Quasi-adaptive response to alkylating agents and Ada-protein functions in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In the exponentially growing E. coli cells we have described in 2005 a new fundamental genetic phenomenon, namely quasi-adaptive response to alkylating compounds, “quasi-Ada”. Phenotypic expression of “quasi-Ada” is similar to the true Ada response, however in contrast it develops in the course of pretreatment of the cells by sublethal dose of non-alkylating agent, an NO-containing dinitrosyl iron complex with glutathione (DNIC_glu). To reveal the mechanisms of quasi-adaptation and its association with the function of the regulatory protein Ada here we used a unique property of dual gene expression regulation of aidB1 gene, a part of Ada-regulon, namely its relative independence from Ada protein in anaerobic conditions. Based on the results of aidB1 gene expression analysis an EPR spectra of E.coli MV2176 cells (aidB1::lacZ) in aerobic and anaerobic conditions after the corresponding treatments we concluded that the function and the spatial structure of ^meAda and [(Cys^?)₂Fe⁺(NO⁺)₂]Ada are identical and thus the nitrosylated protein represents an Ada regulon genes expression regulator during quasi-adaptation development.     

Efficient one-step production of (S)-1-phenyl-1,2-ethanediol from (R)-enantiomer plus NAD+–NADPH in-situ regeneration using engineered Escherichia coli

Background

Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli.

Results

Because the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell.

Conclusions

In this work, we showed that Grx1 and NrdH reduce SSC to L-cysteine, and the generated sulfite is then utilized as the sulfur source to produce additional L-cysteine molecule through the sulfate pathway in E. coli. We also found that co-overexpression of NrdH, CysI, and CysK increases L-cysteine production. Our results propose that the enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from SSC is a novel method for improvement of L-cysteine production.     

A novel riboregulator switch system of gene expression for enhanced microbial production of succinic acid

In this paper, a novel riboregulator Switch System of Gene Expression including an OFF-TO-ON switch and an ON-TO-OFF switch was designed to regulate the expression state of target genes between “ON” and “OFF” by switching the identifiability of ribosome recognition site (RBS) based on the thermodynamic stability of different RNA–RNA hybridizations between RBS and small noncoding RNAs. The proposed riboregulator switch system was employed for the fermentative production of succinic acid using an engineered strain of E. coli JW1021, during which the expression of mgtC gene was controlled at “ON” state and that of pepc and ecaA genes were controlled at the “OFF” state in the lag phase and switched to the “OFF” and “ON” state once the strain enters the logarithmic phase. The results showed that using the strain of JW1021, the yield and productivity of succinic acid can reach 0.91 g g^?1 and 3.25 g L^?1 h^?1, respectively, much higher than those using the strains without harboring the riboregulator switch system.     

Laue crystal structure of <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis> nitrite reductase from a high-yield expression system

The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein “small tetraheme c” replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5–1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 °C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-Å-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).     

Purification and properties of thiosulfate reductase from Desulfovibrio gigas

Thiosulfate reductase of the dissimilatory sulfate-reducing bacterium Desulfovibrio gigas has been purified 415-fold and its properties investigated. The enzyme was unstable during the different steps of purification as well as during storage at-15°C. The molecular weight of thiosulfate reductase estimated from the chromatographic behaviour of the enzyme on Sephadex G-200 was close to 220 000. The absorption spectrum of the purified enzyme exhibited a protein peak at 278 nm without characteristic features in the visible region. Thiosulfate reductase catalyzed the stoichiometric production of hydrogen sulfide and sulfite from thiosulfate, and exhibited tetrathionate reductase activity. It did not show sulfite reductase activity. The optimum pH of thiosulfate reduction occurred between pH 7.4 and 8.0 and its K _m value for thiosulfate was calculated to be 5·10^-4 M. The sensitivity of thiosulfate reductase to sulfhydryl reagent and the reversal of the inhibition by cysteine indicated that one or more sulfhydryl groups were involved in the catalytic activity. The study of electron transport between hydrogenase and thiosulfate reductase showed that the most efficient coupling was obtained with a system containing cytochromes c ₃ (M _r =13000) and c ₃ (M _r =26000).     

