Re- engineering cellular physiology by rewiring high- level global regulatory genes

Similar documents
Practical Bioinformatics

SUPPORTING INFORMATION FOR. SEquence-Enabled Reassembly of β-lactamase (SEER-LAC): a Sensitive Method for the Detection of Double-Stranded DNA

Supplemental data. Pommerrenig et al. (2011). Plant Cell /tpc

High throughput near infrared screening discovers DNA-templated silver clusters with peak fluorescence beyond 950 nm

SUPPLEMENTARY DATA - 1 -

Clay Carter. Department of Biology. QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

SSR ( ) Vol. 48 No ( Microsatellite marker) ( Simple sequence repeat,ssr),

Number-controlled spatial arrangement of gold nanoparticles with

Crick s early Hypothesis Revisited

Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1

Characterization of Pathogenic Genes through Condensed Matrix Method, Case Study through Bacterial Zeta Toxin

Electronic supplementary material

Advanced topics in bioinformatics

SEQUENCE ALIGNMENT BACKGROUND: BIOINFORMATICS. Prokaryotes and Eukaryotes. DNA and RNA

Supplementary Information for

Supplementary Information

Table S1. Primers and PCR conditions used in this paper Primers Sequence (5 3 ) Thermal conditions Reference Rhizobacteria 27F 1492R

Supplemental Figure 1.

Supporting Information

Regulatory Sequence Analysis. Sequence models (Bernoulli and Markov models)

Building a Multifunctional Aptamer-Based DNA Nanoassembly for Targeted Cancer Therapy

The role of the FliD C-terminal domain in pentamer formation and

NSCI Basic Properties of Life and The Biochemistry of Life on Earth

Supporting Information for. Initial Biochemical and Functional Evaluation of Murine Calprotectin Reveals Ca(II)-

TM1 TM2 TM3 TM4 TM5 TM6 TM bp

SUPPLEMENTARY INFORMATION

6.047 / Computational Biology: Genomes, Networks, Evolution Fall 2008

evoglow - express N kit distributed by Cat.#: FP product information broad host range vectors - gram negative bacteria

3. Evolution makes sense of homologies. 3. Evolution makes sense of homologies. 3. Evolution makes sense of homologies

Protein Threading. Combinatorial optimization approach. Stefan Balev.

SUPPLEMENTARY INFORMATION

evoglow - express N kit Cat. No.: product information broad host range vectors - gram negative bacteria

The Trigram and other Fundamental Philosophies

Modelling and Analysis in Bioinformatics. Lecture 1: Genomic k-mer Statistics

Supplemental Table 1. Primers used for cloning and PCR amplification in this study

Supplementary Information

Evolvable Neural Networks for Time Series Prediction with Adaptive Learning Interval

Sex-Linked Inheritance in Macaque Monkeys: Implications for Effective Population Size and Dispersal to Sulawesi

FliZ Is a Posttranslational Activator of FlhD 4 C 2 -Dependent Flagellar Gene Expression

Codon Distribution in Error-Detecting Circular Codes

Why do more divergent sequences produce smaller nonsynonymous/synonymous

Evolutionary dynamics of abundant stop codon readthrough in Anopheles and Drosophila

ChemiScreen CaS Calcium Sensor Receptor Stable Cell Line

Near-instant surface-selective fluorogenic protein quantification using sulfonated

ydci GTC TGT TTG AAC GCG GGC GAC TGG GCG CGC AAT TAA CGG TGT GTA GGC TGG AGC TGC TTC

The 3 Genomic Numbers Discovery: How Our Genome Single-Stranded DNA Sequence Is Self-Designed as a Numerical Whole

Timing molecular motion and production with a synthetic transcriptional clock

part 3: analysis of natural selection pressure

Identification of a Locus Involved in the Utilization of Iron by Haemophilus influenzae

