Zhaohui Xu

Microbial cell-surface display

Cell-surface display allows peptides and proteins to be displayed on the surface of microbial cells by fusing them with the anchoring motifs. The protein to be displayed - the passenger protein - can be fused to an anchoring motif - the carrier protein - by N-terminal fusion, C-terminal fusion or sandwich fusion. The characteristics of carrier protein, passenger protein and host cell, and fusion method all affect the efficiency of surface display of proteins. Microbial cell-surface display has many potential applications, including live vaccine development, peptide library screening, bioconversion using whole cell biocatalyst and bioadsorption.

Incorporating Genomics and Bioinformatics across the Life Sciences Curriculum

Community Page Incorporating Genomics and Bioinformatics across the Life Sciences Curriculum Jayna L. Ditty 1 , Christopher A. Kvaal 2 , Brad Goodner 3 , Sharyn K. Freyermuth 4 , Cheryl Bailey 5 , Robert A. Britton 6 , Stuart G. Gordon 7 , Sabine Heinhorst 8 , Kelynne Reed 9 , Zhaohui Xu 10 , Erin R. Sanders-Lorenz 11 , Seth Axen 12 , Edwin Kim 12 , Mitrick Johns 13 , Kathleen Scott 14 , Cheryl A. Kerfeld 12,15 * 1 Department of Biology, University of St. Thomas, St. Paul, Minnesota, United States of America, 2 Department of Biological Sciences, St. Cloud State University, St. Cloud, Minnesota, United States of America, 3 Department of Biology, Hiram College, Hiram, Ohio, United States of America, 4 Biochemistry Department, University of Missouri- Columbia, Columbia, Missouri, United States of America, 5 Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America, 6 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America, 7 Department of Biology, Presbyterian College, Clinton, South Carolina, United States of America, 8 Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, Mississippi, United States of America, 9 Biology Department, Austin College, Sherman, Texas, United States of America, 10 Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio, United States of America, 11 Department of Microbiology, Immunology and Molecular Genetics, University of California – Los Angeles, Los Angeles, California, United States of America, 12 Department of Energy-Joint Genome Institute, Walnut Creek, California, United States of America, 13 Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois, United States of America, 14 Department of Integrative Biology, University of South Florida, Tampa, Florida, United States of America, 15 Department of Plant and Microbial Biology, University of California Berkley, Berkeley, California, United States of America Introduction Undergraduate life sciences education needs an overhaul, as clearly described in the National Research Council of the National Academies’ publication BIO 2010: Transforming Undergraduate Education for Future Research Biologists. Among BIO 2010’s top recommendations is the need to involve students in working with real data and tools that reflect the nature of life sciences research in the 21st century [1]. Education research studies support the importance of utilizing primary literature, designing and implementing experiments, and analyzing results in the context of a bona fide scientific question [1–12] in cultivating the analytical skills necessary to become a scientist. Incorporating these basic scientific methodologies in under- graduate education leads to increased undergraduate and post-graduate reten- tion in the sciences [13–16]. Toward this end, many undergraduate teaching orga- nizations offer training and suggestions for faculty to update and improve their teaching approaches to help students learn as scientists, through design and discovery (e.g., Council of Undergraduate Research [www.cur.org] and Project Kaleidoscope [ www.pkal.org]). With the advent of genome sequencing and bioinformatics, many scientists now formulate biological questions and inter- pret research results in the context of genomic information. Just as the use of bioinformatic tools and databases changed the way scientists investigate problems, it must change how scientists teach to create new opportunities for students to gain experiences reflecting the influence of genomics, proteomics, and bioinformatics on modern life sciences research [17–41]. Educators have responded by incorpo- rating bioinformatics into diverse life science curricula [42–44]. While these published exercises in, and guidelines for, bioinformatics curricula are helpful and inspirational, faculty new to the area of bioinformatics inevitably need training in the theoretical underpinnings of the algo- rithms [45]. Moreover, effectively inte- grating bioinformatics into courses or independent research projects requires infrastructure for organizing and assessing student work. Here, we present a new platform for faculty to keep current with the rapidly changing field of bioinfor- matics, the Integrated Microbial Genomes Annotation Collaboration Toolkit (IMG- ACT) (Figure 1). It was developed by instructors from both research-intensive and predominately undergraduate institu- tions in collaboration with the Department of Energy-Joint Genome Institute (DOE- JGI) as a means to innovate and update undergraduate education and faculty de- velopment. The IMG-ACT program pro- vides a cadre of tools, including access to a clearinghouse of genome sequences, bioin- formatics databases, data storage, instruc- tor course management, and student notebooks for organizing the results of their bioinformatic investigations. In the process, IMG-ACT makes it feasible to provide undergraduate research opportu- nities to a greater number and diversity of students, in contrast to the traditional mentor-to-student apprenticeship model for undergraduate research, which can be too expensive and time-consuming to provide for every undergraduate. The IMG-ACT serves as the hub for the network of faculty and students that use the system for microbial genome analysis. Open access of the IMG-ACT infrastructure to participating schools en- sures that all types of higher education institutions can utilize it. With the infra- structure in place, faculty can focus their efforts on the pedagogy of bioinformatics, involvement of students in research, and use of this tool for their own research agenda. What the original faculty mem- bers of the IMG-ACT development team present here is an overview of how the IMG-ACT program has affected our Citation: Ditty JL, Kvaal CA, Goodner B, Freyermuth SK, Bailey C, et al. (2010) Incorporating Genomics and Bioinformatics across the Life Sciences Curriculum. PLoS Biol 8(8): e1000448. doi:10.1371/journal.pbio.1000448 Published August 10, 2010 Copyright: s 2010 Ditty et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: No specific funding was received for this work. The Community Page is a forum for organizations and societies to highlight their efforts to enhance the dissemination and value of scientific knowledge. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: IMG-ACT; Integrated Microbial Genomes Annotation Collaboration Toolkit * E-mail: ckerfeld@lbl.gov PLoS Biology | www.plosbiology.org August 2010 | Volume 8 | Issue 8 | e1000448

