Molecular Evidence for Occurrence of Heavy Metal and Antibiotic Resistance Genes Among Predominant Metal Tolerant Pseudomonas sp. and Serratia sp. Prevalent in the Teesta River.

Riverine ecosystems polluted by pharmaceutical and metal industries are potential incubators of bacteria with dual resistance to heavy metals and antibiotics. The processes of co-resistance and cross resistance that empower bacteria to negotiate these challenges, strongly endorse dangers of antibiotic resistance generated by metal stress. Therefore, investigation into the molecular evidence of heavy metal and antibiotic resistance genes was the prime focus of this study. The selected Pseudomonas and Serratia species isolates evinced by their minimum inhibitory concentration and multiple antibiotic resistance (MAR) index showed significant heavy metal tolerance and multi-antibiotic resistance capability, respectively. Consequently, isolates with higher tolerance for the most toxic metal cadmium evinced high MAR index value (0.53 for Pseudomonas sp., and 0.46 for Serratia sp.) in the present investigation. Metal tolerance genes belonging to PIB-type and resistance nodulation division family of proteins were evident in these isolates. The antibiotic resistance genes like mexB, mexF and mexY occurred in Pseudomonas isolates while sdeB genes were present in Serratia isolates. Phylogenetic incongruency and GC composition analysis of PIB-type genes suggested that some of these isolates had acquired resistance through horizontal gene transfer (HGT). Therefore, the Teesta River has become a reservoir for resistant gene exchange or movement via selective pressure exerted by metals and antibiotics. The resultant adaptive mechanisms and altered phenotypes are potential tools to track metal tolerant strains with clinically significant antibiotic resistance traits.

Molecular insight into the expression of metal transporter genes in Chryseobacterium sp. PMSZPI isolated from uranium deposit.

Metal tolerant bacterium Chryseobacterium sp. PMSZPI previously isolated and characterized from uranium ore deposit was studied for elucidating the role of metal transporter genes belonging to the Cation Diffusion Facilitator (CDF), Root-Nodulation-Division (RND) and PIB-type ATPase family in cadmium and uranium tolerance. The bacterium showed tolerance towards cadmium (MIC~6mM) and uranium (MIC~2mM) and was found to harbor metal transporter genes belonging to CDF, RND and PIB-type ATPase family of proteins. Expression studies by real-time PCR showed an upregulation of czcA(RND), czcD(CDF) and cadA(PIB-type ATPase) genes in presence of cadmium or uranium. Higher expression of czcA and czcD was found when the bacterium was treated with cadmium and uranium respectively. This study provides significant insight into the molecular mechanism that plays a role in cadmium and uranium tolerance in bacteria.

Bioprospecting of Plant Growth Promoting Bacilli and Related Genera Prevalent in Soils of Pristine Sacred Groves: Biochemical and Molecular Approach.

Bacillus spp. and related genera native to soils of the pristine sacred groves from Meghalaya, India were characterized using biochemical and 16S rRNA gene analysis which revealed dominance of Bacillus, Paenibacillus, Lysinibacillus and Viridibacillus in the groves. Biochemical estimation was carried out for in vitro testing of plant growth promoting traits present in these isolates. PCR screening were performed for plant growth-promoting related genes involved in the biosynthesis of acid phosphatase (AcPho), indolepyruvate decarboxylase (ipdC), 1-aminocyclopropane-1-carboxylate deaminase (accd) and siderophore biosynthesis protein (asbA). 76% of the sacred grove isolates gave an amplified fragment for AcPho. Three of the isolates gave an amplified fragment for IpdC gene. Apart from 2 isolates, all the other isolates including the reference strains were positive for the amplification of the accd gene indicating their potential to produce ACC deaminase enzyme. 42% of the isolates gave an amplified fragment for asbA gene indicating the potential ability of these isolates to produce the catechol type siderophore, petrobactin. Overall findings indicated multiple PGP genetic traits present in these isolates which suggested that these isolates are capable of expressing multiple PGP traits. Phylogenetic and sequence analysis of accd and asbA genes from the isolates revealed that asbA genes from Paenibacillus taichungiensis SG3 and Paenibacillus tylopili SG24 indicated the occurrence of intergeneric horizontal transfer between Paenibacillus and Bacillus.

Ligand-induced conformation changes drive ATP hydrolysis and function in SMARCAL1.

Mutations and deletions in SMARCAL1, an SWI2/SNF2 protein, cause Schimke immuno-osseous dysplasia (SIOD). SMARCAL1 preferentially binds to DNA molecules possessing double-stranded to single-stranded transition regions and mediates annealing helicase activity. The protein is critical for alleviating replication stress and maintaining genome integrity. In this study, we have analysed the ATPase activity of three mutations ­ A468P, I548N and S579L ­ present in SIOD patients. These mutations are present in RecA-like domain I of the protein. Analysis using active DNA-dependent ATPase A domain (ADAAD), an N-terminal deleted construct of bovine SMARCAL1, showed that all three mutants were unable to hydrolyse ATP. Conformational studies indicated that the α-helix and ß-sheet content of the mutant proteins was altered compared to the wild-type protein. Molecular simulation studies confirmed that major structural changes had occurred in the mutant proteins. These changes included alteration of a loop region connecting motif Ia and II. As motif Ia has been implicated in DNA binding, ligand binding studies were done using fluorescence spectroscopy. These studies revealed that the Kd for protein-DNA interaction in the presence of ATP was indeed altered in the case of mutant proteins compared to the wild-type. Finally, in vivo studies were done to complement the in vitro and in silico studies. The results from these experiments demonstrate that mutations in human SMARCAL1 that result in loss in ATPase activity lead to increased replication stress and therefore possibly manifestation of SIOD.

