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Fulltext Commentary: Complete Genome Sequence of 3-Chlorobenzoate-Degrading Bacterium Cupriavidus necator NH9 and Reclassification of the Strains of the Genera Cupriavidus and Ralstonia Based on Phylogenetic and Whole-Genome Sequence Analyses

Gan HM

Front Microbiol, 2019;10:2011.
PMID: 31555242 DOI: 10.3389/fmicb.2019.02011
Fulltext A glimpse into the genetic basis of symbiosis between Hydrogenophaga and their helper strains in the biodegradation of 4-aminobenzenesulfonate

Kim K, Gan HM

J Genomics, 2017;5:77-82.
PMID: 28775791 DOI: 10.7150/jgen.20216

We report the whole genome sequences of Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2, the first reported bacterial co-culture capable of degrading 4-aminobenzenesulfonate (4-ABS), a recalcitrant industrial waste product. To gain insights into the genetic basis for the syntrophic interaction between this symbiotic pair and also another recently reported Hydrogenophaga associated co-culture, Hydrogenophaga sp. PBC and Ralstonia sp. PBA, we performed detailed genetic analysis of these four strains focusing on the metabolic pathways associated with biotin, para-aminobenzoic acid (pABA), and protocatechuate metabolism. Both assembled Hydrogenophaga draft genomes are missing a majority of the genetic components associated in the biosynthetic pathway of pABA and biotin. Interestingly, a fused pABA synthase was found in R. sp PBA but not in A. radiobacter S2. Furthermore, using whole genome data, the taxonomic classification of R. sp. PBA and A. radiobacter S2 (both previously inferred from 16S rRNA gene) was re-investigated, providing new evidence to propose for their re-classification at the genus and species level, respectively.
The complete mitogenome of the Australian spiny crayfish Euastacus yarraensis (McCoy, 1888) (Crustacea: Decapoda: Parastacidae)

Gan HM, Tan MH, Austin CM

Mitochondrial DNA A DNA Mapp Seq Anal, 2016;27(2):929-30.
PMID: 24938115 DOI: 10.3109/19401736.2014.926490

The mitochondrial genome sequence of the Australian crayfish, Euastacus yarraensis, is documented and compared with other Australian crayfish genera. Euastacus yarraensis has a mitogenome of 15,548 base pairs consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a non-coding AT-rich region. The base composition of E. yarraensis mitogenome is 32.39% for T, 22.45% for C, 34.43% for A, and 10.73% for G, with an AT bias of 66.82%. The mitogenome gene order conforms to what is considered the primitive arrangement for parastacid crayfish.
The complete mitogenome of the red claw crayfish Cherax quadricarinatus (Von Martens, 1868) (Crustacea: Decapoda: Parastacidae)

Gan HM, Tan MH, Austin CM

Mitochondrial DNA A DNA Mapp Seq Anal, 2016;27(1):385-6.
PMID: 24617485 DOI: 10.3109/19401736.2014.895997

The commercial freshwater crayfish Cherax quadricarinatus complete mitochondrial genome was recovered from partial genome sequencing using the MiSeq Personal Sequencer. The mitogenome has 15,869 base pairs consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a non-coding AT-rich region. The base composition of C. quadricarinatus is 32.16% for T, 23.39% for C, 33.26% for A, and 11.19% for G, with an AT bias of 65.42%.
The complete mitogenome of the Macquarie perch, Macquaria australasica Cuvier, 1830 (Teleostei: Percichthyidae)

Gan HM, Tan MH, Austin CM

Mitochondrial DNA A DNA Mapp Seq Anal, 2016;27(1):383-4.
PMID: 24617484 DOI: 10.3109/19401736.2014.895996

The complete mitochondrial genome of the conservationally significant Macquarie perch (Macquaria australasica) was obtained from low-coverage shotgun sequencing using the MiSeq sequencer. The M. australasica mitogenome has 16,496 base pairs (55% A + T content) made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a 819 bp non-coding AT-rich region. This is the first mitogenome sequence for the genus Macquaria, and the third to be reported for the family Percichthyidae.
Fulltext Integrated shotgun sequencing and bioinformatics pipeline allows ultra-fast mitogenome recovery and confirms substantial gene rearrangements in Australian freshwater crayfishes

