rpoD Mutations Research Articles

Repeated Phenotypic Evolution by Different Genetic Routes in Pseudomonas fluorescens SBW25

Repeated evolution of functionally similar phenotypes is observed throughout the tree of life. The extent to which the underlying genetics are conserved remains an area of considerable interest. Previously, we reported the evolution of colony switching in two independent lineages of Pseudomonas fluorescens SBW25. The phenotypic and genotypic bases of colony switching in the first lineage (Line 1) have been described elsewhere. Here, we deconstruct the evolution of colony switching in the second lineage (Line 6). We show that, as for Line 1, Line 6 colony switching results from an increase in the expression of a colanic acid-like polymer (CAP). At the genetic level, nine mutations occur in Line 6. Only one of these—a nonsynonymous point mutation in the housekeeping sigma factor rpoD—is required for colony switching. In contrast, the genetic basis of colony switching in Line 1 is a mutation in the metabolic gene carB. A molecular model has recently been proposed whereby the carB mutation increases capsulation by redressing the intracellular balance of positive (ribosomes) and negative (RsmAE/CsrA) regulators of a positive feedback loop in capsule expression. We show that Line 6 colony switching is consistent with this model; the rpoD mutation generates an increase in ribosomal gene expression, and ultimately an increase in CAP expression.

Adaptive laboratory evolution of a genome-reduced Escherichia coli

Synthetic biology aims to design and construct bacterial genomes harboring the minimum number of genes required for self-replicable life. However, the genome-reduced bacteria often show impaired growth under laboratory conditions that cannot be understood based on the removed genes. The unexpected phenotypes highlight our limited understanding of bacterial genomes. Here, we deploy adaptive laboratory evolution (ALE) to re-optimize growth performance of a genome-reduced strain. The basis for suboptimal growth is the imbalanced metabolism that is rewired during ALE. The metabolic rewiring is globally orchestrated by mutations in rpoD altering promoter binding of RNA polymerase. Lastly, the evolved strain has no translational buffering capacity, enabling effective translation of abundant mRNAs. Multi-omic analysis of the evolved strain reveals transcriptome- and translatome-wide remodeling that orchestrate metabolism and growth. These results reveal that failure of prediction may not be associated with understanding individual genes, but rather from insufficient understanding of the strain’s systems biology.

Mutations in sigma 70 transcription factor improves expression of functional eukaryotic membrane proteins in Escherichia coli

Eukaryotic integral membrane proteins (IMPs) are difficult to study due to low functional expression levels. To investigate factors for efficient biogenesis of eukaryotic IMPs in the prokaryotic model organism Escherichia coli, important, e.g., for isotope-labeling for NMR, we selected for E. coli cells expressing high levels of functional G protein-coupled receptors (GPCRs) by FACS. Utilizing an E. coli strain library with all non-essential genes systematically deleted, we unexpectedly discovered upon whole-genome sequencing that the improved phenotype was not conferred by the deleted genes but by various subtle alterations in the “housekeeping” sigma 70 factor (RpoD). When analyzing effects of the rpoD mutations at the transcriptome level we found that toxic effects incurred on wild-type E. coli during receptor expression were diminished by two independent and synergistic effects: a slower but longer-lasting GPCR biosynthesis and an optimized transcriptional pattern, augmenting growth and expression at low temperature, setting the basis for further bacterial strain engineering.

Compensating the Fitness Costs of Synonymous Mutations.

Synonymous mutations do not change the sequence of the polypeptide but they may still influence fitness. We investigated in Salmonella enterica how four synonymous mutations in the rpsT gene (encoding ribosomal protein S20) reduce fitness (i.e., growth rate) and the mechanisms by which this cost can be genetically compensated. The reduced growth rates of the synonymous mutants were correlated with reduced levels of the rpsT transcript and S20 protein. In an adaptive evolution experiment, these fitness impairments could be compensated by mutations that either caused up-regulation of S20 through increased gene dosage (due to duplications), increased transcription of the rpsT gene (due to an rpoD mutation or mutations in rpsT), or increased translation from the rpsT transcript (due to rpsT mutations). We suggest that the reduced levels of S20 in the synonymous mutants result in production of a defective subpopulation of 30S subunits lacking S20 that reduce protein synthesis and bacterial growth and that the compensatory mutations restore S20 levels and the number of functional ribosomes. Our results demonstrate how specific synonymous mutations can cause substantial fitness reductions and that many different types of intra- and extragenic compensatory mutations can efficiently restore fitness. Furthermore, this study highlights that also synonymous sites can be under strong selection, which may have implications for the use of dN/dS ratios as signature for selection.

