Antioxidant activity in the extracts of two edible aroids.

Two neglected species of Araceae, Alocasia macrorhiza (Linn.) G. Don and Alocasia fornicata (Roxb.) Schott are important as food and ethno medicine in Asia and Africa. Their bioefficacy is documented in the Ayurveda. The solvent extracts of different edible parts of these two species like rhizomes, leaves, roots and stolons were screened for in vitro antioxidant properties using standard procedures. The successive extracts in hexane, benzene, toluene, chloroform, diethyl ether, ethyl acetate and water fraction exhibited IC(50) values in the following order, roots>rhizome>leaves for Alocasia macrorhiza and leaves>stolon for Alocasia fornicate, respectively in 2,2-diphenyl-1-picryl hydrazyl antioxidant inhibition assay. Maximum antioxidant activity was observed in diethyl ether extracts for both species. The IC(50) values were comparable with those of quercetine and ascorbic acid as standards. These results suggest that the two aroid species have antioxidant activity in their edible parts and should be extracted using diethyl ether solvent.

SarA represses agr operon expression in a purified in vitro Staphylococcus aureus transcription system.

Mutation and genetic complementation studies suggested that two chromosomal loci, agr and sar, are involved in the upregulation of several exotoxin genes and the downregulation of a number of surface protein genes in a growth phase-dependent manner in Staphylococcus aureus. We purified recombinant T7-tagged SarA from Escherichia coli and determined its effect on transcription from several S. aureus promoters by using purified RNA polymerase reconstituted with either sigma(A) or sigma(B) from S. aureus. Of the seven sigma(A)-dependent promoters that we tested, SarA repressed transcription from agrP2, agrP3, cna, sarP1, and sea promoters and did not affect sec and znt promoters. Furthermore, SarA had no effect on transcription from the sigma(B)-dependent sarP3 promoter. In vitro experimental data presented in this report suggest that SarA expression is autoregulated.

Characterization of a single-strand origin, ssoU, required for broad host range replication of rolling-circle plasmids.

Single-stranded DNA (ssDNA) promoters are the key components of the single-strand origins (ssos) of replication of rolling-circle (RC) replicating plasmids. The recognition of this origin by the host RNA polymerase and the synthesis of a short primer RNA are critical for initiation of lagging-strand synthesis. This step is thought to be a limiting factor for the establishment of RC plasmids in a broad range of bacteria, because most of the ssos described are fully active only in their natural hosts. A special type of sso, the ssoU, is unique in the sense that it can be efficiently recognized in a number of different Gram-positive hosts. We have experimentally deduced the folded structure and characterized the ssDNA promoter present within the ssoU using P1 nuclease digestion and DNase I protection assays with the Bacillus subtilis and Staphylococcus aureus RNA polymerases. We have also identified the RNA products synthesized from this ssDNA promoter and mapped the initiation points of lagging-strand synthesis in vivo from ssoU-containing plasmids. Through gel mobility shift experiments, we have found that ssDNA containing the ssoU sequence can efficiently interact with the RNA polymerase from two different Gram-positive bacteria, S. aureus and B. subtilis. We have also realigned the narrow and broad host range sso sequences of RC plasmids, and found that they contain significant homology. Our data support the notion that the strength of the RNA polymerase-ssoU interaction may be the critical factor that confers the ability on the ssoU to be fully functional in a broad range of bacteria.

ZntR is an autoregulatory protein and negatively regulates the chromosomal zinc resistance operon znt of Staphylococcus aureus.

