Relating mrna and protein biomarker levels in a Dehalococcoides and Methanospirillum containing community

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1 Applied Microbiology and Biotechnology Electronic Supplementary Material for: Relating mrna and protein biomarker levels in a Dehalococcoides and Methanospirillum containing community Annette R. Rowe a#, Cresten B. Mansfeldt b *, Gretchen L. Heavner b *, and Ruth E. Richardson b Field of Microbiology, 220 Hollister Hall, Cornell University, Ithaca NY, USA a Civil and Environmental Engineering, 220 Hollister Hall, Cornell University, Ithaca NY, USA b # address correspondence to Annette Rowe, Current Address: 562 SHS, 835 Bloom walk, Los Angeles, CA, 90089, Phone: , annettrr@usc.edu *C.B. Mansfeldt and G.L. Heavner contributed equally to this work.

2 Materials and Methods Analysis of Biomarker Translation Start Site Up-stream Sequences Considerable variation in the 50 bp region upstream from biomarkers translation start sites has been observed in DMC (Table S1). Many DMC gene targets do not contain that traditional AGGAGG Shine-Delgarno sequence for ribosome binding. For example, TceA, which has a large protein production per mrna ratio compared to other biomarker proteins quantified does not contain this canonical sequence (Fung et al. 2007). However confidence in the traditional anti-shine-delgarno sequence being present on the DMC ribosome is difficult given the divergence of the DMC 16S rrna gene sequence from the conserved Shine-Delgarno sequence. The data presented in Table S1 support that there is variation in regions up-stream of translation start sites (i.e., ribosome binding sites). This variation may help explain the observed differences in the amount of protein generated per mrna for various DMC targets. Fung JM, Morris RM, Adrian L, Zinder SH (2007) Expression of reductive dehalogenase genes in Dehalococcoides ethenogenes strain 195 growing on tetrachloroethene, trichloroethene, or 2,3-dichlorophenol. Appl Environ Microbiol 73: Zybailov B, Mosley AL, Sardiu ME, Coleman MK, Florens L, Washburn MP (2006) Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. J Proteome Res 5:

3 Table S1. Fifty base pair sequences (green) upstream from Dehalococcoides (DMC) biomarker gene translation start sites (ATG, red). Presumed Shine-Delgarno sequence based on the AGGAGG (black) consensus sequence. Genes at the beginning of operons are denoted. A) DMC 16S rrna gene sequence and fifty base pair downstream (green) sequence provided below to highlight the uncertainty of the anti-shine-delgarno sequence (not identified in the Dehalococcoides 16S rrna gene) based on the consensus sequence binding sequence CCTCCT (red). Gene Locus DMC0079 DMC0318 DMC1545 DMC1559 DMC0380 Annotation 50 bp sequences upstream for start codon Is Gene at the Beginning of Operon Reductive dehalogenase, Yes TceA ACAGACGCCAAAGTGCGAAAAGCTTTTTTTAAATGAGGTATTTTAAAAGTATG Reductive dehalogenase, AGCAGAATTGAATACAGTTTATGTCCAAGTAATCATTAGAGGAGGTTAACATG Yes PceA Reductive dehalogenase, GGTGGCGTTGTGAAAGAACAGGTATGCCGTCAACAAATTAGGAGGATTTTATG Yes putative Reductive dehalogenase, CATGAAACTCAAAACTGTATCTCAAAAGCAAATTCATATCGGAGGAGTAAATG Yes putative Pyridoxal phosphate synthase AGAATATAGCCGAATAGGACAGGAGGGAGAACCCTCAGGGGAGATTAGATATG Yes yaad subunit DMC0374 Ribosome recycling factor CGCCATTTCCGGTCAGCCGATAGGTACATTAATATCCAGCGAGAGTTAAAATG No DMC0603 DNA-directed RNA TTTGTTTTTTAGCGCAACGACGTCCCTTGAGATTATAATAAGGAGCAGACATG Yes polymerase subunit beta DMC0604 DNA-directed RNA CGACCAGGATTTGGCAATGTCATCAAATGATGAAGAGGTATCCGAAAACGATG No polymerase subunit beta' DMC0990 LSU ribosomal protein L12P Unknown CAGCTTGAAGGCTAAATAAAGTAATAAAAATATAATAAGGAGTTATTTTCATG DMC0997 Translation elongation factor TCGAATCTGATTGTGGGCTCCATAATAATAAGGGGGAATAATTAAAAGAAATG Unknown 1A DMC1407 BNR/Asp-box repeat domain AATATGAAGACATTTGCATAAAATAAGTTTAAAAGGAAATTTATAGGTCGATG Yes protein (S-layer) DMC1427 Co-chaperonin GroES CTGCTAAAACATTGCCGCAATAAATTTAGTCAAACTTAGAAGGAGGAATAATG Yes DMC1428 Co-chaperonin GroEL TTATGGCTAAAATCGTAGGCTAAATTTTTTAACGAATATTAGGAGGTATAATG No DMC0112 Dehydrogenase, putative AATACAGGGCGAGGCCCTTTAGAGATTAAAGCACAAGGGGGGCAACAGGAATG Yes DMC0110 [Ni/Fe] hydrogenase, group 1, large subunit ATGCCGCAAAAAAGGGTTAGTAGACCGTAATCTTACCTTTGGAGAAGAAAATG No

