Rapid adaptation often occurs through mutations to the most highly conserved positions of the RNA polymerase core enzyme

Mutations to the genes encoding the RNA polymerase core enzyme (RNAPC) and additional housekeeping regulatory genes were found to be involved in rapid adaptation, in the context of numerous evolutionary experiments, in which bacteria were exposed to diverse selective pressures. This provides a conundrum, as the housekeeping genes that were so often mutated in response to these diverse selective pressures tend to be among the genes that are most conserved in their sequences across the bacterial phylogeny. In order to further examine this apparent discrepancy, we characterized the precise positions of the RNAPC involved in adaptation to a large variety of selective pressures. We found that different positions of the RNAPC are involved in adaptation to various stresses, with very little overlap found between stresses. We further found that RNAPC positions involved in adaptation tended to be more evolutionary conserved, were more likely to occur within defined protein domains, and tended to be closer to the complex's active site, compared to all other RNAPC positions. Finally, we could show that this observed trend of higher conservation of positions involved in rapid adaptation extends beyond the RNAPC to additional housekeeping genes. Combined, our results demonstrate that the positions that change most readily in response to well defined selective pressures exerted in lab environments are also those that evolve most slowly in nature. This suggests that such adaptations may not readily occur in nature, due to their antagonistically pleiotropic effects, or that if they do occur in nature, they are highly transient.

Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein

Promoter recognition by RNA polymerase is a key step in the regulation of gene expression. The bacterial RNA polymerase core enzyme is a complex of five subunits that interacts transitory with one of a set of sigma factors forming the RNA polymerase holoenzyme. The sigma factor confers promoter specificity to the RNA polymerase. In the Gram-positive pathogenic bacterium Streptococcus pneumoniae, most promoters are likely recognized by SigA, a poorly studied housekeeping sigma factor. Here we present a sequence conservation analysis and show that SigA has similar protein architecture to Escherichia coli and Bacillus subtilis homologs, namely the poorly conserved N-terminal 100 residues and well-conserved rest of the protein (domains 2, 3, and 4). Further, we have purified the native (untagged) SigA protein encoded by the pneumococcal R6 strain and reconstituted an RNA polymerase holoenzyme composed of the E. coli core enzyme and the sigma factor SigA (RNAP-SigA). By in vitro transcription, we have found that RNAP-SigA was able to recognize particular promoters, not only from the pneumococcal chromosome but also from the S. agalactiae promiscuous antibiotic-resistance plasmid pMV158. Specifically, SigA was able to direct the RNA polymerase to transcribe genes involved in replication and conjugative mobilization of plasmid pMV158. Our results point to the versatility of SigA in promoter recognition and its contribution to the promiscuity of plasmid pMV158.

Construction and characterization of a functional chimeric laccase from metagenomes suitable as a biocatalyst

Screening of gene-specific amplicons from metagenomes (S-GAM) is an efficient technique for the isolation of homologous genes from metagenomes. Using the S-GAM approach, we targeted multi-copper oxidase (MCO) genes including laccase and bilirubin oxidase (BOX) in soil and compost metagenomes, and successfully isolated novel MCO core regions. These core enzyme genes shared approximately 70% identity with that of the putative MCO from Micromonospora sp. MP36. According to the principle of S-GAM, the N- and C-terminal regions of the deduced products of the mature gene come from the known parent gene, which should be homologous and compatible with the target gene. We constructed two different MCO hybrid genes using Bacillus subtilis BOX and Micromonospora sp. MP36 MCO, to give Bs-mg-mco and Mic-mg-mco, respectively. The constructed chimeric MCO genes were fused with the maltose-binding protein (MBP) gene at the N-terminus for expression in Escherichia coli cells. We found that MBP-Mic-mg-MCO/Mic-mg-MCO possessed the characteristic properties of laccase, although MBP-Bs-mg-MCO had no activity. This novel laccase (Mic-mg-MCO) demonstrated unique substrate specificity, sufficient activity at neutral pH, and high thermal stability, which are suitable properties for its use as a laccase biocatalyst.

Cryo-EM and cellular dissection uncover versatile PME-1 activities in PP2A holoenzyme demethylation and inhibition

Protein phosphatase 2A (PP2A) methylesterase 1 (PME-1) is cancer-promoting but essential for development, and long-believed to remove carboxymethylation, a central modification, from the common PP2A core enzyme but not diverse holoenzymes that target broad cellular signaling by recognizing specific disordered motifs via regulatory subunits. On the contrary, our biochemical dissection and high-resolution cryo-EM structural analysis of a PP2A-B’ holoenzyme-PME-1 complex reveal that PME-1 disordered motifs, including phosphatase substrate-mimicking motifs, tether to the holoenzyme at remote sites, block its substrate binding, and allow large structural shifts in both holoenzyme and PME-1 to enable multiptle dynamic structured contacts and induce methylesterase activation toward the holoenzyme. PME-1 inhibitor and B’-interface mutations differentially modulate cellular PP2A methylation and allow us to uncover cellular PME-1 functions in AKT-p53 signaling. Our studies demonstrate how dynamic structured cores and disordered motifs create versatile activities and lay a foundation for investigating and targeting multifaceted activities toward broad PP2A complexes in cellular signaling.

