The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulated expression. The ctxAB and tcpA genes are known to play a cardinal role in maintaining virulence in Vibrio cholerae, and these genes are believed to be exclusively associated with clinical strains of O1 and O139 serogroups. In this study, we examined the presence of five virulence genes, including ctxAB and tcpA, as well as toxR and toxT, which are involved in the regulation of virulence, in environmental strains of V. cholerae cultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India. PCR analysis revealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes of environmental V. cholerae strains. Sequencing of a part of the tcpA gene carried by an environmental strain showed 97.7% homology to the tcpA gene of the classical biotype of V. cholerae O1. Strains carrying the tcpA gene expressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electron microscopy of the TCP pili. Strains carrying ctxAB genes also produced cholera toxin, determined by monosialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits. Thus, this study demonstrates the presence and expression of critical virulence genes or their homologues in diverse environmental strains of V. cholerae, which appear to constitute an environmental reservoir of virulence genes, thereby providing new insights into the ecology of V. cholerae.Vibrio cholerae is known to be an autochthonous inhabitant of brackish waters and estuarine systems (4, 13). Among the 193 currently recognized O serogroups of V. cholerae (43), only serogroups O1 and O139 have caused epidemics of cholera. The other serogroups of V. cholerae, collectively referred to as non-O1 non-O139 serogroups, have not been associated with epidemics but can cause sporadic diarrhea (30) and are ubiquitously distributed in the aquatic environment (22,26). This sharp distinction between serogroups which can cause cholera and those which are not associated with cholera is related to the observation that more than 95% of the strains belonging to serogroups O1 and O139 produce cholera toxin (CT), which is central to the disease process. In contrast, more than 95% of the strains belonging to non-O1 non-O139 serogroups do not produce CT (15). Another important virulence factor of V. cholerae is the toxin-coregulated pilus (TCP), which is an adhesin that is coordinately regulated with CT production (39). TCP is the only V. cholerae pilus that has been demonstrated to date to have a role in colonization of the gut mucosa of humans (9) and of infant mice (39), the latter being an experimental cholera model.It has been presumed that CT and TCP are exclusively associated with clinical strains of V. cholerae, notably those belonging to serogroups O1 and O139, whereas reports on the incidence of CT among environmental strains of V. cholerae are rare (24). Sim...

Pro-and eukaryotic algal genera, i.e. Lyngbya majuscula, Spirulina subsalsa (Cyanophyceae) and Rhizoclonium hieroglyphicum (Chlorophyceae), were used for bio-recovery of gold (Au) out of aqueous solution. Au (III) spiked with 198 Au was used for the experiment. Batch laboratory experiments indicated quick metabolic independent binding of Au to the algae followed by active accumulation and subsequent reduction. Gold accumulation by different algal genera was found in order of R. hieroglyphicum > L. majuscula > S. subsalsa (3.28, 1.93 and 1.73 mg g -1 , respectively). It was observed that the algal biomass and the media used for the experiment turned purple in colour indicating reduction of Au (III) to Au (0) at intra-and extracellular level. This was confirmed by TEM studies of L. majuscula biomass exposed in HAuCl 4 solution where <20-nm-sized gold particles were found both inside as well as on the surface of the cell. Up to 90-100% of accumulated gold was recovered from the algal biomass by using nitric acid and acidic thiourea solution.

