a-versatile-automated-platform-for-micro-scale-cell-stimulation

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

In budding yeast, Sgs1 is the sole member of the RecQ family of DNA helicases. Like the human Bloom syndrome helicase (BLM), Sgs1 functions during both vegetative growth and meiosis. The sgs1 null mutant sporulates poorly and displays reduced spore viability.

The Sgs1 helicase regulates chromosome synapsis and meiotic crossing over.

The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization. Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruiting-body-like aerial projections whose tips serve as preferential sites for sporulation. To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B. subtilis genome. This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation. This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media. In this way we identified six genes that are involved in the development of multicellular communities. These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function). Further analysis revealed that these six genes play different roles in B. subtilis community development.

Genes involved in formation of structured multicellular communities by Bacillus subtilis.

Gene zur Bildung von strukturierten vielzelligen Gemeinden, die von Bacillus subtilis beteiligt.

Microbes often construct and live within surface-associated multicellular communities known as biofilms. The precise structure, chemistry and physiology of the biofilm all vary with the nature of its resident microbes and local environment. However, an important commonality among biofilms is that their structural integrity critically depends upon an extracellular matrix produced by their constituent cells. Extracellular matrices might be as diverse as biofilms, and they contribute significantly to the organization of the community. This review discusses recent advances in our understanding of the extracellular matrix and its role in biofilm biology.

Biofilms: the matrix revisited.

Biofilme: Die Matrix Revisited.

Wild strains of Bacillus subtilis are capable of forming architecturally complex communities of cells known as biofilms. Critical to biofilm formation is the eps operon, which is believed to be responsible for the biosynthesis of an exopolysaccharide that binds chains of cells together in bundles. We report that transcription of eps is under the negative regulation of SinR, a repressor that was found to bind to multiple sites in the regulatory region of the operon. Mutations in sinR bypassed the requirement in biofilm formation of two genes of unknown function, ylbF and ymcA, and sinI, which is known to encode an antagonist of SinR. We propose that these genes are members of a pathway that is responsible for counteracting SinR-mediated repression. We further propose that SinR is a master regulator that governs the transition between a planktonic state in which the bacteria swim as single cells in liquid or swarm in small groups over surfaces, and a sessile state in which the bacteria adhere to each other to form bundled chains and assemble into multicellular communities.

A master regulator for biofilm formation by Bacillus subtilis.

Ein Master-Regler f&#xFC;r die Biofilmbildung von Bacillus subtilis.

Wild strains of the spore-forming bacterium Bacillus subtilis are capable of forming architecturally complex communities of cells. The formation of these biofilms is mediated in part by the 15-gene exopolysaccharide operon, epsA-O, which is under the direct negative control of the SinR repressor. We report the identification of an additional operon, yqxM-sipW-tasA, that is required for biofilm formation and is under the direct negative control of SinR. We now show that all three members of the operon are required for the formation of robust biofilms and that SinR is a potent repressor of the operon that acts by binding to multiple sites in the promoter region. Genome-wide analysis of SinR-controlled transcription indicates that the epsA-O and yqxM-sipW-tasA operons constitute many of the most strongly controlled genes in the SinR regulon. These findings reinforce the view that SinR is a master regulator for biofilm formation and further suggest that a principal biological function of SinR is to govern the assembly of complex multicellular communities.

Targets of the master regulator of biofilm formation in Bacillus subtilis.

Wilde St&auml;mme von unbegei&szlig;eltes Bakterium Bacillus Subtilis sind in der Lage architektonisch komplexe Gemeinschaften von Zellen zu bilden. Die Bildung dieser Biofilme ist teilweise von der 15-gen Exopolysaccharide Operon vermittelt EpsA-O, die unter der direkten negativen Kontrolle der SinR-Hemmer ist. Wir berichten &uuml;ber die Identifizierung von einer zus&auml;tzlichen Operon, YqxM-SipW-TasA, das erforderliche Biofilm-Bildung und ist unter der direkten negativen Kontrolle der SinR. Wir zeigen nun, dass alle drei Mitglieder von der Operon erforderlich f&uuml;r die Bildung von robusten Biofilme sind und SinR ein potenter Verdr&auml;nger von das Operon ist, die durch Bindung an mehreren Standorten in der Promotor-Region fungiert. Genomweiten Analyse der SinR kontrollierten Transkription bedeutet, dass die EpsA-O und YqxM-SipW-TasA Operone viele der am st&auml;rksten kontrollierten Gene in die SinR Regulon darstellen. Diese Befunde best&auml;tigen, dass SinR ein master Regler f&uuml;r Biofilm-Bildung ist und weiter schlagen vor, da&szlig; die wichtigste biologische Funktion der SinR ist die Montage von komplexen Vielzeller Gemeinschaften zu regieren.