Detection of Benzene, Toluene, Ethyl Benzene, and Xylenes (BTEX) Using Toluene Dioxygenase‐Peroxidase Coupling Reactions

We have developed a simple, whole‐cell bioassay for the detection of bioavailable benzene, toluene, ethyl benzene, and xylenes (BTEX) and similar compounds. A genetically engineered E. coli strain expressing toluene dioxygenase (TDO) and toluene dihydrodiol dehydrogenase (TodD) was constructed, enabling the conversion of BTEX into their respective catechols, which were quickly converted into colored products by a horseradish peroxidase (HRP)‐coupled reaction. The intensity of the color formation was correlated to concentrations of the BTEX compounds. Under the optimized conditions, a detection limit (defined as three times the standard deviation of the response obtained from the blank) of 10, 10, 20, and 50 μM was observed for benzene, toluene, ethyl benzene, and xylene, respectively. The bioassay was selective toward BTEX‐related compounds with no interference observed with commonly used pesticides, herbicides, and organic solvent. The bioassay was very stable with little change in response over a 10‐week period. The excellent stability suggests that the reported bioassay may be suitable for field monitoring of BTEX to identify and track contaminated water and follow the bioremediation progress.

Construction and transformation of a Thermotoga-E. coli shuttle vector

BackgroundThermotoga spp. are attractive candidates for producing biohydrogen, green chemicals, and thermostable enzymes. They may also serve as model systems for understanding life sustainability under hyperthermophilic conditions. A lack of genetic tools has hampered the investigation and application of these organisms. This study aims to develop a genetic transfer system for Thermotoga spp.ResultsMethods for preparing and handling Thermotoga solid cultures under aerobic conditions were optimized. A plating efficiency of ~50% was achieved when the bacterial cells were embedded in 0.3% Gelrite. A Thermotoga-E. coli shuttle vector pDH10 was constructed using pRQ7, a cryptic mini-plasmid found in T. sp. RQ7. Plasmid pDH10 was introduced to T. maritima and T. sp. RQ7 by electroporation and liposome-mediated transformation. Transformants were isolated, and the transformed kanamycin resistance gene (kan) was detected from the plasmid DNA extracts of the recombinant strains by PCR and was confirmed by restriction digestions. The transformed DNA was stably maintained in both Thermotoga and E. coli even without the selective pressure.ConclusionsThermotoga are transformable by multiple means. Recombinant Thermotoga strains have been isolated for the first time. A heterologous kan gene is functionally expressed and stably maintained in Thermotoga.

Linking structural assembly to gene expression: a novel mechanism for regulating the activity of a σ54 transcription factor

In Caulobacter crescentus, the temporal and spatial expression of late flagellar genes is regulated by the σ54 transcriptional activator, FlbD. Genetic experiments have indicated that the trans‐acting factor FliX regulates FlbD in response to the progression of flagellar assembly, repressing FlbD activity until an early flagellar basal body structure is assembled. Following assembly of this structure, FliX is thought to function as an activator of FlbD. Here we have investigated the mechanism of FliX‐mediated regulation of FlbD activity. In vitro transcription experiments showed that purified FliX could function as a repressor of FlbD‐activated transcription. Transcription activated by a gain‐of‐function mutant of FlbD (FlbD‐1204) that is active in vivo in the absence of an early flagellar structure, was resistant to the repressive effects of FliX. DNA binding studies showed that FliX inhibited the interaction of wild‐type FlbD with enhancer DNA but did not effect FlbD‐catalysed ATPase activity. DNA binding activity of FlbD‐1204 was relatively unaffected by FliX indicating that this mutant protein bypasses the transcriptional requirement for early flagellar assembly by escaping FliX‐mediated negative regulation. Gel filtration and co‐immunoprecipitation experiments indicated that FliX formed a stable complex with FlbD. These experiments demonstrate that regulation of FlbD activity is unusual among the well‐studied σ54 transcriptional activators, apparently combining a two‐component receiver domain with additional control imposed via interaction with a partner protein, FliX.