Using global transcription machinery engineering (gTME) to improve ethanol tolerance of Zymomonas mobilis.

BackgroundWith the increasing global crude oil crisis and resulting environmental concerns, the production of biofuels from renewable resources has become increasingly important. One of the major challenges faced during the process of biofuel production is the low tolerance of the microbial host towards increasing biofuel concentrations.ResultsHere, we demonstrate that the ethanol tolerance of Zymomonas mobilis can be greatly enhanced through the random mutagenesis of global transcription factor RpoD protein, (σ70). Using an enrichment screening, four mutants with elevated ethanol tolerance were isolated from error-prone PCR libraries. All mutants showed significant growth improvement in the presence of ethanol stress when compared to the control strain. After an ethanol (9 %) stress exposure lasting 22 h, the rate of glucose consumption was approximately 1.77, 1.78 and 1.39 g L−1 h−1 in the best ethanol-tolerant strain ZM4-mrpoD4, its rebuilt mutant strain ZM4-imrpoD and the control strain, respectively. Our results indicated that both ZM4-mrpoD4 and ZM4-imrpoD consumed glucose at a faster rate after the initial 9 % (v/v) ethanol stress, as nearly 0.64 % of the initial glucose remained after 54 h incubation versus approximately 5.43 % for the control strain. At 9 % ethanol stress, the net ethanol productions by ZM4-mrpoD4 and ZM4-imrpoD during the 30–54 h were 13.0–14.1 g/l versus only 6.6–7.7 g/l for the control strain. The pyruvate decarboxylase activity of ZM4-mrpoD4 was 62.23 and 68.42 U/g at 24 and 48 h, respectively, which were 2.6 and 1.6 times higher than the control strain. After 24 and 48 h of 9 % ethanol stress, the alcohol dehydrogenase activities of ZM4-mrpoD4 were also augmented, showing an approximate 1.4 and 1.3 times increase, respectively, when compared to the control strain. Subsequent quantitative real-time PCR analysis under these stress conditions revealed that the relative expression of pdc in cultured (6 and 24 h) ZM4-mrpoD4 increased by 9.0- and 12.7-fold when compared to control strain.ConclusionsCollectively, these results demonstrate that the RpoD mutation can enhance ethanol tolerance in Z. mobilis. Our results also suggested that RpoD may play an important role in resisting high ethanol concentration in Z. mobilis and manipulating RpoD via global transcription machinery engineering (gTME) can provide an alternative and useful approach for strain improvement for complex phenotypes.

Regulation of sigma factor competition by the alarmone ppGpp.

Many regulons controlled by alternative sigma factors, including sigma(S) and sigma(32), are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of sigma(S)-dependent promoters because underproduction of sigma(70), specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-sigma(70)) restored expression from sigma(S)-dependent promoters in vivo in the absence of ppGpp accumulation. An in vitro transcription/competition assay with reconstituted RNA polymerase showed that addition of ppGpp reduces the ability of wild-type sigma(70) to compete with sigma(32) for core binding and the mutant sigma(70) proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type sigma(70). Similarly, an in vivo competition assay showed that the ability of both sigma(32) and sigma(S) to compete with sigma(70) is diminished in cells lacking ppGpp. Consistently, the fraction of sigma(S) and sigma(32) bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of sigma factors in accordance with cellular demands during physiological stress.

Mutational analysis of the role of the first helix of region 4.2 of the sigma 70 subunit of Escherichia coli RNA polymerase in transcriptional activation by activator protein PhoB.

