Identification of microorganisms responsible for spoilage of tomato (Lycopersicon esculentum) fruit

Tomato contains large amount of water which makes them more susceptible to spoilage by the action of various microorganisms. This makes the storage of this vegetable difficult and its transportation too. Tomato is a prevalent vegetable used both in raw form as salad, for garnishing various food items and added for taste in various cooked items. Thus it forms a major ingredient of food both in Asia as well as European countries. Further tomato is rich source of vitamins especially Vitamin A as it contains its precursor β-carotene which is very essential for vision. It is also a rich source of minerals which makes it an essential component of food. This study was done to find out the organisms which make tomato more susceptible to spoilage. Out of the 30 Rose Bengal agar plates which were inoculated and incubated vigorous growth of fungi was observed in 26 plates with moderate growth on other 4 plates. Also 30 other plates of Nutrient agar were inoculated with the sample and very scanty growth of bacterial colonies was observed in 3-4 plates and mostly occupied by fungal colonies. Thus it was found that fungi were the source of spoilage in most of the samples rather than bacteria. Further morphological studies were done to know the fungal member responsible for the spoilage. Among the fungi, it was found that Aspergillus niger and Fusarium were found in most of the spoiled samples with a few samples containing Penicillium too with A. niger dominating all the plates. Since all these are fungi, it can be related to the severity of the intake of spoiled tomato are developing countries like India where people hardly discard the spoiled foods as spores are relatively heat-resistant and do not perish and spread easily. Also the outcome can be dangerous since these fungi are the source of highly potent mycotoxins which can cause severe food poisoning resulting in fatal outcome

Age dependent differential effect of norepinephrine on the pyramidal neurons of the anterior piriform cortex in mouse model of early odor preference learning

Rodent pups show preference to an odor when it is paired with a tactile stimulation- stroking the back with a paintbrush- only within a critical period of postnatal (P) 10-12 days of age. Norepinephrine (NE) released from the locus coeruleus during stroking plays a crucial role in this learning. Here we established the learning model in mouse pups and showed that it was β-adrenoceptor dependent. Next we investigated the developmental changes in pyramidal cell excitability and NE responsiveness in the anterior piriform cortex layer II neurons. Two concentrations (0.1 and 10 μM) of norepinephrine did not alter intrinsic properties in either group, although there was an age-related difference in those properties. In P8–11 pups, norepinephrine at 0.1 μM presynaptically decreased miniature inhibitory postsynaptic current (IPSC) and increased miniature excitatory postsynaptic current (EPSC) frequencies, opposite to the effect of norepinephrine at 10 μM. This suggested involvement of different receptors with different concentrations. In P14 and older pups both concentrations promoted inhibition

Molecular analysis of GerP and spore-associated proteins of Bacillus cereus

Spores of various strains of Bacillus cereus are the causative agents of emetic and diarrheal foodborne illnesses. Typically, spores will survive thermal treatments that destroy vegetative cells, and then go on to germinate to form the vegetative cells that are associated with toxin production. The spore has to germinate in order to develop into the vegetative cells that produce toxins, hence a thorough understanding of the proteins and molecular mechanisms that underpin spore germination are of great significance from spore control perspectives. A major objective of this thesis was to use molecular genetic and fluorescence microscopy techniques to characterise the location and function of the GerP proteins in Bacillus cereus 14579. The GerP proteins have been identified from mutagenesis studies across the Bacilli as being implicated in spore germination, most likely by impacting upon the permeability of the spore coat. Data presented in this thesis reveal that the various GerP proteins all localise to the same inner-coat vicinity within the spore, as determined via the super-resolution ellipsoid localisation microscopy technique. The study also reveals that only the GerPA protein is required for the localisation of the other GerP proteins in the developing spore. A number of other coat and or germination associated proteins in B. cereus 14579 were examined in the course of this work. These include the GerN and GerT antiporters, which are both shown to have an involvement in inosine mediated spore germination in this strain. However, hypothetical interactions between antiporter proteins and the ‘linker-like’ N-terminal domain of the GerIA inosine-responsive germinant receptor protein appear unlikely since spores engineered with a truncated GerIA receptor subunit germinate normally. The protein encoded at locus BC1245 was also examined in this work, since it too had been implicated in spore germination. Data presented in this thesis indicate that this is not the case, and that the protein is a component of the spore coat. Overall, the work conducted in this project contributes to knowledge of spore assembly, spore structure and mechanisms that underpin germination, which ultimately, should permit the development of improved methodologies for spore control.Cambridge Nehru Scholarship Rajiv Gandhi (UK) Scholarship St. Edmund's Duke of Edinburgh Scholarship Raymond and Beverly Sackler Scholarshi

Orthologues of Bacillus subtilis Spore Crust Proteins Have a Structural Role in the Bacillus megaterium QM B1551 Spore Exosporium.

