Quorum sensing and butanediol fermentation affect colonization and spoilage of carrot slices by Serratia plymuthica
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).
References (27)
- et al.
Food spoilage—interactions between food spoilage bacteria
International Journal of Food Microbiology
(2002) - et al.
Occurrence of proteolytic activity and N-acyl-homoserine lactone signals in the spoilage of aerobically chill-stored proteinaceous raw foods
Journal of Food Protection
(2006) - et al.
Methods for detecting acylated homoserine lactones produced by Gram-negative bacteria and their application in studies of AHL-production kinetics
Journal of Microbiological Methods
(2001) - et al.
Genotypic and phenotypic characterization of a biofilm-forming Serratia plymuthica isolate from a raw vegetable processing line
FEMS Microbiology Letters
(2005) - et al.
Characterization of a luxI/luxR-type quorum sensing system and N-acyl-homoserine lactone-dependent regulation of exo-enzyme and antibacterial component production in Serratia plymuthica RVH1
Research in Microbiology
(2007) - et al.
Presence of acylated homoserine lactones (AHLs) and AHL-producing bacteria in meat and potential role of AHL in spoilage of meat
Applied and Environmental Microbiology
(2004) - et al.
Quorum-sensing-directed protein expression in Serratia proteamaculans B5a
Microbiology
(2003) - et al.
Quorum sensing and production of autoinducer-2 in Campylobacter spp., Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium in foods
Applied and Environmental Microbiology
(2002) - et al.
Influence of food preservation parameters and associated microbiota on production rate, profile and stability of acylated homoserine lactones from food-derived Enterobacteriaceae
International Journal of Food Microbiology
(2003) - et al.
Production of acylated homoserine lactones by psychrotrophic members of the Enterobacteriaceae isolated from foods
Applied and Environmental Microbiology
(1999)
Environmental conditions affect transcription of the pectinase genes of Erwinia chrysanthemi 3937
Journal of Bacteriology
The lux autoinducer regulates the production of exoenzyme virulance determinants in Erwinia carotovora and Pseudomonas aeruginosa
EMBO Journal
Molecular cloning of pectate lyase genes from Erwinia chrysanthemi and their expression in Escherichia coli
Journal of Bacteriology
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