Exploring ionic liquids′ unique stimuli to elucidate uncharacterised cellular and molecular mechanisms in filamentous fungi

The emergence of new fungal pathogens, either of plants or animals, and the increasing number of reported cases of resistant human pathogenic strains to the available antifungal drugs reinforces the need for better understanding the biology of filamentous fungi. Conventional drugs target components of the fungal membrane or cell wall, therefore identifying novel intracellular targets, yet unique to fungi, is a global priority.(...)

Fitness correlates with the extent of cheating in a bacterium.

There is growing awareness of the importance of cooperative behaviours in microbial communities. Empirical support for this insight comes from experiments using mutant strains, termed 'cheats', which exploit the cooperative behaviour of wild-type strains. However, little detailed work has gone into characterising the competitive dynamics of cooperative and cheating strains. We test three specific predictions about the fitness consequences of cheating to different extents by examining the production of the iron-scavenging siderophore molecule, pyoverdin, in the bacterium Pseudomonas aeruginosa. We create a collection of mutants that differ in the amount of pyoverdin that they produce (from 1% to 96% of the production of paired wild types) and demonstrate that these production levels correlate with both gene activity and the ability to bind iron. Across these mutants, we found that (1) when grown in a mixed culture with a cooperative wild-type strain, the relative fitness of a mutant is negatively correlated with the amount of pyoverdin that it produces; (2) the absolute and relative fitness of the wild-type strain in the mixed culture is positively correlated with the amount of pyoverdin that the mutant produces; and (3) when grown in a monoculture, the absolute fitness of the mutant is positively correlated with the amount of pyoverdin that it produces. Overall, we demonstrate that cooperative pyoverdin production is exploitable and illustrate how variation in a social behaviour determines fitness differently, depending on the social environment.

The Role of Specific Amino Acids in the Formation of Ternary Complexes in Nitrogenase Regulation in the Photosynthetic Bacterium Rhodobacter capsulatus

