Genomic analysis of a bacterial strain isolated from the Lonar Lake

-By Kranti Karande

Bacterial isolates of the Rhodococcus genus are widely recognized as being capable of catabolizing a wide variety of aliphatic, aromatic and polyaromatic hydrocarbons. Taxonomically genus Rhodococcus belongs to the Nocardiaceae family under phylum Actinobacteria. The tremendous catabolic diversity and remarkable biotransformation ability of genus Rhodococcus is mainly due to the existence of a diverse array of catabolic genes reported for several enzymatic groups such as monooxygenase, dioxygenase, and hydroxylases.

This study identified the genome sequencing data and the draft genome of the Rhodococcus rhodochrous strain originally isolated from Lonar Lake sediments located in Maharashtra province of India. The strain was identified as Rhodococcus rhodochrous based on the mass spectra of the cell extracts of overnight grown pure cultures and DNA sequencing results.

Specific gene and gene groups responsible for catabolism of various compounds have been identified and assigned to the respective compound. Total 39 genes were assigned to subsystem category ‘Stress response’ and the analysis of the genes for this subsystem category revealed the presence of specific genes. The presence of these specific genes implied that the isolate has the ability to synthesize and uptake ‘Glycine betaine (N,N,N-trimethylglycine)’ which may be the main osmolyte synthesized by this bacterium under osmotic stress conditions usually present in the soda lake environment. 

The genomic data reported in this publication will pave the way for further study of the different catabolic genes implicated in hydrocarbon metabolism in this bacterium.

Reference: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058525/

The microbial diversity of newly diagnosed diabetics is different from that of healthy non-diabetics, although there is no major difference in prediabetics.

– By Kranti Karande

Type 2 diabetes (T2D) is the most common type of diabetes. People who have T2D are said to have insulin resistance. T2D is a chronic condition that affects blood sugar (glucose) absorption and lipid metabolism.

Recently the gut microbiome was identified as an important factor for T2D development. Disruption of the balance between gut microbes has been linked to the development of metabolic diseases, in particular T2D, obesity and cardiovascular disorders. Some of the earlier studies observed dissimialrity between the gut microbiome of diabetics, prediabetics, and healthy nondiabetic individuals, although very few investigated the gut microbiome of treatment-naive individuals with T2D.

In this study, scientists have analyzed ND, PreDMs, NewDMs, and KnownDMs gut microbiome to understand and identify differences in the T2D, and prediabetes-associated microbial community. The scientists also looked at the community changes in microbial association networks and identified genera which are contributing for the transition from healthy (control) to diabetic state here called as driver taxa. They also analyzed the association of a wide array of serum biomarkers with genera, which were differentially abundant and were also found to be contributing for the major changes in the gut microbiome of T2D individuals .

In this study, a total of 102 subjects were studied, and they looked at the gut microbiota of prediabetics (PreDMs) (n= 17), newly diagnosed diabetics (NewDMs) (n = 11), and diabetics on antidiabetic treatment (KnownDMs) (n = 39) and compared them with healthy nondiabetics (ND) (n= 35). Twenty-five different serum biomarkers were measured to assess the status of diabetes and their association with gut microbiota.

The research identified nine separate genera in four sample groups as having differential abundance. Among them, Akkermansia, Blautia, and Ruminococcus were found to be significantly decreased, while Lactobacillus was increased in NewDMs compared to ND and recovered in KnownDMs. Akkermansia was inversely correlated with HbA1c and positively correlated with total antioxidants. Compared to ND, there was increased abundance of Megasphaera, Escherichia, and Acidaminococcus and decreased abundance of Sutterella in KnownDMs.

Among many taxa known to act as community drivers during disease progression, it was observed that genus Sutterella is a common driver taxon among all diabetic groups. On the basis of the results of random forest analysis (methodology) , they discovered that the serum metabolites fasting glucose, HbA1c, methionine, and total antioxidants were highly discriminating factors among studied groups. The compiled data showed that the gut microbial diversity of NewDMs is substantially different from that of ND but not of PreDMs. Interestingly, after anti-diabetic treatment, the microbial diversity of KnownDMs tends to recover toward that of ND.

Gut microbiota is thought to play a role in the development of the disease, and previous studies have documented a microbiome dysbiosis association with T2D. In this study, scientists have attempted to investigate gut microbiota of ND, PreDMs, NewDMs, and KnownDMs. They found that the genera Akkermansia and Blautia decreased significantly in treatment-naive diabetics and were restored in KnownDMs on antidiabetic treatment. Understanding the transition of microbiota and its association with serum biomarkers in diabetics with different disease states may pave the way for new therapeutic approaches for T2D.