A chromosomally encoded znt operon of Staphylococcus aureus consists of two consecutive putative genes designated zntR and zntA. The zntA gene encodes a transmembrane protein that facilitates extrusion of Zn2+ and Co2+, whereas the zntR gene encodes a putative regulatory protein that controls the expression of the znt operon. The zntR gene was amplified using the polymerase chain reaction, cloned into Escherichia coli for overexpression as His-tagged ZntR and purified by Ni2+-affinity column. His-tag-free ZntR was purified to near homogeneity after digestion with enterokinase. Electrophoretic mobility shift assays (EMSAs) indicated that the ZntR bound to a fragment of DNA corresponding to the chromosomal znt promoter region with an affinity of about 8.0 x 10-12 M. The addition of 25 microM Zn2+ or Co2+ in the binding reaction completely or significantly inhibited association of ZntR with the znt promoter. DNase I footprinting assays identified a ZntR binding site encompassing 49 nucleotides in the znt promoter region that contained repeated TGAA sequences. These sequences have been proposed to be the binding sites for SmtB, a metallorepressor protein from the cyanobacterium Synechococcus, to its corresponding operator/promoter. In vitro transcription assays, using S. aureus RNA polymerase, revealed that ZntR represses transcription from the znt promoter in a concentration-dependent fashion. The EMSAs, DNase I footprinting and in vitro transcription assays indicate that ZntR is a trans-acting repressor protein that binds to the znt promoter region and regulates its own transcription together with that of zntA.

Lagging strand replication of rolling-circle plasmids: specific recognition of the ssoA-type origins in different gram-positive bacteria.

Many bacterial plasmids replicate by a rolling-circle mechanism that involves the generation of single-stranded DNA (ssDNA) intermediates. Replication of the lagging strand of such plasmids initiates from their single strand origin (sso). Many different types of ssos have been identified. One group of ssos, termed ssoA, which have conserved sequence and structural features, function efficiently only in their natural hosts in vivo. To study the host specificity of sso sequences, we have analyzed the functions of two closely related ssoAs belonging to the staphylococcal plasmid pE194 and the streptococcal plasmid pLS1 in Staphylococcus aureus. The pLS1 ssoA functioned poorly in vivo in S. aureus as evidenced by accumulation of high levels of ssDNA but supported efficient replication in vitro in staphylococcal extracts. These results suggest that one or more host factors that are present in sufficient quantities in S. aureus cell-free extracts may be limiting in vivo. Mapping of the initiation points of lagging strand synthesis in vivo and in vitro showed that DNA synthesis initiates from specific sites within the pLS1 ssoA. These results demonstrate that specific initiation of replication can occur from the pLS1 ssoA in S. aureus although it plays a minimal role in lagging strand synthesis in vivo. Therefore, the poor functionality of the pLS1 in vivo in a nonnative host is caused by the low efficiency rather than a lack of specificity of the initiation process. We also have identified ssDNA promoters and mapped the primer RNAs synthesized by the S. aureus and Bacillus subtilis RNA polymerases from the pE194 and pLS1 ssoAs. The S. aureus RNA polymerase bound more efficiently to the native pE194 ssoA as compared with the pLS1 ssoA, suggesting that the strength of RNA polymerase-ssoA interaction may play a major role in the functionality of the ssoA sequences in Gram-positive bacteria.

Alternative transcription factor sigmaSB of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar.

A homolog of the multiple-stress-responsive transcription factor sigmaB of Bacillus subtilis was predicted from the DNA sequence analysis of a region of the Staphylococcus aureus chromosome. A hybrid between the coding sequence of the first 11 amino acids of the gene 10 leader peptide of phage T7 (T7.Tag) and the putative sigB gene of S. aureus was constructed and cloned into Escherichia coli BL21(DE3)pLysS for overexpression from a T7 promoter. A homogeneous preparation of the overproduced protein was obtained by affinity chromatography with a T7.Tag monoclonal antibody coupled to agarose. The amino-terminal amino acid sequence of the first 22 residues of the purified protein matched that deduced from the nucleotide sequence. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein, designated sigmaSB, indicated that it migrated as an approximately 39-kDa polypeptide. Promoter-specific transcription from the B. subtilis sigmaB-dependent PB promoter of the sigB operon was stimulated by sigmaSB in a concentration-dependent fashion when reconstituted with the S. aureus core RNA polymerase (RNAP). Specific transcript from the predicted sigmaB-dependent PB promoter of the sigB operon of S. aureus was obtained by the reconstituted RNAP in a runoff transcription reaction. The sar operon of S. aureus contains three promoter elements (P1, P2, and P3) and is known to partly control the synthesis of a number of extracellular toxins and several cell wall proteins. Our in vitro studies revealed that transcription from the P1 promoter is dependent on the primary sigma factor sigmaSA, while that of the P3 promoter is dependent on sigmaSB. As determined by primer extension studies, the 5' end of the sigmaSB-initiated mRNA synthesized in vitro from the sar P3 promoter is in agreement with the 5' end of the cellular RNA.

