Sodium Oxalate

E. coli BW19610 (Howitt et al.,
2006) used for cloning and E. coli Sm10 λ
pir⁺ used for conjugation were routinely grown in
Luria broth (LB) or on LB agar (LA) plates at 37°C. Streptomycin was used at a
concentration of 100 μg/ml to select for suicide vectors. All Y.
enterocolitica strains are derivates of E40 (Sory et al., 1995 (link)), where for biosafety reasons six effector
genes were deleted, as well as the asd gene. They were routinely
grown at 25°C in brain heart infusion (BHI) broth containing 35 μg/ml
nalidixic acid. To allow growth of asd mutant strains, the medium
was supplemented with 50 μg/ml meso-diaminopimelic acid. Shigella
flexneri SC560 (Sansonetti,
1991) were routinely grown at 37°C in BHI containing 100 μg/ml
streptomycin. Mutator plasmid pMK3 was made by amplification of the
asd 5′ region with oligos 3541/3543 and the 3′
region with oligos 3542/3544. The 5′ region was digested with
SalI/EcoRI and the 3′ region with
EcoRI/XbaI. Both fragments together were ligated
into the SalI/XbaI restriction site of pKNG101. To
construct pMA87, flanking regions of about 250 bp just upstream and downstream of
minD were amplified from purified genomic DNA from Y.
enterocolitica E40 using oligonucleotides 6416/6417 and 6418/6419
respectively (Supplementary
file 1C,D). The two fragments were joined by overlapping polymerase chain
reaction (PCR), and the resulting fragment was cloned into the
SalI/XbaI restriction sites of suicide vector
pKNG101 (Kaniga et al., 1991 (link)). To construct
pMA6, full-length yscC with a stop codon was amplified from the
pYVe40 plasmid using primers 5013/5014 and introduced into the
NcoI/EcoRI restriction sites of
pBAD/mycHisA.
Cultures were inoculated at an optical density (OD₆₀₀) of 0.1 in BHI broth
containing sodium oxalate (20 mM) (BHI-OX) supplemented with glycerol (4 mg/ml) and
MgCl₂ (20 mM). After 2 hr of growth at 25°C, induction of the
yop regulon was performed by shifting the culture to 37°C
(Cornelis et al., 1987 (link)). Expression of
the pBAD constructs was induced by adding 0.03% L-arabinose to the culture just
before the shift to 37°C. After 4 hr of incubation at 37°C, cultures were
used for further analysis.

SPR was used to investigate the interactions between LsBOS and LsAAE3. Experiments were performed using the Biacore 8 K (Cytiva) with a series S sensor chip CM5 (Cytiva) and a running buffer of 10 mM Hepes pH 7.4, 150 mM NaCl and 0.05 % tween 20 and a temperature of 25 °C. Initial pH scouting experiments were run which indicated that pH 4 was the optimum pH to use for immobilisation. A standard amine coupling approach was used to activate the chip with reagents 1-ethyl−3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) then LsBOS, at a concentration of 20 nM in 10 mM acetate pH 4.0, was injected over Flow Cell 2 (FC2) for 210 s at 10 μL/min (leaving FC 1 blank as the reference) before blocking flow cells with ethanolamine. LsBOS was immobilised with a response of ~1700. Interaction between LsAAE3 and LsBOS was observed by injecting 1 μM LsAAE3 over FC1 and FC2 for 60 s at a flow rate of 50 μL/min before switching to buffer only flow.
A range of regeneration solutions were tested and 10 mM acetate at pH 4 was found to be the best for removing the bound LsAAE3. However, this was still not ideal, so it was decided to use a single cycle kinetics approach where no regeneration is used between analyte injections. Three start-up cycles were run using only buffer then a blank run with five zero concentration injections of LsAAE3. LsAAE3 was then injected at five increasing concentrations of 2.4, 12, 60, 300 and 1500 nM each with a contact time 120 s and a flow rate 30 µL/min. At the end of all injections buffer was flowed for 600 s to record the dissociation. A concentration dependent response could be seen confirming the interaction. The data were processed and analysed using Biacore Insight Evaluation Software with double-referenced subtraction of the data and then fitted to a simple Langmuir binding model. The association rate (k_on) was determined to be 3.67 × 10³ s⁻¹M⁻¹ dissociation rate (k_off) as 3.61 × 10⁻⁴ s⁻¹ giving an dissociation equilibrium constant (K_D) of 98 nM.
AAE3 activity was determined in the absence of other enzymes and by a coupled enzyme assay^{47 (link)}. Each reaction contained: 10 µL E. coli extract, 100 mM Tris pH 8.0, 2 mM DTT, 5 mM ATP, 10 mM MgCl₂, 0.5 mM CoA, 0.4 mM NADH, 1 mM phosphoenol-pyruvate, 300 µM sodium oxalate, 10 units each myokinase, pyruvate kinase and lactate dehydrogenase, deionised water to 100 µL. Activity was measured by reduction in OD 340 nm over time. LsBOS activity was measured in reactions containing 100 mM Tris pH 8.0, 2 mM DTT, 5 mM ATP, 10 mM MgCl2, 0.5 mM CoA, 300 µM sodium oxalate, 50 µM DAP, and 10 µL each of AAE3 and LsBOS E. coli expression extracts in various combinations, made up to 100 µL with deionised water. Amounts of β-L-ODAP and α-L-ODAP produced were measured using an LCMS procedure^{36 (link)}.