Jan-Willem Veening

A key insight, emerging from discussions and data between the projects PIs, was the importance of switching rates in bistable systems.
While the existence of multiple steady states in bistable systems can be described by universal models (that do not differ between
different systems), switching rates from one stable state to another depend on the molecular details of the system under consideration.

Handling and manipulation of S. pneumoniae using molecular, cell biological and genetic tools.

For cells to accurately read out the genomic content, high fidelity during transcription is required. This is mainly established by the accuracy of the active centre of RNA polymerase (RNAP). Based on in vitro experiments with Escherichia coli RNAP it was also suggested that proofreading of transcription via RNA hydrolysis by RNAP may contribute to overall fidelity and processivity. RNAP’s intrinsic cleavage activity is stimulated by the highly conserved Gre factors suggesting that Gre factors
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During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see (1,2,3)). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard
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Here we develop a set of new tools for S. pneumoniae and as a case study we show that S. pneumoniaea SMC is recruited to oriC by ParB and promotes chromosome segregation.

1. Preparation of B. subtilis cultures

Inoculate cells from -80°C stocks in 10 ml time-lapse microscopy (TLM) medium (62 mM K2HPO4 , 44mM KH2PO4, 15 mM (NH4)2SO4, 6.5 mM sodium citrate, 0.8 mM MgSO4, 0.02 % casamino acids, 27.8 mM glucose, 0.1 mM L-tryptophan, the pH was set to 7 using a KOH solution) supplemented with antibiotics, if necessary.
Grow the cells overnight in a shake flask (30°C, 225 rpm).
The following morning, dilute the cells 1:10 in pre-warmed chemically defined medium (CDM) (62
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S.pneumoniae D39 cells (wild type and delta greA) were grown in C+Y medium and cells were harvested for total RNA isolation at mid-exponential growth (OD600nm 0.3 for wt, 0.25 for delta greA). Total RNA was isolated as described before (Kloosterman et al 2006).
The total RNA samples were examined by capillary electrophoresis.
dephosphorylated with antarctic phosphatase followed by treatment with polynucleotide kinase (PNK).
Afterwards, samples were poly(A)-tailed using poly(A) polymerase. Then a
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Cells were grown to mid-exponential phase (OD600nm ~0.2) in GM17 medium at 37°C (with 0.15 mM ZnSO4 where relevant) and 84 ml of culture was mixed by inverting with 8.4 ml of fixing solution (50 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 30% (v/v) formaldehyde) and incubated at room temperature for 30 min. Cells were disrupted and crosslinked DNA was sheared by sonication. Antibodies coupled to magnetic beads were used to pull down cross-linked complexes. DNA was purified, amplified,
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Strain MK423 was grown under the same conditions as used when growing cells for microscopy analysis; cells of OD600 = 0.4 were diluted 100-fold in C+Y medium with 0.1 mM ZnCl2 and incubated for 2.5 hours until OD600 = 0.15. Cells were then harvested by centrifugation for 5 min at 6500 x g at 4°C. Genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit (Promega) as described previously (Slager et al. 2014 Cell). Fragmentation was performed using Covaris instrument, and libraries
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Strain MK422 was grown under the same conditions as used when growing cells for microscopy analysis; cells of OD600 = 0.4 were diluted 100-fold in C+Y medium with 0.1 mM ZnCl2 and incubated for 2.5 hours until OD600 = 0.15. Cells were then harvested by centrifugation for 5 min at 6500 x g at 4°C. Genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit (Promega) as described previously (Slager et al. 2014 Cell). Fragmentation was performed using Covaris instrument, and libraries
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Strain MK350 was grown under the same conditions as used when growing cells for microscopy analysis; cells of OD600 = 0.4 were diluted 100-fold in C+Y medium with 0.1 mM ZnCl2 and incubated for 2.5 hours until OD600 = 0.15. Cells were then harvested by centrifugation for 5 min at 6500 x g at 4°C. Genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit (Promega) as described previously (Slager et al. 2014 Cell). Fragmentation was performed using Covaris instrument, and libraries
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See wild type sample.

WT 110901_SN865_B_L006_R1_GQC-28-
ATCACG.fastq.gz
100 19.624.852 1,29

llumina fastq format
4 lines for each sequence:
1- Unique identifier, with the following format:
@::::#/
2- Sequence (A, T, C ,G or N (undetermined) only)
3- Orientation (always forward without mapping)
4- Quality value for each base, corresponding to a Phred-like score encoded in ASCII format, with an
offset of of 33 (e.g. “J” gives a value of 41) and is in accordance with sanger FASTQ format.
The sequence file is compressed
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S. pneumoniae RNA-Seq
Barcodes:
HPUra 1: CAGATC
HPUra 2: ACTTGA
Kanamycin: AGTCAA

S. pneumoniae RNA-Seq
Barcodes:
Control 1: CAGATC
Control 2: ACTTGA

E. coli RNA-Seq
Barcodes:
Trimethoprim 1: CCGTCC
Trimethoprim 2: GTAGAG

E. coli RNA-Seq
Barcodes:
Control 1: AGTTCC
Control 2: ATGTCA

E. coli DNA-Seq
Barcodes:
control 1: GTGGCC
control 2: GTTTCG
Trimethoprim 1: CACTCA
Trimethoprim 2: CAGGCG

S. pneumoniae kanamycin DNA PE2

S. pneumoniae kanamycin DNA PE1

S. pneumoniae HPUra 2 DNA PE2

S. pneumoniae HPUra 2 DNA PE1

S. pneumoniae HPUra 1 DNA PE2

S. pneumoniae HPUra 1 DNA PE1

S. pneumoniae Control 2 DNA PE2