Characterization of the primary sigma factor of Staphylococcus aureus.

RNA polymerase (RNAP) isolated from Staphylococcus aureus is deficient in sigma factor and is poorly active in transcription assays. Based on amino acid sequence homology of the Bacillus subtilis vegetative sigma factor sigmaA and the predicted product of the chromosomally located plaC gene of S. aureus, it was hypothesized that plaC could encode the vegetative sigma factor. We cloned plaC under a T7 promoter and overexpressed it in Escherichia coli strain BL21(DE3)pLysE. The overproduced protein, present in inclusion bodies, was solubilized with guanidine hydrochloride, renatured, and purified by DEAE-Sephacel and Sephadex G-75 chromatography. The purified protein, designated sigmaSA, cross-reacted with the B. subtilis anti-sigmaA antibody. E. coli core RNAP, reconstituted with sigmaSA, initiated promoter-specific transcription from the S. aureus promoters hla, sea, and sec and from the E. coli promoters rpoH P1, rpoH P4, and ColE1 RNA-1, which are recognized by the E. coli sigma70. sigmaSA, when added to the purified RNAP from S. aureus, stimulated transcriptional activity of the RNAP up to 72-fold. As determined by primer extension studies, the 5'-ends of the sigmaSA-initiated mRNAs synthesized in vitro from the agr P2 and sea promoters are in general agreement with the 5'-ends of the cellular RNAs. Disruption of the plaC gene on the S. aureus chromosome was lethal. We conclude that plaC encodes the primary sigma factor in S. aureus.

Intracellular inducer Hg2+ concentration is rate determining for the expression of the mercury-resistance operon in cells.

Experiments involving mercury resistance mer operon-lacZ fusions, point mutations in the mercuric ion reductase merA gene, and transcomplementation have revealed that in Hg2+-resistant cells, the inducer Hg2+ concentration is rate determining for activation of transcription. mer operon expression is activated by the presence of nanomolar concentrations of Hg2+ in liquid media only when the mercuric ion reductase function is artificially inactivated in cells, whereas cells with active mercuric ion reductase require micromolar concentrations of Hg2+ for effective induction of the operon.

Purification and characterization of DNA dependent RNA polymerase from Staphylococcus aureus.

DNA dependent RNA polymerase from exponentially growing Staphylococcus aureus cells was purified. An SDS-polyacrylamide gel analysis of the most purified preparation revealed that it consists of beta, beta', alpha, and sigma with apparent molecular masses of 151, 147, 42, and 55 kDa, respectively. The sigma subunit cross reacted with a polyclonal antibody against Bacillus subtilis sigma 43. The cross reacting peptide co-migrated with the B. subtilis sigma 43 subunit. The implications of these results are discussed. Promoter specific in vitro run-off transcripts were obtained using the purified enzyme preparation. Specific conditions for the polymerization reaction are defined.

Purification and characterization of a novel organometallic receptor protein regulating the expression of the broad spectrum mercury-resistant operon of plasmid pDU1358.