Cloning and characterization of the TneDI restriction-modification system of Thermotoga neapolitana

A putative Type II restriction–modification system of Thermotoga neapolitana, TneDI, was cloned into Escherichia coli XL1-Blue MRF′ and characterized. Gene CTN_0339 specifies the endonuclease R.TneDI, while CTN_0340 encodes the cognate DNA methyltransferase M.TneDI. Both enzymes were purified simply by heating the cell lysates of E. coli followed by centrifugation. The enzymes were active over a broad range of temperatures, from 42°C to at least 77°C, with the highest activities observed at 77°C. R.TneDI cleaved at the center of the recognition sequence (CG↓CG) and generated blunt-end cuts. Overexpression of R.TneDI in BL21(DE3) was confirmed by both SDS-PAGE and Western blotting.

Expression of Heterologous Cellulases in Thermotoga sp. Strain RQ2

The ability of Thermotoga spp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA) and Csac_1078 (celB) from Caldicellulosiruptor saccharolyticus were cloned into T. sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA) and TM0070 (xynB), resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried by Thermotoga-E. coli shuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed in E. coli DH5α and T. sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. In E. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas in T. sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost in T. sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study) have ever been expressed in Thermotoga, demonstrating the feasibility of using engineered Thermotoga spp. for efficient cellulose utilization.

A pipeline for completing bacterial genomes using in silico and wet lab approaches

BackgroundDespite the large volume of genome sequencing data produced by next-generation sequencing technologies and the highly sophisticated software dedicated to handling these types of data, gaps are commonly found in draft genome assemblies. The existence of gaps compromises our ability to take full advantage of the genome data. This study aims to identify a practical approach for biologists to complete their own genome assemblies using commonly available tools and resources.ResultsA pipeline was developed to assemble complete genomes primarily from the next generation sequencing (NGS) data. The input of the pipeline is paired-end Illumina sequence reads, and the output is a high quality complete genome sequence. The pipeline alternates the employment of computational and biological methods in seven steps. It combines the strengths of de novo assembly, reference-based assembly, customized programming, public databases utilization, and wet lab experimentation. The application of the pipeline is demonstrated by the completion of a bacterial genome, Thermotoga sp. strain RQ7, a hydrogen-producing strain.ConclusionsThe developed pipeline provides an example of effective integration of computational and biological principles. It highlights the complementary roles that in silico and wet lab methodologies play in bioinformatical studies. The constituting principles and methods are applicable to similar studies on both prokaryotic and eukaryotic genomes.

Bioremediation of soluble heavy metals with recombinant Caulobacter crescentus.

To achieve one-step separation of heavy metal ions from contaminated water, we have developed a novel bioremediation technology based on self-immobilization of the Caulobacter crescentus recombinant strain JS4022/p723-6H, which overexpresses hexahistidine peptide on the surface of the bacterial cells and serves as a whole-cell adsorbent for dissolved heavy metals. Biofilms formed by JS4022/p723-6H are effective at retaining cadmium from bacterial growth media or environmental water samples. Here we provide additional experiment data discussing the application potential of this new technology. Supplementation of calcium to the growth media produced robust JS4022/p723-6H cells by alleviating their sensitivity to chelators. After growth in the presence of 0.3% CaCl2⋅2H2O, double the amount of JS4022/p723-6H cells survived the treatment with 2 mM EDTA. Free cells of JS4022/p723-6H effectively sequestered 51% of the total cadmium from a Lake Erie water sample at pH 5.4, compared to 37% retrieved by the control strain. Similar levels of adsorption were observed at pH 4.2 as well. Cells of JS4022/p723-6H were tolerant of acid treatment for 90 min at pH ≥1.1 or 120 min at pH ≥2.5, which provides an avenue for the convenient regeneration of the bacterial cells with acidic solutions. Designs of possible bioreactors and an operation system are also presented.

Overexpression of a lethal methylase, M.TneDI, in E. coli BL21(DE3)

A pET-based vector pDH21 expressing the methylase, M.TneDI (recognizing CGCG) from Thermotoga was constructed, and transformed into E. coli BL21(DE3). Despite E. coli BL21(DE3) being McrBC positive, 30 transformants were isolated, which were suspected to be McrBC− mutants. The overexpression of M.TneDI was verified by SDS-PAGE analysis. Compared to the previously constructed pJC340 vector, a pACYC184 derivative expressing M.TneDI from a tet promotor, the newly constructed pDH21 vector improved the expression of the methylase about fourfold, allowing complete protection of DNA substrates. This study not only demonstrates a practical approach to overexpressing potential lethal proteins in E. coli but also delivers a production strain of M.TneDI that may be useful in various in vitro methylation applications.