Gan HM, Schultz MB, Austin CM

BMC Evol. Biol., 2014;14:19.
PMID: 24484414 DOI: 10.1186/1471-2148-14-19

Although it is possible to recover the complete mitogenome directly from shotgun sequencing data, currently reported methods and pipelines are still relatively time consuming and costly. Using a sample of the Australian freshwater crayfish Engaeus lengana, we demonstrate that it is possible to achieve three-day turnaround time (four hours hands-on time) from tissue sample to NCBI-ready submission file through the integration of MiSeq sequencing platform, Nextera sample preparation protocol, MITObim assembly algorithm and MITOS annotation pipeline.
Fulltext First genomic insights into carbapenem-resistant Klebsiella pneumoniae from Malaysia

Gan HM, Eng WWH, Dhanoa A

J Glob Antimicrob Resist, 2020 03;20:153-159.
PMID: 31325618 DOI: 10.1016/j.jgar.2019.07.008

OBJECTIVES: Despite the increasing reports of carbapenem-resistant Enterobacteriaceae in Malaysia, genomic resources for carbapenem-resistant clinical strains of Klebsiella pneumoniae (K. pneumoniae) remain unavailable. This study aimed to sequence the genomes of multiple carbapenem-resistant K. pneumoniae strains from Malaysia and to identify the genetic basis for their resistance.
METHODS: Illumina whole genome sequencing was performed on eight carbapenem-resistant K. pneumoniae isolated from a Malaysian hospital. Genetic diversity was inferred from the assembled genomes based on in silico multilocus sequence typing (MLST). In addition, plasmid-derived and chromosome-derived contigs were predicted using the machine learning approach. After genome annotation, genes associated with carbapenem resistance were identified based on similarity searched against the ResFinder database.
RESULTS: The eight K. pneumoniae isolates were grouped into six different sequence types, some of which were represented by a single isolate in the MLST database. Genomic potential for carbapenem-resistance was attributed to the presence of plasmid-localised blaNDM (blaNDM-1/blaNDM-5) or blaKPC (blaKPC-2/blaKPC-6) in these sequenced strains. The majority of these carbapenem resistance genes was flanked by repetitive (transposase or integrase) sequences, suggesting their potential mobility. This study also reported the first blaKPC-6-harbouring plasmid contig to be assembled for K. pneumoniae, and the second for the genus Klebsiella.
CONCLUSION: This study reported the first genomic resources for carbapenem-resistant K. pneumoniae from Malaysia. The high diversity of carbapenem resistance genes and sequence types uncovered from eight isolates from the same hospital is worrying and indicates an urgent need to improve the genomic surveillance of clinical K. pneumoniae in Malaysia.
Fulltext Data on whole-genome sequencing of extended-spectrum beta-lactamases producing Enterobacteriaceae isolates from Malaysia

Gan HM, Eng WWH, Dhanoa A

Data Brief, 2019 Aug;25:104257.
PMID: 31384648 DOI: 10.1016/j.dib.2019.104257

We report the whole genome sequencing data and de novo genome assemblies for eight extended-spectrum beta-lactamases (ESBL) producing Enterobacteriaceae isolates from Malaysia consisting of four Klebsiella pneumoniae, two Enterobacter harmaechei, one Citrobacter freundii and one Escherichia coli. We identified at least one ESBL gene in each genome, with blaCTX-M-15 being the most prevalent ESBL gene in the current genomic sampling.
Fulltext The complete mitochondrial genome of the snakeskin gourami, Trichopodus pectoralis (Regan 1910) (Teleostei: Osphronemidae)

Gan HM, Amornsakun T, Tan MP

Mitochondrial DNA B Resour, 2017 Mar 17;2(1):148-149.
PMID: 33473747 DOI: 10.1080/23802359.2017.1298418

