Revised whole genome and DNA methylome of Mycobacterium marinum type strain ATCC 927T

TL;DR

Researchers updated the genome and DNA methylation data of Mycobacterium marinum to better study its role in disease and tuberculosis mechanisms.

Contribution

A new reference genome and DNA methylome for M. marinum ATCC 927T are provided to enhance research on pathogenesis.

Findings

A revised whole genome sequence for M. marinum ATCC 927T was generated.

The DNA methylome of the same strain was characterized.

The updated data aims to improve understanding of tuberculosis-like disease mechanisms.

Abstract

Mycobacterium marinum, a slow-growing Actinobacterium, typically induces tuberculosis-like disease in fish. Here, we report a new reference sequence for M. marinum ATCC 927T, along with its DNA methylome. This aims to maximize the research potential of this type strain and facilitates investigations into the pathomechanisms of human tuberculosis.

Figures

(A) Whole genome alignment of strain ATCC 927 to its previously sequenced passage variant CCUG 20998. ProgressiveMauve (27) was used to align the indicated genomes in a step-by-step manner for the identification of conserved regions and rearrangements between the two genomes. Significant differences are indicated with arrows and numbered as follows. 1, a truncated ABC transporter permease (QDR78_11875 versus intact CCUG20998_RS12005) due to a sequence deletion, 2, a truncated PE family protein due to a frameshift (QDR78_13350 versus CCUG20998_RS28445); pseudogenes on both genomes. 3, an insertion of six genes (CCUG20998_RS15230–CCUG20998_RS15255) coding for an IS3 family transposase, recombinase (a pseudogene), cytochrome P450, TetR/AcrR family transcriptional regulator, a frameshifted TetR/AcrR family transcriptional regulator, and a frameshifted IS256 family transposase. These genes correspond to plasmid-located genes in ATCC 927T (QDR78_27190–QDR78_27215) coding for TetR/AcrR family transcriptional regulator, TetR/AcrR family transcriptional regulator, cytochrome P450, DUF308 domain-containing protein (a membrane-associated protein), a pseudogene recombinase family protein, and an IS3 family transposase, respectively. 4, a truncated non-ribosomal peptide synthase/polyketide synthase, which is also split into two parts: CCUG20998_RS28465 (AMP-binding protein, 322 amino acids) and CCUG20998_RS28470 (amino acid adenylation domain-containing protein, 1,468 amino acids), versus QDR78_13590 (9,908 amino acids). 5, another non-ribosomal peptide synthase/polyketide synthase: CCUG20998_RS15760 (12,024 amino acids) versus QDR78_15505 (9,880 amino acids). (B) Active and predicted R-M systems found in strain ATCC 927T, along with matched methylation motifs detected through SMRT whole methylome analysis. The motifs GTAYNNNNATC and CTGGAG and their corresponding R-M systems are also found in the Mycobacterium marinum strain MMA1 (REBASE org. #46029). Other PacBio-sequenced strains contain either the first (strain 050012) or the second (Mycobacterium marinum strains E11 and H01) of the R-M system-motif pairs, whereas the R-M system with recognition motif CGACNNNNNNCTGG is unique, with modified adenines (indicated as T for complementary strand) marked in blue. Strain MMA1, a human-infection-associated isolate (26), also possesses an additional Type I system with recognition motif CCACNNNNNNNTCCC that is not found in other strains. No activity was detected for the prophage-encoded methyltransferases. #, numbers of detected m6A type modifications (%, number of detected m6A modifications per total number of all detected modifications).

Tables

TABLE 1. Comparison of the whole genome details of the Mmr ATCC 927T sequenced here with the same strain sequenced earlier by Nanopore/Illumina (NI) (25) and its passage variant CCUG 20998 using PacBio SMRT (PB) (26)e

	Genome							Plasmid					Origin
Name of the strain	Accession no.	Size (bp)	G + C (%)	No. Genes	No. CDSs	No. pgenesa	No. RNAs	Accession no.	Size (bp)	No. Genes	No. tRNAs	G + C (%)
ATCC 927T (NI)	NZ_AP018496.1	6,451,936	65.5	5,744	5,150	537	57	NZ_AP018497.1	127,402d	130	1	63.50	25
CCUG 20998 (PB)	NZ_CP024190.1	6,453,310	65.7	5,411	5,203	152	56	NRb	127,576c	112c	1c	63.96c	26
ATCC 927T (PB)	CP129323	6,455,215	65.7	5,502	5,300	145	57	CP129324	127,576	112	1	63.96	This study

Full text

ANNOUNCEMENT

Mycobacterium marinum (Mmr) strain ATCC 927^T^, a slow-growing Gram-positive bacterium, was originally isolated from spontaneous tuberculosis (TB) in fish (1, 2). Phylogenetic analysis based on 16S rRNA gene sequences revealed 99.3% identity to Mycobacterium tuberculosis (Mtb), which is the leading etiological agent of TB in humans (3, 4). Like Mtb, Mmr also survives inside its natural host (zebrafish) and triggers a positive tuberculin test during infection (5–10), making it an excellent surrogate model for Mtb.