Mechanism of dissimilatory sulfite reduction by Desulfovibrio desulfuricans: purification of a membrane-bound sulfite reductase and coupling iwth cytochrome c 3 and hydrogenase

The localization of the dissimilatory sulfite reductase in Desulfovibrio desulfuricans strain Essex 6 was investigated. After treatment of the cells with lysozyme, 90% of the sulfite reductase activity was found in the membrane fraction, compared to 30% after cell rupture with the French press. Sulfite reductase was purified from the membrane (mSiR) and the soluble (sSiR) fractiion. On SDS-PAGE, both mSiR and sSiR exhibited three bands at 50, 45 and 11 kDa, respectively. From their UV/VIS properties (distinct absorption maxima at 391, 410, 583, 630 nm, enzymes as isolated) and the characteristic red fluorescence in alkaline solution, mSiR and sSiR were identified as desulfoviridin. Sulfite reductase (HSO₃ ^-H₂S) activity was reconstituted by coupling of mSiR to hydrogenase and cytochrome c ₃ from D. desulfuricans. The specific activity of mSiR was 103 nmol H₂ min^-1 mg^-1, and sulfide was the major product (72% of theoretical yield). No coupling was found with sSiR under these conditions. Furthermore, carbon monoxide was used to diferentiate between the membrane-bound and the soluble sulfite reductase. In a colorimetric assay, with photochemically reduced methyl viologen as redox mediator, CO stimulated the activity of sSiR significantly. CO had no effect in the case of mSiR. These studies documented that, as isolated, both forms of sulfite reductase behaved differently in vitro. Clearly, in D. desulfuricans, the six electron conversion HSO₃ ^-H₂S was achieved by a membranebound desulfoviridin without the assistance of artificial redox mediators, such as methyl viologen.Abbreviations SiR sulfite reductase - mSiR sulfite reductase purified from membranes - sSiR sulfite reductase purified from the soluble fraction Enzymes Sulfite reductase, EC 1.8.99.1 Cytochrome c ₃ hydrogenase, EC 1.12.2.1     

Sulfate formation via ATP sulfurylase in thiosulfate- and sulfite-disproportionating bacteria

Disproportionation of thiosulfate or sulfite to sulfate plus sulfide was found in several sulfate-reducing bacteria. Out of nineteen strains tested, eight disproportionated thiosulfate, and four sulfite. Growth with thiosulfate or sulfite as the sole energy source was obtained with three strains (Desulfovibrio sulfodismutans and the strains Bra02 and NTA3); additionally, D. desulfuricans strain CSN grew with sulfite but not with thiosulfate, although thiosulfate was disproportionated. Two sulfur-reducing bacteria, four phototrophic sulfur-oxidizing bacteria (incubated in the dark), and Thiobacillus denitrificans did not disproportionate thiosulfate or sulfite. Desulfovibrio sulfodismutans and D. desulfuricans CSN formed sulfate from thiosulfate or sulfite even when simultaneously oxidizing hydrogen or ethanol, or in the presence of 50 mM sulfate. The capacities of sulfate reduction and of thiosulfate and sulfite disproportionation were constitutively present. Enzyme activities required for sulfate reduction (ATP sulfurylase, pyrophosphatase, APS reductase, sulfite reductase, thiosulfate reductase, as well as adenylate kinase and hydrogenase) were detected in sufficient activities to account for the growth rates observed. ADP sulfurylase and sulfite oxidoreductase activities were not detected. Disproportionation was sensitive to the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) but not to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). It is proposed that during thiosulfate and sulfite disproportionation sulfate is formed via APS reductase and ATP sulfurylase, but not by sulfite oxidoreductase. Reversed electron transport must be assumed to explain the reduction of thiosulfate and sulfite by the electrons derived from APS reductase.Abbreviations CCCP Carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - APS adenosine 5-phosphosulfate (adenylylsulfate)     

Heterologous biosynthesis of triterpenoid dammarenediol-II in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve heterologous biosynthesis of dammarenediol-II, which is the precursor of dammarane-type tetracyclic ginsenosides, by reconstituting the 2,3-oxidosqualene-derived triterpenoid biosynthetic pathway in Escherichia coli.