The narrow spectrum mercury-resistant (mer) operons of transposons Tn21 and Tn501 are inducible by inorganic mercury salts. The major regulatory gene merR is transcribed divergently from the other mer genes, which are cotranscribed. The MerR protein represses its own expression, as well as the expression of the other mer genes in the absence of the inducers. The synthesis of the polycistronic mer message is stimulated by MerR in the presence of the inducers. The MerRBS protein encoded by the broad spectrum mer operon of plasmid pDU1358 was characterized as a novel organomercurial receptor, distinguishing it from the narrow spectrum MerRNS proteins, described above. Several organomercurial compounds directly effected cellular activation of the mer operon transcription via the receptor protein MerRBS, but not by MerRNS. The merR gene from pDU1358 was cloned under the tac promoter, and the overexpressed MerRBS protein was soluble in buffer solutions containing 0.5 M NaCl at pH 7.5, but precipitated when NaCl concentration was reduced to 0.1 M (MerRBS concentrations at or above 0.1 mg/ml). MerRBS was purified to near homogeneity by selective precipitation and solubilization by varying the salt concentration in buffer solutions, followed by Sephadex G-75 column chromatography. Both MerRBS and Tn21-encoded MerRNS bound with DNA fragments containing the pDU1358 mer operator sequence with comparable affinities. In vitro run-off transcription studies revealed that MerRBS activated mer operon expression in the presence of Hg2+ or phenylmercuric acetate. Phenylmercuric acetate did not induce mer operon expression when the MerRNS was used in the assay.

Regulation of the Staphylococcus aureus plasmid pI258 mercury resistance operon.

Experiments involving fusion between the Staphylococcus aureus plasmid pI258-encoded mer operon and the reporter gene beta-lactamase, mutational analysis, and trans-complementation studies have shown that the merR gene of pI258, which shows DNA sequence similarity with known merR genes from other bacteria, regulates the expression of the mer operon in vivo. The merR gene product is a trans-acting protein that activates mer operon transcription in the presence of the inducers Hg2+ and Cd2+. A glutathione-S-transferase-MerR fusion protein specifically bound and protected a 27-nucleotide operator sequence from DNase I digestion. This operator sequence is highly homologous with mer operator sequences of other known systems.

Bacterial resistances to inorganic mercury salts and organomercurials.

Environmental and clinical isolates of mercury-resistant (resistant to inorganic mercury salts and organomercurials) bacteria have genes for the enzymes mercuric ion reductase and organomercurial lyase. These genes are often plasmid-encoded, although chromosomally encoded resistance determinants have been occasionally identified. Organomercurial lyase cleaves the C-Hg bond and releases Hg(II) in addition to the appropriate organic compound. Mercuric reductase reduces Hg(II) to Hg(O), which is nontoxic and volatilizes from the medium. Mercuric reductase is a FAD-containing oxidoreductase and requires NAD(P)H and thiol for in vitro activity. The crystal structure of mercuric ion reductase has been partially solved. The primary sequence and the three-dimensional structure of the mercuric reductase are significantly homologous to those of other flavin-containing oxidoreductases, e.g., glutathione reductase and lipoamide dehydrogenase. The active site sequences are the most conserved region among these flavin-containing enzymes. Genes encoding other functions have been identified on all mercury ion resistance determinants studied thus far. All mercury resistance genes are clustered into an operon. Hg(II) is transported into the cell by the products of one to three genes encoded on the resistance determinants. The expression of the operon is regulated and is inducible by Hg(II). In some systems, the operon is inducible by both Hg(II) and some organomercurials. In gram-negative bacteria, two regulatory genes (merR and merD) were identified. The (merR) regulatory gene is transcribed divergently from the other genes in gram-negative bacteria. The product of merR represses operon expression in the absence of the inducers and activates transcription in the presence of the inducers. The product of merD coregulates (modulates) the expression of the operon. Both merR and merD gene products bind to the same operator DNA. The primary sequence of the promoter for the polycistronic mer operon is not ideal for efficient transcription by the RNA polymerase. The -10 and -35 sequences are separated by 19 (gram-negative systems) or 20 (gram-positive systems) nucleotides, 2 or 3 nucleotides longer than the 17-nucleotide optimum distance for binding and efficient transcription by the Escherichia coli sigma 70-containing RNA polymerase. The binding site of MerR is not altered by the presence of Hg(II) (inducer). Experimental data suggest that the MerR-Hg(II) complex alters the local structure of the promoter region, facilitating initiation of transcription of the mer operon by the RNA polymerase. In gram-positive bacteria MerR also positively regulates expression of the mer operon in the presence of Hg(II).