Ziele der Meister Regulierungsbeh&ouml;rde der Biofilm Formation in Bacillus Subtilis.

Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.

A major protein component of the Bacillus subtilis biofilm matrix.

Mikroben konstruieren strukturell komplexe Vielzellige Gemeinschaften (Biofilme) &uuml;ber die Produktion einer extrazellul&auml;ren Matrix. Hier pr&auml;sentieren wir Beweise von Rasterelektronenmikroskopie zeigt, dass ein wilder Stamm von der Gram-positiven Bakterium Bacillus Subtilis solch eine Matrix erstellt. Genetische, biochemische und zytologische Beweise gibt, dass die Matrix vorwiegend aus einer Protein-Komponente, TasA und eine Exopolysaccharide Komponente besteht. Da TasA oder der Exopolysaccharide ergab eine residuale Matrix, w&auml;hrend der Abwesenheit beider Komponenten zu Totalausfall Form komplexe Vielzellige Gemeinschaften gef&uuml;hrt. Extrazellul&auml;re Komplementation Experimente ergaben, dass eine funktionale Matrix zusammengestellt werden kann, selbst wenn TasA und der Exopolysaccharide von verschiedenen Zellen produziert werden, verst&auml;rkt die Ansicht, die die Komponenten kosteng&Atilde;&frac14;nstig extrazellul&auml;re Matrix Bildung beitragen. Nach der Definition der Hauptbestandteile der Biofilm-Matrix und die Kontrolle von deren Synthese durch die globale Aufsichtsbeh&ouml;rden SinR, pr&auml;sentieren wir ein Arbeitsmodell f&uuml;r wie B. Subtilis wechselt zwischen nomadischen und sesshaften Lebensweise.

Eine gro&szlig;e Protein-Komponente der Bacillus Subtilis Biofilm-Matrix.

Phagocytosis, which is essential for the immune response to pathogens, is initiated by specific interactions between pathogens and cell surface receptors expressed by phagocytes. This study identifies triggering receptor expressed on myeloid cells 2 (TREM-2) and its signaling counterpart DAP12 as a molecular complex that promotes phagocytosis of bacteria. Expression of TREM-2-DAP12 enables nonphagocytic Chinese hamster ovary cells to internalize bacteria. This function depends on actin cytoskeleton dynamics and the activity of the small guanosine triphosphatases Rac and Cdc42. Internalization also requires src kinase activity and tyrosine phosphorylation. In bone marrow-derived macrophages, phagocytosis is decreased in the absence of DAP12 and can be restored by expression of TREM-2-DAP12. Depletion of TREM-2 inhibits both binding and uptake of bacteria. Finally, TREM-2-dependent phagocytosis is impaired in Syk-deficient macrophages. This study highlights a novel role for TREM-2-DAP12 in the immune response to bacterial pathogens.

TREM-2 (triggering receptor expressed on myeloid cells 2) is a phagocytic receptor for bacteria.

The ability to monitor cell signaling events is crucial to the understanding of immune defense against invading pathogens. Conventional analytical techniques such as flow cytometry, microscopy, and Western blot are powerful tools for signaling studies. Nevertheless, each approach is currently stand-alone and limited by multiple time-consuming and labor-intensive steps. In addition, these techniques do not provide correlated signaling information on total intracellular protein abundance and subcellular protein localization. We report on a novel phosphoFlow Chip (pFC) that relies on monolithic microfluidic technology to rapidly conduct signaling studies. The pFC platform integrates cell stimulation and preparation, microscopy, and subsequent flow cytometry. pFC allows host-pathogen phosphoprofiling in 30 min with an order of magnitude reduction in the consumption of reagents. For pFC validation, we monitor the mitogen-activated protein kinases ERK1/2 and p38 in response to Escherichia coli lipopolysaccharide (LPS) stimulation of murine macrophage cells (RAW 264.7). pFC permits ERK1/2 phosphorylation monitoring starting at 5 s after LPS stimulation, with phosphorylation observed at 5 min. In addition, ERK1/2 phosphorylation is correlated with subsequent recruitment into the nucleus, as observed from fluorescence microscopy performed on cells upstream of flow cytometric analysis. The fully integrated cell handling has the added advantage of reduced cell aggregation and cell loss, with no detectable cell activation. The pFC approach is a step toward unified, automated infrastructure for high-throughput systems biology.

Fully integrated microfluidic platform enabling automated phosphoprofiling of macrophage response.

