Development of genetic markers in cyanobacteria and stability of genetically marked strains in soil

Reporter marker GUS (-glucuronidase gene from Escherichia coli) and luc operon from the American firefly were introduced into cyanobacteria and the stability of these markers in soil was examined. To transfer the integrational vector into cyanobacteria, the genomic DNA library of Synechocystis sp. or Anabaena cylindrica maintained in pBR 322, pCY 100 and pCY 101 were transformed with HB 101 containing pRL 528 and selected for Cm^r and Amp^r. These clones of HB 101 containing pRL 528 and the vectors carrying different cyanobacterial chromosomal DNA fragments were used for triparental mating with HB 101 [pRK 2013/pRK 2073] and cyanobacteria. The frequency of transconjugants for integrational vectors was between 2.1 × 10^–5 and 4.0 × 10^–4. The transfer frequency of RSF 1010 based vectors (pDSK 519 and pCY 106) was 1.0–4.5 × 10^–4 in Synechocystis sp. whereas A. cylindrica failed to maintain these vectors. Low frequency transfer (2.0–2.3 × 10^–6) of RK 2 based vectors pVK 100 and pCY 104 was observed in A. cylindrica but these were unable to replicate in Synechocystis sp. The vectors in general were stable at least by 74.9% for 60 days of incubation in BG-11 medium. The markers were less stable in A. cylindrica (74.9–84.2%) compared to Synechocystis sp. (80.1–88.8%) at 60 days of incubation. Integrational vectors were almost 85% stable in both the strains. The RK 2 derivative of pCY 104 was less stable in A. cylindrica (74.9–77.3%) than the RSF 1010-based vector pCY 106 in Synechocystis sp. (80.1–81.0%). A maximum of 64.7% of the markers were lost in soil. The chromosomal markers through integrational vectors were found to be highly stable and 68.2–72.7% of these markers were retained in cyanobacteria at 60 days of incubation. Plasmid markers were less stable, with a loss of 64.7–48.7% at the end of the experiment. In A. cylindrica 58–65% of the RK 2 vector was lost whereas in Synechocystis sp. 49–61% of RSF 1010 was lost at 60 days of incubation.     

Whole Genomic analysis of a clinical isolate of Uropathogenic Escherichia coli strain of Sequence Type - 101 carrying the drug resistance NDM-7 in IncX3 plasmid

The emerging NDM-producing Enterobactereciae is a major threat to public health. The association of NDM-7 with sequence type 101 E.coli is identified in very few numbers. Therefore, it is of interest to analyse the whole genome sequence of NDM-producing uropathogenic E. coli XA31 that was found to carry numerous drug resistance genes of different antibiotic classes. The isolate E. coli belongs to ST-101 carrying blaNDM-7 coexisting with several resistance genes blaOXA-1, blaTEM1-A, blaCTX-M15, aac(6'')-Ib-cr, catB3, tetB. Resfinder predicts this and four other plasmid replicons were identified using the Plasfinder in the CGE platform. The high transferable IncX3 plasmid was found to carry the NDM-7 gene. Thus, we the report the combination of NDM-7-ST101-IncX3 in India. The combination of this epidemic clone with NDM-7 is highly required to develop an effective infection control strategy.     

Use of plant extracts and biocontrol agents for the management of brown spot disease in rice

Fifty plant extracts, four oil cakes and eight antagonistic organisms were tested against Bipolaris oryzae (Cochliobolus miyabeanus), the causal agent of brown spot disease of rice. In vitro studies indicated that two leaf extracts, Nerium oleander and Pithecolobium dulce exerted the higher percent inhibition to mycelial growth (77.4, 75.1%) and spore germination (80.3, 80.0%) of B. oryzae. Among the four oil cake extracts tested in vitro against B. oryzae, neem cake extract showed the maximum inhibition percent to mycelial growth (80.18%) and spore germination (81.13%) of the pathogen followed by mahua cake extract, castor and gingelly cake extract. Trichoderma viride (Tv2) was significantly effective in inhibiting the mycelial growth (62.92%) and spore germination (77.03%) of the pathogen followed by Trichoderma harzianum (Th5) and Trichoderma reesei (Tr3). The promising leaf extracts, oil cake extracts and antagonistic microorganisms were further evaluated for their efficacies in disease management under glasshouse and field conditions. In glasshouse studies, post-infectional spraying of rice plants with neem cake extract, N. oleander leaf extract and T. viride (Tv2) was significantly effective in reducing the incidence of brown spot of rice by 66, 52 and 45 percent respectively. Two rounds of spraying of rice plants with neem cake extract, N. oleander leaf extract and T. viride (Tv2) in the field at initial appearance of disease and 15 days later reduced the incidence of brown spot (70, 53 and 48% disease reduction respectively) and increased the yield by 23, 18 and 15 percent respectively.     

