Diversity of Betaproteobacteria revealed by novel primers suggests their role in arsenic cycling.

High arsenic concentration in groundwater is a severe environmental problem affecting human health, particularly in countries of South and South-East Asia. The Bengal Delta Plain (BDP) distributed within India and Bangladesh is a major arsenic-affected region where groundwater is the primary source of drinking water. Previous studies have indicated that members of the bacterial class Betaproteobacteria constitute a major fraction of the microbial community in many of the aquifers within this region. Bacteria belonging to this class are known to be involved in redox cycling of arsenic as well as other metals such iron and manganese, thereby impacting arsenic mobilization and immobilization. While microbial diversity in arsenic-contaminated environments is generally assessed using universal 16S rRNA gene primers, targeted evaluation of Betaproteobacteria diversity remains poorly constrained. In this study, bacterial diversity was investigated in the groundwater from two shallow aquifers (West Bengal, India) based on 16S rRNA gene clone libraries and sequencing using a custom-designed pair of primers specific to Betaproteobacteria. Specificity of the primers was confirmed in silico as well as by the absence of PCR amplification of other bacterial classes. Four major families (Burkholderiaceae, Comamonadaceae, Gallionellaceae and Rhodocyclaceae) were detected among which members of Burkholderiaceae represented 59% and 71% of the total community in each aquifer. The four OTUs (operational taxonomic units; 97% sequence identity) within Burkholderiaceae were close phylogenetic relatives of bacteria within the genus Burkholderia known to solubilize phosphate minerals. Additionally, the OTUs belonging to Gallionellaceae were closely related to the members of the genera Gallionella and Sideroxydans, known to oxidize iron under microaerophilic conditions. These results suggest that members of Betaproteobacteria can potentially influence iron and phosphorus cycling which can influence biogeochemistry in arsenic-contaminated aquifers of the BDP.

Mechanism of ribosome assisted protein folding: a new insight into rRNA functions.

The peptidyl transferase center (PTC), present in the domain V of 23S rRNA of bacteria can act as a general protein folding modulator. Any general function of a nucleic acid polymer (DNA or RNA) is always related to specific sequence/sequences. The ribosome mediated protein folding also involves a specific interaction between the nucleotides of peptidyl transferase center and the amino acids of an unfolded protein. In this article the mechanism of rRNA assisted protein folding and its significance in the light of high resolution crystal structure of ribosome are discussed.

Protein folding by domain V of Escherichia coli 23S rRNA: specificity of RNA-protein interactions.

The peptidyl transferase center, present in domain V of 23S rRNA of eubacteria and large rRNA of plants and animals, can act as a general protein folding modulator. Here we show that a few specific nucleotides in Escherichia coli domain V RNA bind to unfolded proteins and, as shown previously, bring the trapped proteins to a folding-competent state before releasing them. These nucleotides are the same for the proteins studied so far: bovine carbonic anhydrase, lactate dehydrogenase, malate dehydrogenase, and chicken egg white lysozyme. The amino acids that interact with these nucleotides are also found to be specific in the two cases tested: bovine carbonic anhydrase and lysozyme. They are either neutral or positively charged and are present in random coils on the surface of the crystal structure of both the proteins. In fact, two of these amino acid-nucleotide pairs are identical in the two cases. How these features might help the process of protein folding is discussed.

Protein folding following synthesis in vitro and in vivo: association of newly synthesized protein with 50S subunit of E. coli ribosome.

In the accompanying paper, it was shown that a protein, while reverting to native form from the unfolded state in vitro with the help of bacterial 70S ribosome, split the latter into its subunits (50S and 30S) and remains associated with the 50S subunit. Here, we follow the fate of nascent proteins both in case of in vivo and in vitro translation system. The newly synthesised protein was found to associate with the 50S subunit in both the cases.

In vitro protein folding by E. coli ribosome: unfolded protein splitting 70S to interact with 50S subunit.

