Selective RNA Processing and Stabilization are Multi-Layer and Stoichiometric Regulators of Gene Expression in Escherichia coli.

Selective RNA processing and stabilization (SRPS) facilitates the differential expression of multiple genes in polycistronic operons. However, how the coordinated actions of SRPS-related enzymes affect stoichiometric regulation remains unclear. In the present study, the first genome-wide targetome analysis is reported of these enzymes in Escherichia coli, at a single-nucleotide resolution. A strictly linear relationship is observed between the RNA pyrophosphohydrolase processing ratio and scores assigned to the first three nucleotides of the primary transcript. Stem-loops associated with PNPase targetomes exhibit a folding free energy that is negatively correlated with the termination ratio of PNPase at the 3' end. More than one-tenth of the RNase E processing sites in the 5'-untranslated regions（UTR） form different stem-loops that affect ribosome-binding and translation efficiency. The effectiveness of the SRPS elements is validated using a dual-fluorescence reporter system. The findings highlight a multi-layer and quantitative regulatory method for optimizing the stoichiometric expression of genes in bacteria and promoting the application of SRPS in synthetic biology.

Long-Read Metagenomics of Marine Microbes Reveals Diversely Expressed Secondary Metabolites.

Microbial secondary metabolites play crucial roles in microbial competition, communication, resource acquisition, antibiotic production, and a variety of other biotechnological processes. The retrieval of full-length BGC (biosynthetic gene cluster) sequences from uncultivated bacteria is difficult due to the technical constraints of short-read sequencing, making it impossible to determine BGC diversity. Using long-read sequencing and genome mining, 339 mainly full-length BGCs were recovered in this study, illuminating the wide range of BGCs from uncultivated lineages discovered in seawater from Aoshan Bay, Yellow Sea, China. Many extremely diverse BGCs were discovered in bacterial phyla such as Proteobacteria, Bacteroidota, Acidobacteriota, and Verrucomicrobiota as well as the previously uncultured archaeal phylum "Candidatus Thermoplasmatota." The data from metatranscriptomics showed that 30.1% of secondary metabolic genes were being expressed, and they also revealed the expression pattern of BGC core biosynthetic genes and tailoring enzymes. Taken together, our results demonstrate that long-read metagenomic sequencing combined with metatranscriptomic analysis provides a direct view into the functional expression of BGCs in environmental processes. IMPORTANCE Genome mining of metagenomic data has become the preferred method for the bioprospecting of novel compounds by cataloguing secondary metabolite potential. However, the accurate detection of BGCs requires unfragmented genomic assemblies, which have been technically difficult to obtain from metagenomes until recently with new long-read technologies. We used high-quality metagenome-assembled genomes generated from long-read data to determine the biosynthetic potential of microbes found in the surface water of the Yellow Sea. We recovered 339 highly diverse and mostly full-length BGCs from largely uncultured and underexplored bacterial and archaeal phyla. Additionally, we present long-read metagenomic sequencing combined with metatranscriptomic analysis as a potential method for gaining access to the largely underutilized genetic reservoir of specialized metabolite gene clusters in the majority of microbes that are not cultured. The combination of long-read metagenomic and metatranscriptomic analyses is significant because it can more accurately assess the mechanisms of microbial adaptation to the environment through BGC expression based on metatranscriptomic data.

The Transcriptional Repressor PerR Senses Sulfane Sulfur by Cysteine Persulfidation at the Structural Zn2+ Site in Synechococcus sp. PCC7002.

Cyanobacteria can perform both anoxygenic and oxygenic photosynthesis, a characteristic which ensured that these organisms were crucial in the evolution of the early Earth and the biosphere. Reactive oxygen species (ROS) produced in oxygenic photosynthesis and reactive sulfur species (RSS) produced in anoxygenic photosynthesis are closely related to intracellular redox equilibrium. ROS comprise superoxide anion (O2●-), hydrogen peroxide (H2O2), and hydroxyl radicals (●OH). RSS comprise H2S and sulfane sulfur (persulfide, polysulfide, and S8). Although the sensing mechanism for ROS in cyanobacteria has been explored, that of RSS has not been elucidated. Here, we studied the function of the transcriptional repressor PerR in RSS sensing in Synechococcus sp. PCC7002 (PCC7002). PerR was previously reported to sense ROS; however, our results revealed that it also participated in RSS sensing. PerR repressed the expression of prxI and downregulated the tolerance of PCC7002 to polysulfide (H2Sn). The reporter system indicated that PerR sensed H2Sn. Cys121 of the Cys4:Zn2+ site, which contains four cysteines (Cys121, Cys124, Cys160, and Cys163) bound to one zinc atom, could be modified by H2Sn to Cys121-SSH, as a result of which the zinc atom was released from the site. Moreover, Cys19 could also be modified by polysulfide to Cys19-SSH. Thus, our results reveal that PerR, a representative of the Cys4 zinc finger proteins, senses H2Sn. Our findings provide a new perspective to explore the adaptation strategy of cyanobacteria in Proterozoic and contemporary sulfurization oceans.

Synechococcus sp. PCC7002 Uses Peroxiredoxin to Cope with Reactive Sulfur Species Stress.