Results

By the strategy of synthetic biology, dammarenediol-II biosynthetic pathway was reconstituted in E. coli by co-expression of squalene synthase (SS), squalene epoxidase (SE), NADPH-cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae, and SE from Methylococcus capsulatus (McSE), NADPH-cytochrome P450 reductase (CPR) from Arabidopsis thaliana. Sequences of transmembrane domains were truncated if necessary in each of the genes. Different sources of SE/CPR combinations were tested, during which two CPRs were detected to be new reductase partners of McSE. When the gene encoding dammarenediol-II synthase was co-expressed with the 2,3-oxidosqualene expression modules, dammarenediol-II was detected and the production was 8.63 mg l^?1 in E. coli under the shake-flask conditions.

Conclusions

Two E. coli chassis for production of dammarenediol-II were established which could be potentially applied in other triterpenoid production in E. coli when different oxidosqualene cyclases (OSCs) introduced into the system.

     

Siroheme sulfite reductase isolated from Chromatium vinosum. Purification and investigation of some of its molecular and catalytic properties

Cells of the phototrophic bacterium Chromatium vinosum strain D were shown to contain a siroheme sulfite reductase after autotrophic growth in a sulfide/bicarbonate medium. The enzyme could not be detected in cells grown heterotrophically in a malate/sulfate medium. Siroheme sulfite reductase was isolated from autotrophic cells and obtained in an about 80% pure preparation which was used to investigate some molecular and catalytic properties of the enzyme. It was shown to consist of two different types of subunits with molecular weights of 37,000 and 42,000, most probably arranged in an ₄₄-structure. The molecular weight of the native enzyme was determined to 280,000, 51 atoms of iron and 47 atoms of acid-labile sulfur were found per enzyme molecule. The absorption spectrum indicated siroheme as prosthetic group; it had maxima at 280 nm, 392 nm, 595 nm, and 724 nm. The molar extinction coefficients were determined as 302×10³ cm²xmmol^-1 at 392 nm, 98×10³ cm² xmmol^-1 at 595 nm and 22×10³ cm²x-mmol^-1 at 724 nm. With reduced viologen dyes as electron donor the enzyme reduced sulfite to sulfide, thiosulfate, and trithionate. The turnover number with 59 (2 e^-/enzyme moleculexmin) was low. The pH-optimum was at 6.0. C. vinosum sulfite reductase closely resembled the corresponding enzyme from Thiobacillus denitrificans and also desulfoviridin, the dismilatory sulfite reductase from Desulfovibrio species. It is proposed that C. vinosum catalyses anaerobic oxidation of sulfide and/or elemental sulfur to sulfite in the course of dissimilatory oxidation of reduced sulfur compounds to sulfate.Non-common abbreviations APS adenylyl sulfate - SDS sodium dodecyl sulfate     

Sequence and activity of an endogenous promoter of the assimilatory sulfite reductase gene from Desulfovibrio vulgaris (Hildenborough)

Summary The 5-flanking region of the assimilatory-type sulfite reductase-encoding gene from the anaerobic bacterium, Desulfovibrio vulgaris (Hildenborough), has been isolated and sequenced. The promoter element has been identified and compared with the consensus sequence for Escherichia coli and the sequences of promoter elements identified in other Desulfovibrio strains. The utility of such promoter sequences in E. coli-based expression systems is discussed.     