Folding of unfolded protein on Escherichia coli 70S ribosome is accompanied by rapid dissociation of the ribosome into 50S and 30S subunits. The dissociation rate of 70S ribosome with unfolded protein is much faster than that caused by combined effect of translation and polypeptide release factors known to be involved in the dissociation of ribosome into subunits. The protein then reaches a "folding competent" state on 50S and is released to take up native conformation by itself. Release before attaining the folding competent state or prevention of release by cross-linking it with ribosome, would not allow the protein to get back to its native conformation.

Ribosome-DnaK interactions in relation to protein folding.

Bacterial ribosomes or their 50S subunit can refold many unfolded proteins. The folding activity resides in domain V of 23S RNA of the 50S subunit. Here we show that ribosomes can also refold a denatured chaperone, DnaK, in vitro, and the activity may apply in the folding of nascent DnaK polypeptides in vivo. The chaperone was unusual as the native protein associated with the 50S subunit stably with a 1:1 stoichiometry in vitro. The binding site of the native protein appears to be different from the domain V of 23S RNA, the region with which denatured proteins interact. The DnaK binding influenced the protein folding activity of domain V modestly. Conversely, denatured protein binding to domain V led to dissociation of the native chaperone from the 50S subunit. DnaK thus appears to depend on ribosomes for its own folding, and upon folding, can rebind to ribosome to modulate its general protein folding activity.

23S rRNA assisted folding of cytoplasmic malate dehydrogenase is distinctly different from its self-folding.

The role of the 50S particle of Escherichia coli ribosome and its 23S rRNA in the refolding and subunit association of dimeric porcine heart cytoplasmic malate dehydrogenase (s-MDH) has been investigated. The self-reconstitution of s-MDH is governed by two parallel pathways representing the folding of the inactive monomeric and the dimeric intermediates. However, in the presence of these folding modulators, only one first order kinetics was observed. To understand whether this involved the folding of the monomers or the dimers, subunit association of s-MDH was studied using fluorescein-5-isothiocyanate-rhodamine-isothiocyanate (FITC-RITC) fluorescence energy transfer and chemical cross-linking with gluteraldehyde. The observation suggests that during refolding the interaction of the unstructured monomers of s-MDH with these ribosomal folding modulators leads to very fast formation of structured monomers that immediately dimerise. These inactive dimers then fold to the native ones, which is the rate limiting step in 23S or 50S assisted refolding of s-MDH. Furthermore, the sequential action of the two fragments of domain V of 23S rRNA has been investigated in order to elucidate the mechanism. The central loop of domain V of 23S rRNA (RNA1) traps the monomeric intermediates, and when they are released by the upper stem-loop region of the domain V of 23S rRNA (RNA2) they are already structured enough to form dimeric intermediates which are directed towards the proper folding pathway.

Mutations in domain V of the 23S ribosomal RNA of Bacillus subtilis that inactivate its protein folding property in vitro.

The active site of a protein folding reaction is in domain V of the 23S rRNA in the bacterial ribosome and its homologs in other organisms. This domain has long been known as the peptidyl transferase center. Domain V of Bacillus subtilis is split into two segments, the more conserved large peptidyl transferase loop (RNA1) and the rest (RNA2). These two segments together act as a protein folding modulator as well as the complete domain V RNA. A number of site-directed mutations were introduced in RNA1 and RNA2 of B.subtilis, taking clues from reports of these sites being involved in various steps of protein synthesis. For example, sites like G2505, U2506, U2584 and U2585 in Escherichia coli RNA1 region are protected by deacylated tRNA at high Mg2+ concentration and A2602 is protected by amino acyl tRNA when the P site remains occupied already. Mutations A2058G and A2059G in the RNA1 region render the ribosome Ery(r )in E.coli and Lnc(r )in tobacco chloroplast. Sites in P loop G2252 and G2253 in E.coli are protected against modification by the CCA end of the P site bound tRNA. Mutations were introduced in corresponding nucleotides in B.subtilis RNA1 and RNA2 of domain V. The mutants were tested for refolding using unfolded protein binding assays with unfolded carbonic anhydrase. In the protein folding assay, the mutants showed partial to complete loss of this activity. In the filter binding assay for the RNA-refolding protein complex, the mutants showed an extent of protein binding that agreed well with their protein folding activity.