L'azote est l'un des éléments les plus essentiels dans le monde pour les êtres vivants, car il est essentiel pour la production des éléments de base de la cellule, les acides aminés, les acides nucléiques et les autres constituants cellulaires. L’atmosphère est composé de 78% d'azote gazeux, une source d'azote inutilisable par la plupart des organismes à l'exception de ceux qui possèdent l’enzyme nitrogénase, tels que les bactéries diazotrophique. Ces micro-organismes sont capables de convertir l'azote atmosphérique en ammoniac (NH3), qui est l'une des sources d'azote les plus préférables. Cette réaction exigeant l’ATP, appelée fixation de l'azote, est catalysée par une enzyme, nitrogénase, qui est l'enzyme la plus importante dans le cycle de l'azote. Certaines protéines sont des régulateurs potentiels de la synthèse de la nitrogénase et de son activité; AmtB, DraT, DraG, les protéines PII, etc.. Dans cette thèse, j'ai effectué diverses expériences afin de mieux comprendre leurs rôles détailés dans Rhodobacter capsulatus. La protéine membranaire AmtB, très répandue chez les archaea, les bactéries et les eucaryotes, est un membre de la famille MEP / Amt / Rh. Les protéines AmtB sont des transporteurs d'ammonium, importateurs d'ammonium externe, et ont également été suggéré d’agir comme des senseurs d'ammonium. Il a été montré que l’AmtB de Rhodobacter capsulatus fonctionne comme un capteur pour détecter la présence d'ammonium externe pour réguler la nitrogénase. La nitrogénase est constituée de deux métalloprotéines nommées MoFe-protéine et Fe-protéine. L'addition d'ammoniaque à une culture R. capsulatus conduit à une série de réactions qui mènent à la désactivation de la nitrogénase, appelé "nitrogénase switch-off". Une réaction critique dans ce processus est l’ajout d’un groupe ADP-ribose à la Fe-protéine par DraT. L'entrée de l'ammoniac dans la cellule à travers le pore AmtB est contrôlée par la séquestration de GlnK. GlnK est une protéine PII et les protéines PII sont des protéines centrales dans la régulation du métabolisme de l'azote. Non seulement la séquestration de GlnK par AmtB est importante dans la régulation nitrogénase, mais la liaison de l'ammonium par AmtB ou de son transport partiel est également nécessaire. Les complexes AmtB-GlnK sont supposés de lier DraG, l’enzyme responsable pour enlever l'ADP-ribose ajouté à la nitrogénase par DraT, ainsi formant un complexe ternaire. Dans cette thèse certains détails du mécanisme de transduction du signal et de transport d'ammonium ont été examinés par la génération et la caractérisation d’un mutant dirigé, RCZC, (D335A). La capacité de ce mutant, ainsi que des mutants construits précédemment, RCIA1 (D338A), RCIA2 (G344C), RCIA3 (H193E) et RCIA4 (W237A), d’effectuer le « switch-off » de la nitrogénase a été mesurée par chromatographie en phase gazeuse. Les résultats ont révélé que tous les résidus d'acides aminés ci-dessus ont un rôle essentiel dans la régulation de la nitrogénase. L’immunobuvardage a également été effectués afin de vérifier la présence de la Fe-protéine l'ADP-ribosylée. D335, D388 et W237 semblent être cruciales pour l’ADP-ribosylation, puisque les mutants RCZC, RCIA1 et RCIA4 n'a pas montré de l’ADP-ribosylation de la Fe-protéine. En outre, même si une légère ADP-ribosylation a été observée pour RCIA2 (G344C), nous le considérons comme un résidu d'acide aminé important dans la régulation de la nitrogénase. D’un autre coté, le mutant RCIA3 (H193E) a montré une ADP-ribosylation de la Fe-protéine après un choc d'ammonium, par conséquent, il ne semble pas jouer un rôle important dans l’ADP-ribosylation. Par ailleurs R. capsulatus possède une deuxième Amt appelé AmtY, qui, contrairement à AmtB, ne semble pas avoir des rôles spécifiques. Afin de découvrir ses fonctionnalités, AmtY a été surexprimée dans une souche d’E. coli manquant l’AmtB (GT1001 pRSG1) (réalisée précédemment par d'autres membres du laboratoire) et la formation des complexes AmtY-GlnK en réponse à l'addition d’ammoniac a été examinée. Il a été montré que même si AmtY est en mesure de transporter l'ammoniac lorsqu'il est exprimé dans E. coli, elle ne peut pass’ associer à GlnK en réponse à NH4 +.

Isolation, purification, characterization and application of proteinaceous protease inhibitor from marine bacterium Pseudomonas mendocina BTMW 301

The microorganisms are recognized as important sources of protease inhibitors which are valuable in the fields of medicine, agriculture and biotechnology. The protease inhibitors of microbial origin are found to be versatile in their structure and mode of inhibition that vary from those of other sources. Although surplus of low molecular weight non-protein protease inhibitors from microorganisms have been reported, there is a dearth of reports on proteinaceous protease inhibitors. The search for new metabolites from marine organisms has resulted in the isolation of more or less 10,000 metabolites (Fuesetani and Fuesetani, 2000) many of which are gifted with pharmacodynamic properties. The existence of marine microorganisms was reported earlier, and they were found to be metabolically and physiologically dissimilar from terrestrial microorganisms. Marine microorganisms have potential as important new sources of enzyme inhibitors and consequently a detailed study of new marine microbial inhibitors will provide the basis for future research (Imada, 2004).

