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1 Vaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N 5E3
2 Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Road, Ottawa, Ontario, Canada K1A 0R6
3 Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C&E Trail, Lacombe, Alberta, Canada T4L 1W1
Correspondence
Brenda J. Allan
brenda.allan{at}usask.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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These authors contributed equally to this work.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), with contaminated poultry products representing the leading cause of human infection (Berndtson et al., 1996). Acute C. jejuni infection results in watery to bloody diarrhoea, fever, nausea and vomiting. Although the majority of infections are self-limiting, more serious medical conditions such as reactive arthritis, inflammatory bowel syndrome, Miller–Fisher syndrome (MFS) and Guillain–Barré syndrome (GBS) (Nachamkin et al., 1998) can result.
Unlike many bacterial pathogens, C. jejuni does not grow effectively outside the host. The ability of this bacterium to remain as the most frequent cause of food-borne illness, despite these fastidious growth requirements, is referred to as the Campylobacter conundrum (Jones, 2001). Of particular relevance to food-borne infections is that the growth requirements of C. jejuni do not permit proliferation on contaminated foods under normal storage conditions (Park, 2002). While limited in its ability to proliferate, Campylobacter can survive for extended periods in the environment (Park, 2002) and has been isolated from a number of ecosystems (Altekruse et al., 1999; Bolton et al., 1987; Jones & Bradshaw, 1996; Pearson et al., 1993; Stanley et al., 1998).
For Campylobacter, and other bacteria, the vast majority of growth within natural ecosystems occurs in association with a surface (Kirchman & Mitchell, 1982; Kuhn et al., 1987). In the ecosystem, surface-associated growth can lead to the formation of highly structured, sessile microbial communities known as biofilms. Biofilm development is believed to occur through a series of coordinated stages including (i) transport of micro-organisms to a surface, (ii) initial attachment, (iii) formation of microcolonies, and (iv) formation of mature biofilms (van Loosdrecht et al., 1990). Growth on an agar surface, with increased population densities and the associated challenges, bears functional resemblance to the second and third stages of biofilm formation, prior to the encapsulation that defines the biofilm phenotype.
There have been numerous genomic and proteomic investigations of biofilms involving various species of bacteria (Lazazzera, 2005). While these investigations do not identify a consensus pattern of gene expression, they do present a relatively cohesive picture of the adaptations underlying the phenotype. As considerable variation exists in the specifics of each biofilm, inconsistencies in expressional response are anticipated. Investigations of phenotypic features that are conserved throughout all biofilms, such as immobilization, are more likely to identify conserved cellular response elements.
Establishment of infection is also dependent upon immobilization during adhesion to intestinal cells. As the ability to grow on surfaces and adhere to intestinal epithelial cells is necessary for production of disease (de Melo & Pechere, 1990; Russell & Blake, 1994), growth on the surface of agar has been applied as a model for gene expression during initial stages of host infection (Wang et al., 2004). In these investigations Salmonella typhimurium grown on conventional agar showed surface-specific upregulation of genes associated with virulence, including those for lipopolysaccharide synthesis, iron metabolism and type III secretion (Wang et al., 2004).
Complex phenotypes, including virulence and biofilm formation, are often the cumulative result of parallel responses to different facets of a particular challenge. These phenotypes can be dissected, and associated responses partitioned, through models that incorporate a limited number of the associated stimuli. For example, through examination of responses to surface-associated growth, adaptations associated with immobilization can be distinguished from responses unique to specialized phenotypes such as biofilms.
In this investigation genomic and proteomic approaches were utilized to characterize differential patterns of expression of C. jejuni in response to immobilized growth conditions. Growth on agar resulted in the induction of a discrete number of functional systems directed towards the biological objectives of (i) iron acquisition, (ii) management of oxidative stress, (iii) membrane modifications and (iv) primary active-transport systems. Surface-associated growth also demonstrated a corresponding repression of systems involved in (i) energy production, (ii) motility, (iii) protein synthesis and (iv) non-essential iron-requiring proteins.
