- Volume 1, Issue 1A, 2019

Contemporary metagenomic annotation methods have proven insufficient in our attempts to better understand the complex environments around us. We call the yet to be annotated part of a metagenome it’s ‘dark matter’. The Gene Ontology (GO) is a hierarchical vocabulary used to describe gene product function and a large collection of curated genes with GO annotations already exists. DeepGO utilises deep learning to build models from these curated genes and gene products to predict GO categories for novel proteins. One of the major problems with metagenomic studies today is the process of assembling the environmental DNA sequences into their original genomes. This is difficult, with chimeric metagenomically assembled genomes being common. To avoid this and the computational and time expense, we have modified DeepGO to perform protein function prediction directly from sequence reads with limited protein coding sequence prediction. Three independent models were trained as the following; The first 50 amino acids of a protein were used for training, The last 50 amino acids were used for training, A phasing window of 50 amino acids was used to train across the entirety of a protein sequence. These models were chosen to learn from the different parts of a protein sequence we are likely to capture from only the short unassembled sequence reads. We compared the three models by producing a mock metagenomic community consisting of 6 model bacterial genomes. We evaluated the functions predicted from the unassembled sequence reads and the protein coding sequences predicted from the assembled metagenome.

The rumen bacteria play a major role in lipid metabolism. Bacteria remove the double bonds of unsaturated fatty acids resulting in the production of saturated fatty acids, which are incorporated in milk. Crude olive oil (rich in unsaturated fatty acids) represents a potentially valuable feed source for dairy cows that might enhance the human-health beneficial composition of milk and dairy products. This project studied the effect of supplementing dairy cow diets with olive oil (OO) and palm oil (HVO) on rumen microbiota. For 63 days the animals were fed a control diet (basal diet) with no added lipid and two fat-supplemented diets (30 g kg−1 DM). Rumen sampling were performed at the onset of the experiment and every 21 days for 63 days using a rumen scoop. Total microbial DNA was extracted from ruminal samples for high-throughput sequencing of the 16S rRNA gene through Illumina MiSeq platform. Results revealed the dominance of phyla Firmicutes and Bacteroidetes. Firmicutes was the most prevalent phyla in diet control (75.2 %), OO (71.1 %) and HVO (75.2 %). At genus level Succiniclasticum and Prevotella were the dominant genera, belonging to Firmicutes and Bacteroidetes respectively. Succiniclasticum decrease significantly their relative abundance during OO supplementation (p)

Improved amylases were developed from protoplast fusants of two amylase-producing Apergillusspecies. Twenty regenerated fusants were screened for amylase production on Remazol Brilliant Blue agar. Crude enzymes were produced by solid state fermentation on rice bran were assayed for activity. Three variable factors (temperature, pH and enzyme type) were optimized for amylase activities of parent and selected fusants on a rice bran medium by solid state fermentation. The variables assessed were optimized using the Central Composite Design (CCD) of the Response Surface Methodology (RSM). Amylase activities at room temperature and at 80 °C showed Aspergillusdesignates, T5 (920.21 U ml−1, 966.67 U ml−1), T13 (430 U ml−1, 1011.11 U ml−1) and T14 (500.63 U ml−1, 1012.00 U ml−1) as preferred fusants. Amylases produced by the fusants were observed to be active over the range of pH studied. Fusants T5 and T14 had an optimum acidic and alkaine pH respectively. Optimization studies revealed enzyme T5 at pH 4 and temperature of 40 °C as optimum for amylase production. The statistical tools employed, predicted and compared the optimal conditions for enzyme activities of amylases from parent and fusant strains of Aspergillus revealing the desirability of the fusants over the parents in industrial applications.

Aflatoxin M1 is a metabolite of the most potent aflatoxin, (AFB1) and thus, has been treated and rendered not so toxic, on this basis, its study has been taken for granted. It is the aim of this study to ascertain the toxic nature of AFM1 by determining its effects on microbial flora in the gut of neonatal rats. A dosing experiment was conducted on the neonates, where they were divided into groups and treated with different concentrations of AFM1 using uncontaminated milk as a carrier medium into the rats. The rats were sacrificed; the small and large intestine were harvested and cultured on appropriate selective media for growth of microorganisms. Results show samples from the control group had an uninterrupted microbial community, while the treated group, with increasing doses of AFM1 decreases and depletes the microflora in the gut samples. Lactic acid bacteria were also significantly depleted by AFM1. These findings suggest the capability AFM1 in modifying the gut microbiota in a dose-dependent manner which might result in serious health hazards in neonates.

