Person: Chao, Michael C.
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Chao
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Michael C.
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Chao, Michael C.
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Publication High-resolution definition of the Vibrio cholerae essential gene set with hidden Markov model–based analyses of transposon-insertion sequencing data(Oxford University Press, 2013) Chao, Michael C.; Pritchard, Justin R.; Zhang, Yanjia; Rubin, Eric; Livny, Jonathan; Davis, Brigid M.; Waldor, MatthewThe coupling of high-density transposon mutagenesis to high-throughput DNA sequencing (transposon-insertion sequencing) enables simultaneous and genome-wide assessment of the contributions of individual loci to bacterial growth and survival. We have refined analysis of transposon-insertion sequencing data by normalizing for the effect of DNA replication on sequencing output and using a hidden Markov model (HMM)-based filter to exploit heretofore unappreciated information inherent in all transposon-insertion sequencing data sets. The HMM can smooth variations in read abundance and thereby reduce the effects of read noise, as well as permit fine scale mapping that is independent of genomic annotation and enable classification of loci into several functional categories (e.g. essential, domain essential or ‘sick’). We generated a high-resolution map of genomic loci (encompassing both intra- and intergenic sequences) that are required or beneficial for in vitro growth of the cholera pathogen, Vibrio cholerae. This work uncovered new metabolic and physiologic requirements for V. cholerae survival, and by combining transposon-insertion sequencing and transcriptomic data sets, we also identified several novel noncoding RNA species that contribute to V. cholerae growth. Our findings suggest that HMM-based approaches will enhance extraction of biological meaning from transposon-insertion sequencing genomic data.Publication ARTIST: High-Resolution Genome-Wide Assessment of Fitness Using Transposon-Insertion Sequencing(Public Library of Science, 2014) Pritchard, Justin R.; Chao, Michael C.; Abel, Sören; Davis, Brigid M.; Baranowski, Catherine; Zhang, Yanjia; Rubin, Eric; Waldor, MatthewTransposon-insertion sequencing (TIS) is a powerful approach for deciphering genetic requirements for bacterial growth in different conditions, as it enables simultaneous genome-wide analysis of the fitness of thousands of mutants. However, current methods for comparative analysis of TIS data do not adjust for stochastic experimental variation between datasets and are limited to interrogation of annotated genomic elements. Here, we present ARTIST, an accessible TIS analysis pipeline for identifying essential regions that are required for growth under optimal conditions as well as conditionally essential loci that participate in survival only under specific conditions. ARTIST uses simulation-based normalization to model and compensate for experimental noise, and thereby enhances the statistical power in conditional TIS analyses. ARTIST also employs a novel adaptation of the hidden Markov model to generate statistically robust, high-resolution, annotation-independent maps of fitness-linked loci across the entire genome. Using ARTIST, we sensitively and comprehensively define Mycobacterium tuberculosis and Vibrio cholerae loci required for host infection while limiting inclusion of false positive loci. ARTIST is applicable to a broad range of organisms and will facilitate TIS-based dissection of pathways required for microbial growth and survival under a multitude of conditions.Publication High-resolution genetic analysis of the requirements for horizontal transmission of the ESBL plasmid from Escherichia coli O104:H4(Oxford University Press, 2015) Yamaichi, Yoshiharu; Chao, Michael C.; Sasabe, Jumpei; Clark, Lars; Davis, Brigid M.; Yamamoto, Nozomi; Mori, Hiroshi; Kurokawa, Ken; Waldor, MatthewHorizontal dissemination of the genes encoding extended spectrum beta-lactamases (ESBLs) via conjugative plasmids is facilitating the increasingly widespread resistance of pathogens to beta-lactam antibiotics. However, there is relatively little known about the regulatory factors and mechanisms that govern the spread of these plasmids. Here, we