Recombinase-based reporter system and antisense technology to study gene expression and essentiality in hypoxic nonreplicating mycobacteria

The study of the mechanisms used by Mycobacterium tuberculosis to survive in the absence of growth is hampered by the absence of appropriate genetic tools. Here, we report two strategies, a recombinase-based reporter system and an antisense technology, to study gene expression and essentiality in hypoxic nonreplicating mycobacteria. The recombinase-based reporter system relies on the resolution of an antibiotic marker flanked by the gammadelta-res sites. This system was developed to identify M. tuberculosis promoters, which are specifically expressed under anaerobic conditions. The antisense strategy was designed to study the role of a gene candidate during anaerobic survival. To validate this approach, the dosR, narK2 and rv2466c promoters were selected to drive dosR antisense mRNA expression in quiescent mycobacteria. The conditional knockout strains were found to be attenuated to adapt and survive under anaerobic conditions, as observed for the dosR knockout strain. Together, our work demonstrates that the recombinase-based reporter system and antisense technology represent two genetic tools useful for the identification and characterization of genes essential for the survival of hypoxic nonreplicating M. tuberculosis.     

The Use of Whole-Cell Biosensors to Detect and Quantify Compounds or Conditions Affecting Biological Systems

A new and promising technique in microbial ecology and environmental biology is the use of whole-cell bacterial biosensors. This minireview describes the use of such biosensors for detection and quantification of various compounds and other conditions affecting bacterial expression of different genes. Three types of biosensors (nonspecific, stress-induced, and specific biosensors) are described including their use in different environments. We present tables of published biosensors, including gene fusions, host organisms, and environments in which they are used. We here describe the use of different reporter genes in the construction of biosensors and discuss their use as tools for monitoring the bioavailability of pollutants and their potential use in studying microbial ecology in general.     

Distribution of bacterial growth activity in flow-chamber biofilms.

In microbial communities such as those found in biofilms, individual organisms most often display heterogeneous behavior with respect to their metabolic activity, growth status, gene expression pattern, etc. In that context, a novel reporter system for monitoring of cellular growth activity has been designed. It comprises a transposon cassette carrying fusions between the growth rate-regulated Escherichia coli rrnBP1 promoter and different variant gfp genes. It is shown that the P1 promoter is regulated in the same way in E. coli and Pseudomonas putida, making it useful for monitoring of growth activity in organisms outside the group of enteric bacteria. Construction of fusions to genes encoding unstable Gfp proteins opened up the possibility of the monitoring of rates of rRNA synthesis and, in this way, allowing on-line determination of the distribution of growth activity in a complex community. With the use of these reporter tools, it is demonstrated that individual cells of a toluene-degrading P. putida strain growing in a benzyl alcohol-supplemented biofilm have different levels of growth activity which develop as the biofilm gets older. Cells that eventually grow very slowly or not at all may be stimulated to restart growth if provided with a more easily metabolizable carbon source. Thus, the dynamics of biofilm growth activity has been tracked to the level of individual cells, cell clusters, and microcolonies.     

Selectable marker genes in transgenic plants: applications, alternatives and biosafety

Approximately fifty marker genes used for transgenic and transplastomic plant research or crop development have been assessed for efficiency, biosafety, scientific applications and commercialization. Selectable marker genes can be divided into several categories depending on whether they confer positive or negative selection and whether selection is conditional or non-conditional on the presence of external substrates. Positive selectable marker genes are defined as those that promote the growth of transformed tissue whereas negative selectable marker genes result in the death of the transformed tissue. The positive selectable marker genes that are conditional on the use of toxic agents, such as antibiotics, herbicides or drugs were the first to be developed and exploited. More recent developments include positive selectable marker genes that are conditional on non-toxic agents that may be substrates for growth or that induce growth and differentiation of the transformed tissues. Newer strategies include positive selectable marker genes which are not conditional on external substrates but which alter the physiological processes that govern plant development. A valuable companion to the selectable marker genes are the reporter genes, which do not provide a cell with a selective advantage, but which can be used to monitor transgenic events and manually separate transgenic material from non-transformed material. They fall into two categories depending on whether they are conditional or non-conditional on the presence of external substrates. Some reporter genes can be adapted to function as selectable marker genes through the development of novel substrates. Despite the large number of marker genes that exist for plants, only a few marker genes are used for most plant research and crop development. As the production of transgenic plants is labor intensive, expensive and difficult for most species, practical issues govern the choice of selectable marker genes that are used. Many of the genes have specific limitations or have not been sufficiently tested to merit their widespread use. For research, a variety of selection systems are essential as no single selectable marker gene was found to be sufficient for all circumstances. Although, no adverse biosafety effects have been reported for the marker genes that have been adopted for widespread use, biosafety concerns should help direct which markers will be chosen for future crop development. Common sense dictates that marker genes conferring resistance to significant therapeutic antibiotics should not be used. An area of research that is growing rapidly but is still in its infancy is the development of strategies for eliminating selectable marker genes to generate marker-free plants. Among the several technologies described, two have emerged with significant potential. The simplest is the co-transformation of genes of interest with selectable marker genes followed by the segregation of the separate genes through conventional genetics. The more complicated strategy is the use of site-specific recombinases, under the control of inducible promoters, to excise the marker genes and excision machinery from the transgenic plant after selection has been achieved. In this review each of the genes and processes will be examined to assess the alternatives that exist for producing transgenic plants.     

