DISCUSSION

Microbes often construct and live within surface-associated multicellular communities known as biofilms. Biofilms are enclosed in a extracellular polymeric matrix that forms the scaffold for the three-dimensional architecture of the biofilms and is responsible for adhesion to surfaces and for cohesion in the biofilms (Flemming et al., 2010). Also, biofilms contain bacterial cells that are in a wide range of physiological states. This heterogeneity is considered to be caused by gradients of pH, oxygen, and nutrients in the biofilm matrix (Stewart et al., 2008).

Given that these properties of biofilms have an impact on cellular metabolism, it can be expected that metabolism occurring in biofilms would be different from in planktonic cells.

In this study, in order to gain insight of into the cellular mechanisms of biofilms, I tried to conduct the global metabolite profiling of V. vulnificus biofilms and planktonic cells. The metabolome is much more sensitive to perturbations in the environment than either the transcriptome or the proteome because the metabolites within a cell are an integrated reflection of the cellular phenotype (Sellick et al., 2009). Therefore, metabolomics approach to understand the V. vulnificus biofilms is very significant.

There are three stages of biofilm development: (i) initial attachment, (ii) maturation,

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(iii) dispersion. Here, to characterize mechanisms of mature biofilms, biofilms and planktonic cells were harvested at the peak time point (Fig. 1). Different from the other ‘omics’, metabolomics includes methodological problems derived from heterogeneity in chemical properties (Ohashi et al., 2008). In this reason, metabolome sampling is one of the most important factors determine the quality of metabolomics data (Kim et al., 2013a). Thus, to minimize the losses of metabolites, I selected sample-dependent methods (Fig. 2).

From the experiment, principal component analysis and partial least-squares discriminant analysis revealed significant differences in metabolic profiles between biofilms and planktonic cells (Fig. 3). The levels of 37 metabolites, such as some amino acids, monosaccharides and nucleotides were elevated in biofilms compared to the planktonic counterparts (Table 3). EPM are mainly polysaccharides, proteins and nucleic acids (Flemming et al., 2010). Therefore, most of these metabolites appear to function as building blocks for structural components of extracellular matrix.

Metabolites involved in the glycolysis and pyruvate fermentation were increased in biofilms (Fig. 5). These suggest that V. vulnificus may obtain energy by using glycolysis and pyruvate fermentation, when they are in biofilms. In a mature biofilms, at least three distinct physiological states can be anticipated (Stewart et al., 2008). Especially, cells that are located deeper layer in the biofilms presumably grow by anaerobic metabolism. Therefore, to survive under unfavorable conditions,

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cells in V. vulnificus biofilms may depend on glycolysis and pyruvate fermentation.

In this manner, bacterial cells in V. vulnificus biofilms are possible for long-term survival.

In V. vulnificus biofilms, putrescine biosynthetic pathway related matabolites were greater than in the planktonic cells (Fig. 6). Urea cycle was inactivated, whereas putrescine biosynthetic pathway was activated in V. vulnificus biofilms.

These results suggested that putrescine may have a positive effect on formation of V.

vulnificus biofilms.

Bacteria seem to initiate biofilm development in response to specific environmental cues, such as nutrient availability. Then, these biofilms continue to develop as long as fresh nutrients are provided, but when they are nutrient deprived, they detach from the surface and return to a planktonic mode of growth (George et al., 2000). In the experiment, the amounts and expression levels of the metabolites and genes, respectively, involved in N-acetylglucosamine (GlcNAc) utilization were highly increased in V. vulnificus biofilms. (Fig. 7). Composition analysis of EPS using GC-TOF-MS revealed that GlcNAc is one of the constituent of EPS (Table 5).

Consequently, the combined results suggest that, in mature biofilms, when V.

vulnificus exists in low-nutrient environments, GlcNAc specific PTS may transfer GlcNAc which is derived from EPS. Then, it may be possible to use the GlcNAc as a carbon source.

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PTS consists of components of the carbohydrate-specific and PtsG is GlcNAc specific PTS IIC component (Houot et al., 2010). In this study, to find out whether PtsG affects biofilm forming ability of V. vulnificus, I selected ptsG, for mutant target. Under the condition of low carbon source, biofilm forming activity was reduced in the ptsG mutant compared to the wild type and recovered in the complemented strain (Fig. 9). In these aspects, when bacterial cells in V. vulnificus biofilms are faced with adverse conditions, such as insufficient nutrient, PtsG transports GlcNAc which stems from EPS. Then, GlcNAc appears to be used as a carbon source.

Biofilms formed in industrial settings can be recalcitrant to antimicrobial agents, thereby leading to possible infection of food products (Kumar et al., 1998). This study is expected to give important clues in understanding the cellular mechanisms of V. vulnificus biofilms. Also, in order to conduct more precise analysis of V.

vulnificus biofilms, this study present combined other ‘omics’ study as an improved adaptation over prior microbial metabolomics studies. Through this study, it is revealed that some factors including biofilm-specific physiological pathways are important for V. vulnificus biofilm formation. If we use these factors in order to control biofilms, it can contribute to the food safety.

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마지막으로 패혈증 비브리오균의 biofilm 에서 N-acetylglucosamine utilization 과 관련 metabolite 들과 N-acetylglucosamine specific PTS system (PtsG)과 하위의 해당 경로에 관련된 gene 또한 모두 증가하였다. 이를