A protocol for enumeration of aquatic viruses by epifluorescence microscopy using Anodisc™ 13 membranes

<p>Abstract</p> <p>Background</p> <p>Epifluorescence microscopy is a common method used to enumerate virus-like particles (VLP) from environmental samples and relies on the use of filter membranes with pore sizes < 0.02 μm; the most commonly used protocols employ 25 mm Anodisc™ membranes with a built-in support ring. Other filters with small pore sizes exist, including the 13 mm Anodisc™ membranes without a support ring. However, the use of these membranes for viral enumeration has not been previously reported.</p> <p>Results</p> <p>Here we describe a modified protocol for 13 mm Anodisc membranes that uses a custom filter holder that can be readily constructed in individual investigators' laboratories from commercially available Swinnex^®filter holders. We compared VLP concentrations obtained from phage lysates and seawater samples using both Anodisc membranes, as well as Nuclepore™ small pore-size membranes (0.015 or 0.030 μm). The 13 mm Anodisc membranes gave comparable estimates of VLP abundance to those obtained with the 25 mm Anodisc membranes when similar staining methods were employed. Both Nuclepore membranes typically gave an order of magnitude lower VLP abundance values for environmental samples.</p> <p>Conclusions</p> <p>The 13 mm Anodisc membranes are less costly and require smaller sample volumes than their 25 mm counterpart making them ideal for large-scale studies and sample replication. This method increases the options of reliable approaches available for quantifying VLP from environmental samples.</p

Role of CheB and CheR in the Complex Chemotactic and Aerotactic Pathway of Azospirillum brasilense

It has previously been reported that the alpha-proteobacterium Azospirillum brasilense undergoes methylation-independent chemotaxis; however, a recent study revealed cheB and cheR genes in this organism. We have constructed cheB, cheR, and cheBR mutants of A. brasilense and determined that the CheB and CheR proteins under study significantly influence chemotaxis and aerotaxis but are not essential for these behaviors to occur. First, we found that although cells lacking CheB, CheR, or both were no longer capable of responding to the addition of most chemoattractants in a temporal gradient assay, they did show a chemotactic response (albeit reduced) in a spatial gradient assay. Second, in comparison to the wild type, cheB and cheR mutants under steady-state conditions exhibited an altered swimming bias, whereas the cheBR mutant and the che operon mutant did not. Third, cheB and cheR mutants were null for aerotaxis, whereas the cheBR mutant showed reduced aerotaxis. In contrast to the swimming bias for the model organism Escherichia coli, the swimming bias in A. brasilense cells was dependent on the carbon source present and cells released methanol upon addition of some attractants and upon removal of other attractants. In comparison to the wild type, the cheB, cheR, and cheBR mutants showed various altered patterns of methanol release upon exposure to attractants. This study reveals a significant difference between the chemotaxis adaptation system of A. brasilense and that of the model organism E. coli and suggests that multiple chemotaxis systems are present and contribute to chemotaxis and aerotaxis in A. brasilense

Production of viruses during a spring phytoplankton bloom in the South Pacific Ocean near of New Zealand

Lagrangian studies of virus activity in pelagic environments over extended temporal scales are rare. To address this, viruses and bacteria were examined during the course of a natural phytoplankton bloom in the pelagic South Pacific Ocean east of New Zealand. Daily samples were collected in a mesoscale eddy from year days 263-278 (September 19th-October 4th, 2008). The productive bloom transitioned from a diatom to a pico- and nanoplankton-dominated system, resulting in chlorophyll a concentrations up to 2.43 μg L -1. Virus abundances fluctuated c. 10-fold (1.8 × 10 10-1.3 × 10 11 L -1) over 16 days. The production rates of virus particles were high compared with those reported in other marine systems, ranging from 1.4 × 10 10 to 2.1 × 10 11 L -1 day -1. Our observations suggest viruses contributed significantly to the mortality of bacteria throughout the bloom, with 19-216% of the bacterial standing stock being lysed daily. This mortality released nutrient elements (N, Fe) that likely helped sustain the bloom through the sampling period. Parametric analyses found significant correlations with both biotic (e.g. potential host abundances) and abiotic parameters (e.g. nutrient concentrations, temperature). These observations demonstrate that viruses may be critical in the extended maintenance of regeneration-driven biological production

Molecular Enumeration of an Ecologically Important Cyanophage in a Laurentian Great Lake ▿

Considerable research has shown that cyanobacteria and the viruses that infect them (cyanophage) are pervasive and diverse in global lake populations. Few studies have seasonally analyzed freshwater systems, and little is known about the bacterial and viral communities that coexist during the harsh winters of the Laurentian Great Lakes. Here, we employed quantitative PCR to estimate the abundance of cyanomyoviruses in this system, using the portal vertex g20 gene as a proxy for cyanophage abundance and to determine the potential ecological relevance of these viruses. Cyanomyoviruses were abundant in both the summer and the winter observations, with up to 3.1 × 106 copies of g20 genes ml−1 found at several stations and depths in both seasons, representing up to 4.6% of the total virus community. Lake Erie was productive during both our observations, with high chlorophyll a concentrations in the summer (up to 10.3 μg liter−1) and winter (up to 5.2 μg liter−1). Both bacterial and viral abundances were significantly higher during the summer than during the winter (P < 0.05). Summer bacterial abundances ranged from 3.3 × 106 to 1.6 × 107 ml−1 while winter abundances ranged between ∼3.4 × 105 and 1.2 × 106 ml−1. Total virus abundances were high during both months, with summer abundances significantly higher at most stations, ranging from 6.5 × 107 to 8.8 × 107 ml−1, and with winter abundances ranging from 3.4 × 107 to 6.6 × 107 ml−1. This work confirms that putative cyanomyoviruses are ubiquitous in both summer and winter months in this large freshwater lake system and that they are an abundant component of the virioplankton group