Spatiotemporal dynamics of Vibrio spp. within the Sydney Harbour estuary

© 2016 Siboni, Balaraju, Carney, Labbate and Seymour. Vibrio are a genus of marine bacteria that have substantial environmental and human health importance, and there is evidence that their impact may be increasing as a consequence of changing environmental conditions. We investigated the abundance and composition of the Vibrio community within the Sydney Harbour estuary, one of the most densely populated coastal areas in Australia, and a region currently experiencing rapidly changing environmental conditions. Using quantitative PCR (qPCR) and Vibrio-specific 16S rRNA amplicon sequencing approaches we observed significant spatial and seasonal variation in the abundance and composition of the Vibrio community. Total Vibrio spp. abundance, derived from qPCR analysis, was higher during the late summer than winter and within locations with mid-range salinity (5-26 ppt). In addition we targeted three clinically important pathogens: Vibrio cholerae, V. Vulnificus, and V. parahaemolyticus. While toxigenic strains of V. cholerae were not detected in any samples, non-toxigenic strains were detected in 71% of samples, spanning a salinity range of 0-37 ppt and were observed during both late summer and winter. In contrast, pathogenic V. vulnificus was only detected in 14% of samples, with its occurrence restricted to the late summer and a salinity range of 5-26 ppt. V. parahaemolyticus was not observed at any site or time point. A Vibrio-specific 16S rRNA amplicon sequencing approach revealed clear shifts in Vibrio community composition across sites and between seasons, with several Vibrio operational taxonomic units (OTUs) displaying marked spatial patterns and seasonal trends. Shifts in the composition of the Vibrio community between seasons were primarily driven by changes in temperature, salinity and NO2, while a range of factors including pH, salinity, dissolved oxygen (DO) and NOx (Nitrogen Oxides) explained the observed spatial variation. Our evidence for the presence of a spatiotemporally dynamic Vibrio community within Sydney Harbour is notable given the high levels of human use of this waterway, and the significant increases in seawater temperature predicted for this region

Effect of Reduced Anthropogenic Activities on Water Quality in Lake Vembanad, India

The United Nation’s Sustainable Development Goal Life Below Water (SDG-14) aims to “conserve and sustainably use the oceans, seas, and marine resources for sustainable development”. Within SDG-14, targets 14.1 and 14.2 deal with marine pollution and the adverse impacts of human activities on aquatic systems. Here, we present a remote-sensing-based analysis of short-term changes in the Vembanad-Kol wetland system in the southwest of India. The region has experienced high levels of anthropogenic pressures, including from agriculture, industry, and tourism, leading to adverse ecological and socioeconomic impacts with consequences not only for achieving the targets set out in SDG-14, but also those related to water quality (SDG-6) and health (SDG-3). To move towards the sustainable management of coastal and aquatic ecosystems such as Lake Vembanad, it is important to understand how both natural and anthropogenic processes affect water quality. In 2020, a unique opportunity arose to study water quality in Lake Vembanad during a period when anthropogenic pressures were reduced due to a nationwide lockdown in response to the global pandemic caused by SARS-CoV-2 (25 March–31 May 2020). Using Sentinel-2 and Landsat-8 multi-spectral remote sensing and in situ observations to analyse changes in five different water quality indicators, we show that water quality improved in large areas of Lake Vembanad during the lockdown in 2020, especially in the more central and southern regions, as evidenced by a decrease in total suspended matter, turbidity, and the absorption by coloured dissolved organic matter, all leading to clearer waters as indicated by the Forel-Ule classification of water colour. Further analysis of longer term trends (2013–2020) showed that water quality has been improving over time in the more northern regions of Lake Vembanad independent of the lockdown. The improvement in water quality during the lockdown in April–May 2020 illustrates the importance of addressing anthropogenic activities for the sustainable management of coastal ecosystems and water resources

Lessons learned about the effect of reduced anthropogenic activities on water quality in a large lake system and opportunities towards sustainable management

Despite considerable efforts to protect vulnerable marine, coastal, and freshwater ecosystems, anthropogenic activities remain one of the main causes of poor water quality in rivers, lakes and wetland systems worldwide [1]. To move towards the sustainable management of coastal and aquatic ecosystems, it is important to understand how both natural and anthropogenic processes affect water quality. In 2020, a unique opportunity arose to study water quality in a large lake system in the southwest of India during a period when anthropogenic pressures were reduced due to a nationwide lockdown in response to the COVID-19 pandemic. Using remote sensing and in situ observations to analyse changes in five different water quality indicators, we showed that water quality improved in large areas of Lake Vembanad during the lockdown in 2020 [2]. The lessons learned illustrate that a coordinated response in reducing anthropogenic activities, as seen during the lockdown, could help achieve the targets set out in United Nation’s Sustainable Development Goals 3, 6 and 14 and significantly reduce aquatic pollution and improve water quality by 2030

Characterization of Rad51 from Apicomplexan Parasite <em>Toxoplasma gondii</em>: An Implication for Inefficient Gene Targeting