Production of an aromatic amino acid auxotroph of a trimethoprim-resistant Escherichia coli and deuteration of the aromatic amino acids in its dihydrofolate

Interpretation of the ¹H-NMR spectra of Escherichia coli dihydrofolate reductase is complicated by the large number of overlapping resonances due to protonated aromatic amino acids. Deuteration of the aromatic protons of aromatic amino acid residues is one technique useful for simplifying the ¹H-NMR spectra. Previous attempts to label the dihydrofolate reductase from over-producing strains of Escherichia coli were not completely successful. This labeling problem was solved by transducing via P1 phage a genetic block into the de novo biosynthetic pathway of aromatic amino acids in a trimethoprim resistant strain of E. coli, MB 3746. A new strain, MB 4065, is a very high level producer of dihydrofolate reductase and requires exogenous aromatic amino acids for growth, therefore allowing efficient labeling of its dihydrofolate reductase with exogenous deuterated aromatic amino acid.     

A DNA Region That Complements on Escherichia coli cysG Mutation in Thiobacillus Ferrooxidans

Thiobacillus ferrooxidans AP19-3 has a novel NADH-dependent sulfite reductase in the periplasmic space. The gene responsible for the appearance of NADH-dependent sulfite reductase activity was cloned into a vector plasmid pBR322 to give a 5.7-kb hybrid plasmid, pTHS1, which contains a 1.3-kb DNA fragment of T. ferrooxidans AP19-3. When pTHS1 was used to transform sulfite reductase deficient E. coli mutants, strain AT2455 (cysG), JM246 (cysl), and AT2427 (cysJ), it complemented only the E. coli cysG mutation. Since cysG codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase, the enzyme involved in siroheme synthesis, the results indicate that the DNA region that codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase is present in a T. ferrooxidans 1.3 kb DNA fragment on pTHS1.     

Affinity labeling of purified Ca2+,Mg2+-activated ATPase of Escherichia coli by the 2',3'-dialdehydes of adenosine 5'-di- and triphosphates

The 2′,3′-dialdehydes of ADP and ATP (oADP and oATP), obtained by periodate oxidation of ADP and ATP, inhibited the hydrolytic activity of the purified Ca²⁺.Mg²⁺-activated ATPase of Escherichia coli. Nonspecific labeling of amino groups by these dialdehydes was corrected by carrying out the reactions in the presence of 15 mm ATP. Two types of modification of “ATP-protectable” binding sites by oATP could be detected. The binding of 2 mol “ATP-protectable” oATP/mol ATPase was without affect on ATPase activity and still occurred in the hydrolytically inactive ATPase of an unc A mutant. The binding of a further 3 mol “ATP-protectable” oATP/mol ATPase resulted in almost complete loss of ATPase activity although much of the loss occurred during the binding of the first additional molecule of oATP. This additional ATP-protectable oATP binding did not occur in the unc A mutant and so resembled both the inhibitory effect of oADP on the ATPase activity of normal strains and its lack of binding to the unc A ATPase (P. D. Bragg and C. Hou, 1980, Biochem. Biophys. Res. Commun.95, 952–957). The “ATP-protectable” binding sites for oADP and oATP were located on the α subunit of the ATPase. Binding of oADP or oATP did not result in release of the tightly bound ADP and ATP from the enzyme. We conclude that separate binding sites for oADP and oATP occur on the α subunits of the E. coli ATPase and that the former may be the active site(s) for ATP hydrolysis while the latter are involved in regulation of the ATPase complex.     

Crystal Structure of Adenylylsulfate Reductase from Desulfovibrio gigas Suggests a Potential Self-Regulation Mechanism Involving the C Terminus of the β-Subunit