carried out a high-throughput, transposon insertion site sequencing analysis (TnSeq) to identify genes that enable the maintenance and transmission of pESBL, an R64 (IncI1)-related resistance plasmid that was isolated from Escherichia coli O104:H4 linked to a recent large outbreak of gastroenteritis. With a few exceptions, the majority of the genes identified as required for maintenance and transmission of pESBL matched those of their previously defined R64 counterparts. However, our analyses of the high-density transposon insertion library in pESBL also revealed two very short and linked regions that constitute a previously unrecognized regulatory system controlling spread of IncI1 plasmids. In addition, we investigated the function of the pESBL-encoded M.EcoGIX methyltransferase, which is also encoded by many other IncI1 and IncF plasmids. This enzyme proved to protect pESBL from restriction in new hosts, suggesting it aids in expanding the plasmid's host range. Collectively, our work illustrates the power of the TnSeq approach to enable rapid and comprehensive analyses of plasmid genes and sequences that facilitate the dissemination of determinants of antibiotic resistance.Publication Molecular Dissection of the Essential Features of the Origin of Replication of the Second Vibrio cholerae Chromosome(American Society of Microbiology, 2015) Gerding, Matthew A.; Chao, Michael C.; Davis, Brigid M.; Waldor, MatthewABSTRACT Vibrionaceae family members are interesting models for studying DNA replication initiation, as they contain two circular chromosomes. Chromosome II (chrII) replication is governed by two evolutionarily unique yet highly conserved elements, the origin DNA sequence oriCII and the initiator protein RctB. The minimum functional region of oriCII, oriCII-min, contains multiple elements that are bound by RctB in vitro, but little is known about the specific requirements for individual elements during oriCII initiation. We utilized undirected and site-specific mutagenesis to investigate the functionality of mutant forms of oriCII-min and assessed binding to various mutant forms by RctB. Our analyses showed that deletions, point mutations, and changes in RctB target site spacing or methylation all impaired oriCII-min-based replication. RctB displayed a reduced affinity for most of the low-efficacy origins tested, although its characteristic cooperative binding was generally maintained. Mutations that removed or altered the relative positions of origin components other than RctB binding sites (e.g., AT-rich sequence, DnaA target site) also abolished replicative capacity. Comprehensive mutagenesis and deep-sequencing-based screening (OriSeq) allowed the identification of a previously uncharacterized methylated domain in oriCII that is required for origin function. Together, our results reveal the remarkable evolutionary honing of oriCII and provide new insight into the complex interplay between RctB and oriCII.Publication A Cytosine Methytransferase Modulates the Cell Envelope Stress Response in the Cholera Pathogen(Public Library of Science, 2015) Chao, Michael C.; Zhu, Shijia; Kimura, Satoshi; Davis, Brigid M.; Schadt, Eric E.; Fang, Gang; Waldor, MatthewDNA methylation is a key epigenetic regulator in all domains of life, yet the effects of most bacterial DNA methyltransferases on cellular processes are largely undefined. Here, we used diverse techniques, including bisulfite sequencing, transcriptomics, and transposon insertion site sequencing to extensively characterize a 5-methylcytosine (5mC) methyltransferase, VchM, in the cholera pathogen, Vibrio cholerae. We have comprehensively defined VchM’s DNA targets, its genetic interactions and the gene networks that it regulates. Although VchM is a relatively new component of the V. cholerae genome, it is required for optimal V. cholerae growth in vitro and during infection. Unexpectedly, the usually essential σE cell envelope stress pathway is dispensable in ∆vchM V. cholerae, likely due to its lower activation in this mutant and the capacity for VchM methylation to limit expression of some cell envelope modifying genes. Our work illuminates how an acquired DNA methyltransferase can become integrated within complex cell circuits to control critical housekeeping processes.Publication Protein Complexes