We sequenced and assembled three whole mitogenome sequences of the commercially important snakeskin gourami Trichopodus pectoralis isolated from Malaysia (introduced), Viet Nam (native) and Thailand (native). The mitogenome length range from 16,397 to 16,420 bp. The final partitioned nucleotide alignment consists of 14,002 bp and supports the monophyly of the genus Trichopodus (95% ultrafast bootstrap support) with T. trichopterus forming a sister group with the members of T. pectoralis.
Transcriptome-Guided Identification of Carbohydrate Active Enzymes (CAZy) from the Christmas Island Red Crab, Gecarcoidea natalis and a Vote for the Inclusion of Transcriptome-Derived Crustacean CAZys in Comparative Studies

Gan HM, Austin C, Linton S

Mar Biotechnol (NY), 2018 Oct;20(5):654-665.
PMID: 29995174 DOI: 10.1007/s10126-018-9836-2

The Christmas Island red crab, Gecarcoidea natalis, is an herbivorous land crab that consumes mostly fallen leaf litter. In order to subsist, G. natalis would need to have developed specialised digestive enzymes capable of supplying significant amounts of metabolisable sugars from this diet. To gain insights into the carbohydrate metabolism of G. natalis, a transcriptome assembly was performed, with a specific focus on identifying transcripts coding for carbohydrate active enzyme (CAZy) using in silico approaches. Transcriptome sequencing of the midgut gland identified 70 CAZy-coding transcripts with varying expression values. At least three newly discovered putative GH9 endo-β-1,4-glucanase ("classic cellulase") transcripts were highly expressed in the midgut gland in addition to the previously characterised GH9 and GH16 (β-1,3-glucanase) transcripts, and underscoring the utility of whole transcriptome in uncovering new CAZy-coding transcripts. A highly expressed transcript coding for GH5_10 previously missed by conventional screening of cellulase activity was inferred to be a novel endo-β-1,4-mannase in G. natalis with in silico support from homology modelling and amino acid alignment with other functionally validated GH5_10 proteins. Maximum likelihood tree reconstruction of the GH5_10 proteins demonstrates the phylogenetic affiliation of the G. natalis GH5_10 transcript to that of other decapods, supporting endogenous expression. Surprisingly, crustacean-derived GH5_10 transcripts were near absent in the current CAZy database and yet mining of the transcriptome shotgun assembly (TSA) recovered more than 100 crustacean GH5_10s in addition to several other biotechnological relevant CAZys, underscoring the unappreciated potential of the TSA database as a valuable resource for crustacean CAZys.
Cloning and functional analysis of the genes coding for 4-aminobenzenesulfonate 3,4-dioxygenase from Hydrogenophaga sp. PBC

Gan HM, Shahir S, Yahya A

Microbiology (Reading), 2012 Aug;158(Pt 8):1933-1941.
PMID: 22609751 DOI: 10.1099/mic.0.059550-0

The gene coding for the oxygenase component, sadA, of 4-aminobenzenesulfonate (4-ABS) 3,4-dioxygenase in Hydrogenophaga sp. PBC was previously identified via transposon mutagenesis. Expression of wild-type sadA in trans restored the ability of the sadA mutant to grow on 4-ABS. The inclusion of sadB and sadD, coding for a putative glutamine-synthetase-like protein and a plant-type ferredoxin, respectively, further improved the efficiency of 4-ABS degradation. Transcription analysis using the gfp promoter probe plasmid showed that sadABD was expressed during growth on 4-ABS and 4-sulfocatechol. Heterologous expression of sadABD in Escherichia coli led to the biotransformation of 4-ABS to a metabolite which shared a similar retention time and UV/vis profile with 4-sulfocatechol. The putative reductase gene sadC was isolated via degenerate PCR and expression of sadC and sadABD in E. coli led to maximal 4-ABS biotransformation. In E. coli, the deletion of sadB completely eliminated dioxygenase activity while the deletion of sadC or sadD led to a decrease in dioxygenase activity. Phylogenetic analysis of SadB showed that it is closely related to the glutamine-synthetase-like proteins involved in the aniline degradation pathway. This is the first discovery, to our knowledge, of the functional genetic components for 4-ABS aromatic ring hydroxylation in the bacterial domain.
Fulltext In-silico Taxonomic Classification of 373 Genomes Reveals Species Misidentification and New Genospecies within the Genus Pseudomonas

