One of the fundamental methods for cultivating bacterial strains is conventional plating on solid media, but this method does not reveal the true diversity of the bacterial community. In this study, we develop a new technique and introduce a new device we term, I-tip. The I-tip was developed as an in situ cultivation device that allows microorganisms to enter and natural chemical compounds to diffuse, thereby permitting the microorganisms to grow utilizing chemical compounds in their natural environment. The new method was used to cultivate microorganisms from Baikalian sponges, and the results were compared with conventional plating as well as a pyrosequencing-based molecular survey. The I-tip method produced cultures of 34 species from five major phyla, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, and Gammaproteobacteria, 'missing' only two major phyla detected by pyrosequencing. Meanwhile, standard cultivation produced a smaller collection of 16 species from three major phyla, Betaproteobacteria, Firmicutes, and Gammaproteobacteria, failing to detect over half of the major phyla registered by pyrosequencing. We conclude that the I-tip method can narrow the gap between cultivated and uncultivated species, at least for some of the more challenging microbial communities such as those associated with animal hosts.

Despite numerous studies on bacterial motility, little is known about the regulation of this process by environmental factors in natural isolates. In this study we investigated the control of bacterial motility in response to environmental parameters in two strains isolated from the natural habitat of Lake Baikal. Morphological characterization, carbon source utilization, fermentation analysis, and sequence comparison of 16S rRNA genes showed that these strains belong to two distinct genera, i.e., Enterobacter and Pseudomonas; they were named strains 22 and Y1000, respectively. Both strains swarmed at 25°C and remained motile at low temperatures (4°C), especially the Pseudomonas strain, which further supports the psychrotrophic characteristics of this strain. In contrast, a strong inhibition of motility was observed at above 30°C and with a high NaCl concentration. The existence of flagellar regulatory proteins FlhDC and FleQ was demonstrated in Enterobacter strain 22 and Pseudomonas strain Y1000, respectively, and environmental conditions reduced the expression of the structural genes potentially located at the first level in the flagellar cascade in both organisms. Finally, as in Enterobacter strain 22, a strong reduction in the transcription of the master regulatory gene fleQ was observed in Pseudomonas strain Y1000 in the presence of novobiocin, a DNA gyrase inhibitor, suggesting a link between DNA supercoiling and motility control by environmental factors. Thus, striking similarities observed in the two organisms suggest that these processes have evolved toward a similar regulatory mechanism in polarly flagellated and laterally flagellated (peritrichous) bacteria.Microorganisms are able to survive under a wide range of environmental conditions (e.g., osmolarity, temperature, and nutrient availability) by rapidly adapting their structure and physiology. These mechanisms are based on the existence of multiple regulatory systems in which gene expression is controlled in a coordinate manner in response to environmental stimuli. One example of such a complex process is the regulation of motility and chemotaxis in bacteria (16).More than 80% of the known bacterial species are motile by means of flagella (18). The structure and arrangement of flagella differ from species to species and seem to be related to the specific environments in which the cells live (29). Flagella can be arranged on the cell body in a variety of configurations, including single polar, multiple polar, and many peritrichous (or lateral) configurations. Motility by means of flagella is thought to provide a specific advantage for a bacterium (18), because it helps the bacterium to reach the most favorable environment and to successfully compete with other microorganisms. However, the cost of maintenance of a flagellar motility system is high for bacteria (about 2% of biosynthetic energy expenditure in Escherichia coli) due to the number of genes and the energy required for flagellum synthesis and functioning. As a result, the flagellar system i...

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