Characterization of hyper tolerant bacteria for arsenic oxidation

Groundwater arsenic pollution causes many deaths worldwide. Arsenic levels in ground water are increasing day by day due to human activities, leading to higher threats of arsenic exposure. Arsenic release in drinking water resources causes major health problems.

Researchers at SPPU Pune, NCMR-NCCS Pune and NCL Pune collaboratively attempted to bio-prospect the microorganisms causing arsenic transformation. They used culture-dependent and independent approaches to study the microorganisms from Lonar Lake. Growth and Arsenic oxidation potential of microorganisms at increasing concentration was studied. The study also tried to understand possible pathway of Arsenic oxidation by studying the genes, transcripts and proteins involved.

Soil samples were collected from alkaline Lonar Crater Lake in Maharashtra. DNA was extracted from soil sample using soil DNA extraction kit. Unculturable and culturable diversity of soil sample was studied. Hyper-tolerance towards arsenic was studied. Growth and As(III) oxidation profile, ArsB amplification, enzyme inhibitor assay, As (III) oxidase assay, resting cells assay, Liquid chromatography mass spectrometry analysis , relative quantification of transcripts, microcosm studies and statistical analysis was done.

Bacterial community in the sample set comprised of total 21 phyla. Proteobacteria was predominantly found in the samples followed by Bacteroidetes. The bacterial member Pelagibacteraceae was detected predominantly in the sample followed by Microbacteriaceae, Flavobacteriaceae, Flammeovirgaceae, Vibrionaceae and Rhodobacteraceae with more than 5% abundance in all the samples. The bacterial diversity from Lonar Lake soil exhibited the presence of 10 As(III) oxidizing, 2 As(V) reducing, and 5 arsenic tolerant bacteria. Total 10 different genera were obtained viz. Bacillus, Lysinibacillus, Halomonas, Noviherbaspirillum, Roseomonas, Zobellela, Allidiomarina, Indibacter, Nocardioides, and Oceanimonas.

Arsenic hyper-tolerant Firmicute Bacillus firmus L-148 was isolated from arsenic limiting Lonar lake soil, which tolerated more than 3 M arsenic and could oxidize 75 mM arsenite [As (III)] in 14 days. It oxidized As (III) in presence of heavy metals. B. firmus L-148 was studied at the biochemical, protein, genomic and transcript level for understanding its arsenic oxidizing machinery. This study can be explored for bioremediation of arsenic contaminated water.

The tolerance towards arsenic in bacteria may be due to many reasons like expression of certain genes to combat the deleterious toxic effect. Expression of ArsA can contribute to high tolerance apart from the presence of more than one arsenic transforming operons.  Once it gets delivered, arsenic shows actual level of oxidation . The means of transport of Arsenic was through water pipes and oxidation of samples in the waste water.

Though the potential cultures in this study were isolated from trivial arsenic content environment, they tolerated moderate to high concentration of As(III) and As(V). These findings clearly demonstrated that arsenic tolerance level of bacteria is not correlated to the arsenic content of the environment in which they thrive.


Stress tolerance in bacterial strains of the genus Rhodanobacter isolated from a mixed waste contaminated subsurface

Oak Ridge Integrated Field Research Challenge (ORIFRC) site is characterized by low pH and consists of high nitrate, organics and heavy metals. ORIFRC field laboratory comprises variety of contaminants (uranium, technetium, nitrate, volatile organic carbon species etc.) which are of interest to US Department of Energy. Rhodanobacter is a dominant bacterial species found at this site and ideal for remediation of such mixed contaminated sites.

A collaborative study was conducted by researchers at Florida State University, NCMR-NCCS Pune, University of Illinois, Georgia Institute of Technology and Symbiosis School of Biological Sciences to understand the physiologic basis of stress tolerance in members of the genus Rhodanobacter. The study was conducted in order to understand how bacterial strains of the genus Rhodanobacter survive and dominate in the mixed waste contaminated habitats of the ORIFRC site. To address this, a systematic analysis of relevant phenotypic properties of strains of the genus Rhodanobacter was studied.

Eight strains of Rhodanobacter were isolated from high and low contaminated zones and used for pH and nitrate utilization studies. NaCl, nitrate, nitrite and heavy metal tolerance capacity was studied for the two selected strains of R. denitrificans. Based on metals known to be present at the ORIFRC site, Rhodanobacter strains were tested for tolerance to zinc, cadmium, cobalt, nickel, copper and uranium. To determine the effect of incubation time on growth of R. denitrificans at high metal concentrations, studies were carried out with nickel and uranium since these two metals were found to be present at very high concentrations at the test site.