Tran PN, Savka MA, Gan HM

Front Microbiol, 2017;8:1296.
PMID: 28747902 DOI: 10.3389/fmicb.2017.01296

The genus Pseudomonas has one of the largest diversity of species within the Bacteria kingdom. To date, its taxonomy is still being revised and updated. Due to the non-standardized procedure and ambiguous thresholds at species level, largely based on 16S rRNA gene or conventional biochemical assay, species identification of publicly available Pseudomonas genomes remains questionable. In this study, we performed a large-scale analysis of all Pseudomonas genomes with species designation (excluding the well-defined P. aeruginosa) and re-evaluated their taxonomic assignment via in silico genome-genome hybridization and/or genetic comparison with valid type species. Three-hundred and seventy-three pseudomonad genomes were analyzed and subsequently clustered into 145 distinct genospecies. We detected 207 erroneous labels and corrected 43 to the proper species based on Average Nucleotide Identity Multilocus Sequence Typing (MLST) sequence similarity to the type strain. Surprisingly, more than half of the genomes initially designated as Pseudomonas syringae and Pseudomonas fluorescens should be classified either to a previously described species or to a new genospecies. Notably, high pairwise average nucleotide identity (>95%) indicating species-level similarity was observed between P. synxantha-P. libanensis, P. psychrotolerans-P. oryzihabitans, and P. kilonensis- P. brassicacearum, that were previously differentiated based on conventional biochemical tests and/or genome-genome hybridization techniques.
Two reads to rule them all: Nanopore long read-guided assembly of the iconic Christmas Island red crab, Gecarcoidea natalis (Pocock, 1888), mitochondrial genome and the challenges of AT-rich mitogenomes

Gan HM, Linton SM, Austin CM

Mar Genomics, 2019 Jun;45:64-71.
PMID: 30928201 DOI: 10.1016/j.margen.2019.02.002

Despite recent advances in sequencing technology, a complete mitogenome assembly is still unavailable for the gecarcinid land crabs that include the iconic Christmas Island red crab (Gecarcoidea natalis) which is known for its high population density, annual mass breeding migration and ecological significance in maintaining rainforest structure. Using sequences generated from Nanopore and Illumina platforms, we assembled the complete mitogenome for G. natalis, the first for the genus and only second for the family Gecarcinidae. Nine Nanopore long reads representing 0.15% of the sequencing output from an overnight MinION Nanopore run were aligned to the mitogenome. Two of them were >10 kb and combined are sufficient to span the entire G. natalis mitogenome. The use of Illumina genome skimming data only resulted in a fragmented assembly that can be attributed to low to zero sequencing coverage in multiple high AT-regions including the mitochondrial protein-coding genes (NAD4 and NAD5), 16S ribosomal rRNA and non-coding control region. Supplementing the mitogenome assembly with previously acquired transcriptome dataset containing high abundance of mitochondrial transcripts improved mitogenome sequence coverage and assembly reliability. We then inferred the phylogeny of the Eubrachyura using Maximum Likelihood and Bayesian approaches, confirming the phylogenetic placement of G. natalis within the family Gecarcinidae based on whole mitogenome alignment. Given the substantial impact of AT-content on mitogenome assembly and the value of complete mitogenomes in phylogenetic and comparative studies, we recommend that future mitogenome sequencing projects consider generating a modest amount of Nanopore long reads to facilitate the closing of problematic and fragmented mitogenome assemblies.
Fulltext Nanopore Long-Read Guided Complete Genome Assembly of Hydrogenophaga intermedia, and Genomic Insights into 4-Aminobenzenesulfonate, p-Aminobenzoic Acid and Hydrogen Metabolism in the Genus Hydrogenophaga