We have developed a microfluidic platform that enables, in one experiment, monitoring of signaling events spanning multiple time-scales and cellular locations through seamless integration of cell culture, stimulation and preparation with downstream analysis. A combination of two single-cell resolution techniques-on-chip multi-color flow cytometry and fluorescence imaging provides multiplexed and orthogonal data on cellular events. Automated, microfluidic operation allows quantitatively- and temporally-precise dosing leading to fine time-resolution and improved reproducibility of measurements. The platform was used to profile the toll-like receptor (TLR4) pathway in macrophages challenged with lipopolysaccharide (LPS)-beginning with TLR4 receptor activation by LPS, through intracellular MAPK signaling, RelA/p65 translocation in real time, to TNF-Î± cytokine production, all in one small macrophage population (< 5000 cells) while using minute reagent volume (540 nL/condition). The platform is easily adaptable to many cell types including primary cells and provides a generic platform for profiling signaling pathways.

Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution.

Second-generation sequencing (SGS) has become the preferred method for RNA transcriptome profiling of organisms and single cells. However, SGS analysis of transcriptome diversity (including protein-coding transcripts and regulatory non-coding RNAs) is inefficient unless the sample of interest is first depleted of nucleic acids derived from ribosomal RNA (rRNA), which typically account for up to 95% of total intracellular RNA content. Here we describe a novel microscale hydroxyapatite chromatography (HAC) normalization method to remove eukaryotic and prokaryotic high abundant rRNA species, thereby increasing sequence coverage depth and transcript diversity across non-rRNA populations. RNA-seq analysis of Escherichia coli K-12 and human intracellular total RNA showed that HAC-based normalization enriched for all non-ribosomal RNA species regardless of RNA transcript abundance or length when compared with untreated controls. Microcolumn HAC normalization generated rRNA-depleted cDNA libraries comparable to the well-established duplex specific nuclease (DSN) normalization and Ribo-Zero rRNA-depletion methods, thus establishing microscale HAC as an effective, cost saving, and non-destructive alternative normalization technique.

cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications.

To fully understand the interactions of a pathogen with its host, it is necessary to analyze the RNA transcripts of both the host and pathogen throughout the course of an infection. Although this can be accomplished relatively easily on the host side, the analysis of pathogen transcripts is complicated by the overwhelming amount of host RNA isolated from an infected sample. Even with the read depth provided by second-generation sequencing, it is extremely difficult to get enough pathogen reads for an effective gene-level analysis. In this study, we describe a novel capture-based technique and device that considerably enriches for pathogen transcripts from infected samples. This versatile method can, in principle, enrich for any pathogen in any infected sample. To test the technique's efficacy, we performed time course tissue culture infections using Rift Valley fever virus and Francisella tularensis. At each time point, RNA sequencing (RNA-Seq) was performed and the results of the treated samples were compared with untreated controls. The capture of pathogen transcripts, in all cases, led to more than an order of magnitude enrichment of pathogen reads, greatly increasing the number of genes hit, the coverage of those genes, and the depth at which each transcript was sequenced.

Enriching pathogen transcripts from infected samples: a capture-based approach to enhanced host-pathogen RNA sequencing.

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.

Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material.

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.

Article	Year
The Sgs1 helicase regulates chromosome synapsis and meiotic crossing over. Current biology : CB\| PubMed ID: 14614820	2003
Genes involved in formation of structured multicellular communities by Bacillus subtilis. Journal of bacteriology\| PubMed ID: 15175311	2004
Biofilms: the matrix revisited. Trends in microbiology\| PubMed ID: 15639628	2005
A master regulator for biofilm formation by Bacillus subtilis. Molecular microbiology\| PubMed ID: 15661000	2005
Targets of the master regulator of biofilm formation in Bacillus subtilis. Molecular microbiology\| PubMed ID: 16430695	2006
A major protein component of the Bacillus subtilis biofilm matrix. Molecular microbiology\| PubMed ID: 16430696	2006
TREM-2 (triggering receptor expressed on myeloid cells 2) is a phagocytic receptor for bacteria. The Journal of cell biology\| PubMed ID: 19171755	2009
Fully integrated microfluidic platform enabling automated phosphoprofiling of macrophage response. Analytical chemistry\| PubMed ID: 19323537	2009
Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution. Lab on a chip\| PubMed ID: 22777012	2012
cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications. BioTechniques\| PubMed ID: 23227988	2012
Enriching pathogen transcripts from infected samples: a capture-based approach to enhanced host-pathogen RNA sequencing. Analytical biochemistry\| PubMed ID: 23535274	2013
Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material. RNA biology\| PubMed ID: 23558773	2013
A microfluidic DNA library preparation platform for next-generation sequencing. PloS one\| PubMed ID: 23894387	2013

Steven S. Branda

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