Twenty-five different serum biomarkers were checked and compared with the gut microbiota to assess the different states of diabetes. Targeted 16S rRNA amplicon sequencing was used to assess the microbial diversity, community shuffling, and identification of driver taxa for the disease state. They have investigated relationships between a wide array of serum biomarkers responsible for progression of T2D with significantly diverged and differentially abundant taxa in each study group. Significantly different patterns were observed in the gut microbiota of PreDMs, NewDMs, and KnownDMs compared to ND. In KnownDMs, abundance of some microbial taxa was found to be similar to that of ND group.

Since oxidative stress is known to be involved in the establishment of insulin resistance and diabetic complications , they also measured total antioxidant capacity and lipid peroxides, a marker of oxidative damage to lipids in the blood. Interstingly, they found a significant decrease in total antioxidant capacity and increase in lipid peroxidation in treatment-naive NewDMs but not in PreDMs. In KnownDMs on treatment with metformin, an increase in total antioxidant capacity and decrease in lipid peroxidation were observed.

These findings show differences in the gut microbiome in PreDMs, NewDMs, and KnownDMs compared to ND. In PreDMs, the gut microbiota doesn’t show a significant difference when compared with ND, while in NewDMs, both abundance and diversity have changed significantly, which seems to be restored to some degree in KnownDMs on antidiabetic care.

Reference: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112960/pdf/mSystems.00578-19.pdf

Services at NCMR Pune

PART-II Microbial identification and characterization services

NCMR Pune offers multiple services for microbial identification and characterization. All of these services are listed below:

A. rRNA gene/ ITS sequencing for microbial identification: rRNA gene sequence of bacteria/ Internal Transcribed Sequence (ITS) of fungi is one of the most reliable molecular methods for microbial identification. We offer this sequencing service for identifying bacteria, archaea and fungi using universal primers for each type of organism.
Once the organism is received at NCMR, it goes through the following procedure: DNA isolation, PCR using universal primers for the type of organism, purification of the PCR amplicons, cycle sequencing reactions, purification and run them on an automated capillary-based Sanger DNA Sequencing system. At every step, there is in-house quality check to ensure success of the sequencing reactions. Post sequencing, fragments are manually checked and only good quality sequences are used to form contigs, which are then matched in well-curated databases for assigning closest neighbor as the tentative identification of your submitted organism.

B. Phylogenetic analysis: Phylogenetic analysis based on 16S rRNA gene is a powerful tool to study the evolutionary relationships among micro-organisms. It provides insights of genes or sequences which include relationships, origins, and closest taxonomic identification. These relationships are described by a branching of the tree.

C. Determination of Polar lipids: Lipids are one of the most verdiagram, or tree, with branches joined by nodes and leading to the terminal at the tips of satile compounds present in living system. Bacterial cell membrane is mostly composed of polar lipids like phospholipids, amino lipids and glycolipids etc. Lipids profile of a bacterial species is well suited to be used as a taxonomic character and is required to be included for description of novel taxa.

D. Determination of G+C mol% in DNA: G+C content in DNA of a species of bacteria is a characteristic feature and in bacterial world G+C mol% varies from 24% to 76%. This value is essential for description of a novel species.
There is a linear correlation between the melting temperature of DNA and G+C mol %. NCMR uses fluorimeteric method using double strand specific fluorescent dye and real-time PCR thermocyclers for determination of melting temperature (Tm) and G+C mol % is calculated from the Tm. This service includes isolation of high quality DNA from microbial culture(s), melting temperature experiment (in triplicate) and calculation of the G+C mol% content.

E. DNA-DNA hybridization: DNA-DNA hybridization (DDH) values determine relatedness between bacteria and is one of the minimum criteria for the description of novel taxon when 16S rRNA gene sequence similarity is more than 97%. Since 1960s DDH values have been used for species delineation with 70% as a recommended cut-off. At high temperature DNA gets denatured and when it cooled down gradually, it starts reassociating with the complementary sequences and results into the formation of double stranded DNA again. But when the DNA of two organisms mixed together and denatured, while reassociation of these hybrid DNA the degree of binding is directly proportional to the sequence similarity between the two DNAs. This degree of binding is converted into the percentage hybridisation. NCMR uses fluorimeteric method for the calculation of DDH.
This service includes isolation of high quality DNA from related species, hybridisation experiment (in triplicate) and calculation of the DDH value.

F. Phenotypic characterization of bacteria by conventional method : Phenotypic characterization of bacteria by conventional method, offered by NCMR, includes Morphological, physiological, and biochemical properties.
Morphological characterisation includes: Colony morphology (such as size, shape, colour, margin, opacity and consistency of bacterial colonies), microscopic studies for detecting Gram nature, presence or absence of spore and motility.
Physiological characteristics include: Tolerance of a bacterium to various concentrations of NaCl, pH and temperature.
Biochemical tests include: Catalase, oxidase, H2S production, indole, citrate utilization, MR-VP, nitrate reduction, hydrolysis of various substrates like starch, casein, urea, gelatin, DNA and utilization of various carbohydrates.

