Presence Of Inorganic Sulfate Research Articles

Plant volatile organic compound (E)-2-hexenal facilitates Botrytis cinerea infection of fruits by inducing sulfate assimilation.

Investigation into plant-fungal pathogen interactions is one of the most interesting fields in plant sciences. However, the roles of plant volatile organic compounds in the arms race are still largely unknown. Based on precise quantification of plant volatiles, we discovered that the plant volatile organic compound (E)-2-hexenal, at concentrations that were similar to or lower than those in tissues of strawberry and tomato fruits, upregulates sulfate assimilation in spores and hyphae of the phytopathogenic fungus Botrytis cinerea. This upregulation is independent of the types of sulfur sources in the plant and can be achieved in the presence of inorganic sulfate and organic sulfur sources. Using the fungal deletion mutants, we further found that sulfate assimilation is involved in the infection of tomato and strawberry fruits by B.cinerea, and that the severity of the disease is proportional to the sulfate content in the fruits. Both before and during the infection, (E)-2-hexenal induced utilisation of plant sulfate by B.cinerea facilitates its pathogenesis through enhancing its tolerance to oxidative stress. This work provides novel insights into the role of plant volatiles in plant-fungal pathogen interaction and highlights the importance of sulfur levels in the host in the prevention of grey mould disease.

Construction and Characterization of a New Recombinant Vector to Remove Sulfate Repression of dsz Promoter Transcription in Biodesulfurization of Dibenzothiophene.

Biodesulfurization (BDS) is an environmentally friendly desulfurizing process with the potential of replacing or adding to the current expensive technologies for sulfur removal from fossil fuels. The BDS, however, still suffers from low biocatalyst activity. One reason is repression of dsz promoter transcription in presence of inorganic sulfate that impedes translation of Dsz enzymes required for desulfurization pathway. One approach to solve this problem is replacing the native promoter with a new promoter that is no longer repressed. In this study, dsz genes from desulfurizing strain Rhodococcus sp. FUM94 was cloned in an alkane responsive promoter, pCom8, and expressed in Escherichia coli BL21 (DE3) as a host. The recombinant was not susceptible to inorganic sulfate in the culture medium. Desulfurizing activity of recombinant strain versus wild type indicated that in a sulfate containing medium, BDS yield of recombinant increased from 16.0% ± 0.9 to 34.0% ± 1.9% when dibenzothiophene (DBT) concentration (dissolved in ethanol) increased from 25 to 100 ppm. Also, 2-hydroxy biphenyl (2-HBP) production rate improved 8.5-fold (from 0.302 ± 0.020 to 2.57 ± 0.14 mmol 2-HBP (kg DCW)-1 h-1) at the same DBT concentration range. This is while no 2-HBP production was detected in FUM94 biphasic reaction. In a sulfate-free medium, wild type strain demonstrated desulfurization activity, but decreasing with the increase of DBT concentration dissolved in n-tetradecane. Whereas, the recombinant strain demonstrated increasing desulfurizing activity in a sulfate-containing high DBT concentration environment. Overall, the result of this molecular manipulation can be considered as a step forward toward commercialization of BDS technology.

Regulation of Sulfur Assimilation Pathways in Burkholderia cenocepacia through Control of Genes by the SsuR Transcription Factor

The genome of Burkholderia cenocepacia contains two genes encoding closely related LysR-type transcriptional regulators, CysB and SsuR, involved in control of sulfur assimilation processes. In this study we show that the function of SsuR is essential for the utilization of a number of organic sulfur sources of either environmental or human origin. Among the genes upregulated by SsuR identified here are the tauABC operon encoding a predicted taurine transporter, three tauD-type genes encoding putative taurine dioxygenases, and atsA encoding a putative arylsulfatase. The role of SsuR in expression of these genes/operons was characterized through (i) construction of transcriptional reporter fusions to candidate promoter regions and analysis of their expression in the presence/absence of SsuR and (ii) testing the ability of SsuR to bind SsuR-responsive promoter regions. We also demonstrate that expression of SsuR-activated genes is not repressed in the presence of inorganic sulfate. A more detailed analysis of four SsuR-responsive promoter regions indicated that ~44 bp of the DNA sequence preceding and/or overlapping the predicted -35 element of such promoters is sufficient for SsuR binding. The DNA sequence homology among SsuR "recognition motifs" at different responsive promoters appears to be limited.

