Physiological function of exopolysaccharides produced by Lactococcus lactis

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Abstract

The physiological function of EPS produced by Lactococcus lactis was studied by comparing the tolerance of the non-EPS producing strain L. lactis ssp. cremoris MG1614 and an EPS producing isogenic variant of this strain to several anti-microbial factors. There was no difference in the sensitivity of the strains to increased temperatures, freezing or freeze-drying and the antibiotics, penicillin and vancomycin. A model system showed that EPS production did not affect the survival of L. lactis during passage through the gastrointestinal tract although the EPS itself was not degraded during this passage. The presence of cell associated EPS and EPS in suspension resulted in an increased tolerance to copper and nisin. Furthermore, cell associated EPS also protected the bacteria against bacteriophages and the cell wall degrading enzyme lysozyme. However, it has not been possible, so far, to increase EPS production using the presence of copper, nisin, lysozyme or bacteriophages as inducing factors.

Introduction

Several lactic acid bacteria as well as numerous other micro-organisms produce extracellular polysaccharides (EPSs). EPSs either surround the bacterial cells as a capsule or are excreted into the extracellular environment as slime, although the distinction between these two types of EPSs is not always very clear (Whitfield, 1988).

In the last decade, most studies on EPS produced by lactic acid bacteria have focussed on the influence of physiological growth conditions on the EPS biosynthesis, genetics of EPS biosynthesis, and elucidation of the composition and primary structures of these EPSs. Information about the physiological role of EPSs for the producing lactic acid bacteria themselves is almost completely lacking. Apparently, EPSs have some kind of biological function, because it is very unlikely that bacteria use both substrate and energy for the production of useless metabolites (Dudman, 1977). Some bacteria invest more than 70% of their energy in EPS production, presumably to obtain a selective advantage in the environment (Weiner et al., 1995). In addition EPS synthesis is a relatively stable property and EPS producing organisms have a stable presence in various environments. EPSs are obviously not essential for bacteria because enzymatic or physical removal of EPS does not negatively affect cell growth in vitro and mutants unable to produce EPS appear spontaneously (Schellhaass, 1983). Most proposed functions of EPSs in general are of a protective nature such as protection against dehydration, macrophages, bacteriophages, protozoa, antibiotics and toxic compounds (Whitfield, 1988; Weiner et al., 1995; Roberts, 1996). Other possible functions of EPS include sequestering of essential cations (Weiner et al., 1995) and involvement in adhesion and biofilm formation (Roberts, 1996).

The aim of this study is to obtain more insight into the physiological function of EPS produced by L. lactis ssp. cremoris by comparing the sensitivity of the EPS producing strain NZ4010 and the non-producing isogenic parent strain MG1614 to various anti-microbial factors. The genes necessary for EPS production by strain NZ4010 are encoded on a plasmid and the EPS produced by this strain is composed of glucose, rhamnose, galactose and phosphate in a ratio of 2:2:1:1 (Fig. 1A; Van Kranenburg et al., 1997). Most of the EPS that is produced by L. lactis NZ4010 is excreted into the environment but some of the EPS is attached to the bacteria. The thickness of the layer of this cell-associated EPS depends on the culture conditions that are applied (Looijesteijn and Hugenholtz, 1999). Fig. 1B shows cells of strain NZ4010 with a thick layer of cell-associated EPS. Furthermore, to make a distinction between the protection by cell-associated EPS and by the presence of EPS in the medium, we used cultures of MG1614 supplemented with EPS, as a control. We will refer to these cultures as cultures with EPS in suspension. Knowledge of the biological role of EPS might lead to improvement of the relatively low production yields or to insight into the regulation mechanisms that are involved in EPS biosynthesis by L. lactis.

Section snippets

Bacterial strains, bacteriophages and EPS

Stock cultures of Lactococcus lactis ssp. cremoris were prepared by growing MG1614 (Gasson, 1983) and NZ4010 (Van Kranenburg et al., 1997) in litmus milk with 1% (w/v) glucose and MG1614 harbouring pNZ4030 (Van Kranenburg et al., 1997; Looijesteijn et al., 1999) in GM17 (Terzaghi and Sandine, 1975) containing 5 mg l−1 erythromycin (Duchefa, Haarlem, The Netherlands). Overnight cultures at 20°C were diluted 10 times with the culture medium and stored at −40°C. Before freezing, 10% (v/v) glycerol

Protection against bacteriophages

The EPS producing strain L. lactis NZ4010 was less sensitive to bacteriophages than the non-EPS producing strain MG1614 (Table 1). Apparently the EPS layer around the EPS producing bacteria is responsible for the reduction of the phage sensitivity because addition of EPS did not reduce the sensitivity of strain MG1614 for bacteriophages. To make sure that EPS layer was responsible for the reduction in phage sensitivity and not other phage protection mechanisms that could possibly be encoded on

Discussion

EPS produced by Lactococcus lactis ssp. cremoris NZ4010 cannot be used as an energy source by the producing organism itself (not shown) but it protects the bacteria against several anti-microbial factors such as bacteriophages, metal ions, nisin and lysozyme. For some of these factors the presence of a layer of cell-associated EPS is essential for the protection whereas for other anti-bacterial agents EPS in suspension also protects them.

Lactococcus lactis NZ4010 produces an EPS that is

Conclusion

The presence of EPS produced by L. lactis ssp. cremoris NZ4010 resulted in an increased tolerance to bacteriophages, lysozyme, copper ions and nisin. A layer of cell-associated EPS was essential for the protection against bacteriophages and lysozyme, whereas EPS in suspension also protected the bacteria against copper and nisin. EPS did not have any positive influence on the survival of the bacteria when exposed to increased temperatures, freezing, freeze-drying, penicillin and vancomycin.

Acknowledgements

This work was supported by the Ministry of Economic Affairs, the Ministry of Education, Culture and Science, The Ministry of Agriculture, Nature Management and Fishery in the framework of an industrial relevant research program of The Netherlands Association of Biotechnology Centers in The Netherlands (ABON). We thank Fedde Kingma, Renske van Beresteijn and Ron Meijer for performing the animal trials.

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