4 A) S ribosomal RNA [Dehalococcoides mccartyi 195: NC_002936] GATGAACGCTAGCGGCGTGCCTTATGCATGCAAGTCGAACGGTCTTAAGCAATTAAGATAGTGGCAAACGGGTGAGTAACGCGTAAGTAACCTACCTCTAAGTG GGGGATAGCTTCGGGAAACTGAAGGTAATACCGCATGTGATGGGCTGACATAAGTCGGTTCATTAAAGCCGCAAGGTGCTTGGTGAGGGGCTTGCGTCCGATTA GCTAGTTGGTGGGGTAATGGCCTACCAAGGCTTCGATCGGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAG GCAGCAGCAAGGAATCTTGGGCAATGGGCGAAAGCCTGACCCAGCAACGCCGCGTGAGGGATGAAGGCTTTCGGGTTGTAAACCTCTTTTCACAGGGAAGAATA ATGACGGTACCTGTGGAATAAGCTTCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGAAGCAAGCGTTATCCGGATTTATTGGGCGTAAAGTGAGCGTA GGTGGTCTTTCAAGTTGGATGTGAAATTTCCCGGCTTAACCGGGACGTGTCATTCAATACTGTTGGACTAGAGTACAGCAGGAGAAAACGGAATTCCCGGTGTAG TGGTAAAATGCGTAGATATCGGGAGGAACACCAGAGGCGAAGGCGGTTTTCTAGGTTGTCACTGACACTGAGGCTCGAAAGCGTGGGGAGCGAACAGAATTAGA TACTCTGGTAGTCCACGCCTTAAACTATGGACACTAGGTATAGGGAGTATCGACCCTCTCTGTGCCGAAGCTAACGCTTTAAGTGTCCCGCCTGGGGAGTACGGTC GCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCGTGTGGTTTAATTCGATGCTACACGAAGAACCTTACCAAGATTTGACATGCAT GAAGTAGTGAACCGAAAGGGAAACGACCTGTTAAGTCAGGAGTTTGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGCCGTGAGGTGTTTGGTTAAGTCCTGCA ACGAGCGCAACCCTTGTTGCTAGTTAAATTTTCTAGCGAGACTGCCCCGCGAAACGGGGAGGAAGGTGGGGATGACGTCAAGTCAGCATGGCCTTTATATCTTGG GCTACACACACGCTACAATGGACAGAACAATAGGTTGCAACAGTGTGAACTGGAGCTAATCCCCAAAGCTGTCCTCAGTTCGGATTGCAGGCTGAAACCCGCCT GCATGAAGTTGGAGTTGCTAGTAACCGCATATCAGCAAGGTGCGGTGAATACGTTCTCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGCCGGTAACACTT GAAGTCGATGTGCCAACCGCAAGGAGGCAGTCGCCGAGGGTGGGACTGGTAATTGGGACGAAGTCGTAACAAGGTAGCCGTAGCGGAAGCTGCGGCTGGATCA CCTCCTTTCTAAGGATAATTGGC

5 Fig. S1. Metabolite production in cultures following batch feed of PCE (110 µm) and butyrate (440 µm). Typical concentrations of chloroethenes, ethene, and methane measured via gas chromatography are depicted over time (A). The intermediate metabolite hydrogen, measured via a reduced gas detector is plotted for three separate batch feeds (B). All concentrations are nominal except for hydrogen which depicts the calculated aqueous concentration (Cw) from head space hydrogen measurements.

6 Fig. S2. Time zero peptides per cell for the Methanospirillum (MHU) proteins MvrD and FrcA. Error bars represent 95% confidence intervals for triplicate measurements of two transition ions per peptide. Two peptides (1 and 2) were measured per protein of interest..

7 Fig. S3. Comparison of two protein quantification methods (MRM and NSAF) for key biomarkers: MvrD and FrcA for Methanospirillum (diamonds) and HupL, TceA, PceA and DMC1545 for Dehalococcoides (stars). NSAF = normalized spectral abundance factor (Zybailov et al. 2006).

8 Fig. S4. D. mccartyi str.195 (DMC) cell decay quantified via decline in respiration rate (A) and decline in DMC 16S rrna gene copies (B) and M. hungatei (MHU) cell decay measured via decline in methane production (from hydrogen) (C) and MHU 16S rrna gene copies (D).