Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria.

The molecular mechanisms of ethanol toxicity and tolerance in bacteria, although important for biotechnology and bioenergy applications, remain incompletely understood. Genetic studies have identified potential cellular targets for ethanol and have revealed multiple mechanisms of tolerance, but it remains difficult to separate the direct and indirect effects of ethanol. We used adaptive evolution to generate spontaneous ethanol-tolerant strains of Escherichia coli, and then characterized mechanisms of toxicity and resistance using genome-scale DNAseq, RNAseq, and ribosome profiling coupled with specific assays of ribosome and RNA polymerase function. Evolved alleles of metJ, rho, and rpsQ recapitulated most of the observed ethanol tolerance, implicating translation and transcription as key processes affected by ethanol. Ethanol induced miscoding errors during protein synthesis, from which the evolved rpsQ allele protected cells by increasing ribosome accuracy. Ribosome profiling and RNAseq analyses established that ethanol negatively affects transcriptional and translational processivity. Ethanol-stressed cells exhibited ribosomal stalling at internal AUG codons, which may be ameliorated by the adaptive inactivation of the MetJ repressor of methionine biosynthesis genes. Ethanol also caused aberrant intragenic transcription termination for mRNAs with low ribosome density, which was reduced in a strain with the adaptive rho mutation. Furthermore, ethanol inhibited transcript elongation by RNA polymerase in vitro. We propose that ethanol-induced inhibition and uncoupling of mRNA and protein synthesis through direct effects on ribosomes and RNA polymerase conformations are major contributors to ethanol toxicity in E. coli, and that adaptive mutations in metJ, rho, and rpsQ help protect these central dogma processes in the presence of ethanol.

The poor growth of Rhodospirillum rubrum mutants lacking RubisCO is due to the accumulation of ribulose-1,5-bisphosphate.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the first step of CO(2) fixation in the Calvin-Benson-Bassham (CBB) cycle. Besides its function in fixing CO(2) to support photoautotrophic growth, the CBB cycle is also important under photoheterotrophic growth conditions in purple nonsulfur photosynthetic bacteria. It has been assumed that the poor photoheterotrophic growth of RubisCO-deficient strains was due to the accumulation of excess intracellular reductant, which implied that the CBB cycle is important for maintaining the redox balance under these conditions. However, we present analyses of cbbM mutants in Rhodospirillum rubrum that indicate that toxicity is the result of an elevated intracellular pool of ribulose-1,5-bisphosphate (RuBP). There is a redox effect on growth, but it is apparently an indirect effect on the accumulation of RuBP, perhaps by the regulation of the activities of enzymes involved in RuBP regeneration. Our studies also show that the CBB cycle is not essential for R. rubrum to grow under photoheterotrophic conditions and that its role in controlling the redox balance needs to be further elucidated. Finally, we also show that CbbR is a positive transcriptional regulator of the cbb operon (cbbEFPT) in R. rubrum, as seen with related organisms, and define the transcriptional organization of the cbb genes.

Mutagenesis and functional characterization of the four domains of GlnD, a bifunctional nitrogen sensor protein.

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme (UTase/UR) and is believed to be the primary sensor of nitrogen status in the cell by sensing the level of glutamine in enteric bacteria. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of P(II) protein; P(II) in turn regulates a variety of other proteins. GlnD appears to have four distinct domains: an N-terminal nucleotidyltransferase (NT) domain; a central HD domain, named after conserved histidine and aspartate residues; and two C-terminal ACT domains, named after three of the allosterically regulated enzymes in which this domain is found. Here we report the functional analysis of these domains of GlnD from Escherichia coli and Rhodospirillum rubrum. We confirm the assignment of UTase activity to the NT domain and show that the UR activity is a property specifically of the HD domain: substitutions in this domain eliminated UR activity, and a truncated protein lacking the NT domain displayed UR activity. The deletion of C-terminal ACT domains had little effect on UR activity itself but eliminated the ability of glutamine to stimulate that activity, suggesting a role for glutamine sensing by these domains. The deletion of C-terminal ACT domains also dramatically decreased UTase activity under all conditions tested, but some of these effects are due to the competition of UTase activity with unregulated UR activity in these variants.

