Supplementary data to the manuscript

“Glucosamine as carbon source for amino acid-producing Corynebacterium glutamicum”

by Andreas Uhde#1, Jung-Won Youn#2, Tomoya Maeda1, Lina Clermont1, Christian Matano2, Reinhard Krämer1, Volker F. Wendisch2, Gerd M. Seibold*1 and Kay Marin1

1 Institute of Biochemistry, University of Cologne, Cologne, Germany

2 Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Bielefeld, Germany

# These authors contributed equally to this work

* Corresponding author. Mailing address: Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany. Phone: 49 (0)221 470 6464. Fax: 49 (0)221 470 5091. E-mail:

The supplementary data consist of two tables, three figures and contains the additional references cited within the supplementary data.

Table S1. Strains, plasmids and primers used and constructed in this study.

Strains

Escherichia coli DH5α (Invitrogen)

Corynebacterium glutamicum ATCC13032 (wild type)

Corynebacterium glutamicum M4 This study

Δhpr (Lindner et al., 2011)

ΔptsG Henrich (unpublished)

RES167 ΔptsF (Gaigalat, 2001)

IMptsS This study

IMptsX This study

Plasmids

pDrive (Qiagen, Hilden, Germany)

pDrive_IM_ptsS This study

pDrive_IM_ptsX This study

pVWEx1 (Peters-Wendisch et al., 2001)

pVWEx1-nagAB This study

pEKEx3 (Stansen et al., 2005)

pEKEx3-nagB This study

pEPR1 (Knoppova et al., 2007)

pEPR1_PnagAB_WT This study

pEPR1_PnagAB_M4 This study

pTXB1 (New England Biolabs)

pTXB1-nagB This study

Primer

nagAB_SbfI_fwd GTATCCTGCAGGGAAAGGAGGCCCTTCAGATG

nagAB_SmaI_rev GTACTGCCCGGGAAACGACGGCCAGTGAATTCG

nagA_PR_XbaI_fwd TCTAGAAAGCCATGGTGCTCCTTGT

nagA_PR_BamHI_rev GGATCCTTAGGGGGGTAACAATTCTTCC

Ex-nagB-fw-SbfI GCCCTGCAGGGAAAGGAGGCCCTTCAGATGGACATCATCATCTGCAA

Ex-nagB-rev-BamHI GCGGATCCTAGCGCAGCTTTAATTGCT

IM_ptsX_fwd CGCAGCAATACCTTGGAATG

IM_ptsX_rev GGTTATCGGTGGAGCAATCA

IM_ptsX_ctr CTGAGAATGTGGCTGCTGAG

IM_ptsS_fwd TGCCGTTGCGAAGATGAAGG

IM_ptsS_rev CATTGGCGGCGAAGACAACA

IM_ptsS_ctr GTGATCGCGGACGATAATAC

M13-FP TGTAAAACGACGGCCAGT

pTXB1-nagB-fw-Nde IGCCATATGGACATCATCATCTGCAA

pTXB1-nagB-rev-SapI

GGTGGTTGCTCTTCCGCAGCGCAGCTTTAATTGCTCCA

Table S2. Gene expression differences between C. glutamicum wild type and M4 during growth in complex medium LB

Genea Annotationa mRNA levelb

(M4/WT)

