Temperate viruses can become dormant in their host cells, a process called lysogeny. In every infection, such viruses need to decide between the lytic and the lysogenic cycles, i.e., whether to replicate and lyse their host or to lysogenize and keep the host viable. Here we show that viruses (phages) of the spBeta group use a small-molecule communication system to coordinate lysis-lysogeny decisions. During infection of its Bacillus host cell, the phage produces a 6aa communication peptide that is released to the medium. In subsequent infections, progeny phages measure the concentration of this peptide and lysogenize if the concentration is sufficiently high. We found that different phages encode different versions of the communication peptide, demonstrating a phage-specific peptide communication code for lysogeny decisions. We termed this communication system the “arbitrium” system, and further show that it is encoded by 3 phage genes: aimP, producing the peptide, aimR, the intracellular peptide receptor, and aimX, a negative regulator of lysogeny. The arbitrium system enables an offspring phage to communicate with its predecessors, i.e., to estimate the amount of recent prior infections and hence decide whether to employ the lytic or lysogenic cycle.

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

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We study a modified two-dimensional dilaton gravity theory which is exactly solvable in the semiclassical approximation including back-reaction. The vacuum solutions of this modified theory are asymptotically flat static space-times. Infalling matter forms a black hole if its energy is above a certain threshold. The black hole singularity is initially hidden behind a timelike apparent horizon. As the black hole evaporates by emitting Hawking radiation, the singularity meets the shrinking horizon in finite retarded time to become naked. A natural boundary condition exists at the naked singularity such that for general infalling matter-configurations the evaporating black hole geometries can be matched continuously to a unique static end-state geometry. This end-state geometry is asymptotically flat at its right spatial infinity, while its left spatial infinity is a semi-infinite throat extending into the strong coupling region.

CO2 is converted into biomass almost solely by the enzyme rubisco. The poor carboxylation properties of plant rubiscos have led to efforts that made it the most kinetically characterized enzyme, yet these studies focused on < 5% of its natural diversity. Here, we searched for fast‐carboxylating variants by systematically mining genomic and metagenomic data. Approximately 33,000 unique rubisco sequences were identified and clustered into ≈ 1,000 similarity groups. We then synthesized, purified, and biochemically tested the carboxylation rates of 143 representatives, spanning all clusters of form‐II and form‐II/III rubiscos. Most variants (> 100) were active in vitro, with the fastest having a turnover number of 22 ± 1 s−1—sixfold faster than the median plant rubisco and nearly twofold faster than the fastest measured rubisco to date. Unlike rubiscos from plants and cyanobacteria, the fastest variants discovered here are homodimers and exhibit a much simpler folding and activation kinetics. Our pipeline can be utilized to explore the kinetic space of other enzymes of interest, allowing us to get a better view of the biosynthetic potential of the biosphere.

Toxin-antitoxin (TA) modules, composed of a toxic protein and a counteracting antitoxin, play important roles in bacterial physiology. We examined the experimental insertion of 1.5 million genes from 388 microbial genomes into an Escherichia coli host using over 8.5 million random clones. This revealed hundreds of genes (toxins) that could only be cloned when the neighboring gene (antitoxin) was present on the same clone. Clustering of these genes revealed novel TA families widespread in bacterial genomes, some of which deviate from the classical characteristics previously described for such modules. Introduction of these genes into E. coli validated that the toxin toxicity is mitigated by the antitoxin. Infection experiments with T7 phage showed that two of the new modules can provide resistance against phage. Moreover, our experiments revealed an 'anti-defense' protein in phage T7 that neutralizes phage resistance. Our results expose active fronts in the arms race between bacteria and phage.

The yeast mitochondrial and cytosolic isoenzymes of fumarase, which are encoded by a single nuclear gene (FUMI), follow a unique mechanism of protein subcellular localization and distribution. Translation of all FUMI messages initiates only from the 5'-proximal AUG codon and results in a single translation product that contains the targeting sequence located within the first 32 amino acids of the precursor. All fumarase molecules synthesized in the cell are processed by the mitochondrial matrix signal peptidase; nevertheless, most of the enzyme (80 to 90%) ends up in the cytosol. The translocation and processing of fumarase are cotranslational. We suggest that in Saccharomyces cerevisiae, the single type of initial translation product of the FUMI gene is first partially translocated, and then a subset of these molecules continues to be fully translocated into the organelle, whereas the rest are folded into an import-incompetent state and are released by the retrograde movement of fumarase into the cytosol. (14,34). The MOD5 gene encodes mRNAs that contain two in-frame AUGs, either of which can be used for the initiation of translation. This fact results in two protein species which differ in size and, as in the preceding example, the peptide extension in one of the protein species determines the final location of the longer protein.Recently, it was proposed that the subcellular distribution of rat liver fumarase may also be determined by the initiation of translation at two alternative AUG codons on a single mRNA species (35, 36).In Saccharomyces cerevisiae, a single nuclear gene (FUM]) has been shown to encode both cytosolic and mitochondrial fumarase isoenzymes (41). One study claimed that these isoenzymes have molecular weights roughly estimated at 48,000 and 53,000 for the mitochondrial and cytosolic species, respectively (2), while a more recent study detected only a single size of fumarase molecules in S. cerevisiae (20 FUMJ-lacZ fusions lacking 17 amino acids at the amino terminus are localized exclusively in the cytosol (27, 41).We previously suggested that the translation of both mitochondrial and cytosolic fumarases initiates at the same AUG, consistent with the production of both isoenzymes from a GAL10 promoter situated upstream of a promoterless FUM1 gene (27). Our suggestion also explains the initial observations of Wu and Tzagoloff (42) showing that the vast majority of FUM1 transcripts include the AUG upstream of the proposed signal peptide. This AUG is expected to be an efficient codon for translation initiation, since it is the first from the 5' end of the FUM1 mRNA and since the surrounding nucleotides form a sequence which is similar to the yeast translation initiation consensus sequence (44). In particular, the third nucleotide upstream of this AUG codon (-3 position) is an A, and the first following this AUG codon (+4 position) is a U; these have been identified as optimal for initiation by yeast ribosomes (44). The possibility of a single FUM1 translation product differs from the mode...

Effects of various nutritional and environmental factors on the accumulation of organic acids (mainly L-malic acid) by the filamentous fungus Aspergillus flavus were studied in a 16-L stirred fermentor. Improvement of the molar yield (moles acid produced per moles glucose consumed) of L-malic acid was obtained mainly by increasing the agitation rate (to 350 rpm) and the Fe(z+) ion concentration (to 12 mg/L) and by lowering the nitrogen (to 271 mg/L) and phosphate concentrations (to 1.5 mM) in the medium. These changes resulted in molar yields for L-malic acid and total C(4) acids (L-malic, succinic, and fumaric acids) of 128 and 155%, respectively. The high molar yields obtained (above 100%) are additional evidence for the operation of part of the reductive branch of the tricarboxylic acid cycle in L-malic acid accumulation by A. flavus. The fermentation conditions developed using the above mentioned factors and 9% CaCO(3) in the medium resulted in a high concentration (113 g/L L-malic acid from 120 g/L glucose utilized) and a high overall productivity (0.59 g/L h) of L-malic acid. These changes in acid accumulation coincide with increases in the activities of NAD(+)-malate dehydrogenase, fumarase, and citrate synthase.