32 research outputs found
The Robustness of the <i>Escherichia coli</i> Signal-Transducing UTase/UR-PII Covalent Modification Cycle to Variation in the PII Concentration Requires Very Strong Inhibition of the UTase Activity of UTase/UR by Glutamine
Uridylyltransferase/uridylyl-removing enzyme (UTase/UR)
catalyzes
uridylylation of PII and deuridylylation of PII-UMP, with both activities
regulated by glutamine. In a reconstituted UTase/UR-PII cycle containing
wild-type UTase/UR, the steady-state modification of PII varied from
nearly complete modification to nearly complete demodification as
glutamine was varied, whether the level of PII was saturating or unsaturating,
but when a His-tagged version of UTase/UR was used, the robustness
to variations in PII concentration was lost and the range of PII modification
states in response to glutamine became smaller as the PII concentration
increased. The presence of the His tag on UTase/UR did not alter PII
substrate inhibition of the UT activity and had little effect on the
level of the UT activity but resulted in a slight defect in UR activity.
Importantly, at high PII concentrations, glutamine inhibition of the
UT activity was incomplete. We hypothesized that binding of PII to
the UR active site in the HD domain was responsible for PII substrate
inhibition of the UT activity and, in the His-tagged enzyme, also
weakened glutamine inhibition of the UT activity. Consistent with
this, three different UTase/UR proteins with HD domain alterations
lacked substrate inhibition of UT activity by PII; in one case, the
HD alteration eliminated glutamine regulation of UT activity, while
for the other two proteins, alterations of the HD domain partially
compensated for the effect of the His tag in restoring glutamine regulation
of UT activity. We conclude that very strong inhibition of UT activity
was required for the UTase/UR-PII cycle to display robustness to the
PII concentration, that in the wild-type enzyme PII brings about substrate
inhibition of the UT activity by binding to the HD domain of the enzyme,
and that addition of an N-terminal His tag resulted in an altered
enzyme with subtle changes in the interactions between domains such
that binding of PII to the HD domain interfered with glutamine regulation
of the UT domain
Metabolism of Multiple Aromatic Compounds in Corn Stover Hydrolysate by <i>Rhodopseudomonas palustris</i>
Lignocellulosic
biomass hydrolysates hold great potential as a
feedstock for microbial biofuel production, due to their high concentration
of fermentable sugars. Present at lower concentrations are a suite
of aromatic compounds that can inhibit fermentation by biofuel-producing
microbes. We have developed a microbial-mediated strategy for removing
these aromatic compounds, using the purple nonsulfur bacterium Rhodopseudomonas palustris. When grown photoheterotrophically
in an anaerobic environment, R. palustris removes most of the aromatics from ammonia fiber expansion (AFEX)
treated corn stover hydrolysate (ACSH), while leaving the sugars mostly
intact. We show that R. palustris can
metabolize a host of aromatic substrates in ACSH that have either
been previously described as unable to support growth, such as methoxylated
aromatics, and those that have not yet been tested, such as aromatic
amides. Removing the aromatics from ACSH with R. palustris, allowed growth of a second microbe that could not grow in the untreated
ACSH. By using defined mutants, we show that most of these aromatic
compounds are metabolized by the benzoyl-CoA pathway. We also show
that loss of enzymes in the benzoyl-CoA pathway prevents total degradation
of the aromatics in the hydrolysate, and instead allows for biological
transformation of this suite of aromatics into selected aromatic compounds
potentially recoverable as an additional bioproduct
MOESM4 of Chemical genomic guided engineering of gamma-valerolactone tolerant yeast
Additional file 4. Proteomic profile of Y133 pad1â fdc1â vs Y133 grown in YPD medium plus 1% GVL
MOESM1 of Chemical genomic guided engineering of gamma-valerolactone tolerant yeast
Additional file 1. GVL hydrolysate inhibitors
MOESM7 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032
Additional file 7: Table S6. Comparison of RNA-Seq from anaerobic and aerobic conditions
MOESM8 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032
Additional file 8: Table S7. Proteomic data of ZM4 in ethanol shock and sodium acetate stress conditions
MOESM5 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032
Additional file 5: Table S4. Plasmid gene RNA-Seq counts
MOESM10 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032
Additional file 10: Table S9. Assignments of plasmid genes to expression clusters identified by inter-center expression meta-analysis (see Additional file 1: Figure S3)
MOESM9 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032
Additional file 9: Table S8. Proteomic data of ZM4 in anaerobic and aerobic fermentation in rich and minimal media
MOESM1 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032
Additional file 1: Figure S1. Completion of plasmid sequences by primer walking with a list of the primers used for each plasmid (A). PCR amplification of ZM4 chromosome region containing a 2.4-kb fragment near ZMO0133 locus that is absent in previously reported ZM4 genome sequence (B). A schematic is shown detailing the location of primers used to PCR, and PCR products on agarose gel are also shown. Figure S2. Customized rRNA depletion kit was developed with Life Technologies for Z. mobilis mRNA enrichment, and RNA-Seq result of the percentage of rRNA, tRNA, and mRNA in Z. mobilis total RNA was calculated (A). qRT-PCR measurement of rRNA content before and after rRNA depletion of total RNA using the customized kit (B). rRNA reduction is reported as the fold change in the target rRNA in total RNA relative to depleted RNA. Measurements were collected in WT (Z. mobilis strain 33C derived from Z. mobilis 8b) and MT (a mutant strain of 33C) grown in either rich media with 5% glucose (RMG) or rich media with 5% xylose (RMX) and collected in two biological replicates. Error is reported as standard deviation. Residual rRNA contamination and rRNA depletion efficiency of samples described in (B) was detected by RNA-Seq (C). Error is reported as standard deviation. An example of pairwise replicate correlation of RNA-Seq pseudo read counts (i.e. log2 transformed following addition of La Place constant of 1) for two biological replicates after rRNA depletion (D). Figure S3. Heatmap of RNA-Seq data from 6% and 9% ACSH, anaerobic (AN) and aerobic (AE) conditions. Coloring by condition (left color bar) corresponds to the one used for the Fig. 4. Blue, NREL, fermentor with biomass hydrolysates; black, NREL, flasks with rich RMG medium; light grey, GLBRC, 6% ACSH; Orange, GLBRC, 9% ACSH; light green, Univ. Athens, anaerobic; dark green, Univ. Athens (UA), anaerobic; dark green, UA, aerobic. Top index bar shows expression clusters (see Additional file 6: Table S5 for gene-cluster assignments). Right annotation bar shows generalized factor that is applicable to experimental designs across all the 3 research centers: “Early” and “Late” are growth stages