Tendencias del sector

2.3.1.1 Types of fructan and responsible genera

Bacteria produce fructans in which the majority of fructose units are β(2→6) linked but some branches may contain β(2→1) linkages (Dedonder 1966). These polymers are known as levans. Many Gram-positive and Gram-negative bacteria produce levan, while inulin synthesis with β(2→1) linkages has been reported so far only in the Gram-positive species

Streptococcus mutans, Lactobacillus reuteri and Leuconostoc citreum. Bacterial levan and

inulin are the largest fructans in nature, with a degree of polymerisation (DP) reaching up to 100 000 fructose units (Banguela and Hernández 2006).

Fig. 2.1 Chemical structures of fructans. Redrawn from Banguela et al. (2006) and Vijn and Smeekens

(1999).

Levan producing bacteria include strains belonging to the genera Bacillus, Streptococcus,

Pseudomonas, Erwinia and Actinomyces (Hendry and Wallace 1993). Bacterial levan is

produced extracellularly from sucrose by the action of a single enzyme, levansucrase. Levansucrase activity has been assigned as EC 2.4.1.10 which implies glycosyltransferase status (Webb 1992). Acceptor molecules for the transfructosidase reaction include water (sucrose hydrolysis), glucose, fructans and sucrose (Hernandez et al. 1995). Thus the production of low DP FOS and high DP levan are mediated by a single enzyme. Generally, levansucrases from Gram-positive bacteria catalyse the formation of high DP levan without transient accumulation of FOS, whereas the enzymes from Gram-negative species render high levels of inulin type FOS (1-kestose and nystose) with lower yields of high DP levan (Banguela and Hernández 2006). Similarly to β-fructofuranosidases, levansucrases are classified as glycosyl hydrolases (GH) according to the sequence-based classification of carbohydrate-active enzymes (CAZy), although in family GH68 (Cantarel et al. 2009). In addition to synthesis of levan, bacteria possess endo- and exolevanases as well as non-specific β-fructosidases for the hydrolysis of levan (Vijn and Smeekens 1999).

2.3.1.2 Biological role

The biological role of bacterial fructans has been linked to their interactions with eukaryotes as fructan synthesising bacteria are either plant pathogens or symbionts or are associated with oral and gut floras of humans and animals (Velázquez-Hernández et al. 2009; Vijn and Smeekens 1999). Studies involving gene disruption of fructan synthesising enzymes have contributed to the understanding of the role of microbial fructans and are reviewed extensively by Velázquez-

Hernández et al (2009). Fructans contribute to pathogen virulence by supplying energy stored in reserve carbohydrates and influencing biofilm formation. Levans impose high viscosities on aqueous solutions, and their presumed functions involve protecting cells from desiccation and facilitating adherence to surfaces (Kiska and Macrina 1994a), which supports the idea of fructan involvement in biofilm formation. In agreement with previous studies, it has recently been shown that although levan is not an absolute requirement for the formation of B. subtilis biofilms, it can be a structural and stabilising component of floating biofilms. Additionally it was proposed that when levan was included in the exopolymeric substances (EPS), it could serve as a nutritional reserve (Dogsa et al. 2013). Fructans contribute to biofilm formation by

Lactobacillus spp. in the gastrointestinal tract of birds and mammals. Defects in inulosucrase

activity, which is responsible for the synthesis of polymeric inulin, resulted in reduced competitiveness relative to wild type stains and impacted on colonisation of the mouse gastrointestinal tract by L. reuteri LTH5448 (Walter et al. 2008).

Fructans are also proposed to function in nutrient signalling. The soil and rhizosphere associated B. subtilis is proposed to generate the disaccharide, levanbiose, by the action of extracellular levanase. No cellular transporters have been identified and levanbiose is proposed to accumulate in the extracellular environment. Drawing parallels with sugars implicated as signalling molecules regulating carbon metabolism, growth and development in plant cells, Daguer et al. (2004) proposed that levanbiose could act as a signalling molecule between B. subtilis cells in soil to control survival functions, or between B. subtilis and other microorganisms or plants as part of a molecular dialog to establish corresponding interactions. The biological functions mediated by fructans in bacteria and other organisms reflect an interaction of processes, triggered by the presence of sucrose-related carbohydrates in the cellular environment, that result in the synthesis and/or hydrolysis of the fructans. Transcriptional regulation of responsible coding genes for enzymes involved in the relevant modifications is reasonably well studied, although further research is required to obtain a clearer indication of the global in vivo functioning. In the Gram-negative endophyte,

Gluconacetobacter diazotrophicus, genes involved in fructan metabolism form an operon. The

gene encoding the levansucrase was found to be constitutively expressed while the levanase was induced at low fructose concentrations but repressed in the presence of glucose. The fine regulation of the accumulation of the respective gene products allows the organism to access plant sucrose efficiently (Menéndez et al. 2009). In the Gram-positive B. subtilis, the genes for levan synthesis (sacB, encoding levansucrase) and degradation (levB, encoding endolevanase) are co-transcribed via a sucrose-inducible antitermination mechanism (Crutz et al. 1990; Pereira et al. 2001). Fructose induces exolevanase (sacC) transcription which is controlled by catabolite control protein A (CcpA) mediated carbon catabolite repression (CCR) (Martin-

Verstraete et al. 1999; Martin-Verstraete et al. 1995). Although much is known about the mechanisms involved in the regulation of the levanase operon it is still not clear how this relates to B. subtilis survival in the soil environment. The high level expression of levansucrase in

B. subtilis is thought to be related to its extracellular location as the activity may be rapidly

diluted by diffusion away from the cells and thus higher amounts of the enzyme are required (Pereira et al. 2001). The high molecular weight of levan limits its diffusion away from cells and access to the storage polymer is retained. Levanases are usually cell-associated and hence are required in lower concentrations. The resulting products from their activity are in close proximity to the cells are thus available for assimilation (Martin-Verstraete et al. 1995).

Streptococcus mutans and Actinomyces naeslundii, which inhabit oral environments and

produce dental caries, produce inulin or levan via constitutively expressed fructosyltransferases (Bergeron et al. 2000; Kiska and Macrina 1994b). This may contribute to biofilm formation, persistence in the oral cavity and aid attachment to teeth. Similarly to G. diazatrophicus, fructose activates S. mutans exofructanase transcription but it is suppressed by glucose via a CcpA-independent CCR mechanism (Wen and Burne 2002; Zeng et al. 2006). A. naeslundii secretes a sucrose-inducible exolevanase and a fructose-inducible fructanase (Bergeron and Burne 2001).