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METHODS |
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Genomic analysis.
C. jejuni cells were harvested after 16 h growth on agar or in broth and homogenized in TRIzol (Invitrogen) by passing the mixture through a syringe. Nucleic acids (RNA and DNA) were isolated as recommended by the manufacturer. RNA was quantified spectrophotometrically at 260 nm using an ND-1000 spectrophotometer (Nanodrop). Preparation of complementary DNA (cDNA) and indirect labelling with Cy3 and Cy5 were performed as previously described (Carrillo et al., 2004). Samples were probed with the whole-genome C. jejuni NCTC 11168 microarray (version 3.0). Details of the construction and content of the microarray are available at (http://ibs-isb.nrc-cnrc.gc.ca/ibs/immunochemistry/campychips_e.html). Slides were scanned using a Chipreader (Bio-Rad), and image analysis, signal normalization and data visualization were performed using Array Pro Analyser 4.5 (Media Cybernetics). Net signal intensities were obtained by performing local-ring background subtraction and spots with a signal less than 3SD of background in both channels were excluded from the analysis. The mean signal intensities for duplicate spots were averaged and data from each channel were adjusted by subarray normalization using cross-channel Lowess regression.
ArrayStat (Imaging Research) was used for statistical analysis of the replicated data. A proportional model with offsets, for dependent data, was selected and statistical significance was determined using the pooled common error method with the false discovery rate multiple test correction (nominal alpha=0·05). Four biological replicates were tested, including one comparison in which the Cy3 and Cy5 dyes were swapped to compensate for biases caused by differing chemical properties of the fluorescent dye molecules. Complete results for these experiments are available at the Gene Expression Omnibus Repository (NCBI, http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE3028.
Proteomic analysis
SDS-PAGE of total cellular and detergent-insoluble outer-membrane proteins.
Total cellular proteins were obtained by sonicating cells in 50 mM HEPES buffer (pH 7·4). Detergent-insoluble outer-membrane proteins from the cell pellets were obtained as described previously (Sampathkumar et al., 2004). The extracted proteins were quantified using the dye-binding assay of Bradford (1976) and 8·0 µg of the protein preparation was separated by SDS-PAGE (4·0 % stacking gel and 12·0 % resolving gel) using a Bio-Rad Protean II electrophoresis system. The gels were silver stained according to the manufacturer's instructions (Amersham Biosciences).
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of total cellular proteins.
Sample preparation. Total cellular protein was extracted by sonicating cell pellets in 1·0 ml lysis buffer [8 M urea, 4·0 % (w/v) CHAPS, 40 mM DTT and 2·0 % (v/v) IPG pH 3–10 buffer] once for 15 s at 40 
amplitude on ice. The extracts were centrifuged at 16 000 g for 10 min to remove cell debris. The resulting supernatant was treated with 0·1 vol. buffer containing 50 mM MgCl2, 1 mg DNase I ml–1 and 0·25 mg RNase A ml–1. The reaction was stopped after 15 min at 4 °C with 3 vols ice-cold acetone. Proteins were then precipitated for 2 h at –20 °C. The precipitate was collected by centrifugation at 3700 g for 15 min and resuspended in IPG gel rehydration solution [8·0 M urea, 2 % (w/v) CHAPS, 40 mM DTT, 0·5 % (v/v) pH 3·0–10·0 IPG buffer and 0·01 % (w/v) bromophenol blue]. The dye-binding assay of Bradford (1976) was then performed to quantify the protein concentration.