Gan HM, Lee YP, Austin CM

Front Microbiol, 2017;8:1880.
PMID: 29046667 DOI: 10.3389/fmicb.2017.01880

We improved upon the previously reported draft genome of Hydrogenophaga intermedia strain PBC, a 4-aminobenzenesulfonate-degrading bacterium, by supplementing the assembly with Nanopore long reads which enabled the reconstruction of the genome as a single contig. From the complete genome, major genes responsible for the catabolism of 4-aminobenzenesulfonate in strain PBC are clustered in two distinct genomic regions. Although the catabolic genes for 4-sulfocatechol, the deaminated product of 4-aminobenzenesulfonate, are only found in H. intermedia, the sad operon responsible for the first deamination step of 4-aminobenzenesulfonate is conserved in various Hydrogenophaga strains. The absence of pabB gene in the complete genome of H. intermedia PBC is consistent with its p-aminobenzoic acid (pABA) auxotrophy but surprisingly comparative genomics analysis of 14 Hydrogenophaga genomes indicate that pABA auxotrophy is not an uncommon feature among members of this genus. Of even more interest, several Hydrogenophaga strains do not possess the genomic potential for hydrogen oxidation, calling for a revision to the taxonomic description of Hydrogenophaga as "hydrogen eating bacteria."
Fulltext Characterization of the complete mitochondrial genome of the critically endangered Southern River Terrapin, Batagur affinis affinis (Reptilia: Geoemydidae)

Mohd Salleh MH, Esa Y, Gan HM

Mitochondrial DNA B Resour, 2023;8(7):719-722.
PMID: 37426572 DOI: 10.1080/23802359.2023.2222851

In this study, we report the nearly complete mitochondrial sequence of Batagur affinis affinis. The assembled mitogenome consists of 13 PCGs, 22 tRNA genes, two rRNAs and one near-complete D-loop region. Of the annotated genes, the ND6 subunit gene and eight tRNA genes were encoded on the L-strand, while the remaining genes were dispersed on the H-strand. Except for CO1, which has a GTG start codon, all protein-coding genes begin with ATG. The mitogenome has been deposited in NCBI GenBank under the accession number OQ409915. Phylogenetic tree analysis based on publicly available mitogenomes indicate the sister grouping of B. affinis affinis with B. kachuga.
MitoPhAST, a new automated mitogenomic phylogeny tool in the post-genomic era with a case study of 89 decapod mitogenomes including eight new freshwater crayfish mitogenomes

Tan MH, Gan HM, Schultz MB, Austin CM

Mol Phylogenet Evol, 2015 Apr;85:180-8.
PMID: 25721538 DOI: 10.1016/j.ympev.2015.02.009

The increased rate at which complete mitogenomes are being sequenced and their increasing use for phylogenetic studies have resulted in a bioinformatic bottleneck in preparing and utilising such data for phylogenetic analysis. Hence, we present MitoPhAST, an automated tool that (1) identifies annotated protein-coding gene features and generates a standardised, concatenated and partitioned amino acid alignment directly from complete/partial GenBank/EMBL-format mitogenome flat files, (2) generates a maximum likelihood phylogenetic tree using optimised protein models and (3) reports various mitochondrial genes and sequence information in a table format. To demonstrate the capacity of MitoPhAST in handling a large dataset, we used 81 publicly available decapod mitogenomes, together with eight new complete mitogenomes of Australian freshwater crayfishes, including the first for the genus Gramastacus, to undertake an updated test of the monophyly of the major groups of the order Decapoda and their phylogenetic relationships. The recovered phylogenetic trees using both Bayesian and ML methods support the results of studies using fragments of mtDNA and nuclear markers and other smaller-scale studies using whole mitogenomes. In comparison to the fragment-based phylogenies, nodal support values are generally higher despite reduced taxon sampling suggesting there is value in utilising more fully mitogenomic data. Additionally, the simple table output from MitoPhAST provides an efficient summary and statistical overview of the mitogenomes under study at the gene level, allowing the identification of missing or duplicated genes and gene rearrangements. The finding of new mtDNA gene rearrangements in several genera of Australian freshwater crayfishes indicates that this group has undergone an unusually high rate of evolutionary change for this organelle compared to other major families of decapod crustaceans. As a result, freshwater crayfishes are likely to be a useful model for studies designed to understand the evolution of mtDNA rearrangements. We anticipate that our bioinformatics pipeline will substantially help mitogenome-based studies increase the speed, accuracy and efficiency of phylogenetic studies utilising mitogenome information. MitoPhAST is available for download at https://github.com/mht85/MitoPhAST.
The complete mitogenome of purple mottled shore crab Cyclograpsus granulosus H. Milne-Edwards, 1853 (Crustacea: Decapoda: Grapsoidea)