F. Phenotypic characterization of bacteria by conventional method : Phenotypic characterization of bacteria by conventional method, offered by NCMR, includes Morphological, physiological, and biochemical properties.
Morphological characterization includes: Colony morphology (such as size, shape, color, margin, opacity and consistency of bacterial colonies), microscopic studies for detecting Gram nature, presence or absence of spore and motility.
Physiological characteristics include: Tolerance of a bacterium to various concentrations of NaCl, pH and temperature.
Biochemical tests include: Catalase, oxidase, H2S production, indole, citrate utilization, MR-VP, nitrate reduction, hydrolysis of various substrates like starch, casein, urea, gelatin, DNA and utilization of various carbohydrates.
G. Phenotypic characterization by API: The Analytical Profile Index (API) is a miniaturized panel of biochemical tests compiled for identification of groups of closely related bacteria. Different test panels are available in dehydrated forms which are then reconstituted at the time of use by addition of bacterial suspensions of desired cell density. After proper incubation, test results are scored (after 24 and some time 48 hr) as +/- to generate a seven-digit code (profile). Identity of the bacterium is then derived from the database with the relevant cumulative profile code book or software (apiweb™).
NCMR offers phenotypic characterization by four type of API methods using either one or a combination of strips depending on the users’ requirements.
API 20E: API 20 E is a standardized characterization/identification system for Enterobacteriaceae and other non-fastidious, Gram-negative rods. It consists of 20 microtubes containing dehydrated substrates.
API 20NE: A standardized system characterization/identification of non-fastidious, non-enteric Gram-negative rods (e.g. Pseudomonas, Acinetobacter, Flavobacterium, Moraxella, Vibrio, Aeromonas, etc.), combining 8 conventional and 12 assimilation tests.
API 50 CH: Used for characterization/identification Bacillus and related genera as well as gram-negative rods belonging to Enterobacteriaceae and Vibrionaceae families based on fermentation of 49 carbohydrates.
API ZYM is a semi-quantitative micro-method designed for detection of enzymatic activities.

For more details on these services visit: http://210.212.161.138/services

Services at NCMR Pune

PART-I

National Centre for Microbial Resource, Pune is India’s microbial culture collection centre recognized by World Federation of Culture Collections (WFCC). The main objective of NCMR is to provide authentic microbial cultures to industries as well as academic and research institutes.

Culture collections play an important role in the area of biotechnology and are valuable resources for the sustainable use of microbial diversity and its conservation. But their meaningful exploitation is possible only if the properties of cultures are properly documented and the information is easily accessible. National Centre for Microbial Resource at NCCS Pune established by the Department of Biotechnology, Government of India is one among country’s largest culture collection centres. It is recognized as an International Depository Authority for the deposition of patent cultures under Budapest Treaty and designated national repository under the Biodiversity Act 2002.

NCMR focuses on fundamental research in the fields of microbial diversity, microbial taxonomy, microbial genomics and proteomics etc. One of the main objective of NCMR is providing microbial culture related services. NCMR provides multiple services like culture deposit, culture supply, identification/characterization of microorganisms, genomics/ metagenomics, imaging by scanning electron microscopy, phytoplasma detection, testing for microbial load for food safety certifications etc.

At present, NCMR accepts bacteria, fungi, and plasmids belonging to hazard group 1 and 2. Culture deposition service at NCMR is divided into following categories:

General deposit, IDA deposit and safe deposit.


A. General deposit: Cultures deposited under this category are accessible to public for teaching and /or scientific research and no restriction is imposed. NCMR reserves the right to reject a request for deposit of culture. Unidentified cultures are not accepted for deposit under this category. Once accepted and accessioned by NCMR, a culture cannot be withdrawn from the collection. There is no fee for such deposits.

B. International Depository Authority (IDA): Microorganisms deposited under IDA fulfill the requirement of the deposit for the purposes of patent procedures in all states signatory to the Budapest Treaty. NCMR is the second IDA in India. All IDAs operate as per the regulations of the Budapest treaty. The advantage of depositing microorganisms under IDA is almost similar forms are used and uniform procedures are followed in dealing with such deposits. Confidentiality and security of such deposits are maintained by NCMR. NCMR is obliged to keep the deposits under IDA for the period of 30 years from the date of deposit.

C. Safe deposit: Confidentiality and security is maintained while depositing such cultures. These cultures are not listed in the catalogue. There is an annual fee for such deposits. Depositor needs to sign an agreement with NCMR for making such a deposit.

NCMR supplies cultures listed in NCMR catalog (hard copy/electronic) for teaching and research purposes.

For more information on culture deposit and culture supply services at NCMR, visit following website: http://210.212.161.138/services

In the next article, we will learn about other services offered at NCMR.