Biodesulfurization of dibenzothiophene by recombinant Gordonia alkanivorans RIPI90A

The dszABC genes from newly reported dibenzothiophene biodesulfurizing bacterium, Gordonia alkanivorans RIPI90A were cloned and sequenced. The overall nucleotide sequence similarity between the dszABC genes of G. alkanivorans RIPI90A and those of Rhodococcus erythropolis IGTS8 and Gordonia nitida were 83.1% and 83.2%, respectively. A gene transfer system for G. alkanivorans RIPI90A was established employing the Escherichia coli– Rhodococcus shuttle vector pRSG43 as suitable cloning vector, resulting in transformation efficiencies up to 1.6 × 10 5 CFUs μg −1 plasmid DNA. This stable vector was applied to cloning and efficient expression of the dsz genes under the control of lac promoter. The recombinant strain was able to desulfurize dibenzothiophene in the presence of inorganic sulfate and sulfur-containing amino acids. The maximum desulfurization activity by recombinant resting cells ( 131.8 μ M 2 -hydroxybiphenyl g dry cell weight - 1 h - 1 ) was increased 2.67-fold in comparison to the highest desulfurization activity of native resting cells.

Recombinant Rhodococcus sp. strain T09 can desulfurize DBT in the presence of inorganic sulfate.

The putative Rhodococcus rrn promoter region was cloned from the benzothiophene desulfurizing Rhodococcus sp. strain T09, and the dibenzothiophene desulfurizing gene, dsz, was expressed under the control of the putative rrn promoter in the strain T09 using a Rhodococcus-E.coli shuttle vector. Strain T09 harboring the expression vector, pNT, could desulfurize dibenzothiophene in the presence of inorganic sulfate, methionine, or cysteine, while the Dsz phenotype was completely repressed in recombinant cells carrying the gene under the control of the native dsz promoter under the same conditions. Among the sulfur sources examined, no intermediates were detected and only the final desulfurized product, 2-hydroxy-biphenyl, was produced using ammonium sulfate as the sulfur source.

Lipid peroxidation and protein thiol depletion are not involved in the cytotoxicity of N-hydroxy-2-acetylaminofluorene in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to study the mechanism of cell death induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Exposure to 1.0 mM N-OH-AAF resulted in more than 90% cell death (as measured by LDH leakage) of hepatocytes isolated from male rats within 6 hr. Only 36% of the hepatocytes isolated from female rats died within this period. When inorganic sulfate was omitted from the incubation medium, a 6 hr exposure to 1.0 mM N-OH-AAF resulted in only 40% cell death of male hepatocytes. These findings are in accordance with the sex difference and sulfation dependence of N-OH-AAF hepatotoxicity observed in the rat in vivo. N-OH-AAF decreased glutathione (GSH) in male hepatocytes in a concentration-dependent manner. This GSH consumption was only partly dependent on the presence of inorganic sulfate. No lipid peroxidation was observed during N-OH-AAF exposure; N-OH-AAF even prevented endogenous and diethyl maleate (DEM)-induced lipid peroxidation. No reduction of free protein thiol groups was found after exposure to N-OH-AAF, even after 75% cell death had occurred. A reduction of protein thiols after N-OH-AAF exposure was observed in GSH depleted hepatocytes (obtained by DEM plus vitamin E pretreatment). Under these conditions N-OH-AAF-induced cell death occurred earlier. Therefore, GSH protects against protein thiol depletion by N-OH-AAF in control cells. N-OH-AAF-induced cell death was preceded by a loss of intracellular ATP. It is concluded, therefore, that neither lipid peroxidation nor depletion of protein thiols, but possibly loss of intracellular ATP, is involved in the sulfation-dependent cytotoxic mechanism of N-OH-AAF in isolated rat hepatocytes,

Adenosinetriphosphate sulfurylase from Penicillium chrysogenum: Steady-state kinetics of the forward and reverse reactions, alternative substrate kinetics, and equilibrium binding studies