Uranium (U)-tolerant bacterial diversity from U ore deposit of Domiasiat in North-East India and its prospective utilisation in bioremediation.

Uranium (U)-tolerant aerobic chemo-heterotrophic bacteria were isolated from the sub-surface soils of U-rich deposits in Domiasiat, North East India. The bacterial community explored at molecular level by amplified ribosomal DNA restriction analysis (ARDRA) resulted in 51 distinct phylotypes. Bacterial community assemblages at the U mining site with the concentration of U ranging from 20 to 100 ppm, were found to be most diverse. Representative bacteria analysed by 16S rRNA gene sequencing were affiliated to Firmicutes (51%), Gammaproteobacteria (26%), Actinobacteria (11%), Bacteroidetes (10%) and Betaproteobacteria (2%). Representative strains removed more than 90% and 53% of U from 100 µM and 2 mM uranyl nitrate solutions, respectively, at pH 3.5 within 10 min of exposure and the activity was retained until 24 h. Overall, 76% of characterized isolates possessed phosphatase enzyme and 53% had PIB-type ATPase genes. This study generated baseline information on the diverse indigenous U-tolerant bacteria which could serve as an indicator to estimate the environmental impact expected to be caused by mining in the future. Also, these natural isolates efficient in uranium binding and harbouring phosphatase enzyme and metal-transporting genes could possibly play a vital role in the bioremediation of metal-/radionuclide-contaminated environments.

Occurrence of horizontal gene transfer of P(IB)-type ATPase genes among bacteria isolated from the uranium rich deposit of Domiasiat in North East India.

Uranium (U) tolerant aerobic heterotrophs were isolated from the subsurface soils of one of the pre-mined U-rich deposits at Domiasiat located in the north-eastern part of India. On screening of genomic DNA from 62 isolates exhibiting superior U and heavy metal tolerance, 32 isolates were found to be positive for P(IB)-type ATPase genes. Phylogenetic incongruence and anomalous DNA base compositions revealed the acquisition of P(IB)-type ATPase genes by six isolates through horizontal gene transfer (HGT). Three of these instances of HGT appeared to have occurred at inter-phylum level and the other three instances indicated to have taken place at intra-phylum level. This study provides an insight into one of the possible survival strategies that bacteria might employ to adapt to environments rich in uranium and heavy metals.

Motifs Q and I are required for ATP hydrolysis but not for ATP binding in SWI2/SNF2 proteins.

Active DNA-dependent ATPase A Domain (ADAAD) is a SWI2/SNF2 protein that hydrolyzes ATP in the presence of stem-loop DNA that contains both double-stranded and single-stranded regions. ADAAD possesses the seven helicase motifs that are a characteristic feature of all the SWI2/SNF2 proteins present in yeast as well as mammalian cells. In addition, these proteins also possess the Q motif ~17 nucleotides upstream of motif I. Using site-directed mutagenesis, we have sought to define the role of motifs Q and I in ATP hydrolysis mediated by ADAAD. We show that in ADAAD both motifs Q and I are required for ATP catalysis but not for ATP binding. In addition, the conserved glutamine present in motif Q also dictates the catalytic rate. The ability of the conserved glutamine present in motif Q to dictate the catalytic rate has not been observed in helicases. Further, the SWI2/SNF2 proteins contain a conserved glutamine, one amino acid residue downstream of motif I. This conserved glutamine, Q244 in ADAAD, also directs the rate of catalysis but is not required either for hydrolysis or for ligand binding. Finally, we show that the adenine moiety of ATP is sufficient for interaction with SWI2/SNF2 proteins. The γ-phosphate of ATP is required for inducing the conformational change that leads to ATPase activity. Thus, the SWI2/SNF2 proteins despite sequence conservation with helicases appear to behave in a manner distinct from that of the helicases.

Elucidating the mechanism of DNA-dependent ATP hydrolysis mediated by DNA-dependent ATPase A, a member of the SWI2/SNF2 protein family.

The active DNA-dependent ATPase A domain (ADAAD), a member of the SWI2/SNF2 family, has been shown to bind DNA in a structure-specific manner, recognizing DNA molecules possessing double-stranded to single-stranded transition regions leading to ATP hydrolysis. Extending these studies we have delineated the structural requirements of the DNA effector for ADAAD and have shown that the single-stranded and double-stranded regions both contribute to binding affinity while the double-stranded region additionally plays a role in determining the rate of ATP hydrolysis. We have also investigated the mechanism of interaction of DNA and ATP with ADAAD and shown that each can interact independently with ADAAD in the absence of the other. Furthermore, the protein can bind to dsDNA as well as ssDNA molecules. However, the conformation change induced by the ssDNA is different from the conformational change induced by stem-loop DNA (slDNA), thereby providing an explanation for the observed ATP hydrolysis only in the presence of the double-stranded:single-stranded transition (i.e. slDNA).