A brackishwater isolate of Pseudomonas PS-102, a potential antagonistic bacterium against pathogenic vibrios in penaeid and non-penaeid rearing systems

A Pseudomonas sp PS-102 recovered from Muttukkadu brackish water lagoon, situated south of Chennai, showed significant activity against a number of shrimp pathogenic vibrios. Out of the 112 isolates of bacterial pathogens comprising Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. fluvialis, and Aeromonas spp, 73% were inhibited in vitro by the cell-free culture supernatant of Pseudomonas sp PS-102 isolate. The organism produced yellowish fluorescent pigment on King's B medium, hydrolysed starch and protein, and produced 36.4% siderophore units by CAS assay and 32 μM of catechol siderophores as estimated by Arnow's assay. The PS-102 isolate showed wide ranging environmental tolerance with, temperatures from 25 to 40 °C, pH from 6 to 8, salinity from 0 to 36 ppt, while the antagonistic activity peaked in cultures grown at 30 °C, pH 8.0 and at 5 ppt saline conditions. The antagonistic activity of the culture supernatant was evident even at 30% v / v dilution against V. harveyi. The preliminary studies on the nature of the antibacterial action indicated that the antagonistic principle as heat stable and resistant to proteolytic, lipolytic and amylolytic enzymes. Pseudomonas sp PS 102 was found to be safe to shrimp when PL-9 stage were challenged at 107 CFU ml−1 and by intramuscular injection into of ∼5 g sub-adults shrimp at 105 to 108 CFU. Further, its safety in a mammalian system, tested by its pathogenicity to mice, was also determined and its LD50 to BALB/c mice was found to be 109 CFU. The results of this study indicated that the organism Pseudomonas sp PS 102 could be employed as a potential probiont in shrimp and prawn aquaculture systems for management and control of bacterial infections

A marine bacterium, Micrococcus MCCB 104, antagonistic to vibrios in prawn larval rearing systems

marine bacterium, Micrococcus MCCB 104, isolated from hatchery water, demonstrated extracellular antagonistic properties against Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus, V. fluviallis, V. nereis, V. proteolyticus, V. mediterranei, V. cholerae and Aeromonas sp., bacteria associated with Macrobrachium rosenbergii larval rearing systems. The isolate inhibited the growth of V. alginolyticus during co-culture. The antagonistic component of the extracellular product was heat-stable and insensitive to proteases, lipase, catalase and α-amylase. Micrococcus MCCB 104 was demonstrated to be non-pathogenic to M. rosenbergii larvae

Sequential interactions of Silver-silica nanocomposite (Ag-SiO2NC) with cell wall, metabolism and genetic stability of Psedomonas aeruginosa, a multiple antibiotic resistant bacterium

The study was carried out to understand the effect of silver-silica nanocomposite (Ag-SiO2NC) on the cell wall integrity, metabolism and genetic stability of Pseudomonas aeruginosa, a multiple drugresistant bacterium. Bacterial sensitivity towards antibiotics and Ag-SiO2NC was studied using standard disc diffusion and death rate assay, respectively. The effect of Ag-SiO2NC on cell wall integrity was monitored using SDS assay and fatty acid profile analysis while the effect on metabolism and genetic stability was assayed microscopically, using CTC viability staining and comet assay, respectively. P. aeruginosa was found to be resistant to β-lactamase, glycopeptidase, sulfonamide, quinolones, nitrofurantoin and macrolides classes of antibiotics. Complete mortality of the bacterium was achieved with 80 μgml-1 concentration of Ag-SiO2NC. The cell wall integrity reduced with increasing time and reached a plateau of 70 % in 110 min. Changes were also noticed in the proportion of fatty acids after the treatment. Inside the cytoplasm, a complete inhibition of electron transport system was achieved with 100 μgml-1 Ag-SiO2NC, followed by DNA breakage. The study thus demonstrates that Ag-SiO2NC invades the cytoplasm of the multiple drug-resistant P. aeruginosa by impinging upon the cell wall integrity and kills the cells by interfering with electron transport chain and the genetic stability

Occurrence of filamentous algae and soil characteristics in the paddy fields of Kuttanad and Kole lands of Kerala