Cyanobacteria are a widely distributed group of microorganisms in the ocean, and they often need to cope with the stress of reactive sulfur species, such as sulfide and sulfane sulfur. Sulfane sulfur refers to the various forms of zero-valent sulfur, including persulfide, polysulfide, and element sulfur (S8). Although sulfane sulfur participates in signaling transduction and resistance to reactive oxygen species in cyanobacteria, it is toxic at high concentrations and induces sulfur stress, which has similar effects to oxidative stress. In this study, we report that Synechococcus sp. PCC7002 uses peroxiredoxin to cope with the stress of cellular sulfane sulfur. Synechococcus sp. PCC7002 contains six peroxiredoxins, and all were induced by S8. Peroxiredoxin I (PrxI) reduced S8 to H2S by forming a disulfide bond between residues Cys53 and Cys153 of the enzyme. A partial deletion strain of Synechococcus sp. PCC7002 with decreased copy numbers of the prxI gene was more sensitive to S8 than was the wild type. Thus, peroxiredoxin is involved in maintaining the homeostasis of cellular sulfane sulfur in cyanobacteria. Given that peroxiredoxin evolved before the occurrence of O2 on Earth, its original function could have been to cope with reactive sulfur species stress, and that function has been preserved. IMPORTANCE Cyanobacteria are the earliest microorganisms that perform oxygenic photosynthesis, which has played a key role in the evolution of life on Earth, and they are the most important primary producers in the modern oceans. The cyanobacterium Synechococcus sp. PCC7002 uses peroxiredoxin to reduce high levels of sulfane sulfur. That function is possibly the original role of peroxiredoxin, as the enzyme evolved before the appearance of O2 on Earth. The preservation of the reduction of sulfane sulfur by peroxiredoxin5-type peroxiredoxins may offer cyanobacteria an advantage in the complex environment of the modern oceans.

Temperature Rise Increases the Bioavailability of Marine Synechococcus-Derived Dissolved Organic Matter.

Synechococcus is one group of main primary producers and plays a key role in oceanic carbon fixation and transformation. To explore how the temperature rise affects the bioavailability of Synechococcus-derived dissolved organic matter (SOM) and whether this effect would be altered by the involvement of heterotrophic bacteria, we compared the optical and molecular properties of the SOM of axenic Synechococcus sp. PCC7002 culture (Syn) to that with associated heterotrophic bacteria (SynB) under 15, 18, and 21°C growth temperatures at exponential and decay growth phases. Our results showed that the temperature rise increased the bioavailability of the SOM of both Syn and SynB cultures by lowering the proportion of the hydrogen-poor and double-bond structure-rich humus-like components and highly unsaturated substances, as indicated by the increase of spectral slope ratio (S R ) and biological index (BIX) and decrease of humification index (HIX). Moreover, the involvement of heterotrophic bacteria modified the Synechococcus-derived SOM, together with its intracellular dissolved organic matter (DOM) excludes, lowering the SOM bioavailability. Our results indicated that the warming in climate change scenario may enhance the bioavailability of the Synechococcus-derived SOM although it may be tempered by the involvement of heterotrophic bacteria, providing an insight for preservation of the organic carbon pool in global oceans.

Croceivirga litoralis sp. nov., isolated from coastal surface water, and reclassification of Muricauda lutea as Croceivirga lutea comb. nov.

A novel Gram-stain-negative, aerobic, yellow-pigmented bacterium was isolated from seawater of Aoshan Bay, and designated as strain ASW18T. Strain ASW18T was a long-rod-shaped bacterium without flagellum and lacked gliding ability. Based on 16S rRNA gene phylogeny, strain ASW18T showed the closest relationship to Croceivirga radicis MCCC 1A06690T, with a sequence similarity of 97.0 %. Strain ASW18T was able to grow at 25-40 °C, at pH 5.5-9.5 and with 0.5-9 % (w/v) NaCl. The genomic DNA G+C content of strain ASW18T was 37.3 %. The predominant cellular fatty acids of strain ASW18T were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 1 G. The major polar lipids were phosphatidylethanolamine, phosphatidyldimethylethanolamine, an aminolipid and three unidentified lipids. The respiratory quinone of strain ASW18T was menaquinone with six isoprene units (MK-6). Based on the present polyphasic analysis, strain ASW18T represents a novel species of the genus Croceivirga, for which the name Croceivirga litoralis sp. nov. is proposed; the type strain is ASW18T (=MCCC 1K04203T=KCTC 72852T). In addition, it is also proposed that Muricauda lutea should be reclassified as Croceivirga lutea comb. nov.; the type strain is CSW06T (=CGMCC 1.15761T=JCM 31455T=KCTC 52375T=MCCC 1K03195T).

Synechococcus sp. Strain PCC7002 Uses Sulfide:Quinone Oxidoreductase To Detoxify Exogenous Sulfide and To Convert Endogenous Sulfide to Cellular Sulfane Sulfur.

