Analyses of microbial community structure and function

All the biological information of a cell resides in its genes. Analyses of genes make it possible to clarify what kind of functions they potentially have and when those functions are turned on depending on the environmental conditions. Recent developments and applications of molecular techniques, especially genetic ones, make it possible to determine gene sequences rather easily. The introduction of 'Next generation sequencers' (NGS) in particular have greatly enhanced our ability to obtain massive sequence datasets during a short period of time. The application of molecular techniques to study on marine organisms began with microbes. There are a couple of major reasons for this. First, most marine prokaryotes are difficult or impossible to be cultured by ordinary microbiological methods. Usually less than 1 % of the total population are recovered by such methods. Therefore, techniques that do not depend on culturing methods are indispensable. Second, even cultured microbes require considerable work to identify to species, partly due to the limited morphological characters. Therefore, it takes a lot of time and laborious work to determine what types of microbes are present in nature even for culturable species. Finally, the extraction of nucleic acids is relatively easy for microbes compared with multicellular organisms. Cells can be collected by simple filtration of seawater. Since the introduction of molecular techniques in the 1980sup(th), molecular techniques became commonly used by marine microbiologists because of the reasons stated above. The introduction of culture-independent molecular techniques clarified the following. First, there were many formerly unknown phylogenetic groups in the ocean. Also some groups, such as Archaea, for which their presence in marine environments had previously been unknown were found to commonly occur. Second, cultured microbes do not always represent the entirety of the population. For instance, microbes belonging to the genus Vibrio are often isolated and thoroughly investigated, but this group comprises only a very minor portion of the total population. Third, in marine environments, Bacteroidetes (phylum), alphaproteobacteria (class), gammaproteobacteria (class) usually comprise the major part of populations. In the upper water column, cyanobacteria (phylum) are also commonly found. One particular group, SAR11, that belongs to the alphaproteobacteria, is distributed quite widely over almost all part of the ocean. Fourth, the introduction of NGS clarified that in addition to some dominant phylogenetic groups, there are many minor groups existing at a low concentration of individuals. They are referred to as the, 'rare biosphere'. Apparent species richness or the total number of unique sequences are dependent on those groups. It should be pointed out that it is now technically possible to recover mRNAs from the environment and analyze the sequences of such genes. This enables us to detect possible microbial functions that cannot be elucidated by DNA analyses. The application of metagenomic approaches to investigating marine environments has revealed the presence of many formerly unknown or overlooked sequences, such as those related to nitrogen fixation, anoxygenic photosynthesis, and proteorhodopsin (PR). For example, PR was recognized to be a rhodopsin-like gene flanked with SAR86 16S rDNA. Because prokaryotic rhodopsin was believed to be present only among the Archaea, this finding quickly stimulated further analyses of its actual function and distribution among different phylogenetic groups in aquatic environments. It is now evident that PR is a proton pump powered by solar energy, and is quite widely spread among marine prokaryotes, possibly being present in more than 50 % of surface dwelling populations. Recent works also discovered the presence of a new type of rhodopsin, i.e., a sodium ion pump and a chloride pump. Thus, these genetic analyses have had a considerable impact on research on marine microorganisms, especially their community structures, gene expression and discovery of unknown functions that had been overlooked by more conventional techniques and analyses.