow circles), or WB-1 (filled squares) were shown. The accumulated mortality of ayu injected with WA-1 was higher than that with WA-2. No fish injected with WB-1 died.

Larkin MA, Blackshields G, Brown NP, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23:2947–2948.10.1093/bioinformatics/btm404

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