Acetyl-phosphate (AcP), an intermediate from the phosphotransacetylase-acetate kinase (PTA-AK) pathway, has shown to be critical in pathogenic bacteria for the general metabolism and synthesis of virulence factors. Lysine acetylation is a post-translational modification (PTM) that occurs enzymatically and non-enzymatically by the addition of an acetyl residue from acetyl coenzyme A and AcP, respectively. Neisseria gonorrhoeae, the etiologic agent of gonorrhoea, has been shown to use AcP for lysine acetylation, however, the role that AcP has in the pathogenesis and how acetylation is regulated has not been discerned. The concentration of AcP was altered in N. gonorrhoeae MS11 by interrupting the genes involved in the PTA-AK pathway, pta and ackA, and the gene that encodes for a lysine deacetylase family protein, kdac. AcP concentrations were increased in ΔackA and decreased in Δpta resulting in modulation of lysine acetylation. Growth on glucose, lactate or pyruvate were investigated. In aerobic conditions, ΔackA mutant solely grew in glucose, while the Δpta mutant grew in glucose and lactate. In microaerophilic conditions, ΔackA and Δpta mutants solely grew in presence of glucose. The virulence of ΔackA and Δpta was tested by infecting larvae of Galleria mellonella. WT killed 50 % population (n=15) after 6 days and ΔackA after 24 h, however, Δpta after 6 days it only killed 10 %. Taken together, our results show AcP as an important metabolite for the metabolism and virulence of N. gonorrhoeae.

Bacterial strains were obtained from wheat rhizosphere and screened for zinc solubilization on agar plates. Two strains FA-9 and FA-11 were found efficient for Zn solubilizing activity and were identified as Pseudomonas aeruginosa and Enterobacter sp. by 16S rRNA and gyrase (gyrB) genes analysis, respectively. The strain FA-9 produced a clear zone diameter of 63 mm, 60 mm and 51 mm on agar plate amended with different zinc ores. The strain FA-11 produced a zone diameter of 17 mm with zinc carbonate and 20 mm with zinc oxide while no zone was observed with zinc phosphate. Both strains showed no visible activity with ZnS ore. Similarly, FA-9 and FA-11 increased maximum soluble zinc content (102 µg ml−1 and 45 µg ml−1) from zinc carbonate ore as compared to zinc oxide ore (102 µg ml−1 and 45 µg ml−1) in liquid broth. It was noted that both strains exhibited less potential (7 µg ml−1 and 0.57 µg ml−1) to solubilize ZnS ore in liquid broth. A comparison between agar plate assay and liquid broth quantification shows that agar plate assay does not present the solubilizing potential of ZSB precisely. The strains FA-9 and FA-11 produced auxin with l-tryptophan (3.25 µg ml−1 and 2.86 µg ml−1) and without l-tryptophan (1.23 µg ml−1 and 1.02 µg ml−1). Both strains expressed exo-polysaccharides (EPS) and siderophores activity along with phosphate (P) solubilization, ACC deaminase and antifungal activities. The ACC deaminase and N-fixation activity was confirmed by the amplification of acdS and nifH genes respectively.

Urinary tract infection (UTI) is considered to be one of the most prevalent bacterial infections in the world. It affects urinary tract system including bladder and kidney. Uropathogenic Escherichia coli is more prevalent in females due to their anatomical structure as well as they are more susceptible to recurrent infections. Every woman out of three is affected by UTIs. Gram-negative bacteria are a major cause, particularly Escherichia coli (E. coli). E. coli was considered a main causative agent for 80–90 % of community-acquired infection and for about 40 % of nosocomial UTI. Moreover, it is responsible for 25 % of recurrent infection. Proteomic can be used to analyze and identify complete components of proteins. It can be used to distinguish between bacteria based on synthesized proteins. In addition, proteomic is applicable to identify possible targets of therapy. My study aims To compare protein profiles of E. coli from different UTI patients and identify possible unique protein signature for future biomarker studies. Six Urine samples with E. coli were taken from females aged from 15 to 50 years old. Samples loaded onto Sodium dodecyl sulfate Polyacrylamide gel electrophoresis for separation. Samples with abundant proteins profiles on SDS-PAGE, were selected for run on two dimensional gel electrophoresis. Gels were compared to each other to look for interesting protein spots. Many differences were observed in protein profiles of E. coli isolates in both 1D SDS-PAGE and 2DGE. Two bacterial proteins identified as possible candidate were (OmpA) found in Gram negative bacteria and RNA polymerase-binding transcription factor DksA mostly found in E. coli