Supplemental Figure 1. Phenotype of ProRGA:RGAd17 plants under long day

Pathways and Controls of N 2 O Production in Nitritation Anammox Biomass

Chain-like assembly of gold nanoparticles on artificial DNA templates via Click Chemistry

Encoding of Amino Acids and Proteins from a Communications and Information Theoretic Perspective

Supporting Information. An Electric Single-Molecule Hybridisation Detector for short DNA Fragments

Biosynthesis of Bacterial Glycogen: Primary Structure of Salmonella typhimurium ADPglucose Synthetase as Deduced from the

AtTIL-P91V. AtTIL-P92V. AtTIL-P95V. AtTIL-P98V YFP-HPR

Insects act as vectors for a number of important diseases of

Introduction to Molecular Phylogeny

THE MATHEMATICAL STRUCTURE OF THE GENETIC CODE: A TOOL FOR INQUIRING ON THE ORIGIN OF LIFE

Supplementary Figure 1. Schematic of split-merger microfluidic device used to add transposase to template drops for fragmentation.

It is the author's version of the article accepted for publication in the journal "Biosystems" on 03/10/2015.

Supplementary information. Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization.

Characterization of Multiple-Antimicrobial-Resistant Salmonella Serovars Isolated from Retail Meats

160, and 220 bases, respectively, shorter than pbr322/hag93. (data not shown). The DNA sequence of approximately 100 bases of each

Glucosylglycerate phosphorylase, a novel enzyme specificity involved in compatible solute metabolism

part 4: phenomenological load and biological inference. phenomenological load review types of models. Gαβ = 8π Tαβ. Newton.

Symmetry Studies. Marlos A. G. Viana

Supplementary Materials for

Supporting Information

Evidence for RNA editing in mitochondria of all major groups of

DNA sequence analysis of the imp UV protection and mutation operon of the plasmid TP110: identification of a third gene

Supplemental Figure 1. Differences in amino acid composition between the paralogous copies Os MADS17 and Os MADS6.

Metabolic evidence for biogeographic isolation of the extremophilic bacterium Salinibacter ruber.

Diversity of Chlamydia trachomatis Major Outer Membrane

Using algebraic geometry for phylogenetic reconstruction

Chemical Biology on Genomic DNA: minimizing PCR bias. Electronic Supplementary Information (ESI) for Chemical Communications

codon substitution models and the analysis of natural selection pressure

Evolutionary Analysis of Viral Genomes

Codon-model based inference of selection pressure. (a very brief review prior to the PAML lab)

HADAMARD MATRICES AND QUINT MATRICES IN MATRIX PRESENTATIONS OF MOLECULAR GENETIC SYSTEMS

Electronic Supporting Information for

supplementary information

Motif Finding Algorithms. Sudarsan Padhy IIIT Bhubaneswar

NEW DNA CYCLIC CODES OVER RINGS

Dissertation. presented by: Hung-wei Sung Diploma: Master of Life Science born intaipei, Taiwan Oral examination:

Regulation of the Bacillus subtilis Heat Shock Gene htpg Is under Positive Control

Roles of the outer membrane protein AsmA of Salmonella enterica in control of marrab expression and invasion of epithelial cells

ANALYZING THE DIVERSITY OF A SMALL ANTIBODY MIMIC LIBRARY. Nick Empey. Chapel Hill 2010

Evidence for Evolution: Change Over Time (Make Up Assignment)

A functional homologue of goosecoid in Drosophila

Aoife McLysaght Dept. of Genetics Trinity College Dublin

How DNA barcoding can be more effective in microalgae. identification: a case of cryptic diversity revelation in Scenedesmus

The Cell Cycle & Cell Division. Cell Function Cell Cycle. What does the cell do = cell physiology:

Iron-related fitness epistasis is antagonistic to the evolution of silver resistance in Escherichia coli. 15 th Korea-US Forum on Nanotechnology

evoglow yeast kit distributed by product information Cat.#: FP-21040

evoglow basic kit product information

Appendix B Protein-Signaling Networks from Single-cell Fluctuations and Information Theory Profiling B.1. Introduction

