Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria.

Total bacterial abundances estimated with different epifluorescence microscopy methods (4',6-diamidino-2-phenylindole [DAPI], SYBR Green, and Live/Dead) and with flow cytometry (Syto13) showed good correspondence throughout two microcosm experiments with coastal Mediterranean water. In the Syto13-stained samples we could differentiate bacteria with apparent high DNA (HDNA) content and bacteria with apparent low DNA (LDNA) content. HDNA bacteria, "live" bacteria (determined as such with the Molecular Probes Live/Dead BacLight bacterial viability kit), and nucleoid-containing bacteria (NuCC) comprised similar fractions of the total bacterial community. Similarly, LDNA bacteria and "dead" bacteria (determined with the kit) comprised a similar fraction of the total bacterial community in one of the experiments. The rates of change of each type of bacteria during the microcosm experiments were also positively correlated between methods. In various experiments where predator pressure on bacteria had been reduced, we detected growth of the HDNA bacteria without concomitant growth of the LDNA bacteria, such that the percentage contribution of HDNA bacteria to total bacterial numbers (%HDNA) increased. This indicates that the HDNA bacteria are the dynamic members of the bacterial assemblage. Given how quickly and easily the numbers of HDNA and LDNA bacteria can be obtained, and given the similarity to the numbers of "live" cells and NuCC, the %HDNA is suggested as a reference value for the percentage of actively growing bacteria in marine planktonic environments.

Dominant marine bacterioplankton species found among colony-forming bacteria.

The density of specific aquatic bacteria was determined by use of whole-genome DNA hybridization towards community DNA. From a coastal marine environment (northern Baltic Sea), 48 specific bacteria were isolated on solid media over a 1-year period. Based on the presented hybridization protocol, the total density of the isolates ranged between 7 and 69% of the bacteria determined by acridine orange direct counts. When compared to the number of nucleoid-containing cells, the range increased to 29 to 111%. Thus, our results showed that bacteria able to form colonies on solid media accounted for a large fraction of the bacterioplankton. There were significant changes in the density of the different bacteria over the year, suggesting that bacterioplankton exhibit a seasonal succession analogous to phytoplankton. The bacteria studied were of diverse phylogenetic origin, being distributed among the alpha, beta, and gamma subdivisions of the class Proteobacteria and the cytophaga-flexibacter group. Partial 16S rRNA gene sequence analysis of 29 Baltic Sea isolates as well as of 30 Southern California Bight isolates showed that a majority of the isolates had low similarity (0.85 to 0.95) to reported sequence data. This indicated that the diversity of marine bacteria able to grow on solid media is largely unexplored.

Total counts of marine bacteria include a large fraction of non-nucleoid-containing bacteria (ghosts).

Counts of heterotrophic bacteria in marine waters are usually in the order of 5 x 10(sup5) to 3 x 10(sup6) bacteria ml(sup-1). These numbers are derived from unspecific fluorescent staining techniques (J. E. Hobbie, R. J. Daley, and S. Jasper, Appl. Environ. Microbiol. 33:1225-1228, 1977; K. G. Porter and Y. S. Feig, Limnol. Oceanogr. 25:943-948, 1980) and are subsequently defined as total counts of bacteria. In samples from the Baltic Sea, the North Sea (Skagerrak), and the northeastern Mediterranean Sea, we found that only a minor fraction (2 to 32%) of total counts can be scored as bacteria with nucleoids. Lack of DNA no doubt means inactive cells; therefore, a much lower number of bacteria that grow at rates higher than those previously estimated must be responsible for the measured bacterial production in these seas. The remaining bacterium-sized and/or -shaped particles included in total counts may be cell residues of virus-lysed bacteria (ghosts) or remains of protozoan grazing.