Isoelectric focusing. Protein extracts were subjected to high-resolution 2D-PAGE according to the method described by O'Farrell (1975) and modified by Görg et al. (2000). Equal amounts of proteins (450 µg) were loaded onto a first-dimension gel strip. Isoelectric focusing was performed using an IPGphor electrophoresis unit with the Immobiline DryStrip Kit (Amersham Biosciences). Thirteen-centimetre Immobiline DryStrip gels (pH 4·0–7·0) were rehydrated in 250 µl IPG gel rehydration solution (composition as above except that IPG buffer pH 4·0–7·0 was used) containing 450 µg protein for 16 h at room temperature. Isoelectric focusing was achieved when the total running time yielded 60 kVh at 20 °C. Following isoelectric focusing, the gels were equilibrated twice for 15 min each in 10·0 ml isoelectric focusing gel equilibration buffer [50 mM Tris/HCl (pH 8·8), 6 M urea, 30 % (v/v) glycerol, 2 % (w/v) SDS and 0·002 % (w/v) bromophenol blue] containing 100 mg DTT for the first equilibration and 250 mg iodoacetamide for the second equilibration.
SDS-PAGE. Equilibrated isoelectric-focused strips were placed on top of a uniform 14 % SDS-polyacrylamide gel for second-dimension electrophoresis with the Bio-Rad Protean II xi electrophoresis system. Second-dimension separation was carried out at 25 mA per gel constant current at 4 °C. The gels were stained with Bio-Safe colloidal Coomassie blue G-250 stain according to the manufacturer's (Bio-Rad) instructions.
Analysis of protein spots on 2D-PAGE gels. Individual gels were scanned using an Epson Expression 1680 scanner with a transparency adapter as 8-bit greyscale 300 d.p.i. images. Differentially expressed proteins were then detected and quantified from the images with PDQuest 2-D Analysis Software (Bio-Rad). A twofold difference in expression was set as a threshold during image analysis to detect proteins that were differentially expressed.
Mass spectrometry of proteins.
Protein spots of interest were excised from the gel, destained and subjected to in-gel trypsin digestion according to the established protocols for the MassPrep robotic workstation (Water/Micromass). The samples were then dried in a speed-vac and reconstituted in 75 % acetonitrile containing 5 mg 
-cyano-4-hydroxycinnamic acid ml–1 and eluted directly onto the MALDI plate.
The MALDI-TOF-MS (Voyager DE-STR; Applied Biosystems) was operated in the positive-ion reflectron mode. Four hundred laser shots were averaged and processed with Data Explorer software (Applied Biosystems). The samples were internally calibrated using trypsin autolytic fragments, and database searches carried out with Protein Prospector (UCSF Mass Spectrometry Facility, San Francisco, CA, USA). Sample preparation and MS analysis were performed by the Mass Spectrometry facility at the National Research Council, Plant Biotechnology Institute (Saskatoon, Canada).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), many of the seemingly contradictory results that have emerged from biofilm expression investigations are due to differences in the growth phase. Careful consideration was therefore given to ensuring comparison of bacteria that were at a common stage of growth stage progression.
The growth curve of C. jejuni grown on agar or in broth was determined by plating aliquots from predetermined time points on MH agar and expressed as c.f.u. per 25 ml medium and c.f.u. ml–1 respectively. As shown in Fig. 1, the growth profiles were very similar for both growth conditions. Cell densities in both growth conditions increased steadily up to 12 h and maximum cell densities were achieved at 18 h, following which cell death started to occur (Fig. 1). Thus C. jejuni cells at 16 h of incubation were used in all studies.
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). The absence of a more definitive correlation between the genes and proteins identified is likely to be a consequence of post-transcriptional regulatory events.
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). It is of significance that the proteins identified as being differentially expressed as a consequence of growth on agar through one-dimensional SDS-PAGE are in agreement with the results obtained from the two-dimensional proteomic analysis (Fig. 3). The nine proteins identified as undergoing differential patterns of expression were also identified within the large-scale proteomic analysis (Tables 2 and 4). An additional detergent-insoluble protein of approximately 27 kDa was uniquely expressed during growth on agar; however this protein could not be positively identified.
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; Holmes et al., 2005; Palyada et al., 2004; Ratledge & Dover, 2000; van Vliet et al., 1998). In many species of bacteria Fur is solely responsible for the regulation of iron homeostasis as well as oxidative-stress defence. That C. jejuni, which contains relatively few regulatory proteins, has evolved two separate systems for iron acquisition and prevention of oxidative damage indicates the central role that iron plays in its gene regulation and virulence.
