Rapid multiplex detection and differentiation of Listeria cells by use of fluorescent phage endolysin cell wall binding domains.

The genus Listeria comprises food-borne pathogens associated with severe infections and a high mortality rate. Endolysins from bacteriophages infecting Listeria are promising tools for both their detection and control. These proteins feature a modular organization, consisting of an N-terminal enzymatically active domain (EAD), which contributes lytic activity, and a C-terminal cell wall binding domain (CBD), which targets the lysin to its substrate. Sequence comparison among 12 different endolysins revealed high diversity among the enzyme's functional domains and allowed classification of their CBDs into two major groups and five subclasses. This diversity is reflected in various binding properties, as determined by cell wall binding assays using CBDs fused to fluorescent marker proteins. Although some proteins exhibited a broad binding range and recognize Listeria strains representing all serovars, others target specific serovars only. The CBDs also differed with respect to the number and distribution of ligands recognized on the cells, as well as their binding affinities. Surface plasmon resonance analysis revealed equilibrium affinities in the pico- to nanomolar ranges for all proteins except CBD006, which is due to an internal truncation. Rapid multiplexed detection and differentiation of Listeria strains in mixed bacterial cultures was possible by combining CBDs of different binding specificities with fluorescent markers of various colors. In addition, cells of different Listeria strains could be recovered from artificially contaminated milk or cheese by CBD-based magnetic separation by using broad-range CBDP40 and subsequently identified after incubation with two differently colored CBD fusion proteins of higher specificity.

A small population of planktonic Flavobacteria with disproportionally high growth during the spring phytoplankton bloom in a prealpine lake.

Bacterioplankton growth in temperate Lake Zurich (Switzerland) was studied during the spring phytoplankton bloom by in situ techniques and short-term dilution bioassays. A peak of chlorophyll a (Chl a) concentrations was followed by a rise of bacterial cell numbers and leucine assimilation rates, of the proportions of cells incorporating 5-bromo-2-deoxyuridine (BrdU), and of community net growth rates in dilution cultures. Incorporation of BrdU was low in Betaproteobacteria (2 +/- 1%), indicating that these bacteria did not incorporate the tracer. Pronounced growth of Betaproteobacteria in the enrichments was only observed after the decline of the phytoplankton bloom. An initial peak in the proportions of BrdU-positive Actinobacteria (30%) preceded a distinct rise of their cell numbers during the period of the Chl a maximum. Cytophaga-Flavobacteria (CF) changed little in numbers, but featured high proportions of BrdU-positive cells (28 +/- 12%). Moreover, CF represented > 90% of all newly formed cells in dilution cultures before and during the phytoplankton bloom. One phylogenetic lineage of cultivable Flavobacteria (FLAV2) represented a small (0.5-1%) but highly active population in lake plankton. The growth rates of FLAV2 in dilution cultures doubled during the period of the Chl a maximum, indicating stimulation by phytoplankton exudates. Thus, CF, and specifically Flavobacteria, appeared to be substantially more important for carbon transfer in Lake Zurich spring bacterioplankton than was suggested by their standing stocks. The high in situ growth potential of these bacteria might have been counterbalanced by top-down control.