The present work is the study of filamentous algae in the paddy fields of Kuttanad and Kole lands of Kerala. This investigation was initiated by sampling of filamentous algae in Kuttanad during December 2010 to February 2011. A second phase of sampling was done from November 2011 to February 2012. The sampling periodicity corresponded to the crop growth starting from field preparation through sowing, and continued till the harvest. Sampling locations were selected from the active paddy cultivation regions of the six agronomic zones of Kuttanad. The numbers of sampling locations were proportional to the area of each zone. Algae of the Kole lands were collected during from October 2011 to January 2012. It was observed that blue-green algae dominated in both Kuttanad and Kole lands. Thirty two species of blue-green algae and eight species of green algae were identified from Kuttanad. The highest number of algal species was observed from Kayal lands in Kuttanad throughout the cropping season. Among the thirty two species of blue-green algae twenty five species are nonheterocystous and seven species are heterocystous. Twenty eight species of blue-green and six species of green algae were identified from Kole lands, and highest number of species was observed in Palakkal throughout the cropping season. Among the twenty eight species of blue-green algae collected from Kole lands twenty one species are non-heterocystous, and only seven species are heterocystous filamentous algae. Blooms of Spirogyra were observed during the second phase of sampling in Kuttanad and also in the Kole lands. The results of the germination study revealed that the extract of Spirogyra sp. inhibited seed germination and reduced seedling vigour. The growth of the treated seedlings was evaluated by pot experiments. The results clearly showed that Spirogyra sp. can negatively affect the seed germination, seedling vigour, and the yield of rice.

Differential induction, isolation, physicochemical and molecular characterization of temperate phages of environmental Vibrio cholerae as evidence of phage mediated Horizontal Gene Transfer

