No abstract

Uniformitarian assumptions underlie the oldest evidence for living organisms on Earth, the distinct isotope fractionation between inorganic and organic carbon. Aside from a handful of compelling deviations, the 13C/12C isotopic mean of preserved organic carbon (δ13Corg) has remained remarkably unchanged through time. RuBisCO is the principal carboxylase/oxygenase biomolecular component that is thought to primarily account for the generation of these distinct carbon isotopic signals. However, it is difficult to reconcile a mostly unchanging mean δ13Corg with several known factors that can affect the isotope fractionation of RuBisCO, such as atmospheric composition and the amino acid composition of the enzyme itself, which have each changed markedly over Earth history. Here we report the resurrection and genetic incorporation of a Precambrian-age, Form IB RuBisCO in a modern cyanobacterial host. The isotopic composition of biomass relative to CO2 (ϵp) in ancestral and control strains were much greater when grown under Precambrian CO2 concentrations compared to modern ambient levels, but displaying values within a nominal envelope of modern-day RuBisCO IB enzyme variants. We infer that these isotopic differences derive indirectly from the decreased fitness of the AncIB strain, which includes diminished growth capacity and total cell RuBisCO activity. We argue that to answer the greatest questions of deep-time paleobiology, ancient biogeochemical signals should be reproduced in the laboratory through the synthesis of the geologic record with experimentally-derived constraints on underlying ancient molecular biology.

Carbon isotope biosignatures preserved in the Precambrian geologic record are primarily interpreted to reflect ancient cyanobacterial carbon fixation catalyzed by Form I RuBisCO enzymes. The average range of isotopic biosignatures generally follows that produced by extant cyanobacteria. However, this observation is difficult to reconcile with several environmental (e.g., temperature, pH, and CO2 concentrations) and physiological factors that likely would have differed during the Precambrian and can produce fractionation variability in contemporary organisms that meets or exceeds that observed in the geologic record. To test a range of genetic and environmental factors that may have impacted ancient carbon isotope biosignatures, we engineered a mutant strain of the model cyanobacterium Synechococcus elongatus PCC 7942 that overexpresses RuBisCO and characterized the resultant physiological and isotope fractionation effects. We specifically investigated how both increased atmospheric CO2 concentrations and RuBisCO regulation influence cell growth, oxygen evolution rate, and carbon isotope discrimination in cyanobacteria. We found that >2% CO2 increases the growth rate of wild-type and mutant strains, and that the pool of active RuBisCO enzyme increases with increased expression. At elevated CO2, carbon isotope discrimination (ϵp) is increased by ~8 per mille, whereas RuBisCO overexpression does not significantly affect isotopic discrimination at all tested CO2 concentrations. Our results show that understanding the environmental factors that impact RuBisCO regulation, physiology, and evolution is crucial for reconciling microbially driven carbon isotope fractionation with the geologic record of organic and inorganic carbon isotope signatures.

Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted.

Carbon isotope biosignatures preserved in the Precambrian geologic record are primarily interpreted to reflect ancient cyanobacterial carbon fixation catalyzed by Form I RuBisCO enzymes. The average range of isotopic biosignatures generally follows that produced by extant cyanobacteria. However, this observation is difficult to reconcile with several environmental (e.g., temperature, pH, and CO 2 concentrations), molecular, and physiological factors that likely would have differed during the Precambrian and can produce fractionation variability in contemporary organisms that meets or exceeds that observed in the geologic record. To test a specific range of genetic and environmental factors that may impact ancient carbon isotope biosignatures, we engineered a mutant strain of the model cyanobacterium Synechococcus elongatus PCC 7942 that overexpresses RuBisCO across varying atmospheric CO 2 concentrations. We hypothesized that changes in RuBisCO expression would impact the net rates of intracellular CO 2 fixation versus CO 2 supply, and thus whole-cell carbon isotope discrimination. In particular, we investigated the impacts of RuBisCO overexpression under changing CO 2 concentrations on both carbon isotope biosignatures and cyanobacterial physiology, including cell growth and oxygen evolution rates. We found that an increased pool of active RuBisCO does not significantly affect the 13 C/ 12 C isotopic discrimination (ε p ) at all tested CO 2 concentrations, yielding ε p of ≈ 23‰ for both wild-type and mutant strains at elevated CO 2 . We therefore suggest that expected variation in cyanobacterial RuBisCO expression patterns should not confound carbon isotope biosignature interpretation.A deeper understanding of environmental, evolutionary, and intracellular factors that impact cyanobacterial physiology and isotope discrimination is crucial for reconciling microbially driven carbon biosignatures with those preserved in the geologic record.