Adenylylsulfate reductase (adenosine 5′-phosphosulfate [APS] reductase [APSR]) plays a key role in catalyzing APS to sulfite in dissimilatory sulfate reduction. Here, we report the crystal structure of APSR from Desulfovibrio gigas at 3.1-Å resolution. Different from the α₂β₂-heterotetramer of the Archaeoglobus fulgidus, the overall structure of APSR from D. gigas comprises six αβ-heterodimers that form a hexameric structure. The flavin adenine dinucleotide is noncovalently attached to the α-subunit, and two [4Fe-4S] clusters are enveloped by cluster-binding motifs. The substrate-binding channel in D. gigas is wider than that in A. fulgidus because of shifts in the loop (amino acid 326 to 332) and the α-helix (amino acid 289 to 299) in the α-subunit. The positively charged residue Arg160 in the structure of D. gigas likely replaces the role of Arg83 in that of A. fulgidus for the recognition of substrates. The C-terminal segment of the β-subunit wraps around the α-subunit to form a functional unit, with the C-terminal loop inserted into the active-site channel of the α-subunit from another αβ-heterodimer. Electrostatic interactions between the substrate-binding residue Arg282 in the α-subunit and Asp159 in the C terminus of the β-subunit affect the binding of the substrate. Alignment of APSR sequences from D. gigas and A. fulgidus shows the largest differences toward the C termini of the β-subunits, and structural comparison reveals notable differences at the C termini, activity sites, and other regions. The disulfide comprising Cys156 to Cys162 stabilizes the C-terminal loop of the β-subunit and is crucial for oligomerization. Dynamic light scattering and ultracentrifugation measurements reveal multiple forms of APSR upon the addition of AMP, indicating that AMP binding dissociates the inactive hexamer into functional dimers, presumably by switching the C terminus of the β-subunit away from the active site. The crystal structure of APSR, together with its oligomerization properties, suggests that APSR from sulfate-reducing bacteria might self-regulate its activity through the C terminus of the β-subunit.Sulfate-reducing bacteria (SRB) are a special group of prokaryotes that are found in sulfate-rich environments because of their ability to metabolize sulfate. SRB use sulfate as the final electron acceptor in various anaerobic environments, such as soil, oil fields, the sea, or the innards of animals or even human beings (10, 11, 19, 25, 33). Their ability to degrade sulfate offers protection against environmental pollution. SRB can remove sulfate and toxic heavy atoms from factory waste waters (12). The Desulfovibrio species is a much-studied representative of SRB, and Desulfovibrio gigas has been studied under many diverse conditions to elucidate metabolic pathways (23, 35).Sulfate reduction is one of the oldest forms of cellular metabolism. The reduction can be either assimilatory or dissimilatory. Sulfate is the terminal electron acceptor in dissimilatory reduction and the raw material for the biosynthesis of cysteine in assimilatory reduction. The latter type of reduction occurs in archaebacteria, bacteria, fungi, and plants via various pathways (17). For example, in Escherichia coli, the reduction initially catalyzes sulfate to adenosine 5′-phosphosulfate (APS) by ATP sulfurylase. APS is then phosphorylated by APS kinase to 3′-phosphate APS, which is then further reduced to sulfite by 3′-phosphate APS reductase (APSR). Finally, sulfite is reduced by sulfite reductase to sulfide, which condenses with O-acetylserine by O-acetylserine lyase to form cysteine. For comparison, in dissimilatory sulfate reduction, sulfate is first catalyzed by ATP sulfurylase to APS, which is then directly reduced by APSR to sulfite. Sulfite is subsequently reduced by dissimilatory sulfite reductase to the following three possible products: trithionite (S₃O₆²⁻), thiosulfate (S₂O₃²⁻), or sulfide (S²⁻).Adenylylsulfate reductase, also called APSR, plays an important role in catalyzing APS to AMP and sulfite in the dissimilatory sulfate reduction. APSR was first partially purified and characterized from Desulfovibrio desulfuricans (32). Multiple forms of APSR in Desulfovibrio vulgaris were observed in buffers under varied conditions (1) and were found in the cytoplasm of cells (18). APSR from D. gigas was first purified by Lampreia et al. (21) and showed a molecular mass of 400 kDa comprised of α- and β-subunits, corresponding to the molecular masses of 70 kDa and 23 kDa, respectively. One flavin adenine dinucleotide (FAD) and two [4Fe-4S] clusters per APSR have been observed and characterized by electron paramagnetic resonance and Mössbauer spectroscopy. The enzyme from D. gigas has been described as an α₂β complex involving one FAD and two [4Fe-4S] clusters (20). In D. vulgaris, APSR is apparently an α₂β₂ complex with a molecular mass of 186 kDa; only one Fe-S cluster is found in the αβ-heterodimer (31). Thus, the subunit and quaternary structures of APSR and their constitution of cofactors in terms of FAD and iron-sulfur clusters are still under debate. Only the enzyme from Archaeoglobus fulgidus has benefited from having an X-ray crystal structure. In this APSR, the functional unit has been shown to be the 1:1 αβ-heterodimer, containing two iron-sulfur clusters and one FAD in the structure (7). However, crystal packing shows that the asymmetric unit is an α₂β₂-heterotetramer.The catalytic mechanism of APSR can be divided into the transport of electrons and the cleavage of APS by FAD. Electron input to the FAD catalyzes the cleavage of APS, releasing AMP and sulfite. Although there have been a number of mechanisms proposed to explain the catalytic cleavage of APS to AMP and sulfite (7, 8, 13, 20, 34), many features of the postulated mechanism remain unsettled, including the proteinogenic hydrogen acceptor in the reaction, the conformational change in the enzyme induced by reduction/oxidation of the FAD cofactor, and the reasons for the observed multiple forms of APSR. The divergence between A. fulgidus and Desulfovibrio species also suggests an obvious distinction in the phylogeny of the α- and β-subunits of APSR.To clarify the difference between APSR from A. fulgidus and that from Desulfovibrio species, we have undertaken a structural study of APSR from D. gigas for comparison with the A. fulgidus enzyme. We have isolated and purified APSR directly from massive, anaerobically grown D. gigas cells for structure determination and characterization. The comparison of the structures and sequences revealing the notable differences at the C termini, activity sites, and other regions for the function is discussed. The structure of oxidized APSR from D. gigas provides much direct evidence about the subunit interactions and the role of the quaternary structure in the regulation of the catalytic mechanism.     