Both proteomic (Table 2
) and microarray data (Table 3) demonstrate significant upregulation of genes and proteins with involvement in iron acquisition. These include ChuA, a haemin/haemoglobin uptake system (Cj1614), a possible outer membrane siderophore receptor (Cj0178), components of putative iron-uptake ABC transport systems (Cj0175c, Cj0174c and Cj1663), cfrA, a putative iron-uptake protein (Cj0755) and the periplasmic protein p19 (Cj1659). The induction of iron-uptake molecules, at the levels of both transcription and translation, as a consequence of growth on agar represents the most dramatic alteration in expression; at the level of transcription the three most strongly induced genes code for iron-uptake proteins (Table 3) and a parallel pattern is seen at the level of translation with the unique or highly induced expression of several iron-uptake proteins (Figs 3 and 4).
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The increased requirement for iron during immobilized growth, whether within a biofilm or surface-associated growth, is probably a consequence of the increased cell densities depleting locally available iron stores. These iron-uptake systems show a similar pattern of induction for C. jejuni under iron-limiting conditions (Holmes et al., 2005), as well as during growth within a host (Stintzi et al., 2005).
Induction of iron-uptake systems has not emerged as a conserved response within investigations of biofilms, although it has been reported for select biofilms (Pysz et al., 2004). The availability of iron does however represent a central consideration in the decision to commit to biofilm formation; iron chelation through lactoferrin has been shown to discourage Pseudomonas aeruginosa biofilm formation. The repression of biofilm formation is hypothesized to occur due to the insufficient iron responses to match the localized increases in demand for iron by biofilm bacteria. This appears to be a general phenomenon, with the adhesion of various species of bacteria to biomaterials being suppressed by iron chelators at concentrations which are unable to influence growth in the vegetative state (Ardehali et al., 2002).
Oxidative defence.
In addition to representing a limiting nutrient to bacterial survival, iron imposes an additional challenge to living systems through its involvement in the generation of toxic molecules. Iron can react with oxygen to produce reactive oxygen species like superoxide anions (
), peroxides (RO2) and hydroxyl radicals (OH.) through the Haber–Weiss (
) and Fenton (
) reactions. The absolute requirement for iron for electron transport of microaerophilic organisms like C. jejuni therefore requires the bacteria to maintain a proper iron homeostasis and to coordinate expression of iron-uptake and oxidative stress systems. In contrast to other Gram-negative bacteria, oxidative stress and iron acquisition are regulated separately in C. jejuni (van Vliet et al., 1998). This is of particular significance given the limited number of regulatory systems present within the bacteria.
Oxidative stress proteins often show distinct patterns of induction in response to different kinds of oxidative stress: superoxide stress and peroxide stress. The superoxide stress proteins include manganese-containing superoxide dismutase and endonuclease IV; the peroxide regulon contains catalase (KatA) and alkyl hydroperoxide reductase (AhpC). The regulation of AhpC and KatA expression in C. jejuni has a Fur-independent component, as the expression of these proteins is only partially affected by the mutation of Fur. A second Fur homologue, the peroxide stress regulator PerR, functions to co-regulate ahpC and katA expression (van Vliet et al., 1998).
Parallel to the observation that iron functions as both a signal and an ongoing challenge for formation and survival within a biofilm, oxidative stress also appears to serve as both a trigger and a required adaptive response for biofilm formation and survival. Biofilm formation has been observed in response to oxidative stress in the hyperthermophilic archaeon Archaeoglobus fulgidus (LaPaglia & Hartzell, 1997) and certain aerobic mesophilic biofilms showed increased expression of oxidative stress genes (Golovlev, 2002; Schembri et al., 2003).
As a consequence of growth on agar there was increased expression of the primary oxidative stress defence components, alkyl hydroperoxide reductase (ahpC) and catalase (katA). Increased expression of additional proteins with known involvement in the minimization of oxidative damage, including oxidoreductase (Cj0559), a putative oxidoreductase (Cj0393c) and malate oxidoreductase (Cj1287c), was also observed. The ribosomally associated stress response chaperone, trigger factor (Cj0193c), which has also been implicated in oxidative stress response, exhibited increased expression (Tables 2 and 3).