Eutrophication and deoxygenation possibly occur in coastal waters due to excessive nutrients from agricultural and aquacultural activities, leading to sulfide accumulation. Cyanobacteria, as photosynthetic prokaryotes, play significant roles in carbon fixation in the ocean. Although some cyanobacteria can use sulfide as the electron donor for photosynthesis under anaerobic conditions, little is known on how they interact with sulfide under aerobic conditions. In this study, we report that Synechococcus sp. strain PCC7002 (PCC7002), harboring an sqr gene encoding sulfide:quinone oxidoreductase (SQR), oxidized self-produced sulfide to S0, present as persulfide and polysulfide in the cell. The Δsqr mutant contained less cellular S0 and had increased expression of key genes involved in photosynthesis, but it was less competitive than the wild type in cocultures. Further, PCC7002 with SQR and persulfide dioxygenase (PDO) oxidized exogenous sulfide to tolerate high sulfide levels. Thus, SQR offers some benefits to cyanobacteria even under aerobic conditions, explaining the common presence of SQR in cyanobacteria.IMPORTANCE Cyanobacteria are a major force for primary production via oxygenic photosynthesis in the ocean. A marine cyanobacterium, PCC7002, is actively involved in sulfide metabolism. It uses SQR to detoxify exogenous sulfide, enabling it to survive better than its Δsqr mutant in sulfide-rich environments. PCC7002 also uses SQR to oxidize endogenously generated sulfide to S0, which is required for the proper expression of key genes involved in photosynthesis. Thus, SQR has at least two physiological functions in PCC7002. The observation provides a new perspective for the interplays of C and S cycles.

Cupriavidus necator H16 Uses Flavocytochrome c Sulfide Dehydrogenase To Oxidize Self-Produced and Added Sulfide.

Production of sulfide (H2S, HS-, and S2-) by heterotrophic bacteria during aerobic growth is a common phenomenon. Some bacteria with sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) can oxidize self-produced sulfide to sulfite and thiosulfate, but other bacteria without these enzymes release sulfide into the medium, from which H2S can volatilize into the gas phase. Here, we report that Cupriavidus necator H16, with the fccA and fccB genes encoding flavocytochrome c sulfide dehydrogenases (FCSDs), also oxidized self-produced H2S. A mutant in which fccA and fccB were deleted accumulated and released H2S. When fccA and fccB were expressed in Pseudomonas aeruginosa strain Pa3K with deletions of its sqr and pdo genes, the recombinant rapidly oxidized sulfide to sulfane sulfur. When PDO was also cloned into the recombinant, the recombinant with both FCSD and PDO oxidized sulfide to sulfite and thiosulfate. Thus, the proposed pathway is similar to the pathway catalyzed by SQR and PDO, in which FCSD oxidizes sulfide to polysulfide, polysulfide spontaneously reacts with reduced glutathione (GSH) to produce glutathione persulfide (GSSH), and PDO oxidizes GSSH to sulfite, which chemically reacts with polysulfide to produce thiosulfate. About 20.6% of sequenced bacterial genomes contain SQR, and only 3.9% contain FCSD. This is not a surprise, since SQR is more efficient in conserving energy because it passes electrons from sulfide oxidation into the electron transport chain at the quinone level, while FCSD passes electrons to cytochrome c The transport of electrons from the latter to O2 conserves less energy. FCSDs are grouped into three subgroups, well conserved at the taxonomic level. Thus, our data show the diversity in sulfide oxidation by heterotrophic bacteria.IMPORTANCE Heterotrophic bacteria with SQR and PDO can oxidize self-produced sulfide and do not release H2S into the gas phase. C. necator H16 has FCSD but not SQR, and it does not release H2S. We confirmed that the bacterium used FCSD for the oxidation of self-produced sulfide. The bacterium also oxidized added sulfide. The common presence of SQRs, FCSDs, and PDOs in heterotrophic bacteria suggests the significant role of heterotrophic bacteria in sulfide oxidation, participating in sulfur biogeochemical cycling. Further, FCSDs have been identified in anaerobic photosynthetic bacteria and chemolithotrophic bacteria, but their physiological roles are unknown. We showed that heterotrophic bacteria use FCSDs to oxidize self-produced sulfide and extraneous sulfide, and they may be used for H2S bioremediation.

Snake venom-like waprin from the frog of Ceratophrys calcarata contains antimicrobial function.

A 255-bp cDNA encoding an 84-amino acid residue (aa) precursor protein containing 8 half-cysteines was cloned from the skin of the frog, Ceratophrys calcarata. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 63-aa mature peptide with amino acid sequence, NVTPATKPTPSKPGYCRVMDELILCPDPPLSKDLCKNDSDCPGAQKCCYRTCIMQCLPPIFRE. The mature was named ceratoxin. Ceratoxin shares significant sequence similarity with the toxin family of waprins containing the whey acidic protein-type (WAP) four-disulfide core domain found in snake venoms. Antimicrobial and trypsin-inhibitory abilities of recombinant ceratoxin were tested. Recombinant ceratoxin showed strong antimicrobial activities against wide spectrum of microorganisms including Gram-negative and Gram-positive bacteria and fungi. It had no serine protease-inhibitory activity. The current results suggested that the snake venom-like waprin with antimicrobial activities in the frog skin plays a role in innate immunity.