Regulation of the cadA cadmium resistance determinant of Staphylococcus aureus plasmid pI258.

Regulation of the cadA cadmium and zinc resistance determinant of Staphylococcus aureus plasmid pI258 was demonstrated by using gene fusions and direct measurements of transcription. In growth experiments, cells harboring the intact cadA operon were induced with different cations and challenged by an inhibitory concentration of ZnCl2, a substrate of the CadA resistance system. Uninduced cells did not grow for 8 h after Zn2+ addition, whereas induced cells grew in the presence Zn2+. Cd2+ was a strong inducer, and Bi3+ and Pb2+ also induced well; Co2+ and Zn2+ were weak inducers. A translational beta-lactamase fusion to the cadA gene showed the same induction specificity as that seen with growth experiments with the intact cadA operon. A short beta-lactamase transcriptional fusion to the cadC gene also showed the same pattern of induction, establishing that the cadC gene was not involved in regulation. In Northern (RNA) blot hybridization experiments, a cadmium-inducible, 2.6-kb, operon-length transcript was detected. Primer extension experiments determined that Cd(2+)-inducible transcription of the cadA operon begins at nucleotides 676 and 677 of the published sequence (G. Nucifora, L. Chu, T. K. Misra, and S. Silver, Proc. Natl. Acad. Sci. USA 86: 3544-3548, 1989).

Purification and functional characterization of MerD. A coregulator of the mercury resistance operon in gram-negative bacteria.

Mercury resistance operons (mer) from transposons Tn21, Tn501, and plasmid pDU1358 are highly homologous and inducible with Hg2+. The regulatory gene merR is transcribed from one promoter, which is divergently oriented from the promoter for the other mer genes. MerR, the product of the regulatory gene, negatively regulates its own expression as well as the expression of the other genes. MerR activates transcription of the operon in the presence of inducing concentrations of Hg2+. The most promoter distal gene, merD, which is cotranscribed with the structural genes, down regulates the mer operon. A frame-shift mutation in merD, created by deletion of 3 bp and an insertion of a 16 bp sequence upstream of the major inverted repeats present at the 3' end of the merD sequence, resulted in increased synthesis of the structural gene transcript and higher level of resistance to Hg2+ by a factor of about 2. MerD protein was over-produced using a T7 expression system. The overproduced protein was present in the pellet fraction, when cell lysates were centrifuged at a low speed. Approximately 80% pure MerD protein was recovered from the pellet fraction by extracting with a buffer solution containing 5 M urea. The purified protein migrated as a 13,500 molecular weight protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the N-terminal amino acid sequence corresponded to that deduced from the DNA sequence of merD. MerD bound specifically with the mer promoter sequence. DNase I footprinting experiments identified a common mer operator sequence for MerR and MerD.

Nucleotide sequence and expression of the algE gene involved in alginate biosynthesis by Pseudomonas aeruginosa.

Alginate (Alg), a random polymer of mannuronic acid and glucuronic acid residues, is synthesized and secreted by Pseudomonas aeruginosa primarily during its infection of the lungs of cystic fibrosis patients. The molecular biology and biochemistry of the enzymatic steps leading to the production of the Alg precursor GDP-mannuronic acid have been elucidated, but the mechanism of polymer formation and export of Alg are not understood. We report the nucleotide sequence of a 2.4-kb DNA fragment containing the algE gene, previously designated alg76, encoding the AlgE protein (Mr 54,361) that is believed to be involved in these late steps of Alg biosynthesis. Expression of algE appears to occur from its own promoter. The promoter region contains several direct and inverted repeat sequences and shares structural similarity with promoters of several other alg genes from P. aeruginosa. In addition, the AlgE protein was overproduced from the tac promoter in P. aeruginosa. N-terminal amino acid sequence analysis showed that the polypeptide contains a signal peptide which is cleaved to form the mature protein during AlgE export from the cell cytoplasm.

AlgR3, a protein resembling eukaryotic histone H1, regulates alginate synthesis in Pseudomonas aeruginosa.

