Cbl upregulates cysH for hydrogen sulfide production in Aeromonas veronii

Endogenous hydrogen sulfide (H2S) is generated in many metabolism pathways, and has been recognized as a second messenger against antibiotics and reactive oxygen species (ROS). In Aeromonas veronii, Small Protein B (SmpB) plays an important role in resisting stress. The absence of smpB could trigger sulfate assimilation pathway to adapt the nutrient deficiency, of which was mediated by up-regulation of cbl and cys genes and followed with enhancing H2S production. To figure out the mutual regulations of cbl and cys genes, a series of experiments were performed. Compared with the wild type, cysH was down-regulated significantly in cbl deletion by qRT-PCR. The fluorescence analysis further manifested that Cbl had a positive regulatory effect on the promoter of cysJIH. Bacterial one-hybrid analysis and electrophoretic mobility shift assay (EMSA) verified that Cbl bound with the promoter of cysJIH. Collectively, the tolerance to adversity could be maintained by the production of H2S when SmpB was malfunctioned, of which the activity of cysJIH promoter was positively regulated by upstream Cbl protein. The outcomes also suggested the enormous potentials of Aeromonas veronii in environmental adaptability.

A Competitive O-Acetylserine Sulfhydrylase Inhibitor Modulates the Formation of Cysteine Synthase Complex

Cysteine is the main precursor of sulfur-containing biological molecules in bacteria and contributes to the control of the cell redox state. Hence, this amino acid plays an essential role in microbial survival and pathogenicity and the reductive sulfate assimilation pathway is considered a promising target for the development of new antibacterials. Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS-A), the enzymes catalyzing the last two steps of cysteine biosynthesis, engage in the formation of the cysteine synthase (CS) complex. The interaction between SAT and OASS-A finely tunes cysteine homeostasis, and the development of inhibitors targeting either protein–protein interaction or the single enzymes represents an attractive strategy to undermine bacterial viability. Given the peculiar mode of interaction between SAT and OASS-A, which exploits the insertion of SAT C-terminal sequence into OASS-A active site, we tested whether a recently developed competitive inhibitor of OASS-A exhibited any effect on the CS stability. Through surface plasmon resonance spectroscopy, we (i) determined the equilibrium constant for the Salmonella Typhimurium CS complex formation and (ii) demonstrated that the inhibitor targeting OASS-A active site affects CS complex formation. For comparison, the Escherichia coli CS complex was also investigated, with the aim of testing the potential broad-spectrum activity of the candidate antimicrobial compound.

The Recovery from Sulfur Starvation is Independent from the mRNA Degradation Initiation Enzyme PARN in Arabidopsis

When plants are exposed to sulfur limitation, they upregulate the sulfate assimilation pathway at the expense of growth-promoting measures. Upon cessation of the stress, however, protective measures are deactivated, and growth is restored. In accordance with these findings, transcripts of sulfur-deficiency marker genes are rapidly degraded when starved plants are resupplied with sulfur. Yet it remains unclear which enzymes are responsible for the degradation of transcripts during the recovery from starvation. In eukaryotes, mRNA decay is often initiated by the cleavage of poly(A) tails via deadenylases. As mutations in the poly(A) ribonuclease PARN have been linked to altered abiotic stress responses in Arabidopsis thaliana, we investigated the role of PARN in the recovery from sulfur starvation. Despite the presence of putative PARN-recruiting AU-rich elements in sulfur-responsive transcripts, sulfur-depleted PARN hypomorphic mutants were able to reset their transcriptome to pre-starvation conditions just as readily as wildtype plants. Currently, the subcellular localization of PARN is disputed, with studies reporting both nuclear and cytosolic localization. We detected PARN in cytoplasmic speckles and reconciled the diverging views in literature by identifying two PARN splice variants whose predicted localization is in agreement with those observations.

Environmental Calcium Initiates a Feed-Forward Signaling Circuit That Regulates Biofilm Formation and Rugosity inVibrio vulnificus

ABSTRACTPoor clinical outcomes (disfigurement, amputation, and death) and significant economic losses in the aquaculture industry can be attributed to the potent opportunistic human pathogenVibrio vulnificus.V. vulnificus, as well as the bivalves (oysters) it naturally colonizes, is indigenous to estuaries and human-inhabited coastal regions and must endure constantly changing environmental conditions as freshwater and seawater enter, mix, and exit the water column. Elevated cellular c-di-GMP levels trigger biofilm formation, but relatively little is known regarding the environmental signals that initiate this response. Here, we show that calcium is a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggers expression of thebrpextracellular polysaccharide that enhances biofilm formation. A transposon screen for the loss of calcium-inducedPbrpAexpression revealed CysD, an enzyme in the sulfate assimilation pathway. Targeted disruption of the pathway indicated that the production of a specific metabolic intermediate, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), was required for calcium-inducedPbrpAexpression and that PAPS was separately required for development of the physiologically distinct rugose phenotype. Thus, PAPS behaves as a second messenger inV. vulnificus. Moreover, c-di-GMP and BrpT (the activator ofbrpexpression) acted in concert to bias expression of the sulfate assimilation pathway toward PAPS and c-di-GMP accumulation, establishing a feed-forward regulatory loop to boostbrpexpression. Thus, this signaling network links extracellular calcium and sulfur availability to the intracellular second messengers PAPS and c-di-GMP in the regulation ofV. vulnificusbiofilm formation and rugosity, survival phenotypes underpinning its evolution as a resilient environmental organism.IMPORTANCEThe second messenger c-di-GMP is a key regulator of bacterial physiology. TheV. vulnificusgenome encodes nearly 100 proteins predicted to make, break, and bind c-di-GMP. However, relatively little is known regarding the environmental signals that regulate c-di-GMP levels and biofilm formation inV. vulnificus. Here, we identify calcium as a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggersbrp-mediated biofilm formation. We show that PAPS, a metabolic intermediate of the sulfate assimilation pathway, acts as a second messenger linking environmental calcium and sulfur source availability to the production of another intracellular second messenger (c-di-GMP) to regulate biofilm and rugose colony formation, developmental pathways that are associated with environmental persistence and efficient bivalve colonization by this potent human pathogen.

