Antitoxin MqsA represses curli formation through the master biofilm regulator CsgD

MqsA, the antitoxin of the MqsR/MqsA toxin/antitoxin (TA) system, is a global regulator that reduces expression of several stress response genes (e.g., mqsRA, cspD and rpoS) by binding to the promoter palindromic motif [5′-AACCT (N)3 AGGTT-3′]. We identified a similar mqsRA-like palindrome [5′-AACCT TA AGGTT-3′] 78 bp upstream of the transcription initiation site in the csgD promoter (p-csgD). CsgD is a master regulator for biofilm formation via its control of curli and cellulose production. We show here that MqsA binds to this palindrome in p-csgD to repress csgD transcription. As expected, p-csgD repression by MqsA resulted in reduced transcription from CsgD-regulated curli genes csgA and csgB (encoding the major and minor curlin subunits, respectively). Curli production was reduced in colonies and in planktonic cells upon MqsA production. Hence, MqsA directly represses p-csgD and thereby influences curli formation. This demonstrates that TA systems can impact overall cell physiology by fine-tuning cellular stress responses

Slaying the last unicorn: discovery of histones in the microalga Nanochlorum eucaryotum

Histones are the principal constituents of eukaryotic chromatin. The four core histones (H2A, H2B, H3 and H4) are conserved across sequenced eukaryotic genomes and therefore thought to be universal to eukaryotes. In the early 1980s, however, a series of biochemical investigations failed to find evidence for histones or nucleosomal structures in the microscopic green alga Nanochlorum eucaryotum. If true, derived histone loss in this lineage would constitute an exceptional case that might help us further understand the principles governing eukaryotic gene regulation. To substantiate these earlier reports of histone loss in N. eucaryotum, we sequenced, assembled and quantified its transcriptome. Following a systematic search for histone-fold domains in the assembled transcriptome, we detect orthologues to all four core histones. We also find histone mRNAs to be highly expressed, comparable to the situation in other eukaryotes. Finally, we obtain characteristic protection patterns when N. eucaryotum chromatin is subjected to micrococcal nuclease digestion, indicating widespread formation of nucleosomal complexes in vivo. We conclude that previous reports of missing histones in N. eucaryotum were mistaken. By all indications, Nanochlorum eucaryotum has histone-based chromatin characteristic of most eukaryotes

Fitness landscape of a dynamic RNA structure

RNA structures are dynamic. As a consequence, mutational effects can be hard to rationalize with reference to a single static native structure. We reasoned that deep mutational scanning experiments, which couple molecular function to fitness, should capture mutational effects across multiple conformational states simultaneously. Here, we provide a proof-of-principle that this is indeed the case, using the self-splicing group I intron from Tetrahymena thermophila as a model system. We comprehensively mutagenized two 4-bp segments of the intron that come together to form the P1 extension (P1ex) helix at the 5’ splice site and, following cleavage at the 5’ splice site, dissociate to allow formation of an alternative helix (P10) at the 3’ splice site. Using an in vivo reporter system that couples splicing activity to fitness in E. coli, we demonstrate that fitness is driven jointly by constraints on P1ex and P10 formation and that patterns of epistasis can be used to infer the presence of intramolecular pleiotropy. Importantly, using a machine learning approach that allows quantification of mutational effects in a genotype-specific manner, we show that the fitness landscape can be deconvoluted to implicate P1ex or P10 as the effective genetic background in which molecular fitness is compromised or enhanced. Our results highlight deep mutational scanning as a tool to study transient but important conformational states, with the capacity to provide critical insights into the evolution and evolvability of RNAs as dynamic ensembles. Our findings also suggest that, in the future, deep mutational scanning approaches might help us to reverse-engineer dynamic interactions and critical non-native states from a single fitness landscape

Mechanistic and evolutionary insights from the reciprocal promiscuity of two pyridoxal phosphate-dependent enzymes.

