Siew Woh Choo

MabsBase: a Mycobacterium abscessus genome and annotation database.

Summary Mycobacterium abscessus is a rapidly growing non-tuberculous mycobacterial species that has been associated with a wide spectrum of human infections. As the classification and biology of this organism is still not well understood, comparative genomic analysis on members of this species may provide further insights on their taxonomy, phylogeny, pathogenicity and other information that may contribute to better management of infections. The MabsBase described in this paper is a user-friendly database providing access to whole-genome sequences of newly discovered M. abscessus strains as well as resources for whole-genome annotations and computational predictions, to support the expanding scientific community interested in M. abscessus research. The MabsBase is freely available at http://mabscessus.um.edu.my.

Genome-Wide Analysis of the Binding of the Hox Protein Ultrabithorax and the Hox Cofactor Homothorax in Drosophila

Hox genes encode a family of transcription factors that are key developmental regulators with a highly conserved role in specifying segmental diversity along the metazoan body axis. Although they have been shown to regulate a wide variety of downstream processes, direct transcriptional targets have been difficult to identify and this has been a major obstacle to our understanding of Hox gene function. We report the identification of genome-wide binding sites for the Hox protein Ultrabithorax (Ubx) using a YFP-tagged Drosophila protein-trap line together with chromatin immunoprecipitation and microarray analysis. We identify 1,147 genes bound by Ubx at high confidence in chromatin from the haltere imaginal disc, a prominent site of Ubx function where it specifies haltere versus wing development. The functional relevance of these genes is supported by their overlap with genes differentially expressed between wing and haltere imaginal discs. The Ubx-bound gene set is highly enriched in genes involved in developmental processes and contains both high-level regulators as well as genes involved in more basic cellular functions. Several signalling pathways are highly enriched in the Ubx target gene set and our analysis supports the view that Hox genes regulate many levels of developmental pathways and have targets distributed throughout the gene network. We also performed genome-wide analysis of the binding sites for the Hox cofactor Homothorax (Hth), revealing a striking similarity with the Ubx binding profile. We suggest that these binding profiles may be strongly influenced by chromatin accessibility and provide evidence of a link between Ubx/Hth binding and chromatin state at genes regulated by Polycomb silencing. Overall, we define a set of direct Ubx targets in the haltere imaginal disc and suggest that chromatin accessibility has important implications for Hox target selection and for transcription factor binding in general.

Characterisation ofDrosophila UbxCPTI000601andhthCPTI000378Protein Trap Lines

In Drosophila, protein trap strategies provide powerful approaches for the generation of tagged proteins expressed under endogenous control. Here, we describe expression and functional analysis to evaluate new Ubx and hth protein trap lines generated by the Cambridge Protein Trap project. Both protein traps exhibit spatial and temporal expression patterns consistent with the reported endogenous pattern in the embryo. In imaginal discs, Ubx-YFP is expressed throughout the haltere and 3rd leg imaginal discs, while Hth-YFP is expressed in the proximal regions of haltere and wing discs but not in the pouch region. The Ubx CPTI000601 line is semilethal as a homozygote. No T3/A1 to T2 transformations were observed in the embryonic cuticle or the developing midgut. The homozygous survivors, however, exhibit a weak haltere phenotype with a few wing-like marginal bristles on the haltere capitellum. Although hth CPTI000378 is completely lethal as a homozygote, the hth CPTI000378/hth C1 genotype is viable. Using a hth deletion (Df(3R)BSC479) we show that hth CPTI000378/Df(3R)BSC479 adults are phenotypically normal. No transformations were observed in hth CPTI000378, hth CPTI000378/hth C1, or hth CPTI000378/Df(3R)BSC479 embryonic cuticles. We have successfully characterised the Ubx-YFP and Hth-YFP protein trap lines demonstrating that the tagged proteins show appropriate expression patterns and produce at least partially functional proteins.

Genomic reconnaissance of clinical isolates of emerging human pathogen Mycobacterium abscessus reveals high evolutionary potential

Mycobacterium abscessus (Ma) is an emerging human pathogen that causes both soft tissue infections and systemic disease. We present the first comparative whole-genome study of Ma strains isolated from patients of wide geographical origin. We found a high proportion of accessory strain-specific genes indicating an open, non-conservative pan-genome structure, and clear evidence of rapid phage-mediated evolution. Although we found fewer virulence factors in Ma compared to M. tuberculosis, our data indicated that Ma evolves rapidly and therefore should be monitored closely for the acquisition of more pathogenic traits. This comparative study provides a better understanding of Ma and forms the basis for future functional work on this important pathogen.

