Centrifuge 5424

To measure the chlorophyll a content, 3 × 100 μl of culture was centrifuged at 14,000 rpm (Centrifuge 5424, Eppendorf) for 5 min at RT. The cell pellet was resuspended in 1 ml of 100% methanol, vortexed for 3 min, and centrifuged again at 14,000 rpm (Centrifuge 5424, Eppendorf) for 5 min at RT to spin down cell debris. Using a spectrophotometer, the wavelengths 665, 666, and 750 nm were measured since chlorophyll a absorbs at the wavelengths 440–450 nm and 650–700 nm.
According to the following formula (Lichtenthaler, 1987 (link)), the chlorophyll a content was calculated as follows:

First-strand synthesis was performed in a similar manner as described in Parkhomchuk et al (2009) (link). For first-strand synthesis, 8 μl fragmented mRNAs were mixed with 1 μl of random hexamers (Invitrogen) and 1 μl of 10 mM dNTPs. The samples were incubated at 65°C for 5 min and chilled on ice for 1 min. We added 10 μl of a master mix with final concentrations of 1× RT Buffer (Thermo Fisher Scientific), 10 mM MgCl₂, 20 mM DTT, 4 U/μl RnaseOUT (Thermo Fisher Scientific), and 20 U/μl of SuperScript III RT (Thermo Fisher Scientific) to the RNA samples. The samples were then first incubated at 25°C for 10 min, followed by a 50-min incubation at 50°C. The reaction was stopped by incubating samples at 75°C for 15 min. For dNTP cleanup, 80 μl water, 1 μl glycogen, 10 μl 3 M NaOAc (pH 5.2), and 200 μl cold ethanol were added to the samples. Samples were stored at −80°C for 3–7 d. Samples were centrifuged at 14,000 rpm (Eppendorf Centrifuge 5424) for 20 min at 4°C. Supernatant was removed and 500 μl of cold 75% ethanol was added to the samples. Samples were centrifuged again at 14,000 rpm (Eppendorf Centrifuge 5424) for 10 min at 4°C. Supernatant was removed and samples were resuspended in a mixture composed of 51 μl RNAse-free water, 1 μl of 10× RT Buffer, 1 μl 100 mM DTT, 2 μl of 25 mM MgCl₂. Second-strand synthesis was performed as described in Parkhomchuk et al (2009) (link).

Strain CT-6 was cultivated in PDB medium at 28 °C for 3 days on rotary shakers at 180 rpm. The mycelia were collected by centrifugation followed by genomic DNA extraction using the Wizard^® Genomic DNA Purification Kit (Promega, MD, USA) according to the manufacturer’s instructions. The genomic DNA of strain CT-6 was sequenced using the Illumina HiSeq X Ten platform and the PacBio Sequel II platform. For Illumina sequencing, at least 10 μg of genomic DNA was interrupted to about 400 bp fragments with a Covaris M220 Focused Ultrasonicator (Covaris Inc., Woburn, MA, USA) for sequencing library construction. The sequencing library was constructed according to the NEXTflex™ Rapid DNA-Seq Kit (Illumina, San Diego, CA, USA) method and sequenced on the Illumina HiSeq X Ten platform. For PacBio sequencing, an aliquot of 8 μg of genomic DNA was sheared to 10 kb using a Covaris g-TUBE (Covaris, MA, USA) at 6000 RPM for 60 s using an Eppendorf 5424 centrifuge (Eppendorf, NY, USA). DNA fragments were then end-repaired and ligated with SMRTbell sequencing adapters (Pacific Biosciences, Menlo Park, CA, USA) following the manufacturer’s recommendations. Next, an ~10 kb insert library was prepared and sequenced on one SMRT cell using standard methods.

Cells from cultures of WT and ΔluxTV. harveyi strains grown overnight in LM medium were pelleted by centrifugation at 21,100 × g (Eppendorf 5424 centrifuge) and resuspended in fresh LM medium. Fresh cultures containing 25 mL of LM medium were inoculated with the washed cells at an OD₆₀₀ of 0.005. The cultures were incubated at 30°C with shaking, and RNA was isolated from 3 biological replicates of each strain when the cultures had reached an OD₆₀₀ of 0.1 using the RNeasy minikit (catalog number 74106; Qiagen). RNA-Seq was performed at the Genomics Core Facility at Princeton University, as previously described (60 (link)). Reads were mapped to the V. harveyi BB120 (ATCC BAA-1116) genome using TopHat (61 (link)). Genes with differential expression were identified using DESeq2 (62 (link)), and those exhibiting a log₂ fold change in expression >1, along with a P value <0.01, in the ΔluxT strain compared to WT V. harveyi were designated members of the LuxT regulon.

Strains of interest were inoculated into 500 ml of Luria-Bertani (LB) medium containing 100 μg ml⁻¹ of Ampicillin and were incubated while shaking at 37°C until an OD₆₀₀ of 1.0 was reached. The cultures were induced with 1 mM IPTG followed by incubation at 25°C for four hours. Cells were harvested by centrifugation at 10,000 × g for 10 minutes at 4°C, and processed immediately or frozen at −80°C. Cells were resuspended in 6 ml of 20 mM Hepes, 100 mM NaCl at pH 8.0. Cell suspensions were disrupted by French press at 20,000 psi. Cellular debris was removed by centrifugation at 14,000 × g for 5 minutes at 4°C in a 5424 centrifuge (Eppendorf). Soluble crude cytosol was loaded onto 3 ml of nickel-nitriol-triacetic acid (Ni-NTA) resin (Qiagen). The resin was washed with 10 column volumes of loading buffer containing 500 mM NaCl and then eluted with 20 mM Hepes and 500 mM imidazole (pH 8.0). Purified F. tularensis LpxA-His₆ was desalted using Slide-A-Lyzer Dialysis Cassette (Thermo Scientific) and dialyzed against 20 mM Hepes pH 8.0. Protein concentrations were determined by bicinchoninic acid (BCA) assay. The purification, and acylation of holo-ACP was carried out as described previously, except hydroxypalmitic acid (C16:0) was used in place of hydroxymyrstic acid (C14:0) [22 (link)].

A 300 µL aliquot of a methanol:chloroform (9:1) mixture was added to each sample. Amphipods were then homogenized by manual grinding using a metal spatula (which was cleaned between samples) within the microcentrifuge tubes and placed on dry ice. Samples were then sonicated for 15 min in an ultrasonic water bath and then centrifuged in an Eppendorf 5424 Centrifuge (Macquarie Park, NSW, Australia) at 5500 rpm for 15 min at room temperature. The supernatant was decanted into glass inserts and allowed to evaporate in a fume hood.
Dried extracts were derivatized using 20 µL of methoxyamine in pyridine solution (20 mg/mL). The samples were then vortexed for 30 s and left for 17 h at room temperature. Next, 20 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was added. Samples were vortexed again and left for one hour to react. Analytical-grade hexane was added to each sample (300 µL) prior to GC–MS analysis. A pooled biological quality control (PBQC) was prepared by pooling 7.5 µL of each sample extract, mixed thoroughly and aliquoted into 5 replicates. PBQCs were analysed along with the samples and controls/blanks to assess repeatability, instrument drift and quality control.