The results supported the growth potential of Rhodanobacter in acidic subsurface groundwater conditions and confirmed that under suitable cultivation conditions, isolated R. denitrificans strains can tolerate acidic pH consistent with ORIFRC site pH values. It was also observed that organisms adapted to stress better under conditions of high organic content. The ability of Rhodanobacter strains to grow at extremely low pH and under high nitrate and heavy metals concentrations is responsible for their dominance at the contaminated subsurface of the ORIFRC site. The data indicated that both the strains are well adapted to the eco physiological conditions of the contaminated ORIFRC site.

As bacteria from the genus Rhodanobacter are denitrifiers, their activity in the ORIFRC site subsurface is also linked to carbon and nitrogen cycling and may play a critical role in the bioremediation of uranium. Based on prior findings and the results of the current study, researchers postulated that low pH tolerance and high level of stress tolerance for a range of metals along with denitrification potential gives a selective advantage to members of the genus Rhodanobacter. Bacteria from the genus Rhodanobacter are facultative anaerobes, and this physiologic capability makes them ideal candidates for robust growth in the contaminated subsurface of the ORIFRC site. Due to their enhanced stress tolerance abilities, Rhodanobacter spp. survives at low pH and in the presence of elevated concentrations of heavy metals, nitrate and nitrite.


PAN-India SARS-CoV-2 genome sequencing reveals important insights into the outbreak

The ongoing pandemic of Severe Acute Respiratory Syndrome (SARS-CoV-2) has emerged as a global health problem and has adversely affected the world. The novel corona virus is spreading rapidly creating a threat for humankind. It is estimated that it can spread twice as fast as the 1918 Spanish flu virus. Whole-genome sequencing of pathogens, especially viruses, is a powerful tool to generate rapid information on outbreaks. The results from this technique help in effective understanding of the introduction of the infection ,dynamics of transmission, contact tracing networks and impact of informed outbreak control decisions. This technique has been effective in earlier outbreaks like the Ebola virus.

A collaborative study was conducted by researchers from NIBMG Kalyani, ILS Bhubaneswar, CDFD Hyderabad, NCBS Bengaluru, InStem Bengaluru, NCCS Pune and ICMR-Regional Centre for Medical Research, Bhubaneswar in order to achieve initial goal of completing the sequencing of 1000 SARS-CoV-2 genomes. The nasal and oral swabs were collected from individuals testing positive for COVID-19. The samples were collected from 10 states covering different zones within India. Phylodynamic analysis, mutation analysis and haplotype network analysis was performed. One thousand and fifty two sequences were used for phylodynamic, temporal and geographic mutation patterns and haplotype network analyses. This study will contribute in understanding how the virus is spreading, ultimately helping to restrict transmission, prevent new cases of infection, and provide information for research on how to interevent the spread of infection.

Preliminary results indicated that multiple lineages of SARS-CoV-2 are circulating in India, might have introduced by travel from Europe, USA and East Asia. In particular, there is a predominance of the D614G mutation, which is found to be emerging in almost all regions of the country. Scientists were able to estimate the possible source of country of different varieties of the virus introduced in India because of travel. The virus has also mutated and one of the mutations has attained highest frequencies across most of the states. There are two lineages of the virus named as 20A and 20B which are predominant across the country. The haplotype 20A is most abundant in northern and eastern India, 20B haplotype was abundant in southern and western India. The ancestral haplotypes of 19A and 19B were mostly found in Northern and Eastern India, with 19B being the most abundant in the latter region.

Analysis indicated that the haplotype diversities across India and in each region continued to increase until May 2020, after that it reduced drastically with the emergence of the A2a haplotypes which has overtaken other lineages by June 2020. Such interpretations might enable improved understanding of the virus and hence the health decisions. From the haplotype network, researchers observed that Maharashtra, Karnataka created three distinct haplotype nodes and sequences from Odisha, West Bengal and Uttarakhand sparse in different haplotype nodes. They also observed a haplotype node with the majority of the genomes from West Bengal, Odisha and a small percentage of the samples belonging to Uttarakhand.

Analysis of probable country of origin of these SARS-CoV-2 sequences in India revealed that they had been probably introduced by travel from multiple countries across the globe. 20A, B and C haplotypes were introduced from multiple countries in Europe and also American continents. Interestingly, 20A alone is predicted to have been introduced by travel from Italy, Saudi Arabia, United Kingdom and Switzerland. Similarly, 20B was introduced from the United Kingdom, Brazil, Italy and Greece. In contrast, 19A was introduced from China alone while 19B was introduced by travel from China, Oman and Saudi Arabia.

The number of COVID-19 occurrences in India has increased drastically over the time. Although most of the states have their own strategic lockdown devised to control the outbreak, it will be more efficient if we can include the geographical transmission pattern information in the planning of such strategies. In the current study, scientists have tried to explore the transmission of the infection among different states of India. It is necessary to add more genomic datasets to understand clear picture.


