A huge number of microorganisms are still unknown and uncultivated. SSU rRNA gene-based analyses of complex microbial communities are unveiling their diversity, distribution and abundance in natural environments. How to isolate uncultivated microorganisms is an everlasting preoccupation for microbiologists even in the era of culture-independent functional analyses such as stable isotope labeling, modernized in situ hybridization techniques and community genomics. Conventional isolation procedures are very laborious, and there is significant room for improvement. In this review, we stress several important clues to the systematic isolation and cultivation of uncultured microorganisms.

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A gene for cAMP phosphodiesterase, designated cpdA, was identified in the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120. The predicted amino acid sequence of the gene was similar to the sequences of cAMP phosphodiesterases from Thermosynechococcus elongatus, Escherichia coli and Haemophilus influenzae. The recombinant protein was purified by sequential column chromatography and its biochemical properties were determined. The Anabaena cAMP phosphodiesterase hydrolyzed cAMP and cGMP with similar levels of activity. The K_m value for cAMP was 45 μM and the Vmax was 4.9 mmol min^-1 mg protein^-1. These values are similar to those of Escherichia coli cAMP phosphodiesterase. The enzyme was activated by divalent cations such as Fe²⁺ and Mn²⁺. The tertiary structure of this enzyme was predicted by homology modeling. The deduced structure has two metal-binding sites in the catalytic domain.

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The mucus obtained from the surface of bluegills (Lepomis macrochirus) can be utilized as a source of nutrients by the bacterial strains isolated from the mucus and the adjacent lake water. The isolates from both the mucus and the lake water grew better in 20-fold diluted mucus than in lake water. Almost all mucus isolates (10 of 12 strains) hardly grew at all in lake water, whereas half (6 of 12 strains) of the lake-water isolates were able to grow in lake water, according to the increase in optical density (O.D.) after culturing in mucus or lake water. The isolates grew faster in 20-fold diluted mucus than in lake water, with a greater enhancement of the growth rate for the mucus isolates than the lake-water isolates. The features of the mucus isolates, i.e., little growth in lake water and faster growth in mucus, were deduced to be responsible for the different bacterial flora of the fish mucus. The performance of 20-fold diluted mucus as a source of nutrients was evaluated by comparing it with nutrient broth (NB) medium and 100-fold diluted NB medium (DNB medium). In many strains, the amount of growth (maximum O.D.) was highest in NB, then mucus and lastly DNB, coinciding with the order in terms of the concentrations of glucose equivalent, i.e., NB, 20-fold diluted mucus and DNB. About 60% and 70% of the isolates grew faster in the 20-fold diluted mucus than in the NB and DNB mediums, respectively. The growth yield (increase in O.D. when the unit amount of glucose equivalent was consumed) was on average, ca. 70 times greater in the 20-fold diluted mucus than NB. The growth-rate constant in the mucus did not differ markedly among the isolates belonging to various phylogenetic groups. By contrast, in the NB medium, the isolates closely related to the Actinobacteria and α-Proteobacteria had smaller growth-rate constants and the isolates closely related to γ-Proteobacteria had greater growth-rate constants. The respiratory activity of the isolates was greatly enhanced by adding a small amount of mucus (1% as the final concentration), due to unknown factor(s). Thus, as a source of nutrients, the mucus seemed to be more effective, as well as being able to sustain the growth of various bacterial groups.

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We studied the effects of oil spills on the trophic interactions among microorganisms in marine environments on a mesocosmic scale. When Bunker-A oil or kerosene was introduced into the mesocosm, bacterial numbers increased for the first 3 days, then decreased with a concomitant propagation of either heterotrophic nanoflagellates (HNFs) or viruses. We hypothesized that the sensitivity of HNFs to each oil resulted in this trophic relationship. To test this hypothesis, the rates at which HNFs grazed on bacteria were examined by conducting microcosm experiments in the presence of one of these oils. The rates were markedly reduced by kerosene but slightly increased by Bunker-A oil. The removal of HNFs by filtration led to a fluctuation in the numbers of bacteria and viruses that was quite similar to that in the mesocosm with kerosene. We conclude that the inhibitory effect of oil components on unicellular microorganisms is one of the key factors controlling the microbial food-web in oil-contaminated seawater.

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Catellibacterium nectariphilum was recently proposed as a new genus and species within the `Rhodobacter group' in the α-3 subgroup of Proteobacteria. This species did not show significant growth on a nutrient medium, and required unknown growth factor(s) for vigorous growth. The growth factor(s) would be diffused in the supernatant of the enrichment culture of the activated sludge from which C. nectariphilum was isolated. We have searched for microorganisms that produce growth factor(s) for C. nectariphilum, and obtained three bacterial strains, designated GF9, GF20 and GF22, from the same sludge. A phylogenetic analysis based on the 16S rRNA gene sequence indicated that these strains were closely related to each other, and all belonged to the genus Sphingomonas. Addition of a supernatant prepared from a culture of strain GF9 clearly increased the cell yield and growth rate of C. nectariphilum. Growth-promoting activity in other species of the family Sphingomonadaceae was also measured. The results indicated that various Sphingomonas species and their relatives had a similar ability and it was widely distributed within the family Sphingomonadaceae. The growth factor(s) produced by Sphingomonas species have not been identified, but were heat-stable, non-peptides and low-molecular weight (below 1,000 Da) compound(s).

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Some bacterial strains that did not have tannase activity formed vague halos around their colonies on tannin-treated media. These halos were due to alkaline dissociation of the tannin-protein complex of the media during bacterial growth. All tannase-positive results obtained by the plate screening method should therefore be confirmed using a more analytical method.

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Characteristic patterns of expansion were generated by bacterial cell populations of Serratia marcescens on media with different concentrations of nutrient and agar. These patterns were classified as Eden-like, dense-branching-morphology-like, flower-like, concentric ring-like and diffusion-limited aggregation-like. Although flower-like was specific to Serratia marcescens, the other patterns were exhibited by Bacillus subtilis. Through macro- and microscopic tracing of the processes generating these patterns, physico-chemical principles of bacterial growth, collaborative and independent properties of bacteria, structural organization for population expansion, and the division of labor among bacterial cells (i.e., wall composer, pressure generator, and logistic supporter) were brought to light.

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