Genomic Approaches to Understanding Hox Gene Function

For many years, biologists have sought to understand how the homeodomain-containing transcriptional regulators encoded by Hox genes are able to control the development of animal morphology. Almost a century of genetics and several decades of molecular biology have defined the conserved organization of homeotic gene clusters in animals and the basic molecular properties of Hox transcription factors. In contrast to these successes, we remain relatively ignorant of how Hox proteins find their target genes in the genome or what sets of genes a Hox protein regulates to direct morphogenesis. The recent deployment of genomic methods, such as whole transcriptome mRNA expression profiling and genome-wide analysis of protein-DNA interactions, begins to shed light on these issues. Results from such studies, principally in the fruit fly, indicate that Hox proteins control the expression of hundreds, if not thousands, of genes throughout the gene regulatory network and that, in many cases, the effects on the expression of individual genes may be quite subtle. Hox proteins regulate both high-level effectors, including other transcription factors and signaling molecules, as well as the cytodifferentiation genes or Realizators at the bottom of regulatory hierarchies. Insights emerging from mapping Hox binding sites in the genome begin to suggest that Hox binding may be strongly influenced by chromatin accessibility rather than binding site affinity. If this is the case, it indicates we need to refocus our efforts at understanding Hox function toward the dynamics of gene regulatory networks and chromatin epigenetics.

Support from Phylogenomic Networks and Subspecies Signatures for Separation of Mycobacterium massiliense from Mycobacterium bolletii

ABSTRACT Mycobacterium abscessus subspecies classification has important clinical implications. We used phylogenomic network and amino acid analyses to provide evidence for the separation of Mycobacterium bolletii and Mycobacterium massiliense into two distinct subspecies which can potentially be differentiated rapidly by their protein signatures.

Erratum to: Comparing the genomes of Helicobacter pylori

Correction: Comparing the genomes of Helicobacter pylori clinical strain UM032 and mice-adapted derivatives Yalda Khosravi, Vellaya Rehvathy, Wei Yee Wee, Susana Wang, Primo Baybayan, Siddarth Singh, Meredith Ashby, Junxian Ong, Arlaine Anne Amoyo, Shih Wee Seow, Siew Woh Choo, Tim Perkins, Eng Guan Chua, Alfred Tay, Barry James Marshall, Mun Fai Loke, Khean Lee Goh, Sven Pettersson and Jamuna Vadivelu

A phylogenomic approach to bacterial subspecies classification: proof of concept in Mycobacterium abscessus.

BackgroundMycobacterium abscessus is a rapidly growing mycobacterium that is often associated with human infections. The taxonomy of this species has undergone several revisions and is still being debated. In this study, we sequenced the genomes of 12 M. abscessus strains and used phylogenomic analysis to perform subspecies classification.ResultsA data mining approach was used to rank and select informative genes based on the relative entropy metric for the construction of a phylogenetic tree. The resulting tree topology was similar to that generated using the concatenation of five classical housekeeping genes: rpoB, hsp65, secA, recA and sodA. Additional support for the reliability of the subspecies classification came from the analysis of erm41 and ITS gene sequences, single nucleotide polymorphisms (SNPs)-based classification and strain clustering demonstrated by a variable number tandem repeat (VNTR) assay and a multilocus sequence analysis (MLSA). We subsequently found that the concatenation of a minimal set of three median-ranked genes: DNA polymerase III subunit alpha (polC), 4-hydroxy-2-ketovalerate aldolase (Hoa) and cell division protein FtsZ (ftsZ), is sufficient to recover the same tree topology. PCR assays designed specifically for these genes showed that all three genes could be amplified in the reference strain of M. abscessus ATCC 19977T.ConclusionThis study provides proof of concept that whole-genome sequence-based data mining approach can provide confirmatory evidence of the phylogenetic informativeness of existing markers, as well as lead to the discovery of a more economical and informative set of markers that produces similar subspecies classification in M. abscessus. The systematic procedure used in this study to choose the informative minimal set of gene markers can potentially be applied to species or subspecies classification of other bacteria.

Roles of cofactors and chromatin accessibility in hox protein target specificity

BackgroundThe regulation of specific target genes by transcription factors is central to our understanding of gene network control in developmental and physiological processes yet how target specificity is achieved is still poorly understood. This is well illustrated by the Hox family of transcription factors as their limited in vitro DNA-binding specificity contrasts with their clear in vivo functional specificity.ResultsWe generated genome-wide binding profiles for three Hox proteins, Ubx, Abd-A and Abd-B, following transient expression in Drosophila Kc167 cells, revealing clear target specificity and a striking influence of chromatin accessibility. In the absence of the TALE class homeodomain cofactors Exd and Hth, Ubx and Abd-A bind at a very similar set of target sites in accessible chromatin, whereas Abd-B binds at an additional specific set of targets. Provision of Hox cofactors Exd and Hth considerably modifies the Ubx genome-wide binding profile enabling Ubx to bind at an additional novel set of targets. Both the Abd-B specific targets and the cofactor-dependent Ubx targets are in chromatin that is relatively DNase1 inaccessible prior to the expression of Hox proteins/Hox cofactors.ConclusionsOur experiments demonstrate a strong role for chromatin accessibility in Hox protein binding and suggest that Hox protein competition with nucleosomes has a major role in Hox protein target specificity in vivo.

Draft Genome Sequence of Mycobacterium bolletii Strain M24, a Rapidly Growing Mycobacterium of Contentious Taxonomic Status

The whole-genome sequence of Mycobacterium bolletii M24, isolated from the bronchoalveolar lavage fluid of a Malaysian patient, is reported here. The circular chromosome of 5,507,730 bp helped to clarify the taxonomic position of this organism within the M. abscessus complex and revealed the presence of proteins potentially important for pathogenicity in a human host.