A regulatory mutation (alg52) in a Pseudomonas aeruginosa alginate-negative mutant (strain 8882) is complemented efficiently by the gene algR2 and somewhat inefficiently by a second gene termed algR3. algR3 and algR2 are located on a 4.4-kilobase-pair HindIII-BamHI fragment, which has been completely sequenced. algR2 has previously been characterized. Introduction of kanamycin-resistance cassettes and deletion-subcloning experiments involving various open reading frames in the HindIII-BamHI fragment have localized the algR3 gene, which encodes a 340-amino acid polypeptide. This highly basic regulatory protein contains 17% lysine and 36% alanine. The predicted amino acid sequence shows no significant similarity with any bacterial proteins and yet is highly similar to the sea urchin Lytechinus pictus histone H1 subtype of protein. Promoter localization by reverse transcriptase mapping of the algR3 gene shows the presence of Escherichia coli sigma 70 recognition sequences, and coupled transcription/translation experiments in E. coli demonstrate the presence of a 39-kDa polypeptide encoded by the cloned algR3 gene.

Cloning, nucleotide sequence, and expression of the chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505.

The chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505 was cloned into broad-host-range vector pSUP104. The hybrid plasmid containing an 11.1-kilobase insert conferred chromate resistance and reduced uptake of chromate in P. aeruginosa PAO1. Resistance to chromate was not expressed in Escherichia coli. Contiguous 1.6- and 6.3-kilobase HindIII fragments from this plasmid hybridized to pUM505 but not to P. aeruginosa chromosomal DNA and only weakly to chromate resistance plasmids pLHB1 and pMG6. Further subcloning produced a plasmid with an insert of 2,145 base pairs, which was sequenced. Analysis of deletions revealed that a single open reading frame was sufficient to determine chromate resistance. This open reading frame encodes a highly hydrophobic polypeptide, ChrA, of 416 amino acid residues that appeared to be expressed in E. coli under control of the T7 promoter. No significant homology was found between ChrA and proteins in the amino acid sequence libraries, but 29% amino acid identity was found with the ChrA amino acid sequence for another chromate resistance determinant sequenced in this laboratory from an Alcaligenes eutrophus plasmid (A. Nies, D. Nies, and S. Silver, submitted for publication).

Nucleotide sequence of a regulatory region controlling alginate synthesis in Pseudomonas aeruginosa: characterization of the algR2 gene.

Alginate (Alg), an exopolysaccharide with strong gelling properties, is produced by Pseudomonas aeruginosa primarily during its infection of the cystic fibrosis (CF) lung. The alg genes are normally not expressed in other environments. The promoter for a critical Alg biosynthetic gene, algD, encoding GDP-mannose dehydrogenase, is activated only under conditions reminiscent of the CF lung (i.e., under high osmolarity), and at least two regulatory genes, algR1 and algR2, have been implicated in this activation process. The physical mapping of a 4.4-kb region harboring algR2 has been accomplished and the complete nucleotide sequence of this fragment, including that of algR2, is presented. The cloning and complementation experiments also demonstrate the presence, on this fragment, of regulatory gene(s) different from algR1 and algR2. The expression of the algR2 gene allows a high level of activation of the algD promoter in Escherichia coli, in the presence of algR1 in a high osmotic environment, suggesting that the AlgR2 and AlgR1 proteins act cooperatively to activate the algD promoter. Hyperexpression of the algR2 gene from the tac promoter also allows the conversion of nonmucoid cells of strain 8822, a spontaneous revertant of the mucoid CF isolate strain 8821, back to mucoidy, but not that of the clinical isolate, strain PAO1.

Down regulation of the mercury resistance operon by the most promoter-distal gene merD.

The effect of the merD gene on the expression of the mer operon was determined from the rates of accumulation of merA-lacZ fusion protein in the presence and absence of an active merD gene in trans. In the presence of the merD gene, beta-galactosidase activity was 2- to 4-fold lower. The merD gene was cloned in a T7 promoter expression vector and the MerD protein product was visualized by autoradiography.