Supplemental Metabolomics Procedures
Metabolomics Data Analysis and Statistics
For each sample, raw data files were converted to .cdf format, and matrix of molecular features as
defined by retention time and mass (m/z) was generated using XCMS software in R (Smith et al., 2006)
for feature detection and alignment. Raw peak areas were normalized to total ion signal in R, outlier
injections were detected based on total signal and PC1 of a principle component analysis, and the mean
area of the chromatographic peak was calculated among replicate injections (n=2). Features were
grouped based on a novel clustering tool, RAMClust (manuscript in preparation), which groups features
into “spectral clusters” based on coelution and covariance across the full dataset, whereby spectra are
used to determine the identity of observed compounds in the experiment. The resulting spectral
clusters are comparable to spectra obtained from other GC‐MS deconvolution algorithms, such as in
AMDIS software (Stein, 1999). The peak areas for each feature in a spectral cluster were condensed via
the weighted mean of all features in a spectrum into a single quantitative value for each compound. An
alkane standard mix (C7‐C40) was used to calculate retention indices for each spectral cluster. Spectral
clusters were annotated based on spectral and retention index matching using NISTv12, Golm, and
Massbank metabolite databases.. Analysis of variance was conducted on each compound using the aov
function in R, and p‐values were adjusted for false positives using the p.adjust in R (Benjamini and
Hochberg, 1995). Reported compounds include those with an experimental mass spectrum matching an
authentic standard (Metabolomics Standards Initiative level 2)(Sumner et al., 2007) those with only
molecular class information (level 3), and unknowns (level 4). The retention index (RI) was reported for
metabolites identified at level 3 or 4, and matches to Golm Metabolome Database (GMD) unknowns are
reported using a GMD identifier.
Cited references:
Benjamini, Y., and Hochberg, Y. (1995). Controlling the false discovery rate: a practical and powerful
approach to multiple testing. J Roy Stat Soc B Met, 57, 289–300.
Smith, C. A., Want, E. J., O’Maille, G., Abagyan, R., and Siuzdak, G. (2006). XCMS: processing mass
spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification.
Anal. Chem., 78, 779–87.
Stein, S. E. (1999). An integrated method for spectrum extraction and compound identification from gas
chromatography/mass spectrometry data. J Am Soc Mass Spectr, 10, 770–781.
Sumner, L. W., Amberg, A., Barrett, D., Beale, M. H., Beger, R., Daykin, C. A., Fan, T. W.‐M., Fiehn, O.,
Goodacre, R., Griffin, J. L., Hankemeier, T., Hardy, N., Harnly, J., Higashi, R., Kopka, J., Lane, A. N., Lindon,
J. C., Marriott, P., Nicholls, A. W., Reily, M. D., Thaden, J. J., and Viant, M. R. (2007). Proposed minimum
reporting standards for chemical analysis Chemical Analysis Working Group (CAWG) Metabolomics
Standards Initiative (MSI). Metabolomics, 3, 211–221.
Supplementary Figure Legends
Supplementary Figure 1. Southern blot of wild type and urease mutant DNA digested with XhoI.
Expected arrangements of XhoI-digested DNA is shown before and after recombination with the ShBle
knockout. Due to incomplete DNA digestion, a substantial band appears at the top of each lane
corresponding to undigested genomic DNA. DNA from line 11-5 appears not to have digested at all
under these conditions. Despite this technical difficulty, the lower bands are consistent with the
hypotheses described in the text and the larger band in 11-4 helps to explain the doublet in the BamHIdigested Southern blot as a potential duplication in the 3’ region of urease gene.
Supplementary Figure 2. Phenotypic analysis of mutants created by pTALEN-Ble. Growth of four strains
on nitrate-containing medium (A) or urea-containing medium (B) shows a clearly identifiable phenotype
after transformation of the TALEN only (without the additional KO-plasmid).
Supplementary Figure 3. Phenotypic analysis of incomplete or monoallelic mutants grown on nitrate
and urea. While no growth on urea-containing medium was observed for the complete or biallelic
mutants (Fig. 7), there was no significant decrease in growth rate for the incomplete or monoallelic
mutants. Growth rates (fold change in chlorophyll a fluorescence per day) are shown in parentheses in
the figure legend.
Supplementary Figure 4. Plasmid map and Sequence for pTH
Supplementary Figure 5. Plasmid map and Sequence for pTH+Urease_TALENs