Effect of perturbation of ATP level on the activity and regulation of nitrogenase in Rhodospirillum rubrum.

Nitrogenase activity in Rhodospirillum rubrum and in some other photosynthetic bacteria is regulated in part by the availability of light. This regulation is through a posttranslational modification system that is itself regulated by P(II) homologs in the cell. P(II) is one of the most broadly distributed regulatory proteins in nature and directly or indirectly senses nitrogen and carbon signals in the cell. However, its possible role in responding to light availability remains unclear. Because P(II) binds ATP, we tested the hypothesis that removal of light would affect P(II) by changing intracellular ATP levels, and this in turn would affect the regulation of nitrogenase activity. This in vivo test involved a variety of different methods for the measurement of ATP, as well as the deliberate perturbation of intracellular ATP levels by chemical and genetic means. To our surprise, we found fairly normal levels of nitrogenase activity and posttranslational regulation of nitrogenase even under conditions of drastically reduced ATP levels. This indicates that low ATP levels have no more than a modest impact on the P(II)-mediated regulation of NifA activity and on the posttranslational regulation of nitrogenase activity. The relatively high nitrogenase activity also shows that the ATP-dependent electron flux from dinitrogenase reductase to dinitrogenase is also surprisingly insensitive to a depleted ATP level. These in vivo results disprove the simple model of ATP as the key energy signal to P(II) under these conditions. We currently suppose that the ratio of ADP/ATP might be the relevant signal, as suggested by a number of recent in vitro analyses.

Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover.

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.

Identification and functional characterization of NifA variants that are independent of GlnB activation in the photosynthetic bacterium Rhodospirillum rubrum.

The activity of NifA, the transcriptional activator of the nitrogen fixation (nif) gene, is tightly regulated in response to ammonium and oxygen. However, the mechanisms for the regulation of NifA activity are quite different among various nitrogen-fixing bacteria. Unlike the well-studied NifL-NifA regulatory systems in Klebsiella pneumoniae and Azotobacter vinelandii, in Rhodospirillum rubrum NifA is activated by a direct protein-protein interaction with the uridylylated form of GlnB, which in turn causes a conformational change in NifA. We report the identification of several substitutions in the N-terminal GAF domain of R. rubrum NifA that allow NifA to be activated in the absence of GlnB. Presumably these substitutions cause conformational changes in NifA necessary for activation, without interaction with GlnB. We also found that wild-type NifA can be activated in a GlnB-independent manner under certain growth conditions, suggesting that some other effector(s) can also activate NifA. An attempt to use Tn5 mutagenesis to obtain mutants that altered the pool of these presumptive effector(s) failed, though much rarer spontaneous mutations in nifA were detected. This suggests that the necessary alteration of the pool of effector(s) for NifA activation cannot be obtained by knockout mutations.

Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum.

The AmtB protein transports uncharged NH(3) into the cell, but it also interacts with the nitrogen regulatory protein P(II), which in turn regulates a variety of proteins involved in nitrogen fixation and utilization. Three P(II) homologues, GlnB, GlnK and GlnJ, have been identified in the photosynthetic bacterium Rhodospirillum rubrum, and they have roles in at least four overlapping and distinct functions, one of which is the post-translational regulation of nitrogenase activity. In R. rubrum, nitrogenase activity is tightly regulated in response to addition or energy depletion (shift to darkness), and this regulation is catalysed by the post-translational regulatory system encoded by draTG. Two amtB homologues, amtB(1) and amtB(2), have been identified in R. rubrum, and they are linked with glnJ and glnK, respectively. Mutants lacking AmtB(1) are defective in their response to both addition and darkness, while mutants lacking AmtB(2) show little effect on the regulation of nitrogenase activity. These responses to darkness and appear to involve different signal transduction pathways, and the poor response to darkness does not seem to be an indirect result of perturbation of internal pools of nitrogen. It is also shown that AmtB(1) is necessary to sequester detectable amounts GlnJ to the cell membrane. These results suggest that some element of the AmtB(1)-P(II) regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by P(II) is proposed. Other results demonstrate a degree of specificity in interaction of AmtB(1) with the different P(II) homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which P(II) proteins regulate processes involved in nitrogen acquisition and utilization.