Recent Developments on the Origin and Nature of Reductive Sulfurous Off-Odours in Wine

Reductive sulfurous off-odors are still one of the main reasons for rejecting wines by consumers. In 2008 at the International Wine Challenge in London, approximately 6% of the more than 10,000 wines presented were described as faulty. Twenty-eight percent were described as faulty because they presented “reduced characters” similar to those presented by “cork taint” and in nearly the same portion. Reductive off-odors are caused by low volatile sulfurous compounds. Their origin may be traced back to the metabolism of the microorganisms (yeasts and lactic acid bacteria) involved in the fermentation steps during wine making, often followed by chemical conversions. The main source of volatile sulfur compounds (VSCs) are precursors from the sulfate assimilation pathway (SAP, sometimes named as the “sulfate reduction pathway” SRP), used by yeast to assimilate sulfur from the environment and incorporate it into the essential sulfur-containing amino acids methionine and cysteine. Reductive off-odors became of increasing interest within the last few years, and the method to remove them by treatment with copper (II) salts (sulfate or citrate) is more and more questioned: The effectiveness is doubted, and after prolonged bottle storage, they reappear quite often. Numerous reports within the last few years and an ongoing flood of publications dealing with this matter reflect the importance of this problem. In a recent detailed review, almost all relevant aspects were discussed on a scientific data basis, and a “decision tree” was formulated to support winemakers handling this problem. Since we are dealing with a very complicated matter with a multitude of black spots still remaining, these advices can only be realized using specific equipment and special chemicals, not necessarily found in small wineries. The main problem in dealing with sulfurous compounds arises from the high variability of their reactivities. Sulfur is a metalloid with a large valence span across eight electron transformations from S (−II) up to S (+VI). This allows it to participate in an array of oxidation, reduction and disproportionation reactions, both abiotic and linked to microbial metabolism. In addition, sulfur is the element with the most allotropes and a high tendency to form chains and rings, with different stabilities of defined species and a high interconvertibility among each other. We suppose, there is simply a lack of knowledge of what is transferred during filling into bottles after fermentation and fining procedures. The treatment with copper (II) salts to remove sulfurous off-odors before filling rather increases instead of solving the problem. This paper picks up the abundant knowledge from recent literature and tries to add some aspects and observations, based on the assumption that the formation of polythionates, hitherto not taken into consideration, may explain some of the mystery of the re-appearance of reductive off-odors.

Sulfate assimilation pathway intermediate phosphoadenosine 5′-phosphosulfate acts as a signal molecule affecting production of curli fibres in Escherichia coli

The enterobacterium Escherichia coli can utilize a variety of molecules as sulfur sources, including cysteine, sulfate, thiosulfate and organosulfonates. An intermediate of the sulfate assimilation pathway, adenosine 5′-phosphosulfate (APS), also acts as a signal molecule regulating the utilization of different sulfur sources. In this work, we show that inactivation of the cysH gene, leading to accumulation of phosphoadenosine 5′-phosphosulfate (PAPS), also an intermediate of the sulfate assimilation pathway, results in increased surface adhesion and cell aggregation by activating the expression of the curli-encoding csgBAC operon. In contrast, curli production was unaffected by the inactivation of any other gene belonging to the sulfate assimilation pathway. Overexpression of the cysH gene downregulated csgBAC transcription, further suggesting a link between intracellular PAPS levels and curli gene expression. In addition to curli components, the Flu, OmpX and Slp proteins were also found in increased amounts in the outer membrane compartment of the cysH mutant; deletion of the corresponding genes suggested that these proteins also contribute to surface adhesion and cell surface properties in this strain. Our results indicate that, similar to APS, PAPS also acts as a signal molecule, albeit with a distinct mechanism and role: whilst APS regulates organosulfonate utilization, PAPS would couple availability of sulfur sources to remodulation of the cell surface, as part of a more global effect on cell physiology.

New Insights into Sulfur Metabolism in Yeasts as Revealed by Studies of Yarrowia lipolytica

ABSTRACTYarrowia lipolytica, located at the frontier of hemiascomycetous yeasts and fungi, is an excellent candidate for studies of metabolism evolution. This yeast, widely recognized for its technological applications, in particular produces volatile sulfur compounds (VSCs) that fully contribute to the flavor of smear cheese. We report here a relevant global vision of sulfur metabolism inY. lipolyticabased on a comparison between high- and low-sulfur source supplies (sulfate, methionine, or cystine) by combined approaches (transcriptomics, metabolite profiling, and VSC analysis). The strongest repression of the sulfate assimilation pathway was observed in the case of high methionine supply, together with a large accumulation of sulfur intermediates. A high sulfate supply seems to provoke considerable cellular stress via sulfite production, resulting in a decrease of the availability of the glutathione pathway's sulfur intermediates. The most limited effect was observed for the cystine supply, suggesting that the intracellular cysteine level is more controlled than that of methionine and sulfate. Using a combination of metabolomic profiling and genetic experiments, we revealed taurine and hypotaurine metabolism in yeast for the first time. On the basis of a phylogenetic study, we then demonstrated that this pathway was lost by some of the hemiascomycetous yeasts during evolution.