In toxigenic Vibrio cholerae, cholera toxin is encoded by the CTX prophage, which consists of a core region carrying ctxAB genes and genes required for CTX⌽ morphogenesis, and an RS2 region encoding regulation, replication, and integration functions. Integrated CTX⌽ is often flanked by another genetic element known as RS1 which carries all open reading frames (ORFs) found in RS2 and an additional ORF designated rstC. We identified a single-stranded circularized form of the RS1 element, in addition to the CTX⌽ genome, in nucleic acids extracted from phage preparations of 32 out of 83 (38.5%) RS1-positive toxigenic V. cholerae strains analyzed. Subsequently, the corresponding double-stranded replicative form (RF) of the RS1 element was isolated from a representative strain and marked with a kanamycin resistance (Km r ) marker in an intergenic site to construct pRS1-Km. Restriction and PCR analysis of pRS1-Km and sequencing of a 300-bp region confirmed that this RF DNA was the excised RS1 element which formed a novel junction between ig1 and rstC. Introduction of pRS1-Km into a V. cholerae O1 classical biotype strain, O395, led to the production of extracellular Km r transducing particles, which carried a single-stranded form of pRS1-Km, thus resembling the genome of a filamentous phage (RS1-Km⌽). Analysis of V. cholerae strains for susceptibility to RS1-Km⌽ showed that classical biotype strains were more susceptible to the phage compared to El Tor and O139 strains. Nontoxigenic (CTX ؊ ) O1 and O139 strains which carried genes encoding the CTX⌽ receptor toxin-coregulated pilus (TCP) were also more susceptible (>1,000-fold) to the phage compared to toxigenic El Tor or O139 strains. Like CTX⌽, the RS1⌽ genome also integrated into the host chromosomes by using the attRS sequence. However, only transductants of RS1-Km⌽ which also harbored the CTX⌽ genome produced a detectable level of extracellular RS1-Km⌽. This suggested that the core genes of CTX⌽ are also required for the morphogenesis of RS1⌽. The results of this study showed for the first time that RS1 element, which encodes a site-specific recombination system in V. cholerae, can propagate horizontally as a filamentous phage, exploiting the morphogenesis genes of CTX⌽.Toxigenic Vibrio cholerae strains are lysogens of a filamentous bacteriophage designated CTX⌽ (20), which carries the ctxAB genes encoding cholera toxin (CT). CTX⌽ is unusual among filamentous phages because the phage genome encodes the functions necessary for a site-specific integration system and thus can integrate into the V. cholerae chromosome at a specific attachment site known as attRS, forming stable lysogens (7,9,20). A typical CTX⌽ genome has two regions, the core and the RS2. The 4.6-kb core region encodes CT as well as the functions that are required for the virion morphogenesis, whereas the 2.5-kb RS2 region encodes the regulation, replication, and integration functions of the CTX⌽ genome (21). In toxigenic V. cholerae, particularly in El Tor and O139 strains, the CTX⌽ genome is integra...

We have shown that the domain V of bacterial 23 S rRNA could fold denatured proteins to their active state. This segment of 23 S rRNA could further be split into two parts. One part containing mainly the central loop of domain V could bind denatured human carbonic anhydrase I stably. This association could be reversed by adding the other part of domain V. The released enzyme was directed in such a way by the central loop of domain V that it could now fold by itself to active form. This agrees with our earlier observation that proteins fold within the cell posttranslationally, a process that is completed after release of the newly synthesized polypeptide from the ribosome (Chattopadhyay, S., Pal, S., Chandra, S., Sarkar, D., and DasGupta, C. (1999) Biochim. Biophys. Acta 1429, 293-298).We have identified the ribosome as a general protein folding modulator on the basis of its ability to successfully fold all the denatured proteins that we have tried so far (e.g. lactate dehydrogenase, glucose-6-phosphate dehydrogenase, horseradish peroxidase, restriction endonucleases, alkaline phosphatase, malate dehydrogenase, ␤-lactamase, carbonic anhydrase, ␤-galactosidase, etc.) (1-6). This in vitro protein folding activity has been found to reside in the domain V of the 23 S rRNA in 50 S particle of the ribosome. This activity of ribosome has also been identified in vivo by showing slow posttranslational activation of the enzyme ␤-galactosidase in Escherichia coli that was synthesized just prior to the addition of the 30 S specific protein synthesis inhibitors kasugamycin and streptomycin. This posttranslational activation, however, was immediately arrested by adding antibiotics that bind to domain V of 23 S rRNA of 50 S ribosomal particle (1). The important question then is whether biological entities like molecular chaperons (7,8) and ribosomes fold proteins to their active states following a pathway basically similar to spontaneous folding or whether there will there be a paradigm shift in our understanding of protein folding in the cell when we know how ribosome (2-6, 9 -11), which synthesizes the polypeptide, also folds it to active form.We identified the protein folding activity in the large loop of domain V of 23 S rRNA of bacterial ribosome (4, 9, 10). We have reported in a number of publications (1-6, 10) that the 70 S bacterial ribosomes, the 80 S wheat germ and rat liver ribosomes, the 50 S bacterial ribosomal subunit, its 23 S rRNA, as well as the 660-nt 1 domain V of 23 S rRNA could all fold denatured proteins, and at the end of the reaction they were found to dissociate completely from the proteins without the assistance of any co-factor. This implied that there were at least two steps in these reactions: (a) interaction with unfolded proteins to fold them and (b) dissociation from the folded proteins. We took the 660-nt-long domain V RNA from Bacillus subtilis and further split it into two smaller pieces that acted in a particular sequence on the unfolded protein to fold it. Here we present the role of these t...

BackgroundMolecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone ‘foldases’ that are distinct from chaperone’ holdases’ that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC) located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA) is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains.ResultsWe demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII) and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein.ConclusionThe ribosome can behave like a ‘holdase’ chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.