and Proteolytic Activation of the Cell Wall Hydrolase RipA Regulate Septal Resolution in Mycobacteria(Public Library of Science, 2013) Chao, Michael C.; Kieser, Karen; Minami, Shoko; Mavrici, Daniela; Aldridge, Bree; Fortune, Sarah; Alber, Tom; Rubin, EricPeptidoglycan hydrolases are a double-edged sword. They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria. How bacteria ensure that peptidoglycan hydrolases function only in the correct spatial and temporal context remains largely unknown. Here, we demonstrate that dysregulation converts the essential mycobacterial peptidoglycan hydrolase RipA to an autolysin that compromises cellular structural integrity. We find that mycobacteria control RipA activity through two interconnected levels of regulation in vivo—protein interactions coordinate PG hydrolysis, while proteolysis is necessary for RipA enzymatic activity. Dysregulation of RipA protein complexes by treatment with a peptidoglycan synthase inhibitor leads to excessive RipA activity and impairment of correct morphology. Furthermore, expression of a RipA dominant negative mutant or of differentially processed RipA homologues reveals that RipA is produced as a zymogen, requiring proteolytic processing for activity. The amount of RipA processing differs between fast-growing and slow-growing mycobacteria and correlates with the requirement for peptidoglycan hydrolase activity in these species. Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development.Publication Platelet-Derived Growth Factor Receptor β Is Critical for Zebrafish Intersegmental Vessel Formation(Public Library of Science, 2010) Wiens, Katie M.; Lee, Hyunju; Shimada, Hiroyuki; Metcalf, Anthony E.; Chao, Michael C.; Lien, Ching-LingBackground: Platelet-derived growth factor receptor β (PDGFRβ) is a tyrosine kinase receptor known to affect vascular development. The zebrafish is an excellent model to study specific regulators of vascular development, yet the role of PDGF signaling has not been determined in early zebrafish embryos. Furthermore, vascular mural cells, in which PDGFRβ functions cell autonomously in other systems, have not been identified in zebrafish embryos younger than 72 hours post fertilization. Methodology/Principal Findings: In order to investigate the role of PDGFRβ in zebrafish vascular development, we cloned the highly conserved zebrafish homolog of PDGFRβ. We found that pdgfrβ is expressed in the hypochord, a developmental structure that is immediately dorsal to the dorsal aorta and potentially regulates blood vessel development in the zebrafish. Using a PDGFR tyrosine kinase inhibitor, a morpholino oligonucleotide specific to PDGFRβ, and a dominant negative PDGFRβ transgenic line, we found that PDGFRβ is necessary for angiogenesis of the intersegmental vessels. Significance/Conclusion: Our data provide the first evidence that PDGFRβ signaling is required for zebrafish angiogenesis. We propose a novel mechanism for zebrafish PDGFRβ signaling that regulates vascular angiogenesis in the absence of mural cells.Publication Interaction and Modulation of Two Antagonistic Cell Wall Enzymes of Mycobacteria(Public Library of Science, 2010) Hett, Erik Christian; Chao, Michael C.; Rubin, EricBacterial cell growth and division require coordinated cell wall hydrolysis and synthesis, allowing for the removal and expansion of cell wall material. Without proper coordination, unchecked hydrolysis can result in cell lysis. How these opposing activities are simultaneously regulated is poorly understood. In Mycobacterium tuberculosis, the resuscitation-promoting factor B (RpfB), a lytic transglycosylase, interacts and synergizes with Rpf-interacting protein A (RipA), an endopeptidase, to hydrolyze peptidoglycan. However, it remains unclear what governs this synergy and how it is coordinated with cell wall synthesis. Here we identify the bifunctional peptidoglycan-synthesizing enzyme, penicillin binding protein 1 (PBP1), as a RipA-interacting protein. PBP1, like RipA, localizes both at the poles and septa of dividing cells. Depletion of the ponA1 gene, encoding PBP1 in M. smegmatis, results in a severe growth defect and abnormally shaped cells, indicating that PBP1 is necessary for viability and cell wall stability. Finally, PBP1 inhibits the synergistic hydrolysis of peptidoglycan by the