Membrane modification.
Our characterization of sessile C. jejuni indicates the upregulation of enzymes involved in modifications of the lipid, carbohydrate and protein components of the membrane. These include the upregulation of ADP-L-glycero-D-manno-heptose-6-epimerase (waaD), as well as two putative membrane-modification enzymes, tetraacyldisaccharide 4'-kinase (lpxK) and lipopolysaccharide heptosyltransferase (waaC) (Table 3). Notably, our analysis does not reveal the induction of a large number of the enzymes with established roles in the formation of the biofilm-associated extracellular matrix, supportive of growth on agar as a model of mid-to-late-phase biofilm formation.
ABC transport systems.
Many of the membrane proteins that undergo differential expression as a consequence of growth on agar are associated with the primary active transport of amino acids. To our knowledge there have been no previous connections proposed between either low-iron adaptation or biofilm formation and amino acid transport and metabolism, although amino acid starvation was identified as a trigger for biofilm formation for Candida albicans (Garcia-Sanchez et al., 2004). The induction of ABC transport systems which are not specific to amino acid transport has been reported as a consequence of biofilm formation (Pysz et al., 2004) although the functional relevance remains unclear.
As many of the induced transporters identified in our investigation are ABC-type primary active-transport systems one possibility is that they may have roles in mediating the efflux of antibiotics and antimicrobial agents such that the induction of these systems may be related to the increased resistance to antibiotics which is a hallmark of biofilm bacteria. While the sheltering of bacteria within the biofilm extracellular matrix may provide the initial line of defence against antibiotics this may be supplemented through the ability to actively transport antibiotics out of the bacteria.
Other transport systems.
In addition to the ABC transport systems, growth on agar also resulted in the induction of both phosphate and molybdate-transport systems (Table 3).
The induction of molydbate uptake systems, and corresponding repression of molydbate-requiring enzymes such as periplasmic nitrate reductase, has been reported from investigations of C. jejuni in iron-limiting conditions (Holmes et al., 2005; Palyada et al., 2004). This parallels our observation of induction of a putative molybdenum-transport ATP-binding protein, a molybdate-binding lipoprotein and a periplasmic phosphate-binding protein, as well as the repression of periplasmic nitrate reductase and molybdopterin oxidoreductase under sessile growth conditions. Molybdate metabolism appears to be closely coordinated with that of iron, so the patterns of expression we observe may be a secondary consequence of iron limitation during immobilized growth.
Systems repressed during growth on agar
Growth on agar results in the repression of a variety of cellular systems as determined by their induction during growth in broth. The overall objectives represented by these systems indicate a trend towards a reduction in metabolism, motility and protein synthesis. There is also a general repression of non-essential, iron-containing enzymes, presumably as a measure to conserve iron for essential processes.
Non-essential iron-containing proteins.
Levels of several non-essential iron-containing proteins were decreased in iron-limiting conditions at the levels of transcription (Table 5) and translation (Table 4). For example, non-haem protein (Cj0012c), Ni/Fe hydrogenase (Cj1267c), ferredoxin, cytochrome c peroxidase and a member of the succinate dehydrogenase complex (Cj0437) all show decreased levels of expression. The alterations in levels of expression of these proteins were some of the most dramatically observed at the level of translation (Fig. 5). The pattern of genes induced in broth bears close resemblance to those reported to be induced upon the addition of iron to C. jejuni which have been cultured in iron-limited media (Holmes et al., 2005; Palyada et al., 2004).
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). This probably serves a dual function in conservation of bioavailable iron, as well as in the minimization of reactive by-products of oxidative metabolism. This is consistent with patterns of expression reported by others under low-iron conditions.
Motility.