Meet Dr. Neetha Joseph- Scientist at NCMR-NCCS Pune

Dr. Neetha Joseph’s research interest is in microbial systematics, ecology and community analysis. She is affiliated with NCMR-NCCS Pune from last 8 years. She is in-charge of FAME analysis service and curator of Firmicutes. It was a great pleasure to interact with Dr. Neetha and to know more about her as a person and her work.

Kranti: Dr. Neetha, you have worked with coastal environment micro-organisms during your PhD. At a personal level, what motivated you to enter into microbiology research?
Dr. Neetha: Kranti, my native place is in Kerala, a beautiful coastal area in India. Kochi is a lovely place with lot of Backwaters and Estuaries. When I finished my post-graduation, I got an opportunity to join at National Institute of Oceanography (NIO) where most of the research work is related to Ocean and Estuaries. Nutrient enrichment due to various anthropogenic activities is the most widespread problem in estuaries around the world. Significant spatial and temporal variability of physico-chemical and geochemical characteristics and productivity patterns are the important characteristics of estuaries. Microbial communities are involved in mineralization of organic matter; therefore, I was interested in understanding the response of these sedimentary microbial communities to these regional and seasonal changes using signature biomolecules (Phospholipid Fatty Acids – PLFA) as a means of identifying the specific group of microorganisms in the natural ecosystems .

Kranti: Everybody has someone in their life who inspires them to achieve something. Who is your inspiration in science?
Dr. Neetha: My PhD guide at NIO, Kochi is my inspiration in Science. She inspired me a lot! She encouraged me in various aspects of science and helped in boosting my confidence.

Kranti: Which methods and tools you use in your research?
Dr. Neetha: Microbial communities are involved in mineralization of organic matter in estuarine sediment. To understand the response of these microbial communities to various physiochemical and geochemical factors using signature biomolecules (Phospholipid Fatty Acids – PLFA) as a means of identifying the specific group of microorganisms in the natural ecosystems. Phospholipids are mainly found in the cell membrane, not in storage lipids and have a rapid turnover in aquatic sediments. So it provides a measure of viable cellular biomass in an ecosystem.  Different physiological and functional groups of microorganisms in sediments were described using PLFA analysis.
The extracted PLFAs were analyzed using gas chromatography (Agilent 7890 Series, USA) with a cross-linked phenyl – methyl siloxane capillary column (25 m, 0.2 mm) and FID. Identification of the FAMEs was carried out by comparison of retention time and equivalent chain length with known standards like Eukary calibration mixture – 1201A (Eukary6 method, Version: 3.7) and MIDI peak identification software (MIDI Inc., Newark, DE).

Kranti: You are contributing to microbiology related services offered at NCMR Pune. What are those services ?
Dr. Neetha: I am in – charge for FAME analysis service and curator of Firmicutes at NCMR. Under FAME analysis, the bacterial (aerobic and anaerobic) or yeast samples are identified based on their cell membrane fatty acids. Also cell membrane fatty acids are analyzed for novel taxa along with their closely related type strains for publication.

Kranti: Are journals necessary in the age of internet? Don’t you think research should be done not just to publish a paper but also to have real life impacts?  
Dr. Neetha: We know that nowadays we can extract all the information we require via internet. But we cannot compare the beauty of reading a book or journal with internet. Yes, I totally agree that we should do research not only to publish a paper but also to have real life impacts.

Kranti: Being a woman in science, what are the challenges that you’ve faced?
Dr. Neetha: Being a woman in science, the major challenge I face is to manage family, children and their education along with my research work. Another challenge is to get time to spend for research along with my routine services and other commitments.

Kranti: How do you maintain the balance of your family and work-life?
Dr. Neetha: For that I should thank my husband and children for their co-operation and moral support throughout my career.

Kranti: What advice would you like to give to young women who want to pursue research?
Dr. Neetha: If you have an actual interest in science along with sincerity, dedication and hardworking nature, you will be able to succeed in your research career. As a woman, you should be able to manage your time and having patience is also equally important to succeed in your life.

Kranti: Would you share with us any memorable incident/moment of your research life?
Dr. Neetha: In the year 2000, I got an opportunity to participate in Cochin – Alleppey – Mangalore Cruise on board CRV Sagar Paschimi, under DOD, COMAPS Programme. It was a rare experience and golden memory in my research life.

Kranti: Most of the scientist’s children opt for career in science. Do you want your child to become a scientist too? 
Dr. Neetha: Yes, if they are showing real interest in science and research, definitely I will encourage him or her to opt for career in Science.