Genome sequence analysis of the Indian strain Mannheimia haemolytica serotype A2 from ovine pneumonic pasteurellosis

Mannheimia haemolytica is a leading causative agent of pasteurellosis in ruminants. Genome of M. haemolytica strains from different hosts has been sequenced worldwide to understand its pathogenesis. There are only few reports on the isolation of M. haemolytica in India with limited information on its molecular characteristics. The present study focuses on genome sequence analysis of a M. haemolytica strain isolated from pneumonic sheep. Mannheimia haemolytica A2 strain NIVEDI/MH/1 was isolated and identified by species and serotype-specific PCRs. Whole genome sequencing was performed using the Ion Torrent Personal Genome Machine. A comparative genomic analysis was performed to understand the virulence determinants of the Indian strain and its phylogenetic relationship with other global strains. Sequence data revealed a draft genome of 2,211,426 bp size with 41.3% GC content, assembled into 17 contigs, and contained 2379 genes. Five genomic islands identified in the genome showed high sequence identity with other respiratory pathogens of the Pasteurellaceae family. Phylogenetic analysis showed M. haemolytica A2 NIVEDI/MH/1 is very close to a M. haemolytica A2 strain from pneumonic calf. Further, the analysis revealed the presence of virulence, metal-, and multidrug resistance genes needed for pathogenesis and survival of the bacteria during infection. Also, we identified the presence of type I-C and type II-C of CRISPR-Cas arrays in the present sequenced genome. The study emphasizes the role of M. haemolytica in respiratory infections of ruminants in the Indian subcontinent and indicates the role of vertical and horizontal gene pools in pathogenicity and survivability of the bacteria.     

Identification of Recombination and Positively Selected Genes in Brucella

Brucella is a facultative intracellular bacterium belongs to the class alpha proteobacteria. It causes zoonotic disease brucellosis to wide range of animals. Brucella species are highly conserved in nucleotide level. Here, we employed a comparative genomics approach to examine the role of homologous recombination and positive selection in the evolution of Brucella. For the analysis, we have selected 19 complete genomes from 8 species of Brucella. Among the 1599 core genome predicted, 24 genes were showing signals of recombination but no significant breakpoint was found. The analysis revealed that recombination events are less frequent and the impact of recombination occurred is negligible on the evolution of Brucella. This leads to the view that Brucella is clonally evolved. On other hand, 56 genes (3.5 % of core genome) were showing signals of positive selection. Results suggest that natural selection plays an important role in the evolution of Brucella. Some of the genes that are responsible for the pathogenesis of Brucella were found positively selected, presumably due to their role in avoidance of the host immune system.     

Patterns and drivers of fish extirpations in rivers of the American Southwest and Southeast

Effective conservation of freshwater biodiversity requires spatially explicit investigations of how dams and hydroclimatic alterations among climate regions may interact to drive species to extinction. We investigated how dams and hydroclimatic alterations interact with species ecological and life history traits to influence past extirpation probabilities of native freshwater fishes in the Upper and Lower Colorado River (CR), Alabama‐Coosa‐Tallapoosa (ACT), and Apalachicola‐Chattahoochee‐Flint (ACF) basins. Using long‐term discharge data for continuously gaged streams and rivers, we quantified streamflow anomalies (i.e., departure “expected” streamflow) at the sub‐basin scale over the past half‐century. Next, we related extirpation probabilities of native fishes in both regions to streamflow anomalies, river basin characteristics, species traits, and non‐native species richness using binomial logistic regression. Sub‐basin extirpations in the Southwest (n = 95 Upper CR, n = 130 Lower CR) were highest in lowland mainstem rivers impacted by large dams and in desert springs. Dampened flow seasonality, increased longevity (i.e., delayed reproduction), and decreased fish egg sizes (i.e., lower parental care) were related to elevated fish extirpation probability in the Southwest. Sub‐basin extirpations in the Southeast (ACT n = 46, ACF n = 22) were most prevalent in upland rivers, with flow dependency, greater age and length at maturity, isolation by dams, and greater distance upstream. Our results confirm that dams are an overriding driver of native fish species losses, irrespective of basin‐wide differences in native or non‐native species richness. Dams and hydrologic alterations interact with species traits to influence community disassembly, and very high extirpation risks in the Southeast are due to interactions between high dam density and species restricted ranges. Given global surges in dam building and retrofitting, increased extirpation risks should be expected unless management strategies that balance flow regulation with ecological outcomes are widely implemented.