Tan MH, Gan HM, Lee YP, Austin CM

Mitochondrial DNA A DNA Mapp Seq Anal, 2016 11;27(6):3981-3982.
PMID: 25541307

The mitochondrial genome sequence of the purple mottled shore crab, Cyclograpsus granulosus, is documented (GenBank accession number: LN624373), which makes it the third for genera of the superfamily Grapsoidea. Cyclograpsus granulosus has a mitogenome of 16,300 bp consisting of 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and a non-coding AT-rich region. The base composition of the C. granulosus mitogenome is 36.15% for T, 19.54% for C, 33.14% for A and 11.17% for G, with an AT bias of 69.29%. The mitogenome gene order is atypical for the brachyuran crabs, but is identical to species of the genus Eriocheir from the same family.
The complete mitogenome of the porcelain crab Petrolisthes haswelli Miers, 1884 (Crustacea: Decapoda: Anomura)

Tan MH, Gan HM, Lee YP, Austin CM

Mitochondrial DNA A DNA Mapp Seq Anal, 2016 11;27(6):3983-3984.
PMID: 25541305

The mitochondrial genome sequence of the porcellanid crab, Petrolisthes haswelli is provided, making it the second for the family Porcellanidae and the third for the superfamily Galatheoidea. Petrolisthes haswelli has a mitogenome of 15,348 bp consisting of 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and a non-coding AT-rich region. The base composition of the P. haswelli mitogenome is 35.66% for T, 18.65% for C, 34.35% for A and 11.34% for G, with an AT bias of 70.01%. The mitogenome gene order is identical to the mitogenome of Neopetrolisthes maculatus, the only other species of the family with a sequenced mitogenome.
The complete mitogenome of the ghost crab Ocypode ceratophthalmus (Pallas, 1772) (Crustacea: Decapoda: Ocypodidae)

Tan MH, Gan HM, Lee YP, Austin CM

Mitochondrial DNA A DNA Mapp Seq Anal, 2016 05;27(3):2123-4.
PMID: 25423512 DOI: 10.3109/19401736.2014.982587

The mitochondrial genome sequence of the ghost crab, Ocypode ceratophthalmus, is documented (GenBank accession number: LN611669) in this article. This is the first mitogenome for the family Ocypodidae and the second for the order Ocypodoidea. Ocypode ceratophthalmus has a mitogenome of 15,564 base pairs consisting of 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and a non-coding AT-rich region. The base composition of the O. ceratophthalmus mitogenome is 35.78% for T, 19.36% for C, 33.73% for A and 11.13% for G, with an AT bias of 69.51% and the gene order is the typical arrangement for brachyuran crabs.
The complete mitogenome of the soldier crab Mictyris longicarpus (Latreille, 1806) (Crustacea: Decapoda: Mictyridae)

Tan MH, Gan HM, Lee YP, Austin CM

Mitochondrial DNA A DNA Mapp Seq Anal, 2016 05;27(3):2121-2.
PMID: 25423510 DOI: 10.3109/19401736.2014.982585

The Mictyris longicarpus (soldier crab) complete mitochondrial genome sequence is reported making it the first for the family Mictyridae and the second for the superfamily Ocypodoidea. The mitogenome is 15,548 base pairs made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs and a non-coding AT-rich region. The soldier crab mitogenome gene order is characteristic of brachyuran crabs with a base composition of 36.58% for T, 19.15% for C, 32.43% for A and 11.83% for G, with an AT bias of 69.01%.

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