Effectiveness of some crown compounds on inhibition of polyphenoloxidase in model systems and in apple

Enzymatic browning is (in most cases) an undesirable reaction which usually impairs the sensory properties and chemical changes in raw fruits and vegetables after mechanical operations (such as peeling, coring or slicing). A great emphasis is put on research to develop new methods to prevent enzymatic browning especially in fresh-cut (minimally processed) fruits and vegetables. The inhibition effect of crown compounds, macrocyclic ethers, benzo-18-crown-6 with sorbic acid and benzo-18-crown-6 with potassium sorbate, on polyphenoloxidase (PPO) activity was studied. The effectiveness of these compounds was evaluated by using 3,4-dihydroxy phenylalanine (L-DOPA), and chlorogenic acid (3-o-caffeoyl-D-quinic acid), the most widespread natural PPO substrates in fruits and vegetables, as well as browning inhibition substances on the cut surface of apples. Results showed that crown compounds used in this study were effective, both as inhibitors of the oxidation of phenolic compounds (PPO substrates) in model solutions and as inhibitors of enzyme discolorations of real systems (fresh-cut apples). In the earlier published papers (V UKOVI C 'et al., 1999) the synthesis of crown compound used in this study was presented

Conformational spread as a mechanism for cooperativity in the bacterial flagellar switch

The bacterial flagellar switch that controls the direction of flagellar rotation during chemotaxis has a highly cooperative response. This has previously been understood in terms of the classic two-state, concerted model of allosteric regulation. Here, we used high-resolution optical microscopy to observe switching of single motors and uncover the stochastic multistate nature of the switch. Our observations are in detailed quantitative agreement with a recent general model of allosteric cooperativity that exhibits conformational spread—the stochastic growth and shrinkage of domains of adjacent subunits sharing a particular conformational state. We expect that conformational spread will be important in explaining cooperativity in other large signaling complexes

Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorous predation

Modification of essential bacterial peptidoglycan (PG) containing cell walls can lead to antibiotic resistance, for example β-lactam resistance by L,D-transpeptidase activities. Predatory Bdellovibrio bacteriovorus are naturally antibacterial and combat infections by traversing, modifying and finally destroying walls of Gram-negative prey bacteria, modifying their own PG as they grow inside prey. Historically, these multi-enzymatic processes on two similar PG walls have proved challenging to elucidate. Here, with a PG labelling approach utilizing timed pulses of multiple fluorescent D-amino acids (FDAAs), we illuminate dynamic changes that predator and prey walls go through during the different phases of bacteria:bacteria invasion. We show formation of a reinforced circular port-hole in the prey wall; L,D-transpeptidaseBd mediated D-amino acid modifications strengthening prey PG during Bdellovibrio invasion and a zonal mode of predator-elongation. This process is followed by unconventional, multi-point and synchronous septation of the intracellular Bdellovibrio, accommodating odd- and even-numbered progeny formation by non-binary division

Fast, Multiphase Volume Adaptation to Hyperosmotic Shock by Escherichia coli

All living cells employ an array of different mechanisms to help them survive changes in extra cellular osmotic pressure. The difference in the concentration of chemicals in a bacterium's cytoplasm and the external environment generates an osmotic pressure that inflates the cell. It is thought that the bacterium Escherichia coli use a number of interconnected systems to adapt to changes in external pressure, allowing them to maintain turgor and live in surroundings that range more than two-hundred-fold in external osmolality. Here, we use fluorescence imaging to make the first measurements of cell volume changes over time during hyperosmotic shock and subsequent adaptation on a single cell level in vivo with a time resolution on the order of seconds. We directly observe two previously unseen phases of the cytoplasmic water efflux upon hyperosmotic shock. Furthermore, we monitor cell volume changes during the post-shock recovery and observe a two-phase response that depends on the shock magnitude. The initial phase of recovery is fast, on the order of 15–20 min and shows little cell-to-cell variation. For large sucrose shocks, a secondary phase that lasts several hours adds to the recovery. We find that cells are able to recover fully from shocks as high as 1 Osmol/kg using existing systems, but that for larger shocks, protein synthesis is required for full recovery

Coevolved mutations reveal distinct architectures for two core proteins in the bacterial flagellar motor

Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) "torque" helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could be the convertor element that provides mechanistic and species diversity.JK was supported by Medical Research Council grant U117581331. SK was supported by seed funds from Lahore University of Managment Sciences (LUMS) and the Molecular Biology Consortium

Molecular imaging of glycan chains couples cell-wall polysaccharide architecture to bacterial cell