Vibrio cholerae experiences frequent feast and famine conditions. In the absence of all the three nutrient components (Carbon, Nitrogen, and Phosphorous), the cells could manage starvation and survive up to 6 months with only 1 log reduction in viability. Addition of carbon or nitrogen to the starvation media resulted in 3 log reduction in cell number. Simultaneous addition of carbon and nitrogen (Phosphorous starvation) reduced the cell viability to a below detectable level. The suboptimal growth conditions; non-metabolizable source of carbon and nitrogen; variable C: N ratio; inhibition of metabolism and cell division, increased the cell survival under phosphate starvation. However, when all the three components are limited, the cells did not initiate active metabolism and could conserve the energy for long-term survival. This observation suggests that integration of carbon, nitrogen and phosphorus sensing is imperfect in V. cholerae and it cannot down-regulate the metabolism during phosphorous limitation. The carbon and nitrogen could prime the cells to accelerate the rate of metabolism, irrespective of the presence of phosphorous, thereby creating an energetically unfavorable situation. The lack of crucial component phosphorus fails to activate a stringent response that results in increased futile cycling of nutrients, loss of ATP and cell death. The Vibrio genus was found to be less efficient in surviving phosphate starvation than E. coli and S. Typhimurium. The two-component system CreC (a response regulator of phosphorous) is absent in V. cholerae and may be responsible for the lack of stringent response to phosphorus starvation.

Campylobacter jejuniis is the most common cause of bacterial gastroenteritis worldwide. It is often considered a ‘commensal’ in chickens where it rapidly colonises the caecum of young chicks and is present in around 80 % of farmed poultry. C. jejuni has a small genome and most strains are unable to transport or metabolize glucose. Instead they use amino acids, TCA cycle intermediates and short chain fatty acids as energy and carbon sources. Recently, a WGS project identified an unusual group of C. jejuni (RG-2 group) within a bank of C. jejuni strains isolated from farm associated Norway rats. These strains had acquired an entire locus (glc) of seven genes which enable uptake and metabolism of glucose via the Entner Doudoroff (ED) pathway. This project is addressing the impact of these genes on metabolism and niche survival of these strains. Campylobacter are microaerophilic bacteria. Good growth of C. jejuni NCTC 11168 and of aglcnegative Norway rat C. jejuni isolate was recorded in the presence of 5 % O2up to 13 %in a Whitley M35 workstation. In contrast, two glcpositive strains of C. jejuni, Dg275 and Dg95, exhibited the unusual ability to grow well in an atmosphere of 16 % oxygen, as monitored by c.f.u. and OD600. Current studies are also focusing on the contribution of glucose utilization via the ED pathway to influence survival and growth in oxygen.

The survival of enteric pathogens in the multi-nutrient environment involves interaction with other organisms. The fact that competition for nutrient resources has shaped the networking of metabolic pathways is well known. The cAMP receptor protein i.e. CRP is a keystone regulatory protein, connecting various metabolic pathways. Our aim was to study the importance of CRP in nutrient uptake and utilization in Salmonella Typhimurium under intra-species and inter-species nutritive competition. The crp gene knockout (Δcrp) was co-cultured with the wild-type or other pathogens. The Δcrp failed to compete with the wild-type Salmonella Typhimurium, Escherichia coli, Vibrio cholerae and Staphylococcus aureus in nutrient intensive media. However, the survival of the co-cultured Δcrp was unaffected in nutrient-poor media. These results suggest that CRP is necessary for the effective acquisition of readily available nutrients as found in rich media in co-culture. The role of released antimicrobials or surface proteins of the co-cultured strains was also overruled by culturing the mutant in the supernatant of these organisms and separating the cultures in the same media respectively. The co-cultured Δcrp showed an enhanced survival when overall metabolism was reduced with low temperature and antibiotics like chloramphenicol. A circumstantial evidence that CRP manages the global and limits the futile metabolism is provided by this study. The absence of CRP doesn’t affect the survival of the standalone culture. However, the role of this protein becomes obvious in a bacterial community setup. Therefore, CRP is crucial for Salmonella to survive in an intestinal and external environment.

