Quorum sensing and butanediol fermentation affect colonization and spoilage of carrot slices by Serratia plymuthica

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Abstract

In this work we investigated the role of quorum sensing and specific quorum-sensing dependent properties in the colonization and spoilage of carrot slices by Serratia plymuthica RVH1, a strain isolated previously from a vegetable washing and cutting machine in an industrial kitchen. Disinfected carrot slices were inoculated by immersion in a bacterial suspension and then placed in a Petri dish with a shallow layer of the same bacterial suspension. Subsequently, visible spoilage of the air-exposed upper side of the slices and the evolution of bacterial numbers and pH of the surrounding suspension were recorded during 19 days. A knockout mutant in the N-acyl-homoserine lactone (AHL) synthase splI was clearly compromised in its ability to colonize the surface of the carrot and cause browning, and the addition of synthetic AHL could restore this phenotype. To examine in more detail which properties contribute to this phenomenon, we isolated mutants deficient in the production of extracellular proteases and in butanediol fermentation, both of which are regulated by quorum sensing in S. plymuthica RVH1. The protease-deficient mutant (lipB) was not affected in the carrot slice spoilage assay. Since RVH1 does not produce pectinolytic enzymes, this suggests that hydrolytic enzymes do not play a major role in produce spoilage by this organism. On the other hand, a budB mutant with inactive butanediol fermentation pathway showed strongly enhanced growth on the carrot slices, in spite of a reduced survival in the surrounding medium. To explain these results, we hypothesize that a response is induced in the carrot slices that suppresses bacterial colonization and outgrowth, similar to the defense response induced by volatile butanediol pathway products in intact plants.

Introduction

Our understanding of the virulence and pathogenesis of foodborne pathogens, as well as their transmission, survival, stress response and interactions with other microbiota has made significant progress. Comparatively little is known about the ecology of food spoilage bacteria and the biochemical mechanisms behind spoilage at the molecular level. However, there is growing evidence that bacterial spoilage is a cell density dependent phenomenon regulated at the genetic level by quorum sensing. In this form of bacterial cell-to-cell communication, bacteria sense their population density by monitoring self-made signal molecules, and respond to it by activating or repressing certain target genes. Typically, quorum sensing affects phenotypes that rely on the coordinated behaviour of a relatively large number of cells, such as biofilm formation and virulence (Miller and Bassler, 2001). Interestingly, several bacteria which are typically associated with food spoilage produce N-acyl-homoserine lactones (AHLs), the major type of quorum sensing signal molecules (Bruhn et al., 2004, Cloak et al., 2002, Flodgaard et al., 2003, Gram et al., 1999, Gram et al., 2002, Liu et al., 2006, Ravn et al., 2001), and AHLs can be detected in a number of food products which carry large bacterial populations such as meat and bean sprouts (Bruhn et al., 2004, Gram et al., 2002, Rasch et al., 2005). Furthermore, several enzymes believed to play a role in spoilage such as protease, lipase, cellulase and pectinase, are produced under control of quorum sensing systems in a variety of bacteria (Christensen et al., 2003, Jones et al., 1993, Liu et al., 2007, Thomson et al., 2000). More direct evidence came from the demonstration that spoilage of bean sprouts by a Pectobacterium strain or milk by Serratia proteamaculans was delayed or reduced when bacterial AHL production was knocked out by mutation (Christensen et al., 2003, Rasch et al., 2005). However, not all studies support a role for quorum sensing in spoilage. For example, vacuum-packed meat inoculated with Hafnia alvei wild type spoiled at the same rate as with its AHL-deficient mutant (Bruhn et al., 2004). Also, the use of quorum sensing inhibitors which interfere with the quorum signalling cascade, did not convincingly reduce spoilage of bean sprouts by Pectobacterium (Rasch et al., 2007). More work is clearly needed to determine the role of quorum sensing in various food spoilage systems. In addition, the precise role of quorum sensing, i.e. which quorum sensing regulated functions or pathways actually contribute to spoilage, remains largely unknown.

Serratia plymuthica RVH1 was isolated from a raw vegetable processing line in an industrial kitchen by our research group (Van Houdt et al., 2004). It produces several AHLs and its quorum sensing system has recently been characterized. SplI, a Vibrio fisheri LuxI homolog, is responsible for the production of AHLs, while SplR, a LuxR homolog, senses the AHL concentration and acts as a repressor of several genes when cell density increases and signal molecules accumulate (Van Houdt et al., 2005, Van Houdt et al., 2007). The quorum sensing dependent phenotypes that have been identified in S. plymuthica RVH1 include the production of an extracellular chitinase, protease and nuclease, the production of an antibacterial compound and, most recently, also butanediol fermentation (Van Houdt et al., 2006, Van Houdt et al., 2007).

In this study we demonstrate that the deterioration of carrot slices by S. plymuthica RVH1 is dependent on quorum sensing, and we subsequently analyse the involvement of some known quorum sensing regulated properties by making use of specific mutants. We report on an unexpected and previously undocumented role of butanediol fermentation in this process.

Section snippets

Bacterial strains and growth conditions

All bacterial strains used in this work are listed in Table 1 and were grown for 21 h in LB medium (Luria Bertani, 10 g/l trypton, 5 g/l yeast extract, 5 g/l NaCl) at 30 °C to obtain stationary phase cultures. All S. plymuthica strains were derived from the RVH1 strain and were kanamycin (Km) resistant due to a resistance marker. The previously constructed strain RVH1-Gfp was chosen as ‘wild type’ strain in this work because it is also Km resistant due to a transposon with a gfp (green

Quorum sensing influences spoilage of carrot slices by S. plymuthica RVH1

In our experimental setup, S. plymuthica RVH1-Gfp wild type readily colonized the carrot slices, as a mucoid patch became visible on the air exposed surface after 5 days (Fig. 1A). When streaked out, this mucoid material yielded kanamycin resistant bacterial colonies, indicating that it was formed by the inoculated S. plymuthica strain. The formation of the mucoid patches was accompanied by a browning reaction on the carrot surface, and will be designated as carrot spoilage when it affects 1/4

Discussion

In this study it was demonstrated that quorum sensing affects spoilage of carrot slices by S. plymuthica RVH1-Gfp by promoting colonization and mucoid bacterial outgrowth accompanied with tissue browning. An AHL-negative mutant was severely attenuated in its ability to cause spoilage, while complementation with synthetic signal molecules restored the wild type phenotype. These results are in accordance with previous studies that also demonstrated a correlation between quorum sensing and

Acknowledgments

Author P.M. was supported by a doctoral fellowship from the Flemish Institute for the Promotion of Scientific Technological Research (IWT), and author A.A. by a postdoctoral fellowship from the Research Foundation Flanders (FWO-Vlaanderen).

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