Transcription of the genes belonging to the phosphate (pho) regulon in Escherichia coli requires the specific activator protein PhoB, in addition to RNA polymerase containing the major sigma factor, sigma 70, which is encoded by rpoD. We previously isolated two mutant sigma 70s (D570G and E575K) that were specifically defective in transcribing the pho genes. The mutated sites were located near and within the first helix of the helix-turn-helix (HTH) motif or region 4.2 of sigma 70. To study further the role of the first helix of the HTH motif of sigma 70 in transcriptional activation by PhoB, we made a series of rpoD mutations that alter the motif and purified the mutant sigma 70 proteins. RNA polymerases containing the mutant sigma 70s Y571A, T572L, V576T, K578E and F580V showed reduced in vitro transcription from the pstS promoter, a representative pho promoter, in the presence of PhoB, whereas RNA polymerase containing another mutant sigma 70 (E574K) showed enhanced transcription from the promoter. Transcription from the activator-independent tac promoter and the pBR-P4 promoter, which is independent of PhoB and requires cAMP-CRP (cAMP receptor protein) for transcription, was affected at most only marginally by these sigma 70 mutations. These results provide further evidence that the first helix plays an important role in the specific interaction between RNA polymerase and PhoB protein bound to the pho promoters in transcriptional activation.

Role of the sigma 70 subunit of RNA polymerase in transcriptional activation by activator protein PhoB in Escherichia coli.

Transcription of the genes belonging to the phosphate (pho) regulon in Escherichia coli, which are induced by phosphate starvation, requires the specific activator protein PhoB in addition to the RNA polymerase holoenzyme containing the major sigma-factor sigma 70. To study the mechanism of transcriptional activation and identify the subunit of RNA polymerase involved in specific interaction with PhoB, we attempted to isolate rpoA and rpoD mutants that are specifically defective in the expression of the pho genes. We isolated two rpoD mutants with such properties, but no rpoA mutant with similar properties. The rpoD mutations altered amino acids within and near the first helix of the putative helix-turn-helix (HTH) motif in the carboxy-terminal region of sigma 70. Activities of the pho promoters in vivo were severely reduced in these mutants, whereas those of the PhoB-independent promoters were affected only marginally at most. The reconstituted mutant RNA polymerase holoenzymes were severely defective in transcribing the pstS gene, one of the pho genes, whereas they were efficient in transcribing the PhoB-independent promoters. Phosphorylated PhoB, which binds to the pho promoters with high affinity, mediated the specific binding of the wild-type holoenzyme to the pstS promoter, but it did not mediate the binding of the mutant holoenzymes. These results suggest that PhoB promotes specific interaction between RNA polymerase and the pho promoters for transcriptional activation, and the first helix of the putative HTH motif plays an essential role in the interaction, probably by making direct contact with PhoB.

Changes in conserved region 2 of Escherichia coli σ70 affecting promoter recognition

We describe a mutation in rpoD, the gene encoding the sigma 70 subunit of RNA polymerase, which alters the promoter specificity of the holoenzyme in vivo. The mutant sigma causes a substantial and specific increase in the activity of mutant ant and lac promoters with a T.A to C.G substitution at position -12, the first position of the -10 hexamer. The rpoD mutation is a single base-pair substitution causing a Gln----His change at position 437, which is in a domain of conserved region 2.4 that is predicted to form an alpha-helix. Gln437 would lie one turn of the alpha-helix away from Thr440, which was previously implicated in recognition of position -12. The rpoD-QH437 mutation described here lends further support to the model that region 2.4 of sigma is involved in recognition of the 5' end of the -10 hexamer. In addition, two rpoD mutations with non-specific effects on promoter recognition are described.

Altered promoter recognition by mutant forms of the σ70 subunit of Escherichia coli RNA polymerase

We have systematically assayed the in vivo promoter recognition properties of 13 mutations in rpoD, the gene that encodes the σ 70 subunit of Escherichia coli RNA polymerase holoenzyme, using transcriptional fusions to 37 mutant and wild-type promoters. We found three classes of rpoD mutations: (1) mutations that suggest contacts between amino acid side-chains of σ 70 and specific bases in the promoter; (2) mutations that appear to affect either sequence independent contacts to promoter DNA or isomerization of the polymerase; and (3) mutations that have little or no effect on promoter recognition. Our results lead us to suggest that a sequence near the C terminus of σ 70, which is similar to the helix-turn-helix DNA binding motif of phage and bacterial DNA binding proteins, is responsible for recognition of the −35 region, and that a sequence internal to σ 70, in a region which is highly conserved among σ factors, recognizes the −10 region of the promoter. rpoD mutations that lie in the recognition helix of the proposed helix-turn-helix motif affect interactions with specific bases in the −35 region, while mutations in the upstream helix, which is thought to contact the phosphate backbone, have sequence-independent effects on promoter recognition.