The bacterial multidrug resistance regulator BmrR distorts promoter DNA to activate transcription

The MerR-family proteins represent a unique family of bacteria transcription factors (TFs), which activate transcription in a manner distinct from canonical ones. Here, we report a cryo-EM structure of a B. subtilis transcription activation complex comprising B. subtilis six-subunit (2αββ‘ωε) RNA Polymerase (RNAP) core enzyme, σA, a promoter DNA, and the ligand-bound B. subtilis BmrR, a prototype of MerR-family TFs. The structure reveals that RNAP and BmrR recognize the upstream promoter DNA from opposite faces and induce four significant kinks from the −35 element to the −10 element of the promoter DNA in a cooperative manner, which restores otherwise inactive promoter activity by shortening the length of promoter non-optimal −35/−10 spacer. Our structure supports a DNA-distortion and RNAP-non-contact paradigm of transcriptional activation by MerR TFs.

Identification of Integrator-PP2A complex (INTAC), an RNA polymerase II phosphatase

The 14-subunit metazoan-specific Integrator contains an endonuclease that cleaves nascent RNA transcripts. Here, we identified a complex containing Integrator and protein phosphatase 2A core enzyme (PP2A-AC), termed INTAC. The 3.5-angstrom-resolution structure reveals that nine human Integrator subunits and PP2A-AC assemble into a cruciform-shaped central scaffold formed by the backbone and shoulder modules, with the phosphatase and endonuclease modules flanking the opposite sides. As a noncanonical PP2A holoenzyme, the INTAC complex dephosphorylates the carboxy-terminal repeat domain of RNA polymerase II at serine-2, -5, and -7 and thus regulates transcription. Our study extends the function of PP2A to transcriptional regulation and reveals how dual enzymatic activities—RNA cleavage and RNA polymerase II dephosphorylation—are structurally and functionally integrated into the INTAC complex.

Adaptations accumulated under prolonged resource exhaustion are highly transient

Many non-sproulating bacterial species can survive for years within exhausted growth media in a state termed long-term stationary phase (LTSP). We have been carrying out evolutionary experiments aimed at elucidating the dynamics of genetic adaptation under LTSP. We showed that Escherichia coli adapts to prolonged resource exhaustion through the highly convergent acquisition of mutations. In the most striking example of such convergent adaptation, we observed that across all independently evolving LTSP populations, over 90% of E. coli cells carry mutations to one of three specific sites of the RNA polymerase core enzyme (RNAPC). These LTSP adaptations reduce the ability of the cells carrying them to grow once fresh resources are again provided. Here, we examine how LTSP populations recover from costs associated with their adaptation, once resources are again provided to them. We demonstrate that due to the ability of LTSP populations to maintain high levels of standing genetic variation during adaptation, costly adaptations are very rapidly purged from the population once they are provided with fresh resources. We further demonstrate that recovery from costs acquired during adaptation under LTSP occurs much more rapidly than would be possible if LTSP adaptations had fixed during the time populations spent under resource exhaustion. Finally, we previously reported that under LTSP, some clones develop a mutator phenotype, greatly increasing their mutation accumulation rates. Here, we show that the mechanisms, by which populations recover from costs associated with fixed adaptations, may depend on mutator status.

Structural basis of reiterative transcription from the pyrG and pyrBI promoters by bacterial RNA polymerase

Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains eight poly-G RNA bases synthesized using three C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak–trigger–freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. Lastly, we determined a structure of a transcript initiation complex at the pyrBI promoter and proposed an alternative mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme.

Hemerythrins enhance aerobic respiration in Methylomicrobium alcaliphilum 20ZR, a methane-consuming bacterium

ABSTRACT Numerous hemerythrins, di-iron proteins, have been identified in prokaryote genomes, but in most cases their function remains elusive. Bacterial hemerythrin homologs (bacteriohemerythrins, Bhrs) may contribute to various cellular functions, including oxygen sensing, metal binding and antibiotic resistance. It has been proposed that methanotrophic Bhrs support methane oxidation by supplying oxygen to a core enzyme, particulate methane monooxygenase. In this study, the consequences of the overexpression or deletion of the Bhr gene (bhr) in Methylomicrobiam alcaliphillum 20ZR were investigated. We found that the bhrknockout (20ZRΔbhr) displays growth kinetics and methane consumption rates similar to wild type. However, the 20ZRΔbhr accumulates elevated concentrations of acetate at aerobic conditions, indicating slowed respiration. The methanotrophic strain overproducing Bhr shows increased oxygen consumption and reduced carbon-conversion efficiency, while its methane consumption rates remain unchanged. These results suggest that the methanotrophic Bhr proteins specifically contribute to oxygen-dependent respiration, while they have minimal, if any, input of oxygen for the methane oxidation machinery.

Precursor-directed biosynthesis of catechol compounds in Acinetobacter bouvetii DSM 14964

Genome mining for VibH homologs reveals several Acinetobacter species with a gene cluster putatively encoding biosynthesis of catechol siderophores with an amine core. Enzyme flexibility allows for in vivo synthesis of non-native catechol compounds.