Distribution of Bacterial Growth Activity in Flow-Chamber Biofilms

In microbial communities such as those found in biofilms, individual organisms most often display heterogeneous behavior with respect to their metabolic activity, growth status, gene expression pattern, etc. In that context, a novel reporter system for monitoring of cellular growth activity has been designed. It comprises a transposon cassette carrying fusions between the growth rate-regulated Escherichia coli rrnBP1 promoter and different variant gfp genes. It is shown that the P1 promoter is regulated in the same way in E. coli and Pseudomonas putida, making it useful for monitoring of growth activity in organisms outside the group of enteric bacteria. Construction of fusions to genes encoding unstable Gfp proteins opened up the possibility of the monitoring of rates of rRNA synthesis and, in this way, allowing on-line determination of the distribution of growth activity in a complex community. With the use of these reporter tools, it is demonstrated that individual cells of a toluene-degrading P. putida strain growing in a benzyl alcohol-supplemented biofilm have different levels of growth activity which develop as the biofilm gets older. Cells that eventually grow very slowly or not at all may be stimulated to restart growth if provided with a more easily metabolizable carbon source. Thus, the dynamics of biofilm growth activity has been tracked to the level of individual cells, cell clusters, and microcolonies.     

Genetically engineered microbial biosensors for in situ monitoring of environmental pollution

Microbial biosensors are compact, portable, cost effective, and simple to use, making them seem eminently suitable for the in situ monitoring of environmental pollution. One promising approach for such applications is the fusion of reporter genes with regulatory genes that are dose-dependently responsive to the target chemicals or physiological signals. Their biosensor capabilities, such as target range and sensitivity, could be improved by modification of regulatory genes. Recent uses of such genetically engineered microbial biosensors include the development of portable biosensor kits and high-throughput cell arrays on chips, optic fibers, or other platforms for on-site and on-line monitoring of environmental pollution. This mini-review discusses recent advances in microbial biosensors and their future prospects, with a focus on the development and application of genetically modified microbial biosensors for in situ environmental monitoring.     

A resource of vectors and ES cells for targeted deletion of microRNAs in mice

The 21-23 nucleotide, single-stranded RNAs classified as microRNAs (miRNA) perform fundamental roles in diverse cellular and developmental processes. In contrast to the situation for protein-coding genes, no public resource of miRNA mouse mutant alleles exists. Here we describe a collection of 428 miRNA targeting vectors covering 476 of the miRNA genes annotated in the miRBase registry. Using these vectors, we generated a library of highly germline-transmissible C57BL/6N mouse embryonic stem (ES) cell clones harboring targeted deletions for 392 miRNA genes. For most of these targeted clones, chimerism and germline transmission can be scored through a coat color marker. The targeted alleles have been designed to be adaptable research tools that can be efficiently altered by recombinase-mediated cassette exchange to create reporter, conditional and other allelic variants. This miRNA knockout (mirKO) resource can be searched electronically and is available from ES cell repositories for distribution to the scientific community.     