The poor growth of Rhodospirillum rubrum mutants lacking PII proteins is due to an excess of glutamine synthetase activity.

The P(II) family of proteins is found in all three domains of life and serves as a central regulator of the function of proteins involved in nitrogen metabolism, reflecting the nitrogen and carbon balance in the cell. The genetic elimination of the genes encoding these proteins typically leads to severe growth problems, but the basis of this effect has been unknown except with Escherichia coli. We have analysed a number of the suppressor mutations that correct such growth problems in Rhodospirillum rubrum mutants lacking P(II) proteins. These suppressors map to nifR3, ntrB, ntrC, amtB(1) and the glnA region and all have the common property of decreasing total activity of glutamine synthetase (GS). We also show that GS activity is very high in the poorly growing parental strains lacking P(II) proteins. Consistent with this, overexpression of GS in glnE mutants (lacking adenylyltransferase activity) also causes poor growth. All of these results strongly imply that elevated GS activity is the causative basis for the poor growth seen in R. rubrum mutants lacking P(II) and presumably in mutants of some other organisms with similar genotypes. The result underscores the importance of proper regulation of GS activity for cell growth.

GlnD is essential for NifA activation, NtrB/NtrC-regulated gene expression, and posttranslational regulation of nitrogenase activity in the photosynthetic, nitrogen-fixing bacterium Rhodospirillum rubrum.

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme and is thought to be the primary sensor of nitrogen status in the cell. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of P(II) proteins, which in turn regulate a variety of other proteins. We report here the characterization of glnD mutants from the photosynthetic, nitrogen-fixing bacterium Rhodospirillum rubrum and the analysis of the roles of GlnD in the regulation of nitrogen fixation. Unlike glnD mutations in Azotobacter vinelandii and some other bacteria, glnD deletion mutations are not lethal in R. rubrum. Such mutants grew well in minimal medium with glutamate as the sole nitrogen source, although they grew slowly with ammonium as the sole nitrogen source (MN medium) and were unable to fix N(2). The slow growth in MN medium is apparently due to low glutamine synthetase activity, because a DeltaglnD strain with an altered glutamine synthetase that cannot be adenylylated can grow well in MN medium. Various mutation and complementation studies were used to show that the critical uridylyltransferase activity of GlnD is localized to the N-terminal region. Mutants with intermediate levels of uridylyltransferase activity are differentially defective in nif gene expression, the posttranslational regulation of nitrogenase, and NtrB/NtrC function, indicating the complexity of the physiological role of GlnD. These results have implications for the interpretation of results obtained with GlnD in many other organisms.

Identification of critical residues in GlnB for its activation of NifA activity in the photosynthetic bacterium Rhodospirillum rubrum.

The P(II) regulatory protein family is unusually widely distributed, being found in all three domains of life. Three P(II) homologs called GlnB, GlnK, and GlnJ have been identified in the photosynthetic bacterium Rhodospirillum rubrum. These have roles in at least four distinct functions, one of which is activation of the nitrogen fixation-specific regulatory protein NifA. The activation of NifA requires only the covalently modified (uridylylated) form of GlnB. GlnK and GlnJ are not involved. However, the basis of specificity for different P(II) homologs in different processes is poorly understood. We examined this specificity by altering GlnJ to support NifA activation. A small number of amino acid substitutions in GlnJ were important for this ability. Two (affecting residues 45 and 54) are in a loop called the T-loop, which contains the site of uridylylation and is believed to be very important for contacts with other proteins, but other critical residues lie in the C terminus (residues 95-97 and 109-112) and near the N terminus (residues 3-5 and 17). Because many of the residues important for P(II)-NifA interaction lie far from the T-loop in the known x-ray crystal structures of P(II) proteins, our results lead to the hypothesis that the T-loop of GlnB is flexible enough to come into proximity with both the C- and N-terminal regions of the protein to bind NifA. Finally, the results show that the level of P(II) accumulation is also an important factor for NifA activation.