For bacteria, mechanisms of resistance to redox stress are utilised to increase survival. Although B. henselae has the ability to resist redox stress, its genome sequence is characterised by a paucity of genes responsible for redox-stress resistance, particularly hydrogen peroxide degradation systems. However, our results show a surprisingly high resistance to peroxides, given the lack of peroxide disposal systems. To determine how B. henselae achieves resistance to H₂O₂ stress, the potential role of MbfA (membrane-bound bacterioferritin), which in other α-Proteobacteria is believed to function as an iron exporter, was investigated. The results show that B. henselae has the ability to export iron and that this export activity is promoted by H₂O₂as export was inhibited by exogenous catalase and anaerobiosis. The form of iron exported was largely ferric. The impact of the iron export process on the resistance to, and degradation of, H₂O₂, by B. henselae was determined and the results showed that B. henselae mediates a rapid consumption of exogenously supplied H₂O₂. This degradation was entirely inhibited when iron chelators were included along with the H₂O₂. The resistance of B. henselae to NO was also tested since NO is generated by phagocytic host cells along with H₂O₂, and is suggested to potentiate the toxicity of H₂O₂ towards engulfed bacteria through inhibition of haem-dependent catalases and alkylhydroperoxidases. Our results suggest that NO does not cause a marked increase in H₂O₂ toxicity for B. henselae, in contrast to E. coli (a haem-catalayse/peroxidase dependent bacterium).

Streptomyces coelicolor is a non-pathogenic soil saprophytic bacterium and is a model organism for antibiotic production. This species contains a single copy of a nine gene cluster known as the mammalian cell entry (mce) operon. This operon was originally characterised in Mycobacterium tuberculosis as an important virulence factor acting in invasion and survival within macrophages and encodes an ABC transporter for cholesterol import. As the function of the mceoperon in S. coelicoloris currently unknown, this study aims to characterise the operon through deletion of the mcelocus and resulting impact on bacterial morphology and survival. SEM images demonstrate that spores of a mcedeletion mutant (Δmce) display a wrinkled, and ‘fragile’ phenotype, with spores appearing to germinate whilst on the spore chain. Heat kill assays show that the deletion of the mce operon result in S. coelicolor spores which are less tolerant to temperatures of 60, 70, 80, 90 and 100°C compared to WT S. coelicolor spores. Heat activation of Δmce spores was also consistently absent at all temperatures tested. The spores of a Δmce mutant also exhibit a precocious germination phenotype seen on SEM images confirmed with germination assays.

The contemplation of present research work to get over scantiness of amino acid by amelioration ofmicroorganism. As a matter of fact the use of chemical and physical factors symbolize a new era in the excerption of microorganism for proficient of producing a desired product. In this workbiosynthetic pathway of valine and their regulation was observed in Bacillus cereus, Corynebacterium and Pseudomonas fluoresence. The contrivance of operation of ems mutants was analyze in different fermentation media at 24, 48, 72 and 96 h incubation under optimum condition by acidic ninhydrin method. Optical density was recorded by spectrophotometer 470 nm for Valine. The effects of carbon and nitrogen sources and growth factors on the production of valine were studied. In this studies mutant evince that mutation confabulate upon the bacteria the ability to produce valine many times more than its parental strain within the same span of incubation period. The objective of this work to step up headway to search unconventional sources to overcome nutritional exhortation of valine.

The interaction between transcription factors, promoter elements and RNA polymerase is crucial to bacterial adaptation to physical and chemical changes in the environment. Transcription factor discovery has strongly relied on the isolation of regulatory mutants followed by biochemical confirmation. However, highly pleiotropic regulatory mutations can often be deleterious, unstable and difficult to select, or could directly or indirectly affect gene expression at multiple levels. Here, we describe a biochemical approach to identify trans-acting regulatory proteins independent of their cellular function that could be used as an alternative to genetic screens. The method consists in (i) incubating bacterial lysates with an immobilized DNA encompassing a promoter as defined by RNA-Seq data (ii) pull down of bound proteins and (iii) liquid chromatography tandem mass spectrometry (LC/MS/MS). We tested the usefulness of this approach by identifying proteins binding to the Vibrio cholerae rpoS promoter that drives the expression of the general stress response regulator RpoS. The approach identified several proteins binding to the rpoS promoter that included the factor for inversion stimulation (FIS), and the master quorum sensing regulator HapR. Binding of both purified proteins to the rpoS promoter was confirmed by electrophoresis mobility shift assays. The role of fis and hapR on rpoS expression was examined in strains containing a chromosomally-integrated rpoS-lacZ fusion. Deletion of fis had little effect while HapR appeared to enhance rpoS expression.