Lecture IV A. Shannon s theory of noisy channels and molecular codes

Yeast two-hybrid system survey of interactions between LEE-encoded proteins of enteropathogenic Escherichia coli

Constitutive Signal Transduction by Mutant Ssy5p and Ptr3p Components of the SPS Amino Acid Sensor System in Saccharomyces cerevisiae

Transcription:

Re- engineering cellular physiology by rewiring high- level global regulatory genes Stephen Fitzgerald 1,2,, Shane C Dillon 1, Tzu- Chiao Chao 2, Heather L Wiencko 3, Karsten Hokamp 3, Andrew DS Cameron 2, Charles J Dorman 1 1 Department of Microbiology, Moyne InsKtute of PrevenKve Medicine, Trinity College Dublin, Dublin 2, Ireland 2 Department of Biology, University of Regina, Saskatchewan, Canada 3 Department of GeneKcs, Smurfit InsKtute, Trinity College Dublin, Dublin 2, Ireland Supplementary informakon

SL1344 reference hns stpa SL1344RX de novo assembly conkg borders Figure S1 Visual alignment with Mauve, showing the wild type SL1344 genome (top) and SL1344RX (bottom). Aligned coloured blocks represent regions of homologous sequence that are free from genomic rearrangement. Strain SL1344RX aligns with wild type, with breaks observed at the positions for hns and stpa. These small sections each align to the opposite position, showing the intended reciprocal exchanges were correctly carried out and were detected by sequencing. Vertical red lines indicate the borders of contigs used for genome assembly with both hns and stpa embedded within large contigs.

Figure S2 Visualization of the regions adjoining hns (top two tracks) and stpa (bottom two tracks) in SL1344RX shows no DNA sequence variants were found in either de novo assembly.

1 0 1 2 a b c 1.0 0.5 0.0 0.5 1.0 1.5 2 1 0 1 8 9 10 11 12 13 14 8 9 10 11 12 13 8 9 10 11 12 13 expression intensity expression intensity d e f expression intensity 1 0 1 2 1 0 1 2 2 1 0 1 2 ctrl hns ctrl Fig S3 Sample MA-plots (a c) and corresponding boxplots (d f) for H-NS (a and d), StpA (b and e) and RpoS (c and f) regulated genes. H-NS, StpA or RpoS regulated genes (blue stars) were plotted together with an equivalent number of control non -H-NS, -StpA or RpoS regulated genes (black stars). Loess curves displaying the average value across sample (blue line) and control (black line) datasets were fitted. Ten separate control gene sets were used for comparison with each regulon and significant changes in gene expression (P < 0.01) identified by t-test. Significant changes in expression of both the StpA and RpoS regulons were observed in each of ten comparisons but no significant changes were observed across the H-NS regulon. stpa ctrl rpos

Mass spectrometric analysis Mutant and wild type cells were harvested as for the Western blots and lysed with bead beating. Proteins were extracted using acetone precipitation. After resolubilization in 50 mm ammonium bicarbonate buffer the protein sample was digested with trypsin (Sigma-Aldrich). The resulting peptides were analysed with a Synapt G2 HDMS (Waters) coupled to a Nanoacquity (Waters) nano-lc with an Acquity UPLC T3HSS column (75 mm x 200 mm). Several runs in sensitive as well as resolution mode (scan length 0.7-1.2 s) were performed using dataindependent-acquisition (MSE) with leu-enkephaline as lockspray for mass correction. Spectra were subsequently analysed with the ProteinLynx Global Server version 3.02 (Waters). Searches were allowed to reiterate until a false discovery rate of 4% was reached. Identified masses corresponding to StpA were further used to create an inclusion list for subsequent data-dependent analysis.

