High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis

Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called “MultiCLEM,” allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique.

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Novel Whole-Cell Antibiotic Biosensors for Compound Discovery

Applied and Environmental Microbiology ◽

10.1128/aem.00586-07 ◽

2007 ◽

Vol 73 (20) ◽

pp. 6436-6443 ◽

Cited By ~ 73

Author(s):

Andreas Urban ◽

Stefan Eckermann ◽

Beate Fast ◽

Susanne Metzger ◽

Matthias Gehling ◽

...

Keyword(s):

Bacillus Subtilis ◽

High Throughput ◽

High Throughput Screening ◽

Protein Biosynthesis ◽

Firefly Luciferase ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Environmental Biology ◽

Drug Candidates ◽

Novel Drug

ABSTRACT Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of ≤25 μg/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis.

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Systematic sequencing of the 283 kb 210 -232 region of the Bacillus subtilis genome containing the skin element and many sporulation genes

Microbiology ◽

10.1099/13500872-142-11-3103 ◽

1996 ◽

Vol 142 (11) ◽

pp. 3103-3111 ◽

Cited By ~ 30

Author(s):

M. Mizuno ◽

S. Masuda ◽

K.-i. Takemaru ◽

S. Hosono ◽

T. Sato ◽

...

Keyword(s):

Bacillus Subtilis ◽

Subtilis Genome ◽

Sporulation Genes

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Mutation in yaaT Leads to Significant Inhibition of Phosphorelay during Sporulation in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.184.20.5545-5553.2002 ◽

2002 ◽

Vol 184 (20) ◽

pp. 5545-5553 ◽

Cited By ~ 20

Author(s):

Shigeo Hosoya ◽

Kei Asai ◽

Naotake Ogasawara ◽

Michio Takeuchi ◽

Tsutomu Sato

Keyword(s):

Bacillus Subtilis ◽

Fluorescent Protein ◽

Early Stage ◽

Response Regulator ◽

Significant Inhibition ◽

Open Reading Frame ◽

Histidine Kinases ◽

Reading Frame ◽

Green Fluorescent ◽

Sporulation Genes

ABSTRACT In the course of a Bacillus subtilis functional genomics project which involved screening for sporulation genes, we identified an open reading frame, yaaT, whose disruptant exhibits a sporulation defect. Twenty-four hours after the initiation of sporulation, most cells of the yaaT mutant exhibited stage 0 of sporulation, indicating that the yaaT mutation blocks sporulation at an early stage. Furthermore, the mutation in yaaT led to a significant decrease in transcription from a promoter controlled by Spo0A, a key response regulator required for the initiation of sporulation. However, neither the level of transcription of spo0A, the activity of σH, which transcribes spo0A, nor the amount of Spo0A protein was severely affected by the mutation in yaaT. Bypassing the phosphorelay by introducing an spo0A mutation (sof-1) into the yaaT mutant suppressed the sporulation defect, suggesting that the yaaT mutation interferes with the phosphorelay process comprising Spo0F, Spo0B, and histidine kinases. We also observed that mutation of spo0E, which encodes the phosphatase that dephosphorylates Spo0A-P, suppressed the sporulation defect in the yaaT mutant. These results strongly suggest that yaaT plays a significant role in the transduction of signals to the phosphorelay for initiation of sporulation. Micrographs indicated that YaaT-green fluorescent protein localizes to the peripheral membrane, as well as to the septum, during sporulation.

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New vectors for co-expression of proteins: Structure of Bacillus subtilis ScoAB obtained by high-throughput protocols

Protein Expression and Purification ◽

10.1016/j.pep.2007.01.013 ◽

2007 ◽

Vol 53 (2) ◽

pp. 396-403 ◽

Cited By ~ 31

Author(s):

Lucy Stols ◽

Min Zhou ◽

William H. Eschenfeldt ◽

Cynthia Sanville Millard ◽

James Abdullah ◽

...

Keyword(s):

Bacillus Subtilis ◽

High Throughput

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Expression of Biofilm-Degrading Enzymes in Plants and Automated High-Throughput Activity Screening Using Experimental Bacillus subtilis Biofilms

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.708150 ◽

2021 ◽

Vol 9 ◽

Author(s):

P. Opdensteinen ◽

S. J. Dietz ◽

B. B. Gengenbach ◽

J. F. Buyel

Keyword(s):