A mutant Escherichia coliσ70 subunit of RNA polymerase with altered promoter specificity

A mutation is described that alters the promoter specificity of σ 70, the primary sigma factor of Escherichia coli RNA polymerase. In strains carrying both the mutant and wild-type sigma gene ( rpoD), the mutant sigma causes a large increase in the activity of mutant P22 ant promoters with A · T or C · G instead of the wild-type, consensus G · C base-pair at position −33, the third position of the consensus −35 hexamer 5′-TTGACA-3′. There is little or no effect on the activities of the wild-type and 23 other mutant ant promoters, including one with T · A at −33. The mutant sigma also activates E. coli lac promoters with A · T or C · G, but not T · A, at the corresponding position. The rpoD mutation ( rpoD-RH588) changes a CGT codon to CAT. The corresponding change in σ 70 is Arg588 → His. This residue is in a region that is conserved among most sigma factors, a region that is also homologous with the helix-turn-helix motif of DNA-binding proteins. These results suggest that this region of σ 70 is directly involved in recognition of the −35 hexamer.

Mutations in rpoD that increase expression of genes in the mal regulon of Escherichia coli K-12

The σ subunits of eubacterial RNA polymerases determine the site selectivity of initiation of transcription at promoters. Mutations in rpoD, the gene that encodes σ 70, the major σ factor in Escherichia coli, should be useful in determining the molecular details of the process of transcription initiation. However, such mutations are likely to be deleterious or lethal, since σ 70 is an essential gene product. We designed a system for the rapid isolation and fine structure mapping of mutations in rpoD, which allows selection of mutations that would otherwise be deleterious to the cell. We used this system to isolate a new class of mutations in rpoD, mutations that relieve the requirement for CAP-cAMP for initiation at promoters in the mal regulon. These mutations, which we designate rpoD(Mal) mutations, occur in two clusters in the rpoD gene within regions previously suggested by amino acid sequence comparisons to be important for σ structure or function. We cannot distinguish whether the rpoD(Mal) mutations affect mal expression by altering interaction between RNA polymerase and mal promoters or between RNA polymerase and the accessory transcription factor MalT. However, the effects of the mutations on activator-independent transcription from the lac promoter (4 rpoD(Mal) mutations decrease CAP-independent expression of the lac promoter in vivo) suggest that the regions of σ identified by our mutations may be directly involved in promoter recognition.

Mutations in rpoD, the gene encoding the σ70 subunit of Escherichia coli RNA polymerase, that increase expression of the lac operon in the absence of CAP-cAMP

We have isolated a new class of mutations in rpoD, the gene encoding the σ 70 subunit of Escherichia coli RNA polymerase, that alter the transcription initiation properties of RNA polymerase holoenzyme. The rpoD(Lac) mutations increase expression of the lac operon in the absence of CAP-cAMP, allowing a strain lacking adenyl cyclase to grow on lactose. Four of the six alleles isolated have three- to fivefold increases in the amount of lac mRNA and β-galactosidase per cell. We show that these four mutations increase transcription initiation from the same promoter used by wild-type RNA polymerase. The mutations were mapped and sequenced. One mutation occurs in the codon for amino acid 389 of the σ 70 polypeptide. The remaining five mutations are clustered, affecting residues 570, 571 and 575. These five mutations are within or near a proposed helix-turn-helix motif in the C terminus of σ 70.

Mutations in the sigma subunit of E. coli RNA polymerase which affect positive control of transcription.

The sigma subunits of bacterial RNA polymerases are required for the selective initiation of transcription. We have isolated and characterized mutations in rpoD, the gene which encodes the major form of sigma in E. coli, which affect the selectivity of transcription. These mutations increase the expression of araBAD up to 12-fold in the absence of CAP-cAMP. Expression of lac is unaffected, while expression of malT-activated operons is decreased. We determined the DNA sequence of 17 independently isolated mutations, and found that they consist of three different changes in a single CGC arginine codon at position 596 in the sigma polypeptide.