Using flow cytometry to quantify microbial heterogeneity

Flow cytometry is a powerful technique for the study of single cells, and thus it is of particular utility in the study of heterogeneity in microbial populations. This review seeks to highlight the role of flow cytometric analyses in studies of microbial heterogeneity, drawing wherever possible on recently published research articles. Whilst microbial heterogeneity is well documented in both natural and laboratory environments, the underlying causes are less well understood. Possible sources for the heterogeneity that is observed in microbial systems are discussed, together with the flow cytometric tools that aid its study. The role of flow cytometry in molecular biology is discussed with reference to gene reporter systems, which enable heterogeneity of gene expression to be monitored. With the recent sequencing of a variety of microbial genomes, it is anticipated that flow cytometry will have an increasing role to play in studying the effects of gene expression and mutation on heterogeneity, and in resolving the interactions of genetics and physiology.     

pLR: A lentiviral backbone series to stable transduction of bicistronic genes and exchange of promoters

Gene transfer based on lentiviral vectors allow the integration of exogenous genes into the genome of a target cell, turning these vectors into one of the most used methods for stable transgene expression in mammalian cells, in vitro and in vivo. Currently, there are no lentivectors that allow the cloning of different genes to be regulated by different promoters. Also, there are none that permit the analysis of the expression through an IRES (internal ribosome entry site) - reporter gene system. In this work, we have generated a series of lentivectors containing: (1) a malleable structure to allow the cloning of different target genes in a multicloning site (mcs); (2) unique site to exchange promoters, and (3) IRES followed by one of two reporter genes: eGFP or DsRed. The series of the produced vectors were named pLR (for lentivirus and RSV promoter) and were fairly efficient with a strong fluorescence of the reporter genes in direct transfection and viral transduction experiments. This being said, the pLR series have been found to be powerful biotechnological tools for stable gene transfer and expression.     

Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes

A general purpose transformation vector, designated pPha-T1, was constructed for use with the diatom Phaeodactylum tricornutum Bohlin. This vector harbors the sh ble cassette for primary selection on medium containing the antibiotic zeocin, and a multiple cloning site flanked by the P. tricornutum fcp A promoter. pPha-T1 was used to establish the utility of three selectable marker genes and two reporter genes for P. tricornutum transformation. The nat and sat-1 genes confer resistance to the antibiotic nourseothricin, and npt II confers resistance to G418. Each of these genes was effective as a selectable marker for identifying primary transformants. These markers could also be used for dual selections in combination with the sh ble gene. The reporter genes uid A and gfp were also introduced into P. tricornutum using pPha-T1. Gus expression in some transformants reached 15 μg·μg⁻¹ of total soluble protein and permitted excellent cell staining, while GFP fluorescence was readily visible with standard fluorescence microscopy. The egfp gene, which has optimal codon usage for expression in human cells, was the only version of gfp that produced a strong fluorescent signal in P. tricornutum. The codon bias of the egfp gene is similar to that of P. tricornutum genes. This study suggests that codon usage has a significant effect on the efficient expression of reporter genes in P. tricornutum. The results presented here demonstrate that a variety of selectable markers and reporter genes can be expressed in P. tricornutum , enhancing the potential of this organism for exploring basic biological questions and industrial applications.     

Engineering More Stable,Selectable Marker-Free Autoluminescent Mycobacteria by One Step

In our previous study, we demonstrated that the use of the autoluminescent Mycobacterium tuberculosis as a reporter strain had the potential to drastically reduce the time, effort, animals and costs consumed in evaluation of the activities of drugs and vaccines in live mice. However, the strains were relatively unstable and lost reporter with time without selection. The kanamycin selection marker used wasn’t the best choice as it provides resistance to amino glycosides which are an important class of second line drugs used in tuberculosis treatment. In addition, the marker could limit utility of the strains for screening of new potential drugs or evaluating drug combinations for tuberculosis treatment. Limited selection marker genes for mycobacterial genetic manipulation is a major drawback for such a marker-containing strain in many research fields. Therefore, selectable marker-free, more stable autoluminescent mycobacteria are highly needed. After trying several strategies, we created such mycobacterial strains successfully by using an integrative vector and removing both the resistance maker and integrase genes by Xer site-specific recombination in one step. The corresponding plasmid vectors developed in this study could be very convenient in constructing other selectable marker-free, more stable reporter mycobacteria with diverse applications.     