The resurgence of the enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries like India. The southern Indian state of Kerala is endemic to cholera. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. Marine aquaculture settings and mangrove environments of Kerala serve as reservoirs for V. cholerae. The non-O1/non-O139 environmental isolates of V. cholerae with incomplete ‘virulence casette’ are to be dealt with caution as they constitute a major reservoir of diverse virulence genes in the marine environment and play a crucial role in pathogenicity and horizontal gene transfer. The genes coding cholera toxin are borne on, and can be infectiously transmitted by CTXΦ, a filamentous lysogenic vibriophages. Temperate phages can provide crucial virulence and fitness factors affecting cell metabolism, bacterial adhesion, colonization, immunity, antibiotic resistance and serum resistance. The present study was an attempt to screen the marine environments like aquafarms and mangroves of coastal areas of Alappuzha and Cochin, Kerala for the presence of lysogenic V. cholerae, to study their pathogenicity and also gene transfer potential. Phenotypic and molecular methods were used for identification of isolates as V. cholerae. The thirty one isolates which were Gram negative, oxidase positive, fermentative, with or without gas production on MOF media and which showed yellow coloured colonies on TCBS (Thiosulfate Citrate Bile salt Sucrose) agar were segregated as vibrios. Twenty two environmental V. cholerae strains of both O1 and non- O1/non-O139 serogroups on induction with mitomycin C showed the presence of lysogenic phages. They produced characteristic turbid plaques in double agar overlay assay using the indicator strain V. cholerae El Tor MAK 757. PCR based molecular typing with primers targeting specific conserved sequences in the bacterial genome, demonstrated genetic diversity among these lysogen containing non-O1 V. cholerae . Polymerase chain reaction was also employed as a rapid screening method to verify the presence of 9 virulence genes namely, ctxA, ctxB, ace, hlyA, toxR, zot,tcpA, ninT and nanH, using gene specific primers. The presence of tcpA gene in ALPVC3 was alarming, as it indicates the possibility of an epidemic by accepting the cholera. Differential induction studies used ΦALPVC3, ΦALPVC11, ΦALPVC12 and ΦEKM14, underlining the possibility of prophage induction in natural ecosystems, due to abiotic factors like antibiotics, pollutants, temperature and UV. The efficiency of induction of prophages varied considerably in response to the different induction agents. The growth curve of lysogenic V. cholerae used in the study drastically varied in the presence of strong prophage inducers like antibiotics and UV. Bacterial cell lysis was directly proportional to increase in phage number due to induction. Morphological characterization of vibriophages by Transmission Electron Microscopy revealed hexagonal heads for all the four phages. Vibriophage ΦALPVC3 exhibited isometric and contractile tails characteristic of family Myoviridae, while phages ΦALPVC11 and ΦALPVC12 demonstrated the typical hexagonal head and non-contractile tail of family Siphoviridae. ΦEKM14, the podophage was distinguished by short non-contractile tail and icosahedral head. This work demonstrated that environmental parameters can influence the viability and cell adsorption rates of V. cholerae phages. Adsorption studies showed 100% adsorption of ΦALPVC3 ΦALPVC11, ΦALPVC12 and ΦEKM14 after 25, 30, 40 and 35 minutes respectively. Exposure to high temperatures ranging from 50ºC to 100ºC drastically reduced phage viability. The optimum concentration of NaCl required for survival of vibriophages except ΦEKM14 was 0.5 M and that for ΦEKM14 was 1M NaCl. Survival of phage particles was maximum at pH 7-8. V. cholerae is assumed to have existed long before their human host and so the pathogenic clones may have evolved from aquatic forms which later colonized the human intestine by progressive acquisition of genes. This is supported by the fact that the vast majority of V. cholerae strains are still part of the natural aquatic environment. CTXΦ has played a critical role in the evolution of the pathogenicity of V. cholerae as it can transmit the ctxAB gene. The unusual transformation of V. cholerae strains associated with epidemics and the emergence of V. cholera O139 demonstrates the evolutionary success of the organism in attaining greater fitness. Genetic changes in pathogenic V. cholerae constitute a natural process for developing immunity within an endemically infected population. The alternative hosts and lysogenic environmental V. cholerae strains may potentially act as cofactors in promoting cholera phage ‘‘blooms’’ within aquatic environments, thereby influencing transmission of phage sensitive, pathogenic V. cholerae strains by aquatic vehicles. Differential induction of the phages is a clear indication of the impact of environmental pollution and global changes on phage induction. The development of molecular biology techniques offered an accessible gateway for investigating the molecular events leading to genetic diversity in the marine environment. Using nucleic acids as targets, the methods of fingerprinting like ERIC PCR and BOX PCR, revealed that the marine environment harbours potentially pathogenic group of bacteria with genetic diversity. The distribution of virulence associated genes in the environmental isolates of V. cholerae provides tangible material for further investigation. Nucleotide and protein sequence analysis alongwith protein structure prediction aids in better understanding of the variation inalleles of same gene in different ecological niche and its impact on the protein structure for attaining greater fitness of pathogens. The evidences of the co-evolution of virulence genes in toxigenic V. cholerae O1 from different lineages of environmental non-O1 strains is alarming. Transduction studies would indicate that the phenomenon of acquisition of these virulence genes by lateral gene transfer, although rare, is not quite uncommon amongst non-O1/non-O139 V. cholerae and it has a key role in diversification. All these considerations justify the need for an integrated approach towards the development of an effective surveillance system to monitor evolution of V. cholerae strains with epidemic potential. Results presented in this study, if considered together with the mechanism proposed as above, would strongly suggest that the bacteriophage also intervenes as a variable in shaping the cholera bacterium, which cannot be ignored and hinting at imminent future epidemics.

Use of entomopathogenic bacterium Pseudomonas putida (Enterobacteriaceae) and it secretions against the Greater Wax Moth Galleria mellonella pupae

The bacterium from Pseudomonas putida from Steinernema abbasi and its metabolic secretions caused the mortality of the Galleria mellonella pupae. Experiments were conducted in sand and filter paper on time exposure, temperature, moisture, dose and time of penetration of bacterium in pupae and tested stored or dried toxic metabolites using G. mellonella pupae as a test target organism. Death of pupae was probably due to the toxic metabolites. Pseudomonas putida cells were recovered from the haemocoele when bacterial cells were applied to the G. mellonella pupae indicating that bacterial cells can enter the haemocoele in the absence of nematode vector. Penetration of bacterium was found rapidly after application on G. mellonella pupae. Pseudomonas putida or its toxic secretions can be used as a microbial control for insect control. The experimental results indicate that there is possibility of using P. putida and its toxic secretions as a biopesticide and can contribute in the development of new microbial and biological control against insect pests.