The kinetics of the forward ATP sulfurylase-catalyzed reaction were examined using a new assay based on 32 PP i released from [γ- 32P]MgATP in the presence of inorganic sulfate. Replots yielded V maxf = 6.6 units mg protein −1, K mA = 0.13 mM, K ia = 0.33 mM, and K mB = 0.55 mM, where A = MgATP and B = SO 4 2−. Thiosulfate, a dead-end inhibitor of the reaction, was competitive with sulfate and noncompetitive with respect to MgATP. The ratio k cat K m A was determined for several alternative inorganic substrates, B, where A = MgATP and B = SO 4 2−, SeO 4 2−, MoO 4 2−, WO 4 2−, or CrO 4 2−. For SO 4 2− and SeO 4 2−, the ratio was 5–6.5 × 10 4 m −1 s −1; for the others, the ratio was 5.8–7.3 × 10 5 m −1 s −1. The results support a random addition of MgATP and inorganic substrate. The kinetics of the reverse reaction were examined using a new assay based on 35SO 4 2− release from [ 35S]APS (adenosine 5′-phosphosulfate) in the presence of Mg PP i. Reciprocal plots were linear, intersecting below the horizontal axis. Replots yielded V maxr = 50 units mg protein −1, K mQ = 0.3 μM, K iq = 0.04 μM, and K mP = 4 μM, where Q = APS and P = PP i; (total of all species). MgATP and SO 4 2− were both competitive with APS and noncompetitive with respect to Mg PP i. Taken together with earlier results suggesting that APS is competitive with both MgATP and SO 4 2− and that Mg PP i is noncompetitive with respect to both substrates, the qualitative results point to a random A-B, ordered P-Q kinetic mechanism. The Scatchard plot for [ 35S]APS binding was curved, indicating either negative cooperativity or more than a single class of sites. [γ- 32P]MgATP displayed half-site saturation in the presence of saturating FSO 3 −.

Sulfoconjugation and Glucuronidation of Salicylamide in Isolated Rat Hepatocytes

Sulfoconjugation and glucuronidation of salicylamide by isolated hepatocytes were examined with various concentrations of salicylamide and sodium sulfate. The ratio of sulfate to glucuronide formed changed markedly, depending on the concentrations of salicylamide and inorganic sulfate in the medium. The apparent Km value of sulfoconjugation for salicylamide was 0.006 mM, while its Vmax value varied depending on the concentration of inorganic sulfate (e.g., 2.1 and 0.5 nmoles/min/106 cells at 1.2 and 0.5 mM inorganic sulfate, respectively). The Km and Vmax values of glucuronidation for salicylamide were 0.19 mM and 1.28 nmoles/min/106 cells, respectively, in the absence of sodium sulfate. Glucuronidation was suppressed in the presence of inorganic sulfate. The suppression could be attributable to the competitive consumption of salicylamide by sulfotransferase. Additional in vivo experiments revealed that an extra amount of salicylamide markedly lowered the blood inorganic sulfate levels of rats. The significance of the finding is discussed in conjunction with the variation of the Vmax value of sulfoconjugation with the inorganic sulfate concentration.

Regulation of arylsulfatase synthesis by sulfur compounds in Klebsiella aerogenes.

In Klebsiella aerogenes, arylsulfatase synthesis was repressed by inorganic sulfate, sulfite, sulfide, thiosulfate, and cysteine, but not by methionine under normal growth conditions. We isolated cysteine-requiring mutants (Cys minus), and mutants (AtsS minus, AtsR minus) in which the regulation of arylsulfatase synthesis was altered. In the cysteine auxotroph, enzyme synthesis was also repressed by inorganic sulfate or cysteine. Kinetic studies on mutants of the cysteine auxotroph showed that inorganic sulfate repressed arylsulfatase synthesis and that this was not due to cysteine formed by reduction of sulfate. Arylsulfatase synthesis in the AtsS minus mutant was not repressed by inorganic sulfate but was repressed by cysteine. This mutant strain had a normal level of inorganic sulfate transport. Another mutant strain, defective in the inorganic sulfate transport system, synthesized arylsulfatase in the presence of inorganic sulfate but not in the presence of cysteine. The AtsS minus mutant could synthesize the enzyme in the presence of inorganic sulfate but not cysteine. The AtsR minus mutant could synthesize the enzyme in the presence of either inorganic sulfate or cysteine. These results suggest that there are two independent functional corepressors of arylsulfatase synthesis in K. aerogenes.