Inhibition of Mycobacterium smegmatis gene expression and growth using antisense peptide nucleic acids

Antisense agents that inhibit genes at the mRNA level are attractive tools for genome-wide studies and drug target validation. The approach may be particularly well suited to studies of bacteria that are difficult to manipulate with standard genetic tools. Antisense peptide nucleic acids (PNA) with attached carrier peptides can inhibit gene expression in Escherichia coli and Staphylococcus aureus. Here we asked whether peptide-PNAs could mediate antisense effects in Mycobacterium smegmatis. We first targeted the gfp reporter gene and observed dose- and sequence-dependent inhibition at low micromolar concentrations. Sequence alterations within both the PNA and target mRNA sequences eliminated inhibition, strongly supporting an antisense mechanism of inhibition. Also, antisense PNAs with various attached peptides showed improved anti-gfp effects. Two peptide-PNAs targeted to the essential gene inhA were growth inhibitory and caused cell morphology changes that resemble that of InhA-depleted cells. Therefore, antisense peptide-PNAs can efficiently and specifically inhibit both reporter and endogenous essential genes in mycobacteria.     

Transient expression assays in grapevine: a step towards genetic improvement

In the past few years, the usefulness of transient expression assays has continuously increased for the characterization of unknown gene function and metabolic pathways. In grapevine (Vitis vinifera L.), one of the most economically important fruit crops in the world, recent systematic sequencing projects produced many gene data sets that require detailed analysis. Due to their rapid nature, transient expression assays are well suited for large‐scale genetic studies. Although genes and metabolic pathways of any species can be analysed by transient expression in model plants, a need for homologous systems has emerged to avoid the misinterpretation of results due to a foreign genetic background. Over the last 10 years, various protocols have thus been developed to apply this powerful technology to grapevine. Using cell suspension cultures, somatic embryos, leaves or whole plantlets, transient expression assays enabled the study of the function, regulation and subcellular localization of genes involved in specific metabolic pathways such as the biosynthesis of phenylpropanoids. Disease resistance genes that could be used for marker‐assisted selection in conventional breeding or for stable transformation of elite cultivars have also been characterized. Additionally, transient expression assays have proved useful for shaping new tools for grapevine genetic improvement: synthetic promoters, silencing constructs, minimal linear cassettes or viral vectors. This review provides an update on the different tools (DNA constructs, reporter genes, vectors) and methods (Agrobacterium‐mediated and direct gene transfer methods) available for transient gene expression in grapevine. The most representative results published thus far are then described.     

基因捕捉及其在植物基因分离和功能基因组学上的应用

基因捕捉是一种报告基因的随机整合技术。基因捕捉系统已成为分离基因、鉴定基因功能的重要手段。基因捕捉(gene traps)包括增强子捕捉(enhancer trap)、启动子捕捉(promoter trap)和基因捕捉(gene trap),通称为基因捕捉(gcne traps)。在增强子捕捉中,报告基因与一个基本启动子融合,这个启动子不能使报告基因表达,但可被临近的增强子激活。在启动子捕捉和基因捕捉中,报告基因的启动子被去除,融合基因只有以正确的方向插入到转录单元内才能表达。对基因捕捉系统的结构特征、构建方法、应用范围、研究现状和应用前景等作了系统论述,并对有关问题进行了讨论。     

Use of green fluorescent protein-based reporters to monitor balanced production of antifungal compounds in the biocontrol agent Pseudomonas fluorescens CHA0

AIMS: To develop reporter constructs based on stable and unstable variants of the green fluorescent protein (GFP) for monitoring balanced production of antifungal compounds that are crucial for the capacity of the root-colonizing Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogenic fungi. METHODS AND RESULTS: Pseudomonas fluorescens CHA0 produces the three antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT) and pyrrolnitrin (PRN). The gfp[mut3] and gfp[AAV] reporter genes were fused to the promoter regions of the DAPG, PLT and PRN biosynthetic genes. The reporter fusions were then used to follow the kinetics of expression of the three antifungal metabolites in a microplate assay. DAPG and PLT were found to display an inverse relationship in which each metabolite activates its own biosynthesis while repressing the synthesis of the other metabolite. PRN appears not to be involved in this balance. However, the microbial and plant phenolic metabolite salicylate was found to interfere with the expression of both DAPG and PLT. CONCLUSIONS: The results obtained provide evidence that P. fluorescens CHA0 may keep the antifungal compounds DAPG and PLT at a fine-tuned balance that can be affected by certain microbial and plant phenolics. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, the present study is the first to use stable and unstable GFP variants to study antibiotic gene expression in a biocontrol pseudomonad. The developed reporter fusions will be a highly valuable tool to study in situ expression of this bacterial biocontrol trait on plant roots, i.e. at the site of pathogen suppression.