5% (w/v) unless indicated otherwise. The cultures were incubated under shaking at 30 °C, and growth was monitored every hour. For growth experiments on agar plates, cells were precultured as described previously and diluted to an OD600 nm of 0.5 in CGXII medium. From this, a twofold dilution series in CGXII was prepared and 2 μL of each dilution SAHA HDAC nmr spotted onto CGXII agar plates containing the appropriate carbon source. Agar plates were incubated for 24–48 h at 30 °C. For gene inactivation in C. glutamicum,
a vector integration method was used (Elleling & Reyes, 2005; Jolkver et al., 2009). To produce an insertion in cg2937, an c. 500 bp fragment of the target gene was amplified by PCR using the oligonucleotides 5′-ACTCGCCGCAATTTCCTCCG-3′ and 5′-CGAGGCGTTCGCTGATGATG-3′ and cloned into the pDRIVE vector (Qiagen), which was verified by PCR. Transformation into C. glutamicum was performed using electroporation (2.5 kV, 5 ms), and positive colonies were selected on kanamycin-containing agar plates, and chromosomal integration was confirmed by PCR using primers binding c. 100 bp before
the start codon (5′-GTCTGATGTCTGATGTATAT-3′) and the appropriate M13 primer for the pDRIVE vector (5′-AACAGCTATGACCATG-3′). Cells were precultured as described previously for the growth experiment unless annotated otherwise. Cells were washed three times in CGXII media containing no carbon source. Cells were diluted to an OD600 nm of 1 in CGXII media supplemented with 10 mM glucose and incubated Cobimetinib ic50 on ice until the uptake assay was performed. Before the measurement, cells were incubated under stirring at 30 °C for energizing. The assay was started by adding 2 μM [14C] Neu5Ac. Every minute over 5 min, 100-μL samples were taken and cells were collected by rapid filtration (0.45 μm pore size; Millipore). Cells were washed with 5 mL CGXII medium, and the radioactivity retained on the filter discs was determined by liquid scintillation counting. The number of colony-forming units in each culture was also determined using serial dilutions onto BHI plates and incubation at 30 °C overnight
followed by counting. We identified orthologues of known sialic acid catabolism genes from E. coli using the ncbi blastp and identified similar sialic acid clusters across the Corynebacteriaciae using XBase (Chaudhuri et al., 2008) and the SEED (Overbeek et al., 2005). Amisulpride To verify the initial phenotype array data, which suggested that C. glutamicum ATCC 13032 could grow on Neu5Ac (Holder et al., 2011), we grew the same strain on a minimal CGXII medium with 0.5 % Neu5Ac as the sole carbon source. No growth was observed in the absence of an added carbon source, but there was clear growth with Neu5Ac, albeit with a long lag phase compared with growth on glucose (Fig. 1a solid symbols). After 24 h growth, the final growth yield was very high with glucose, giving an OD600 nm of c. 17, while the value for Neu5Ac was c. 7.