<div><p>Repairing double strand breaks (DSBs) is absolutely essential for the survival of obligate intracellular parasite <em>Toxoplasma gondii</em>. Thus, DSB repair mechanisms could be excellent targets for chemotherapeutic interventions. Recent genetic and bioinformatics analyses confirm the presence of both homologous recombination (HR) as well as non homologous end joining (NHEJ) proteins in this lower eukaryote. In order to get mechanistic insights into the HR mediated DSB repair pathway in this parasite, we have characterized the key protein involved in homologous recombination, namely TgRad51, at the biochemical and genetic levels. We have purified recombinant TgRad51 protein to 99% homogeneity and have characterized it biochemically. The ATP hydrolysis activity of TgRad51 shows a higher <em>K_M</em> and much lower <em>k_cat</em> compared to bacterial RecA or Rad51 from other related protozoan parasites. Taking yeast as a surrogate model system we have shown that TgRad51 is less efficient in gene conversion mechanism. Further, we have found that TgRad51 mediated gene integration is more prone towards random genetic loci rather than targeted locus. We hypothesize that compromised ATPase activity of TgRad51 is responsible for inefficient gene targeting and poor gene conversion efficiency in this protozoan parasite. With increase in homologous flanking regions almost three fold increments in targeted gene integration is observed, which is similar to the trend found with ScRad51. Our findings not only help us in understanding the reason behind inefficient gene targeting in <em>T. gondii</em> but also could be exploited to facilitate high throughput knockout as well as epitope tagging of <em>Toxoplasma</em> genes.</p> </div

Latitudinal Dynamics of Vibrio along the Eastern Coastline of Australia

The marine genus of bacteria, Vibrio, includes several significant human and animal pathogens, highlighting the importance of defining the factors that govern their occurrence in the environment. To determine what controls large-scale spatial patterns among this genus, we examined the abundance and diversity of Vibrio communities along a 4000 km latitudinal gradient spanning the Australian coast. We used a Vibrio-specific amplicon sequencing assay to define Vibrio community diversity, as well as quantitative PCR and digital droplet PCR to identify patterns in the abundances of the human pathogens V. cholera, V. parahaemolyticus and V. vulnificus. The hsp60 amplicon sequencing analysis revealed significant differences in the composition of tropical and temperate Vibrio communities. Over 50% of Vibrio species detected, including the human pathogens V. parahaemolyticus and V. vulnificus, displayed significant correlations with either temperature, salinity, or both, as well as different species of phytoplankton. High levels of V. parahaemolyticus and V. vulnificus were detected in the tropical site at Darwin and the subtropical Gold Coast site, along with high levels of V. parahaemolyticus at the subtropical Sydney site. This study has revealed the key ecological determinants and latitudinal patterns in the abundance and diversity of coastal Vibrio communities, including insights into the distribution of human pathogens, within a region experiencing significant ecological shifts due to climate change

Gene targeting efficiency of TgRad51 is independent of Ku80 function.

<p>(A) Schematic diagram showing knockout strategy for <i>CHL1</i> gene. Varying lengths of flanking homologous sequences on either side are indicated. (B) Efficiency of gene knockout with increasing flanking homology in <i>KU80</i> proficient (closed- circle, triangle or square) and KU80 deficient (open- circle, triangle or square) cells. The lengths of the flanking homologous stretches are indicated on the X-axis. These experiments are done at least 3 times and the mean values with standard deviations are plotted.</p

Alignment of TgRad51 with <i>Plasmodium falciparum</i> (PfRad51), human (HsRad51), <i>Saccharomyces cerevisiae</i> (ScRad51), <i>Schizosaccharomyces pombe</i> (SpRad51), <i>Trypanosoma bricei</i> (TbRad51) and <i>Leishmania major</i> (LmRad51) using Clustal method (Meg align, DNA star).

<p>Shaded areas represent identical amino acids. The two DNA binding Walker motifs are boxed.</p

Gene conversion efficiency of <i>TgRAD51</i> is poor compared to that of <i>ScRAD51</i>.

<p>(A) Schematic diagram of DSB repair choice experiment. <i>URA3</i> and <i>KANMX</i> represent wild type alleles. A HO endonuclease site is incorporated in <i>ura3::HOcs</i> mutant allele. Once the HO induced DSB is repaired by gene conversion (GC), the mutant <i>ura3::HOcs</i> allele is converted into wild type <i>URA3</i> allele. A single strand annealing (SSA) event leads to deletion of the intervening sequence (containing <i>KANMX</i> gene) and merging of <i>ura3::HOcs</i> and <i>URA3</i> alleles to create the wild type <i>URA3</i> allele. (B) Bar diagram showing the percentage of cells survived after induction of DSB. The relevant genotypes are marked on the X-axis. The white bars represent fraction of the cells survived by repairing the DSB using GC mechanism, where as the hatched bars denote the fraction of the survivors that employed SSA mechanism. Each bars represent mean value ± SD from 4 different experiments. (C) Western blots showing the abundance of ScRad51 and TgRad51 proteins. Different lanes are marked with the respective genotypes. Actin is the loading control.</p

Gene targeting efficiency of TgRAD51 increases with increase in stretch of homologous sequences.

<p>(A) Schematic diagram showing molecular events leading to targeted integration of ADE2 gene at ADH4 locus versus random integrations. KANMX is a second selectable marker retained only in case of random integration via non-homologous recombination mechanism. (B) Bar diagram showing efficiency of gene targeting at the ADH4 locus in cells harboring ScRad51 or TgRad51 as the sole recombinase. The mean value from four independent experiments is plotted with standard deviations. (C) Schematic diagram showing knockout strategy for <i>SBA1</i> gene. Varying lengths of flanking homologous sequences on either side are indicated. (D) Efficiency of gene knockout with increasing flanking homology. The lengths of the flanking homologous stretches are indicated on the X-axis. These experiments are done at least 3 times and the mean values with standard deviations are plotted.</p