The repression of genes involved in motility is a logical consequence of an immobilized growth state and has been observed during biofilm formation for both P. aeruginosa (Whitely et al., 2001) and Bacillus subtilis (Stanley et al., 2003). In our investigation we report the repression of flagellin (FlaB), the flagellar biosynthesis protein (FlhA) and a putative RNA polymerase sigma factor for the flagellar operon (FliA) in the sessile state (Tables 4 and 5). The repression of biomolecules involved in motility would function to conserve the significant energy expenditure associated with flagella production, consistent with the general downregulation of energy-production systems observed during immobilized growth.
While the repression of motility genes during immobilized growth is consistent with the majority of biofilm expression studies, this has not been a unanimous conclusion. Indeed some groups have reported the induction of motility biomolecules during biofilm formation. There are suggestions however that the lack of repression of motility systems in these investigations reflects differences in experimental design rather than discrepancies in biological response. For studies of B. subtilis biofilms, in which one of the two investigations observed repression of flagellar genes, the biofilms were grown in similar conditions; however, the planktonic cells were not (Ren et al., 2004a; Stanley et al., 2003). Similarly, investigations of Escherichia coli biofilms that did not observe repression of motility genes through microarray analysis (Beloin & Ghigo, 2005; Ren et al., 2004b; Schembri et al., 2003) did show repression when assayed with a lacZ transcriptional fusion (Prigent-Combaret et al., 1999). Thus, repression of genes involved in motility probably represents a conserved theme amongst biofilms.
Ribosomal synthesis.
Repression of ribosomal proteins during immobilized growth emerges as one of the most definitive conclusions from our investigations, with 30 of the 53 ribosomal proteins identified as being repressed (Table 6). This is consistent with the overall trend of adaptation in the sessile state towards a dormant lifestyle with reduced energy production and expenditure.
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). The suggestion that the expression of ribosomal proteins is activated in biofilms is surprising as this presumably reflects increased growth rate.
Conclusions
While growth within the permissive conditions of liquid media is often taken as the reference point of bacterial behaviour these conditions do not reflect the challenges encountered by bacteria in the natural environment. There is considerable evidence that bacteria spend a significant portion of their natural existence in an immobilized state through the formation of microcolonies and biofilms. The cellular adaptations that accompany survival in a sedentary state are therefore of significant biological interest. For pathogenic bacteria, such as C. jejuni, immobilization takes on further significance as it represents a prerequisite step to the formation of biofilms, as well as in the establishment of the early stages of virulence. Growth on agar is proposed to function as a limited model of a developmental stage towards each of these phenotypes.
Through genomic and proteomic analysis we have established expression profiles for C. jejuni during immobilized and planktonic growth. Relative to growth in broth, growth on agar results in the induction of systems directed towards the biological objectives of iron acquisition, management of oxidative stress and modifications to both the lipid and protein composition of the membranes. During growth on agar there is a corresponding repression of systems involved in energy production, motility and protein synthesis, in particular of non-essential iron-requiring proteins.
The ability of a single growth condition to function as a model for two distinct phenotypes is a consequence of the common denominator within each of these processes in the requirement for an immobilized growth state. This is consistent with the emerging hypothesis that specific bacterial phenotypes do not result from the expression of phenotype-specific genes but rather from the expression of particular subsets of genes that can be induced under a variety of conditions (Lazazzera, 2005).
Food-borne pathogenic bacteria such as C. jejuni can experience rapid transitions from a relatively dormant phase of environmental survival to life within a host. This transition is associated with dramatic shifts in nutrient availability as well as host immune defences. The systems induced during surface-associated growth bestow a phenotype that facilitates survival both within the environment, as well as upon encountering the host, through the prioritizing of systems to withstand two of the most significant challenges associated with the host: iron limitation and oxidative stress.
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ACKNOWLEDGEMENTS |
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Published with permission of the Director of VIDO as manuscript no. 411.
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Received 3 August 2005;
revised 21 October 2005;
accepted 10 November 2005.
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) or on agar (
). At the indicated times, samples were removed to determine the viable cell counts present by plating serial dilutions on MH agar.