A comprehensive understanding of bacteria phenotypes requires tools that are able to characterise structure and function across multiple length scales, from communities and individual cells down to single molecules. Multimodal sensing combines multiple transduction technologies in parallel to probe different properties simultaneously, and thereby increase the range of measurable interactions, the amount of information that can be extracted, and improve detection accuracy. Electrophotonics is a multimodal sensing approach that combines electrochemical and photonic techniques in a single, integrated device that provides enhanced quantitative measurements of chemical reactions. We present a new electrophotonic device based on a Si3N4 guided mode resonant (GMR) structure with an integrated indium tin oxide (ITO) electrode. The GMR structure is sensitive to refractive index changes at the sensor surface, enabling label free, real time detection of biomolecules, microorganisms and imaging of molecular interactions with micrometre-scale resolution to provide spatial information about surface binding interactions. The ITO electrode has been shown to be compatible with voltammetry-based techniques for interrogating redox behaviour along with electrochemical impedance spectroscopy. We demonstrate the wide range of microbiological applications of electrophotonic technology including the characterisation of redox active protein electron transfer and surface adsorption, bacterial adherence and growth on chemically functionalised surfaces, and label free parallel detection of clinically relevant biomarkers. We believe that the multimodal measurements with this novel technology can provide new approaches to investigate and understand microbial biology.

D-serine is an amino acid that has become a focus in recent years due to its unique role in many biological processes. It is a host metabolite in humans with diverse roles in neurotransmission and signalling. Previous work in our group showed that D-serine can play a critical role in controlling expression of pathogenic virulence factors in bacteria, specifically Escherichia coli, as well as impacting microbial community composition through niche specificity. In enterohaemorrhagic E. coli (EHEC), the presence of D-serine results in down regulation of the type 3 secretion system (T3SS), resulting in inability to colonise. In contrast, uropathogenic E. coli (UPEC) can catabolise this metabolite and colonise the bladder where D-serine is present in high concentrations, leading to a urinary tract infections (UTI). Hence, in different pathotypes, D-serine can act as a positive (treat) or negative (trick) environmental stimulus. UPEC and neonatal meningitis E. coli (NMEC) strains often carry the DsdXCA operon which allows for the metabolism of D-serine. This locus is responsible for the detoxification of D-serine, allowing for tolerance of D-serine as a carbon source. My work has focused on understanding the role of D-serine as a signal for gene expression in UPEC and NMEC through the action of the regulator, DsdC. Using global approaches I have characterised the binding sites of DsdC across the chromosome revealing new insights into how this protein contributes to UPEC and NMEC pathogenesis. The work is important as it helps us understand how specific pathogens sense their environment and cause disease.

The dominance and pathogenicity of Helicobacter pylori in the gastric niche is eminent. Urease test is used to diagnose the infection of H. pylori. However, nowadays several reports indicated the emergence of organisms other than H. pylori (Non H. pylori bacteria– NHPs). We have reported urease positive, Ochrobactrum intermedium from the non-ulcer dyspeptic individual. Therefore such urease positive bacteria raise a question on the reliability of urease test. Since 2005, nearly 35 cases are reported for the presence of Ochrobactrum spp. from clinical specimens. Their high level of resistance to antibiotics and phylogenetic relationship to anthropozoonotic pathogen, Brucella makes them a probable human pathogen. Therefore, it is necessary to study their prevalence, survival mechanism and function in the human stomach. To check their pervasiveness in the Indian population, 218 urease positive gastric biopsy samples were processed and 62 Ochrobactrum spp. isolates were identified with and without H. pylori. Population study of Ochrobactrum spp. was also done using multilocus sequence typing (MLST) which revealed 45 sequence type and clonal population. As the prevalence of Ochrobactrum spp. is approx. 30 %, their survival mechanism was studied under in vitro gastric conditions (acidic pH, urea, microaerophilic environment), using microarray and RNA seq methods. The differential gene expression analysis showed 2 different acid resistance mechanism, i.e. Urease dependent acid resistance mechanism like H. pylori, and Amino acid dependent acid resistance mechanism. Our finding necessitates further detailed investigation of such NHPs in gastric environment and role of such bacteria in gastric niche and warrants further refinement of urease based diagnosis.