A 10 10 10 11 SL1344 2Xhns c.f.u/ml 10 9 10 8 10 7 10 6 SL1344 2XstpA 0 1 2 3 4 5 Time (h) 6 7 8 B SL1344 2Xhns DnaK control RpoS C SL1344 2XstpA DnaK control RpoS Figure S4 Expression of the RpoS sigma factor in SL1344 2XstpA and in SL1344 2Xhns as a function of growth. The growth cycles of SL1344 2XstpA and SL1344 2Xhns were determined by colony counts (a). RpoS expression was monitored by Western blotting and was first detected after 3 h and 4 h of growth in SL1344 2Xhns (b) and SL1344 2XstpA (c), respectively (n=1).

Table S1 High stringency (>95% variant read frequency) and low stringency (>45% variant) SNPs idenkfied in SL1344RX.

Table S2 Differentially expressed genes in SL1344RX STM Iden?fier Gene Name Log 2 Ra?o P- value EMBL assigned func?on STM1729 ycif 5.10 2.85E- 07 conserved hypothekcal protein STM1730 ycie 4.24 1.26E- 07 conserved hypothekcal protein STM1267 3.14 1.27E- 04 hypothekcal protein STM4561 osmy 3.13 2.09E- 05 PutaKve periplasmic protein STM0465 ybay 2.93 7.43E- 04 conserved hypothekcal lipoprotein STM2795 ygau 2.69 2.30E- 03 conserved hypothekcal protein STM0831 dps 2.68 2.88E- 03 DNA proteckon during starvakon protein STM0562 cusa 2.66 8.14E- 02 cakon efflux protein (pseudogene) - sope 2.63 3.31E- 02 Type III secrekon system effector protein STM1119 wrba 2.46 1.24E- 02 trp repressor binding protein STM1731 katn 2.44 1.59E- 03 catalase STM2164 yehy 2.28 4.29E- 03 hypothekcal permease transmembrane component STM1091 sopb 2.25 1.48E- 02 Type III secrekon system effector protein. STM2884 sipc 2.21 5.36E- 02 Pathogenicity island 1 Type III secrekon system effector protein STM1929 otsb 2.17 8.09E- 04 trehalose phosphatase STM1565 rpsv 2.16 1.74E- 03 30S ribosomal protein S22 STM1500 ynfd 2.09 1.35E- 01 hypothekcal exported protein STM2883 sipd 2.08 5.82E- 02 SPI- 1 Type III secrekon system translocon protein STM2474 tktb 2.06 2.63E- 03 transketolase 2 STM3571 @sy 2.02 1.49E- 02 cell division protein STM2577 acps 2.01 9.21E- 03 Holo- [acyl- carrier- protein] synthase STM1311 osme 2.01 2.48E- 03 osmokcally inducible lipoprotein E precursor STM0759 ybgs 1.98 4.68E- 03 hypothekcal exported protein STM0326 dhaf - 2.01 1.21E- 02 glycerol dehydratase reackvakon factor large subunit (pseudogene) STM4392 prib - 2.38 4.49E- 02 primosomal replicakon protein N STM2434-2.40 1.88E- 02 hypothekcal protein

Table S3 Differentially expressed RpoS regulated genes in SL1344RX STM Identifier Gene Name Fold Change P-value EMBL assigned function STM1729 ycif 5.10 2.85E-07 conserved hypothetical protein STM1730 ycie 4.24 1.26E-07 conserved hypothetical protein STM4561 osmy 3.13 2.09E-05 Putative periplasmic protein STM2795 ygau 2.69 2.30E-03 conserved hypothetical protein STM0831 dps 2.68 2.88E-03 DNA protection during starvation protein STM1119 wrba 2.46 1.24E-02 trp repressor binding protein STM1731 katn 2.44 1.59E-03 catalase STM2164 yehy 2.28 4.29E-03 hypothetical permease transmembrane component STM1929 otsb 2.17 8.09E-04 trehalose phosphatase STM1565 rpsv 2.16 1.74E-03 30S ribosomal protein S22 STM2474 tktb 2.06 2.63E-03 transketolase 2 STM1311 osme 2.01 2.48E-03 osmotically inducible lipoprotein E precursor STM0465 ybay 2.93 7.43E-04 conserved hypothetical lipoprotein