We revised the whole-genome sequence of ATCC 927^T^ using PacBio SMRT, identified the methylation motifs, and matched them to corresponding restriction-modification (R-M) systems. Freeze-dried ATCC 927^T^, from the American Type Culture Collection, was cultured in Middlebrook 7H9 broth containing 10% Albumin−Dextrose−Catalase (Thermo Fisher Scientific) and 0.5% glycerol for 7 days at 29°C. The genomic DNA was extracted using the MagAttract HMW DNA Kit (Qiagen, Nordic). Library preparation, quality control, sequencing, and assembly were performed by Novogene (UK) using their standard operating procedures. PacBio libraries were prepared using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences), following the standard PacBio protocol (11) and selecting DNA fragments > 17 kb using the BluePippin system (Sage Sciences). The libraries sequenced on a PacBio Sequel II instrument using CLR mode generated a total of 334,798 reads with an average length of 77,683 bp and an N50 value of 120,575  bp. Default parameters were used for all the following software unless otherwise specified. Base calling and demultiplexing were performed using SMRT Link V11.1 software (11). Canu-1.6 (12) and HGAP-4 (13) were used for de novo assembly of the PacBio long reads, incorporating pre-processing steps for Canu and as a separate step for HGAP. Alignment of pre-assembled reads, Arrow-2.3 (14), and Circlator-1.5.3 (15) were used for error correction, polishing, and circular assembly processes, respectively. The completeness of the assemblies was assessed using BUSCO 4.0.2 (16). The resulting two scaffolds with read depths of 499× for the chromosome and 309× for the plasmid were annotated at NCBI using the Prokaryotic Genome Annotation Pipeline PGAP-6.5 (17–19). Prophage sequences were predicted using the PHASTER server (20), and SEQWARE methylome analysis at REBASE (21) was conducted as previously described (22, 23). Due to the high sequence identity (99.63%) of the plasmid to the previously published plasmid sequence of ATCC 927^T^ (24), the plasmid was named pMMRN. Table 1 lists all central genomic data of ATCC 927^T^ in comparison to other related Mmr genomes. Figure 1 illustrates the major differences between ATCC 927^T^ and its passage variant CCUG 20998, also defined by PacBio SMRT (25), as well as indicates the methylation motifs with their corresponding active and predicted R-M systems. In summary, this study provides a new reference sequence for ATCC 927^T^ and reports the first in-depth DNA methylome analysis for this non-tuberculous bacterial model.

(A) Whole genome alignment of strain ATCC 927 to its previously sequenced passage variant CCUG 20998. ProgressiveMauve (27) was used to align the indicated genomes in a step-by-step manner for the identification of conserved regions and rearrangements between the two genomes. Significant differences are indicated with arrows and numbered as follows. 1, a truncated ABC transporter permease (QDR78_11875 versus intact CCUG20998_RS12005) due to a sequence deletion, 2, a truncated PE family protein due to a frameshift (QDR78_13350 versus CCUG20998_RS28445); pseudogenes on both genomes. 3, an insertion of six genes (CCUG20998_RS15230–CCUG20998_RS15255) coding for an IS3 family transposase, recombinase (a pseudogene), cytochrome P450, TetR/AcrR family transcriptional regulator, a frameshifted TetR/AcrR family transcriptional regulator, and a frameshifted IS256 family transposase. These genes correspond to plasmid-located genes in ATCC 927T (QDR78_27190–QDR78_27215) coding for TetR/AcrR family transcriptional regulator, TetR/AcrR family transcriptional regulator, cytochrome P450, DUF308 domain-containing protein (a membrane-associated protein), a pseudogene recombinase family protein, and an IS3 family transposase, respectively. 4, a truncated non-ribosomal peptide synthase/polyketide synthase, which is also split into two parts: CCUG20998_RS28465 (AMP-binding protein, 322 amino acids) and CCUG20998_RS28470 (amino acid adenylation domain-containing protein, 1,468 amino acids), versus QDR78_13590 (9,908 amino acids). 5, another non-ribosomal peptide synthase/polyketide synthase: CCUG20998_RS15760 (12,024 amino acids) versus QDR78_15505 (9,880 amino acids). (B) Active and predicted R-M systems found in strain ATCC 927T, along with matched methylation motifs detected through SMRT whole methylome analysis. The motifs GTAYNNNNATC and CTGGAG and their corresponding R-M systems are also found in the Mycobacterium marinum strain MMA1 (REBASE org. #46029). Other PacBio-sequenced strains contain either the first (strain 050012) or the second (Mycobacterium marinum strains E11 and H01) of the R-M system-motif pairs, whereas the R-M system with recognition motif CGACNNNNNNCTGG is unique, with modified adenines (indicated as T for complementary strand) marked in blue. Strain MMA1, a human-infection-associated isolate (26), also possesses an additional Type I system with recognition motif CCACNNNNNNNTCCC that is not found in other strains. No activity was detected for the prophage-encoded methyltransferases. #, numbers of detected m6A type modifications (%, number of detected m6A modifications per total number of all detected modifications).