The exosporium of Bacillus megaterium QM B1551 spores is morphologically distinct from exosporia observed for the spores of many other species. Previous work has demonstrated that unidentified genes carried on one of the large indigenous plasmids are required for the assembly of the Bacillus megaterium exosporium. Here, we provide evidence that pBM600-encoded orthologues of the Bacillus subtilis CotW and CotX proteins, which form the crust layer in spores of that species, are structural components of the Bacillus megaterium QM B1551 spore exosporium. The introduction of plasmid-borne cotW and orthologous cotX genes to the PV361 strain, which lacks all indigenous plasmids and produces spores that are devoid of an exosporium, results in the development of spores with a rudimentary exosporium-type structure. Additionally, purified recombinant CotW protein is shown to assemble at the air-water interface to form thin sheets of material, which is consistent with the idea that this protein may form a basal layer in the Bacillus megaterium QM B1551 exosporium.IMPORTANCE When starved of nutrients, some bacterial species develop metabolically dormant spores that can persist in a viable state in the environment for several years. The outermost layers of spores are of particular interest since (i) these represent the primary site for interaction with the environment and (ii) the protein constituents may have biotechnological applications. The outermost layer, or exosporium, in Bacillus megaterium QM B1551 spores is of interest, as it is morphologically distinct from the exosporia of spores of the pathogenic Bacillus cereus family. In this work, we provide evidence that structurally important protein constituents of the Bacillus megaterium exosporium are different from those in the Bacillus cereus family. We also show that one of these proteins, when purified, can assemble to form sheets of exosporium-like material. This is significant, as it indicates that spore-forming bacteria employ different proteins and mechanisms of assembly to construct their external layers.Julia Manetsberger was the recipient of an EPSRC studentship, awarded from the departmental block DTG

Novel cortex lytic enzymes in Bacillus megaterium QM B1551 spores.

Present models for spore germination in Bacillus species include a requirement for either the SleB or CwlJ cortex lytic enzymes to efficiently depolymerise the spore cortex. Previous work has demonstrated that B. megaterium spores may differ to other species in this regard, since sleB cwlJ null mutant spores complemented with the gene in trans for the non-peptidoglycan lysin YpeB can efficiently degrade the cortex. Here, we identify two novel cortex lytic enzymes, encoded at the BMQ_2391 and BMQ_3234 loci, which are essential for cortex hydrolysis in the absence of SleB and CwlJ. Ellipsoid localisation microscopy places the BMQ_3234 protein within the inner-spore coat, a region of the spore that is populated by other cortex lytic enzymes. The findings reinforce the idea that there is a degree of variation in mechanisms of cortex hydrolysis across the Bacillales, raising potential implications for environmental decontamination strategies based upon targeted inactivation of components of the spore germination apparatus.BAR was the recipient of a scholarship from the Sultanate of Oman government. AG was the recipient of a Cambridge Nehru Scholarship and received support from the Raymond and Beverly Sackler Foundation

SpoVT: From Fine-Tuning Regulator in Bacillus subtilis to Essential Sporulation Protein in Bacillus cereus.

Sporulation is a highly sophisticated developmental process adopted by most Bacilli as a survival strategy to withstand extreme conditions that normally do not support microbial growth. A complicated regulatory cascade, divided into various stages and taking place in two different compartments of the cell, involves a number of primary and secondary regulator proteins that drive gene expression directed toward the formation and maturation of an endospore. Such regulator proteins are highly conserved among various spore formers. Despite this conservation, both regulatory and phenotypic differences are observed between different species of spore forming bacteria. In this study, we demonstrate that deletion of the regulatory sporulation protein SpoVT results in a severe sporulation defect in Bacillus cereus, whereas this is not observed in Bacillus subtilis. Although spores are initially formed, the process is stalled at a later stage in development, followed by lysis of the forespore and the mother cell. A transcriptomic investigation of B. cereus ΔspoVT shows upregulation of genes involved in germination, potentially leading to premature lysis of prespores formed. Additionally, extreme variation in the expression of species-specific genes of unknown function was observed. Introduction of the B. subtilis SpoVT protein could partly restore the sporulation defect in the B. cereus spoVT mutant strain. The difference in phenotype is thus more than likely explained by differences in promoter targets rather than differences in mode of action of the conserved SpoVT regulator protein. This study stresses that evolutionary variances in regulon members of sporulation regulators can have profound effects on the spore developmental process and that mere protein homology is not a foolproof predictor of similar phenotypes

Proteins Encoded by the gerP Operon Are Localized to the Inner Coat in Bacillus cereus Spores and Are Dependent on GerPA and SafA for Assembly.