Bacillus Subtilis ◽

Plant Cell ◽

High Throughput ◽

High Throughput Screening ◽

Scale Up ◽

Growth Media ◽

Subcellular Targeting ◽

Degrading Enzymes ◽

Depth Filtration ◽

Activity Screening

Biofilm-forming bacteria are sources of infections because they are often resistant to antibiotics and chemical removal. Recombinant biofilm-degrading enzymes have the potential to remove biofilms gently, but they can be toxic toward microbial hosts and are therefore difficult to produce in bacteria. Here, we investigated Nicotiana species for the production of such enzymes using the dispersin B-like enzyme Lysobacter gummosus glyco 2 (Lg2) as a model. We first optimized transient Lg2 expression in plant cell packs using different subcellular targeting methods. We found that expression levels were transferable to differentiated plants, facilitating the scale-up of production. Our process yielded 20 mg kg−1 Lg2 in extracts but 0.3 mg kg−1 after purification, limited by losses during depth filtration. Next, we established an experimental biofilm assay to screen enzymes for degrading activity using different Bacillus subtilis strains. We then tested complex and chemically defined growth media for reproducible biofilm formation before converting the assay to an automated high-throughput screening format. Finally, we quantified the biofilm-degrading activity of Lg2 in comparison with commercial enzymes against our experimental biofilms, indicating that crude extracts can be screened directly. This ability will allow us to combine high-throughput expression in plant cell packs with automated activity screening.

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Matrix linear models for high-throughput chemical genetic screens

10.1101/468140 ◽

2018 ◽

Author(s):

Jane W. Liang ◽

Robert J. Nichols ◽

Śaunak Sen

Keyword(s):

High Throughput ◽

Linear Models ◽

Natural Extension ◽

Attractive Alternative ◽

Genetic Screens ◽

Computationally Efficient ◽

Model Framework ◽

Chemical Genetic ◽

Link Type ◽

Univariate Approach

AbstractWe develop a flexible and computationally efficient approach for analysing high throughput chemical genetic screens. In such screens, a library of genetic mutants is phenotyped in a large number of stresses. The goal is to detect interactions between genes and stresses. Typically, this is achieved by grouping the mutants and stresses into categories, and performing modified t-tests for each combination. This approach does not have a natural extension if mutants or stresses have quantitative or non-overlapping annotations (eg. if conditions have doses, or a mutant falls into more than one category simultaneously). We develop a matrix linear model framework that allows us to model relationships between mutants and conditions in a simple, yet flexible multivariate framework. It encodes both categorical and continuous relationships to enhance detection of associations. To handle large datasets, we develop a fast estimation approach that takes advantage of the structure of matrix linear models. We evaluate our method’s performance in simulations and in an E. coli chemical genetic screen, comparing it with an existing univariate approach based on modified t-tests. We show that matrix linear models perform slightly better than the univariate approach when mutants and conditions are classified in non-overlapping categories, and substantially better when conditions can be ordered in dosage categories. Our approach is much faster computationally and is scalable to larger datasets. It is an attractive alternative to current methods, and provides a natural framework extensible to larger, and more complex chemical genetic screens. A Julia implementation of matrix linear models and the code used for the analysis in this paper can be found at https://bitbucket.org/jwliang/mlm_packages and https://bitbucket.org/jwliang/mlm_gs_supplement, respectively.

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Evolutionary Analysis of the Bacillus subtilis Genome Reveals New Genes Involved in Sporulation

Molecular Biology and Evolution ◽

10.1093/molbev/msaa035 ◽

2020 ◽

Vol 37 (6) ◽

pp. 1667-1678 ◽

Cited By ~ 1

Author(s):

Lei Shi ◽

Abderahmane Derouiche ◽

Santosh Pandit ◽

Shadi Rahimi ◽

Aida Kalantari ◽

...

Keyword(s):

Bacillus Subtilis ◽

Representative Sample ◽

Genome Mining ◽

Evolutionary Analysis ◽

Evolutionary Time ◽

Time Points ◽

New Genes ◽

Sporulation Process ◽

Subtilis Genome ◽

Sporulation Genes

Abstract Bacilli can form dormant, highly resistant, and metabolically inactive spores to cope with extreme environmental challenges. In this study, we examined the evolutionary age of Bacillus subtilis sporulation genes using the approach known as genomic phylostratigraphy. We found that B. subtilis sporulation genes cluster in several groups that emerged at distant evolutionary time-points, suggesting that the sporulation process underwent several stages of expansion. Next, we asked whether such evolutionary stratification of the genome could be used to predict involvement in sporulation of presently uncharacterized genes (y-genes). We individually inactivated a representative sample of uncharacterized genes that arose during the same evolutionary periods as the known sporulation genes and tested the resulting strains for sporulation phenotypes. Sporulation was significantly affected in 16 out of 37 (43%) tested strains. In addition to expanding the knowledge base on B. subtilis sporulation, our findings suggest that evolutionary age could be used to help with genome mining.

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