cg3237 / sodA, superoxide dismutase / 2.3
cg3207 / pheA, prephenate dehydratase / 0.4
cg3169 / pck, phosphoenolpyruvate carboxykinase (GTP) / 2.3
cg3009 / hypothetical protein / 2.4
cg3008 / porA, Porin / 2.5
cg2958 / butA, L-2.3-butanediol dehydrogenase / 2.2
cg2929 / nagA, putative N-acetylglucosamine-6-phosphate deacetylase / 55.3
cg2928 / nagB, glucosamine-6-phosphate deaminase / 57.2
cg2256 / ABC-type transporter, ATPase component / 0.4
cg1931 / putative secretedprotein / 2.4
cg1821 / hypothetical protein / 2.2
cg1791 / gapA,glyceraldehyde-3-phosphate dehydrogenase / 2.3
cg1685 / tatX, Sec-independent protein secretion pathway component / 2.4
cg1682 / trypsin-like serine protease / 2.2
cg1368 / atpD, F0F1-type ATP synthase beta subunit / 2.6
cg1341 / narI, nitrate reductase gamma subunit / 0.2
cg0990 / rpmG, ribosomal protein L33 / 2.8
cg0949 / gltA, citrate synthase / 2.4
cg0821 / Hypothetical protein / 2.5
cg0810 / Hypothetical protein / 3.0
cg0768 / ABC-type transporter, ATPase component / 2.1
cg0631 / rpsE, ribosomal protein S5 / 3.2
cg0628 / rpsH, ribosomal protein S8 / 2.2
cg0602 / rpIP, ribosomal protein L16/L10E / 2.3
cg0601 / rpsC, ribosomal protein S3 / 2.5
cg0589 / ABC-type transporter, ATPase component / 2.4
cg0563 / rplK, ribosomal protein L11 / 2.6
cg0478 / Hypothetical protein / 0.3
cg0477 / Hypothetical protein / 0.1
cg0476 / murB2, putative UDP-N-acetylmuramate dehydrogenase / 0.4

aGene identifiers and annotations are given according to BX927147.

bThe mRNA levels were derived from a single cultivation.

Legends to the supplementary figures

Fig. S1. Growth (open triangles) and substrate depletion (circles) of C. glutamicum M4 mutant strain with a substrate mixture of 10 gl-1 glucose (solid circles) and 10 gl-1 glucosamine (open circles).

Fig. S2. Genomic locus of the M4 mutation site (boxed) upstream of the nagA gene in C. glutamicum. P1 and P2 are transcriptional start sites as they were determined by Engels et al., 2008.

Fig. S3. Growth of C. glutamicum mutant strains in CGXII minimal medium plus10 gl-1 glucosamine. Wild type (solid circles), ΔptsH (open triangles), ΔptsF (solid squares), ΔptsG (solid triangles), IMptsS (open circles) and IMptsX (open squares).

Figure S1

Figure S2

______

caacagcagg cctcaagtcc gaagataatt aacctaaatc cgtagacata

P1 P2

* *

agacatcata cgtcctatgc ttgctggaag gaagcaaata acctcagaaa

C

gatggcagaa gtggtgcatt atcaagaaaa tgcaggtcaa gcagttaaaa

|------nagA------►

______

Figure S3

References

Engels, V., T. Georgi & V. F. Wendisch, (2008) ScrB (Cg2927) is a sucrose-6-phosphate hydrolase essential for sucrose utilization by Corynebacterium glutamicum. FEMS Microbiol Lett 289: 80-89.

Gaigalat, L., (2001) Master Thesis. In.: University of Bielefeld, pp.

Knoppova, M., M. Phensaijai, M. Vesely, M. Zemanova, J. Nesvera & M. Patek, (2007) Plasmid vectors for testing in vivo promoter activities in Corynebacterium glutamicum and Rhodococcus erythropolis. Curr Microbiol 55: 234-239.

Lindner, S. N., G. M. Seibold, A. Henrich, R. Krämer & V. F. Wendisch, (2011) Phosphotransferase System-Independent Glucose Utilization in Corynebacterium glutamicum by Inositol Permeases and Glucokinases. Appl Environ Microbiol 77: 3571-3581.

Peters-Wendisch, P. G., B. Schiel, V. F. Wendisch, E. Katsoulidis, B. Mockel, H. Sahm & B. J. Eikmanns, (2001) Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. J Mol Microb Biotech 3: 295-300.

Stansen, C., D. Uy, S. Delaunay, L. Eggeling, J. L. Goergen & V. F. Wendisch, (2005) Characterization of a Corynebacterium glutamicum lactate utilization operon induced during temperature-triggered glutamate production. Appl Environ Microbiol 71: 5920-5928.

8