Orange protein from <Emphasis Type="Italic">Desulfovibrio alaskensis</Emphasis> G20: insights into the Mo–Cu cluster protein-assisted synthesis

A novel metalloprotein containing a unique [S₂MoS₂CuS₂MoS₂]^3? cluster, designated as Orange Protein (ORP), was isolated for the first time from Desulfovibrio gigas, a sulphate reducer. The orp operon is conserved in almost all sequenced Desulfovibrio genomes and in other anaerobic bacteria, however, so far D. gigas ORP had been the only ORP characterized in the literature. In this work, the purification of another ORP isolated form Desulfovibrio alaskensis G20 is reported. The native protein is monomeric (12443.8 ± 0.1 Da by ESI–MS) and contains also a MoCu cluster with characteristic absorption bands at 337 and 480 nm, assigned to S–Mo charge transfer bands. Desulfovibrio alaskensis G20 recombinant protein was obtained in the apo-form from E. coli. Cluster reconstitution studies and UV–visible titrations with tetrathiomolybdate of the apo-ORP incubated with Cu ions indicate that the cluster is incorporated in a protein metal-assisted synthetic mode and the protein favors the 2Mo:1Cu stoichiometry. In Desulfovibrio alaskensis G20, the orp genes are encoded by a polycistronic unit composed of six genes whereas in Desulfovibrio vulgaris Hildenborough the same genes are organized into two divergent operons, although the composition in genes is similar. The gene expression of ORP (Dde_3198) increased 6.6 ± 0.5 times when molybdate was added to the growth medium but was not affected by Cu(II) addition, suggesting an involvement in molybdenum metabolism directly or indirectly in these anaerobic bacteria.     