Table S4 Bacterial Strains and plasmids Strain or Plasmid Relevant characteristic Resistance Source/reference S. Typhimurium SL1344 rpsl hisg46 Hoiseth and Stocker(1981) hns-flag hns::3xflag tag Km R This work stpa-flag stpa::3x FLAG tag Km R This work 2X hns P stpa -hns Km R This work 2X stpa P hns -stpa Tc R This work SL1344RX P stpa -hns P hns -stpa Km R Tc R This work SL1344RX hns-flag P stpa -hns-3xflag P hns -stpa Km R Tc R This work SL1344RX stpa-flag P stpa -hns P hns -stpa-3xflag Km R Tc R This work pkd46 λ genes gam, bet and exo under the control of the arabinose inducible pbad promoter Amp R Datsenko and Wanner (2000) psub11 pgp704 derivative containing the Km R cassette with flanking FRT sites from pkd4 and upstream 3xFLAG-tag sequence Km R Uzzau et al (2001) Km R = Kanamycin resistant, Tet R = Tetracycline resistant, Amp R = Ampicillin resistant.

Table S5 Oligonucleotide primer sequences hns.rt.fw 5'-gca caa ctg aat tta tcg ct-3' hns.rt.rv 5'-cat tct ctt gcc tgc gca-3' stpa.rt.fw 5'-aat cgc ata caa tac cgc-3' stpa.rt.rv 5'-ggc tcg cga att ctc cat tg-3' hns.flag.fw 5'-aga aca agg taa gca act gga aga ttt cct gat caa gga aga cta caa aga cca tga cgg-3' hns.flag.rv 5'-aaa aat ccc gcc agc ggc ggg att tta agc atc cag gaa gca tat gaa tat cct cct tag-3' stpa.flag.fw 5'-ggc gct ggc ggc ggg gaa atc tct gga tga ttt ctt aat cga cta caa aga cca tga cgg-3' stpa.flag.rv 5'-cgg att aga aaa aca act taa atg tga aag tgg gtc tta aca tat gaa tat cct cct tag-3' stpa.tet.fw 5'-gga tag ctt tta aca gat ggc gct tcg ttt cct gtc tta aga ccc act ttc aca tt-3' stpa.tet.rv 5'-atg agc atg gcg caa cgt tcc ctg agt ccc ggt taa cta agc act tgt ctc ctg-3' hns.kan.fw 5'-ctt cct gga tgc tta aaa tcc cgc cgc tgg cgg gat cat atg aat atc ctc ctt ag-3' hns.kan.rv 5'-gcc tgg ggt cgt cag cgg aga act cag gca aaa aaa gac tac aaa gac cat gac gg-3' stpa.int.fw 5'- aat agt ttt ttg ttt tct gcg tta aaa ggt ttt tat tga tat gag cga agc act taa aat-3' stpa.int.rv 5'-gac agg aaa cga agc gcc atc tgt taa aag cta tcc gtg att acg ccc cgc cct gcc act-3' hns.int.fw 5'-gct caa caa acc acc cca ata taa gtt tga gat tac tac aat gaa ttt gat gtt aca gaa-3' hns.int.rv 5'-atc ccg cca gcg gcg gga ttt taa gca tcc agg aag taa att acg ccc cgc cct gcc act-3' RT = RT-qPCR, flag = 3X FLAG-tag epitope tagging, tet = integration of tetra, kan = integration of neor, int = integration at stpa or hns loci

2024 © sciencedocbox.com Privacy Policy | Terms of Service | Feedback