RipA-RpfB complex in vitro. These data reveal a post-translational mechanism for regulating cell wall hydrolysis and synthesis through protein–protein interactions between enzymes with antagonistic functions.Publication lin-12 Notch functions in the adult nervous system of C. elegans(BioMed Central, 2005) Chao, Michael C.; Larkins-Ford, Jonah; Tucey, Tim M; Hart, Anne CBackground: Notch signaling pathways are conserved across species and traditionally have been implicated in cell fate determination during embryonic development. Notch signaling components are also expressed postdevelopmentally in the brains of adult mice and Drosophila. Recent studies suggest that Notch signaling may play a role in the physiological, rather than developmental, regulation of neurons. Here, we investigate a new non-developmental role for Caenorhabditis elegans lin-12 Notch signaling in neurons regulating the spontaneous reversal rate during locomotion. Results: The spontaneous reversal rate of C. elegans during normal locomotion is constant. Both lin-12 gain and loss of function mutant animals had significantly increased reversal rates compared to wild type controls. These defects were caused by lin-12 activity, because the loss of function defect could be rescued by a wild type lin-12 transgene. Furthermore, overexpression of lin-12 recapitulated the gain-of-function defect. Increasing or decreasing lin-12 activity in the postdevelopmental adult animal was sufficient to rapidly and reversibly increase reversals, thereby excluding a developmental role for lin-12. Although lin-12 is expressed in the vulval and somatic gonad lineages, we find that these tissues play no role in regulating reversal rates. In contrast, altering lin-12 activity specifically in the nervous system was sufficient to increase reversals. These behavioral changes require components of the canonical lin-12 signaling cascade, including the ligand lag-2 and the transcriptional effector lag-1. Finally, the C. elegans AMPA/kainate glutamate receptor homolog glr-1 shows strong genetic interactions with lin-12, suggesting that glr-1 and/or other glutamate gated channels may be targets of lin-12 regulation. Conclusion: Our results demonstrate a neuronal role for lin-12 Notch in C. elegans and suggest that lin-12 acutely regulates neuronal physiology to modulate animal behavior, without altering neuronal cell fate specification or neurite outgrowth. This is consistent with a role for Notch signaling in neurological disease with late onset symptoms.Publication A mycobacterial enzyme essential for cell division synergizes with resuscitation-promoting factor(Public Library of Science, 2008) Hett, Erik Christian; Chao, Michael C.; Deng, Lynn L.; Rubin, EricThe final stage of bacterial cell division requires the activity of one or more enzymes capable of degrading the layers of peptidoglycan connecting two recently developed daughter cells. Although this is a key step in cell division and is required by all peptidoglycan-containing bacteria, little is known about how these potentially lethal enzymes are regulated. It is likely that regulation is mediated, at least partly, through protein–protein interactions. Two lytic transglycosylases of mycobacteria, known as resuscitation-promoting factor B and E (RpfB and RpfE), have previously been shown to interact with the peptidoglycan-hydrolyzing endopeptidase, Rpf-interacting protein A (RipA). These proteins may form a complex at the septum of dividing bacteria. To investigate the function of this potential complex, we generated depletion strains in M. smegmatis. Here we show that, while depletion of rpfB has no effect on viability or morphology, ripA depletion results in a marked decrease in growth and formation of long, branched chains. These growth and morphological defects could be functionally complemented by the M. tuberculosis ripA orthologue (rv1477), but not by another ripA-like orthologue (rv1478). Depletion of ripA also resulted in increased susceptibility to the cell wall–targeting β-lactams. Furthermore, we demonstrate that RipA has hydrolytic activity towards several cell wall substrates and synergizes with RpfB. These data reveal the unusual essentiality of a peptidoglycan hydrolase and suggest a novel protein–protein interaction as one way of regulating its activity.