Enzymes that utilize the cofactor pyridoxal 5′-phosphate play essential roles in amino acid metabolism in all organisms. The cofactor is used by proteins that adopt at least five different folds, which raises questions about the evolutionary processes that might explain the observed distribution of functions among folds. In this study, we show that a representative of fold type III, the Escherichia coli alanine racemase (ALR), is a promiscuous cystathionine β-lyase (CBL). Furthermore, E. coli CBL (fold type I) is a promiscuous alanine racemase. A single round of error-prone PCR and selection yielded variant ALR(Y274F), which catalyzes cystathionine β-elimination with a near-native Michaelis constant (Km = 3.3 mM) but a poor turnover number (kcat ≈10 h−1). In contrast, directed evolution also yielded CBL(P113S), which catalyzes L-alanine racemization with a poor Km (58 mM) but a high kcat (22 s−1). The structures of both variants were solved in the presence and absence of the L-alanine analogue, (R)-1-aminoethylphosphonic acid. As expected, the ALR active site was enlarged by the Y274F substitution, allowing better access for cystathionine. More surprisingly, the favorable kinetic parameters of CBL(P113S) appear to result from optimizing the pKa of Tyr-111, which acts as the catalytic acid during L-alanine racemization. Our data emphasize the short mutational routes between the functions of pyridoxal 5′-phosphate-dependent enzymes, regardless of whether or not they share the same fold. Thus, they confound the prevailing model of enzyme evolution, which predicts that overlapping patterns of promiscuity result from sharing a common multifunctional ancestor

de novo synthesis of a bacterial toxin/antitoxin system

The prevalence of toxin/antitoxin (TA) systems in almost all genomes suggests they evolve rapidly. Here we show that an antitoxin from a type V system (GhoS, an endoribonuclease specific for the mRNA of the toxin GhoT) can be converted into a novel toxin (ArT) simply by adding two mutations. In contrast to GhoS, which increases growth, the new toxin ArT decreases growth dramatically in Escherichia coli. Transmission electron microscopy analysis revealed that the nucleoid in ArT-producing cells is concentrated and appears hollow. Whole-transcriptome profiling revealed ArT cleaves 50 additional transcripts, which shows that the endoribonuclease activity of GhoS has been broadened as it was converted to ArT. Furthermore, we evolved an antitoxin for the new toxin ArT from two unrelated antitoxin templates, the protein-based antitoxin MqsA and RNA-based antitoxin ToxI, and showed that the evolved MqsA and ToxI variants are able to counteract the toxicity of ArT. In addition, the de novo TA system was found to increase persistence, a phenotype commonly associated with TA systems. Therefore, toxins and antitoxins from disparate systems can be interconverted

Reversing methanogenesis to capture methane for liquid biofuel precursors

Background Energy from remote methane reserves is transformative; however, unintended release of this potent greenhouse gas makes it imperative to convert methane efficiently into more readily transported biofuels. No pure microbial culture that grows on methane anaerobically has been isolated, despite that methane capture through anaerobic processes is more efficient than aerobic ones. Results Here we engineered the archaeal methanogen Methanosarcina acetivorans to grow anaerobically on methane as a pure culture and to convert methane into the biofuel precursor acetate. To capture methane, we cloned the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable organism, anaerobic methanotrophic archaeal population 1 (ANME-1) from a Black Sea mat, into M. acetivorans to effectively run methanogenesis in reverse. Starting with low-density inocula, M. acetivorans cells producing ANME-1 Mcr consumed up to 9 ± 1 % of methane (corresponding to 109 ± 12 µmol of methane) after 6 weeks of anaerobic growth on methane and utilized 10 mM FeCl3 as an electron acceptor. Accordingly, increases in cell density and total protein were observed as cells grew on methane in a biofilm on solid FeCl3. When incubated on methane for 5 days, high-densities of ANME-1 Mcr-producing M. acetivorans cells consumed 15 ± 2 % methane (corresponding to 143 ± 16 µmol of methane), and produced 10.3 ± 0.8 mM acetate (corresponding to 52 ± 4 µmol of acetate). We further confirmed the growth on methane and acetate production using 13C isotopic labeling of methane and bicarbonate coupled with nuclear magnetic resonance and gas chromatography/mass spectroscopy, as well as RNA sequencing. Conclusions We anticipate that our metabolically-engineered strain will provide insights into how methane is cycled in the environment by Archaea as well as will possibly be utilized to convert remote sources of methane into more easily transported biofuels via acetate