Pathogenicity of the bacterium Pseudomonas putida from the entomopathogenic nematode Steinernema abbasi and its secretion against Galleria mellonella larvae

The Entomopathogenic bacterium Pseudomonas putida from Steinernema abbasi and its metabolic secretions were lethal to the Galleria mellonella larvae. Different laboratory experiments on time interval, substrate, moisture, temperature, dose, penetration of cells, stored and dried metabolites were conducted in sand and filter paper bioassays. It was concluded that death was probably due to the toxic metabolites. This bacterium and its metabolites were found very effective at 30 degree C. Penetration of bacterium was rapid after application on G. mellonella larvae. P. putida cells were recovered from the haemocoele when suspensions containing bacterial cells were applied to the G. mellonella indicating that bacterial symbionts do have a free-living existence and can enter the haemocoele in the absence of nematode vector. Stored metabolite and dried metabolites were found persistent for long time. This bacterium or its toxic secretions can be used for insect control that can be important component of integrated pest management against different insect pests. P. putida and its secretions are suggested as the most appropriate suspension to apply against insect pest control program in tropical ecological regions.

Pathogenicity of the bacterium Xenorhabdus nematophila isolated from the entomopathogenic nematode Steinernema carpocapsae and its secretions against Galleria mellonella larvae

The entomopathogenic bacterium, Xenorhabdus nematophila was isolated from the hemolymph of Galleria mellonella infected with Steinernema carpocapsae. The bacterial cells and its metabolic secretions have been found lethal to the Galleria larvae. Toxic secretion in broth caused 95% mortality within 4 d of application whereas the bacterial cells caused 93% mortality after 6 d. When filter and sand substrates were compared, the later one was observed as appropriate. Similarly, bacterial cells and secretion in broth were more effective at 14% moisture and 25 °C temperature treatments. Maximum insect mortality (100%) was observed when bacterial concentration of 4×106 cells/ml was used. Similarly, maximum bacterial cells in broth (95%) were penetrated into the insect body within 2 h of their application. However, when stored bacterial toxic secretion was applied to the insects its efficacy declined. On the other hand, when the same toxic secretion was dried and then dissolved either in broth or water was proved to be effective. The present study showed that the bacterium, X. nematophila or its toxic secretion can be used as an important component of integrated pest management against Galleria.

Antifungal activity of bacterium symbiotically associated with Steinernema abbasi towards Fusarium oxysporum

Fusarium oxysporum f.sp. lycopersici (Fol) is the causal agent of the Fusarium wilt disease of tomato. Soil fumigant (mainly methyl bromide) applications are in use for its control. With the increasing environmental awareness, biological control methods are under investigation for their effectiveness, including the use of antagonists. Pseudomonas oryzihabitans (=Flavimonas oryzihabitans), a symbiont of the entomopathogenic nematode Steinernema abbasi was investigated as an antagonism of a Fol isolate in two laboratory and two glasshouse experiments. Bacteria and cell-free filtrate antifungal activity were tested both in dual cultures and in broth culture. In pot experiments, suspensions of bacteria in five concentrations (106, 105, 104, 103 and 102 cells/ml) were tested for their ability to control the pathogen at 25±3°C. In all tests the bacterium significantly inhibited the growth of Fol mycelium in vitro. Similar results were obtained when the bacterium was also tested against Fusarium oxysporum f.sp. radicis lycopersici and against Rhizoctonia solani. Moreover, when it was introduced into the soil, it was able to suppress the Fusarium wilt of tomato.

Biological control of Rhizoctonia solani Damping-off with a bacterium symbiotically associated with Steinernema abbasi

Rhizoctonia solani is a causal agent of damping-off of may cultivated plants. An isolate of the bacterium Pseudomonas oryzihabitans, symbiotically associated with the entomopathogenic nematode Steinernema abbasi, strongly inhibited the pathogen in vitro. The bacterium was firmly attached onto fungus mycelia and degraded the cell walls of the pathogen. In greenhouse experiments, bacterial suspension in sterile water applied in the soil, effectively controlled damping-off of radish.