Biopolymer composite cell walls maintain cell shape and resist forces in plants, fungi and bacteria. Peptidoglycan, a crucial antibiotic target and immunomodulator, performs this role in bacteria. The textbook structural model of peptidoglycan is a highly ordered, crystalline material. Here we use atomic force microscopy (AFM) to image individual glycan chains in peptidoglycan from Escherichia coli in unprecedented detail. We quantify and map the extent to which chains are oriented in a similar direction (orientational order), showing it is much less ordered than previously depicted. Combining AFM with size exclusion chromatography, we reveal glycan chains up to 200 nm long. We show that altered cell shape is associated with substantial changes in peptidoglycan biophysical properties. Glycans from E. coli in its normal rod shape are long and circumferentially oriented, but when a spheroid shape is induced (chemically or genetically) glycans become short and disordered

Novel Methods for Analysing Bacterial Tracks Reveal Persistence in Rhodobacter sphaeroides

Tracking bacteria using video microscopy is a powerful experimental approach to probe their motile behaviour. The trajectories obtained contain much information relating to the complex patterns of bacterial motility. However, methods for the quantitative analysis of such data are limited. Most swimming bacteria move in approximately straight lines, interspersed with random reorientation phases. It is therefore necessary to segment observed tracks into swimming and reorientation phases to extract useful statistics. We present novel robust analysis tools to discern these two phases in tracks. Our methods comprise a simple and effective protocol for removing spurious tracks from tracking datasets, followed by analysis based on a two-state hidden Markov model, taking advantage of the availability of mutant strains that exhibit swimming-only or reorientating-only motion to generate an empirical prior distribution. Using simulated tracks with varying levels of added noise, we validate our methods and compare them with an existing heuristic method. To our knowledge this is the first example of a systematic assessment of analysis methods in this field. The new methods are substantially more robust to noise and introduce less systematic bias than the heuristic method. We apply our methods to tracks obtained from the bacterial species Rhodobacter sphaeroides and Escherichia coli. Our results demonstrate that R. sphaeroides exhibits persistence over the course of a tumbling event, which is a novel result with important implications in the study of this and similar species

Physiology of Escherichia coli at high osmolarity and its use in industrial ethanol production

Biofuels are becoming increasingly important in the light of climate change, increasing energy demands and higher fuel prices. Their production must be carefully balanced against the production of foods and use of fresh water, both of which are consumed by crop based biofuels such as corn ethanol. One proposed solution is to instead use waste materials such as plant matter including wood offcuts and plant trimmings. This waste can be turned into syngas (a mix of CO and H₂) and converted to ethanol using microorganisms. Production of ethanol using microorganisms however, is complicated as the ethanol produced by the cells becomes toxic at higher concentrations, inhibiting their growth and further production. The usual method of keeping the toxicity down to allow further production is to continuously distil ethanol off at low concentrations and consequently, a high cost. Since the mechanisms of ethanol damage to microbes are similar to those that occur during osmotic challenge: damage to the membrane, cytoplasmic dehydration, and protein unfolding, I hypothesized that we can use knowledge of osmoregulatory mechanisms to increase the resistance of cells to ethanol damage and decrease distillation costs. While working under this hypothesis I had to address some of the challenges one faces when understanding the physiology and growth of microbes, and for the purpose I have developed a number of useful techniques; a method for calibrating optical densities to cell number, a neural network for identifying cells and determining their concentrations via microscope imaging and a simple particle diffusion simulation for correcting errors due to confinement of particles within cells. In addition, I have produced a simplified model of industrial production to help evaluate economic impacts that changes to the growth of microbes and the plant process may have. To study any useful links between osmolarity and ethanol resistance, I chose to use Escherichia coli as the model organism due to the large amount of data available on its osmoregulatory mechanisms. It has been long known that when bacteria do grow at high but not lethal osmolarity, they grow at a reduced rate which, even if it increases the ethanol resistance, may have a detrimental effect on the desired production rates. So therefore, in addition to testing the ethanol tolerance of the bacteria under different osmotic conditions, and as a second focus of this project, I have tried to understand why the reduction in growth rates occurs, with the hope of mitigating this effect. This will offer a better understanding of osmotic growth and provide useful insights for industrial bio-production. To this end, I have tried to discern some of the possible reasons for this slower growth by measuring various cell physiological parameters such as batch-culture yield, cytoplasmic diffusion and proteome allocation using my newly developed techniques. I have found a reduction in the cell yield with increasing osmolarity of 50% with an increase of 1Osm of osmotic agent, a slight decrease in cytoplasmic diffusion and a slight decrease in RNA content at high osmolarity. I have also proposed a coarse-grained model of proteome partitioning to help integrate these results and explain growth at high osmolarity. It is still to be determined if, as a whole, the changes observed explain fully the reduction in growth. When it comes to ethanol resistance, and contrary to my hypothesis, I found that increasing the osmolarity of the medium with sucrose or NaCl reduced the ethanol resistance. However, I found that the proW gene provides significant ethanol resistance, indicating glycine betaine, or another substrate for this transporter, is highly useful as a protectant. And this transporter is a potential candidate for overexpression. A reduction in growth temperature also provides significant solvent tolerance at the expense of a reduction in growth rate and hence production.Restricted Acces