Kanako Tago

Single-cell analysis and isolation for microbiology and biotechnology: methods and applications

Various single-cell isolation techniques, including dilution, micromanipulation, flow cytometry, microfluidics, and compartmentalization, have been developed. These techniques can be used to cultivate previously uncultured microbes, to assess and monitor cell physiology and function, and to screen for novel microbiological products. Various other techniques, such as viable staining, in situ hybridization, and those using autofluorescence proteins, are frequently combined with these single-cell isolation techniques depending on the purpose of the study. In this review article, we summarize currently available single-cell isolation techniques and their applications, when used in combination with other techniques, in microbiological and biotechnological studies.

Isolation of functional single cells from environments using a micromanipulator: application to study denitrifying bacteria

We developed a novel method to isolate functionally active single cells from environmental samples and named it the functional single-cell (FSC) isolation method. This method is based on a combination of substrate-responsive direct viable counts, live-cell staining with 5-carboxyfluorescein diacetate acetoxymethyl ester, and micromanipulation followed by cultivation in a medium. To evaluate this method, we applied it to study a denitrifying community in rice paddy soil. Similar denitrifier counts were obtained by the conventional most probable number analysis and our FSC isolation method. Using the FSC isolation method, 37 denitrifying bacteria were isolated, some of which harbored copper-containing nitrite reductase gene (nirK). The 16S rRNA gene analysis showed that members belonging to the genera Azospirillum and Ochrobactrum may be the major denitrifiers in the rice paddy soil. These results indicate that the FSC isolation method is a useful tool to obtain functionally active single cells from environmental samples.

Complete Genome Sequence of the Denitrifying and N2O-Reducing Bacterium Azoarcus sp. Strain KH32C

We report the finished and annotated genome sequence of a denitrifying and N(2)O-reducing betaproteobacterium, Azoarcus sp. strain KH32C. The genome is composed of one chromosome and one megaplasmid and contains genes for plant-microbe interactions and the gene clusters for aromatic-compound degradations.

Complete genome sequence of the denitrifying and N(2)O-reducing bacterium Pseudogulbenkiania sp. strain NH8B.

Pseudogulbenkiania sp. strain NH8B is a Neisseriales bacterium isolated from an agricultural field. This strain has strong denitrification and N(2)O reduction activities. Here, we report the finished and annotated genome sequence of this organism.

Advantages of functional single-cell isolation method over standard agar plate dilution method as a tool for studying denitrifying bacteria in rice paddy soil

We recently established a method for isolating functional single cells from environmental samples using a micromanipulator (Functional single-cell (FSC) isolation), and applied it to the study of denitrifying bacteria in rice paddy soil (Ashida et al. 2010. Appl Microbiol Biotechnol 85:1211–1217). To further examine the advantages and possible disadvantages of the FSC method, we isolated denitrifying bacteria from the same rice paddy soil sample using both FSC and standard agar plate dilution (APD) methods and compared in this study. The proportion of denitrifying bacteria in the total isolates was more than 6-fold larger with FSC isolation (57.1%) compared with the APD method (9.2%). Denitrifying bacteria belonging to Alphaproteobacteria and Bacilli were commonly isolated using both methods, whereas those belonging to Betaproteobacteria, which had been found to be active in the denitrification-inductive paddy soil, were isolated only with the FSC method. On the other hand, Actinobacteria were only isolated using the APD method. The mean potential denitrification activity of the FSC isolates was higher than that of the APD isolates. Overall, FSC isolation was confirmed to be an excellent method for studying denitrifying bacteria compared with the standard agar plate dilution method.

Microbial populations in various paddy soils respond differently to denitrification-inducing conditions, albeit background bacterial populations are similar

Abstract Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for the denitrification in various rice paddy soils are not well known. In the present study, key denitrifiers were identified in six rice paddy soils by comparing the bacterial community structures in soils with strong denitrifying activity with those in soils without the activity. The effect of crop rotation on the denitrifier populations was also examined. Our study revealed three notable findings: (1) community structures of total bacteria were similar among the six rice paddy soils with different soil characteristics, (2) the populations that responded to denitrification-inducing conditions differed with soil type (Andosol, Gley soil or Gray lowland soil), (3) the populations that responded to denitrification-inducing conditions were similar within each type of soil, regardless of the cropping system (e.g. continuous or rotation).

Taxonomic composition of denitrifying bacterial isolates is different among three rice paddy field soils in Japan

Abstract Denitrification is one of the important nitrogen transformation processes in rice (Oryza sativa L.) paddy soil. We have performed both culture-dependent and -independent studies to investigate the key players in denitrification in rice paddy soil. Our previous works suggested the possibility that community composition of denitrifying populations is different among soils. To confirm this possibility, in the present study, denitrifying bacteria were isolated from rice paddy field soils (Kumamoto and Tokyo) in Japan by the functional single-cell isolation method, and examined for their taxonomic position. Combined with our previously isolated strains from Niigata paddy field soil, our results clearly indicated that the taxonomic composition of the denitrifying bacterial community is different at the genus level among three soils. A significant difference in mean potential denitrification activity of the isolates at the genus level was also found. Soil and/or other environmental factors could shape the community structure of denitrifiers in rice paddy soil.