The germination of Bacillus spores is triggered by certain amino acids and sugar molecules which permeate the outermost layers of the spore to interact with receptor complexes that reside in the inner membrane. Previous studies have shown that mutations in the hexacistronic gerP locus reduce the rate of spore germination, with experimental evidence indicating that the defect stems from reduced permeability of the spore coat to germinant molecules. Here, we use the ellipsoid localization microscopy technique to reveal that all six Bacillus cereus GerP proteins share proximity with cortex-lytic enzymes within the inner coat. We also reveal that the GerPA protein alone can localize in the absence of all other GerP proteins and that it has an essential role for the localization of all other GerP proteins within the spore. Its essential role is also demonstrated to be dependent on SafA, but not CotE, for localization, which is consistent with an inner coat location. GerP-null spores are shown also to have reduced permeability to fluorescently labeled dextran molecules compared to wild-type spores. Overall, the results support the hypothesis that the GerP proteins have a structural role within the spore associated with coat permeability.IMPORTANCE The bacterial spore coat comprises a multilayered proteinaceous structure that influences the distribution, survival, and germination properties of spores in the environment. The results from the current study are significant since they increase our understanding of coat assembly and architecture while adding detail to existing models of germination. We demonstrate also that the ellipsoid localization microscopy (ELM) image analysis technique can be used as a novel tool to provide direct quantitative measurements of spore coat permeability. Progress in all of these areas should ultimately facilitate improved methods of spore control in a range of industrial, health care, and environmental sectors.1. Engineering and Physical Sciences Research Council Centre for Doctoral Training in Sensor Technologies and Applications (EP/L015889/1) 2. Cambridge Nehru Scholarship 3. Raymond and Beverly Sackler Foundation 4. MedImmun

Pheromone-Induced Odor Associative Fear Learning in Rats

Alarm pheromones alert conspecifics to the presence of danger. Can pheromone communication aid in learning specific cues? Such facilitation has an evident evolutionary advantage. We use two associative learning paradigms to test this hypothesis. The first is stressed cage mate-induced conditioning. One pair-housed adult rat received 4 pairings of terpinene + shock over 30 min. Ten minutes after return to the home cage, its companion rat was removed and exposed to terpinene. Single-housed controls were exposed to either terpinene or shock only. Companion rats showed terpinene-specific freezing, which was prevented by β-adrenoceptor blockade. Using Arc to index neuronal activation in response to terpinene re-exposure, stressed cage-mate induced associative learning was measured. Companion rats showed increased neuronal activity in the accessory olfactory bulb, while terpinene + shock-conditioned rats showed increased activity in the main olfactory bulb. Both groups had enhanced activity in the anterior basolateral amygdala and central amygdala. To test involvement of pheromone mediation, in the 2nd paradigm, we paired terpinene with soiled bedding from odor + shock rats or a rat alarm pheromone. Both conditioning increased rats’ freezing to terpinene. Blocking NMDA receptors in the basolateral amygdala prevented odor-specific learning suggesting shock and pheromone-paired pathways converge in the amygdala. An alarm pheromone thus enables cue-specific learning as well as signalling danger

Locus Coeruleus Activation Patterns Differentially Modulate Odor Discrimination Learning and Odor Valence in Rats

The locus coeruleus (LC) produces phasic and tonic firing patterns that are theorized to have distinct functional consequences. However, how different firing modes affect learning and valence encoding of sensory information are unknown. Here, we show bilateral optogenetic activation of rat LC neurons using 10-Hz phasic trains of either 300 ms or 10 s accelerated acquisition of a similar odor discrimination. Similar odor discrimination learning was impaired by noradrenergic blockade in the piriform cortex (PC). However, 10-Hz phasic light-mediated learning facilitation was prevented by a dopaminergic antagonist in the PC, or by ventral tegmental area (VTA) silencing with lidocaine, suggesting a LC–VTA–PC dopamine circuitry involvement. Ten-hertz tonic stimulation did not alter odor discrimination acquisition, and was ineffective in activating VTA DA neurons. For valence encoding, tonic stimulation at 25 Hz induced conditioned odor aversion, whereas 10-Hz phasic stimulations produced an odor preference. Both conditionings were prevented by noradrenergic blockade in the basolateral amygdala (BLA). Cholera Toxin B retro-labeling showed larger engagement of nucleus accumbens-projecting neurons in the BLA with 10-Hz phasic activation, and larger engagement of central amygdala projecting cells with 25-Hz tonic light. These outcomes argue that the LC activation patterns differentially influence both target networks and behavior