Characterization of virulence factors and phylogenetic group determination of <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from diarrheic and non-diarrheic calves from Brazil

This study aimed to detect virulence factors, pathovars, and phylogenetic groups of Escherichia coli strains obtained from feces of calves with and without diarrhea up to 70 days old and to determine the association between occurrence of diarrhea, phylogenetic groups, and pathovars. Phylo-typing analysis of the 336 E. coli strains isolated from calves with Clermont method showed that 21 (6.25 %) belong to phylogroup A, 228 (67.85 %) to phylogroup B1, 2 (0.6 %) to phylogroup B2, 5 (1.49 %) to phylogroup C, 57 (16.96 %) to phylogroup E, and 3 (0.9 %) to phylogroup F. Phylogroup D was not identified and 20 strains (5.95 %) were assigned as “unknown.” The distribution of phylogenetic groups among pathovars showed that NTEC belong to phylogroups B1 (17) and C (4); EPEC to phylogroups B1 (6) and E (8); STEC to phylogroups A (5), B1 (56), B2 (2), C (1), and E (15); EHEC to phylogroups B1 (95) and E (5); and ETEC to phylogroups A (3), B1 (7), and E (10). The EAST-1 strains were phylogroups A (13), B1 (47), E (19), and F (3); E. coli strains of “unknown” phylogroups belonged to pathovars EPEC (1), EHEC (2), STEC (7), and EAST-1 strains (6). ETEC was associated with diarrhea (P = 0.002). Our study did not find association between the phylogenetic background and occurrence of diarrhea (P = 0.164) but did find some relationship in phylogenetic group and pathovar. The study showed that EHEC and STEC are classified as phylogroup B1, EAST-1 phylogroup A, ETEC, and EPEC phylogroup E.     

The control of sulphate reduction in bacteria

1. An enzyme from Escherichia coli 9723 that reduces adenosine 3′-phosphate 5′-sulphatophosphate to inorganic sulphite is described. Extracts of E. coli K₁₂ and Bacillus subtilis 1379 contain a similar enzyme. 2. This reductase and sulphite reductase (EC 1.8.1.2) of E. coli 9723, E. coli K₁₂ and of B. subtilis are repressed by growth in the presence of l-cystine. Cysteine synthase (EC 4.2.1.22) is unaffected. 3. Growth of E. coli 9723 on inorganic sulphite represses the sulphate-activating enzymes (EC 2.7.7.4 and 2.7.1.25) almost completely but has little effect on sulphite reductase. Growth on 0·042–0·056mm-l-cystine gives a similar result. 4. Such differential repression by cyst(e)ine prevents E. coli, when growing on sulphite, from synthesizing unnecessary enzymes.     

Overexpression, purification and immunodetection of DsrD from Desulfovibrio vulgaris Hildenborough

Dissimilatory sulfite reductase (DsrAB) of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough is an ₂₂ tetramer of 180 kDa, encoded by the dsr operon. In addition to the dsrA and dsrB genes, this operon contains a gene (dsrD) encoding a protein of only 78 amino acids. Although, the function of DsrD is currently unknown, the presence of a dsrD gene has been demonstrated in a variety of sulfate-reducing bacteria and archaea. DsrD was expressed in Escherichia coli at a very high level and purified to homogeneity. Protein blotting experiments, using antisera raised against purified DsrD, demonstrated that it is expressed constitutively in D. vulgaris and does not copurify with DsrAB. Spectroscopic analysis of DsrD indicated that it does not bind either sulfite or sulfide, the substrate and product, respectively of the reaction catalyzed by DsrAB. Thus, although the conservation of this protein and its demonstrated presence in D. vulgaris, suggest an essential function in dissimilatory sulfite reduction, this function remains to be elucidated.     

Oxidation-reduction properties of maize ferredoxin: sulfite oxidoreductase

Oxidation-reduction titrations have been carried out on the wild-type, ferredoxin-dependent sulfite reductase from maize and two site-specific variants of the enzyme. E(m) values have been determined for the siroheme and [4Fe-4S] cluster prosthetic groups of the enzyme, which titrate as independent, one-electron carriers. Visible-region difference spectra suggest that reduction of the [4Fe-4S] cluster significantly perturbs the spectrum of the reduced siroheme group of the enzyme. The effects of siroheme axial ligation, by either cyanide or phosphate ligands, on the redox properties of sulfite reductase have also been examined. For comparison, the effects of phosphate and cyanide on the redox properties of the ferredoxin-dependent nitrite reductase of spinach chloroplasts, an enzyme with the same prosthetic group arrangement as sulfite reductase, have been examined.