Measurement of bacterial random motility and chemotaxis coefficients: I. Stopped-flow diffusion chamber assay

Bacterial chemotaxis, the directed movement of a cell population in response to a chemical gradient, plays a critical role in the distribution and dynamic interaction of bacterial populations in nonmixed systems. Therefore, in order to make reliable predictions about the migratory behavior of bacteria within the environment, a quantitative characterization of the chemotactic response in terms of intrinsic cell properties is needed.The design of the stopped-flow diffusion chamber (SFDC) provides a well-characterized chemical gradient and reliable method for measuring bacterial migration behavior. During flow through the chamber, a step change in chemical concentration is imposed on a uniform suspension of bacteria. Once flow is stopped, diffusion causes a transient chemical gradient to develop, and bacteria respond by forming a band of high cell density which travels toward higher concentrations of the attractant. Changes in bacterial spatial distributions observed through light scattering are recorded on photomicrographs during a 10-min period. Computer-aided image analysis converts absorbance of the photographic negatives to a digital representation of bacterial density profiles. A mathematical model (part II) is used to quantitatively characterize these observations in terms of intrinsic cell parameters: a chemotactic sensitivity coefficient, mu(0), from the aggregate cell density accumulated in the band and a random motility coefficient, mu, from population dispersion in the absence of a chemical gradient.Using the SFDC assay and an individual-cell-based mathematical model, we successfully determined values for both of these population parameters for Escherichia coli K12 responding to fucose. The values obtained were mu = 1.1 +/- 0. 4 x 10(-5) cm(2)/s and chi(o) = 8 +/- 3 +/- 10(-5) cm(2)/s. We have demonstrated a method capable of determining these parameter values from the now validated mathematical model which will be useful for predicting bacterial migration in application systems.     

Advantages of distinguishing the active fraction in bacterioplankton assemblages: some examples

The difficulty of distinguishing between active and dormant or dead bacterial cells is an important problem for the aquatic microbiologist.Active cells can be detected under the microscope by the presence of an intact electron transport system able to reduce the colourless INT [2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride] to an optically dense intracellular deposit.An improvement of this method has been applied to Lake Geneva and to a fish pond in the Ivory Coast. The portion of INT-reducing bacterial cells ranged from 1 to 71%, depending on place, depth, season and time of the day. In all cases bacterial activity, determined by uptake of ³H Thymidine or ¹⁴C glucose, and frequency of dividing cells were better correlated with the number of INT-reducing cells than with the total number of cells. This means that counts of cells able to reduce INT have a better metabolic significance than total cell counts. Some examples are developed which show the advantages of applying this method in cases where it is useful to distinguish active cells in a bacterial assemblage.     

Bottom-up effects of bream (Abramis brama L.) in Lake Balaton

Enclosures (17 m³) were used in the mesotrophic area of Lake Balaton to determine the impact of benthivorous bream (Abramis brama L.) on the lower trophic levels during summers of 1984–86. In enclosures with a fish biomass similar to the biomass in the eutrophic area of the lake, the number of phytoplankton species was highest. In enclosures with a low fish biomass the phytoplankton was dominated by the greens. A high biomass of bream in the mesotrophic basin caused bacterial production corresponding to that of the eutrophic part of the lake. Crustaceans were dominated by copepods and were unable to control phytoplankton peaks. Bottom-up effects of bream were more obvious than top-down effects and seem to be more important in the possible control of water quality.     

Preferential feeding behaviour of Daphnia magna

Preferential feeding behaviour by Daphnia magna was shown when Daphnia were fed on a mixture of ¹⁴C-labelled algae (Chlorella vulgaris or Scenedesmus quadricauda) and ³H-labelled bacteria (Escherichia coli). Daphnia consumption of bacteria was equal or higher in the presence of algae. On the other hand, in the presence of bacteria, algal consumption decreased by 40–70% compared to algal consumption in the absence of bacteria. ¹⁴C radioactive uptake was in good agreement with the chlorophyll content and demonstrates the preferential feeding behaviour of Daphnia.     

Nematode Temperature Responses: a Niche Dimension In Populations of Bacterial-Feeding Nematodes

The optimum temperatures for population development were determined for six species of bacterial-feeding nematodes from among eight temperatures, ranging from 5 to 40 C. Four of the species are cohabiting species. The range of temperatures over which population development occurs (temperature niche breadth) is different for the cohabiting species. This difference may be a means of reducing competition between species, thus increasing temperatures over which habitats can be exploited.     

Evaluation of the potential of a bead mill for the release of intracellular bacterial enzymes

The suitability of bead mills for the release of intracellular bacterial enzymes has been studied using the Dyno-Mill Model KDL. The effect of cell concentration, bead size and agitation speed on the release of beta-lactamase from Enterobacter cloacae P99 was examined. Scale-up considerations included, the best operational values for these parameters were 1 g cell paste suspended in 2.5 ml buffer, 0.25 mm diameter glass beads and 15 ms ⁻¹ agitation speed. These conditions proved suitable for the release of enzymes from other Gram-negative bacteria in both batch and continuous processes.     

Terminal deoxyribonucleotidyl transferase activity in acute undifferentiated leukemia.

Guanosine, rather than its methylated derivative, was found to be the inverted nucleoside present in the 5′ terminal capping structure of insect oocyte messenger RNA. Since methylation of the terminal guanosine at the 7 position is necessary for the initiation of protein synthesis in eukaryotes, this evidence suggests that the translational inactivity of the mRNA prior to fertilization may be associated with the absence of methylation.     

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.     

Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection

Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation.