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HrcA and DnaK are important for static and continuous-flow biofilm formation and disinfectant resistance in Listeria monocytogenes

Stijn van der Veen, Tjakko Abee

  • 1Top Institute Food and Nutrition (TIFN), Nieuwe Kanaal 9A, 6709 PA Wageningen, The Netherlands
  • 2Laboratory of Food Microbiology, Wageningen University and Research Centre, Bomenweg 2, 6703 HD Wageningen, The Netherlands
  • Correspondence
    Stijn van der Veen
    Stijn.vanderVeen{at}wur.nl

    • Microbiology 2010; 156(12):3782–3790 · https://doi.org/10.1099/mic.0.043000-0

    View at publisher PubMed

    Abstract

    The food-borne pathogen Listeria monocytogenes is able to form biofilms in food processing environments. Since biofilms are generally difficult to eradicate during clean-up procedures, they pose a major risk for the food industry. Stress resistance mechanisms involved in L. monocytogenes biofilm formation and disinfectant resistance have, to our knowledge, not been identified thus far. In this study, we investigated the role of hrcA, which encodes the transcriptional regulator of the class I heat-shock response, and dnaK, which encodes a class I heat-shock response chaperone protein, in static and continuous-flow biofilm formation and resistance against benzalkonium chloride and peracetic acid. Induction of both hrcA and dnaK during continuous-flow biofilm formation was observed using quantitative real-time PCR and promoter reporters. Furthermore, in-frame deletion and complementation mutants of hrcA and dnaK revealed that HrcA and DnaK are required to reach wild-type levels of both static and continuous-flow biofilms. Finally, disinfection treatments of planktonic-grown cells and suspended static and continuous-flow biofilm cells of wild-type and mutants showed that HrcA and DnaK are important for resistance against benzalkonium chloride and peracetic acid. In conclusion, our study revealed that HrcA and DnaK are important for L. monocytogenes biofilm formation and disinfectant resistance.

    • Abbreviation: EGFP, enhanced GFP.

    Edited by: D. Demuth

    INTRODUCTION

    The food-borne pathogen Listeria monocytogenes is frequently encountered in food processing facilities (Chasseignaux et al., 2002; Pritchard et al., 1995; Tompkin, 2002), where it most likely survives in the form of biofilms. Biofilms are generally considered to be more resistant to antimicrobial agents and disinfectants than planktonic cells (Lewis, 2001; Mah & O'Toole, 2001; Robbins et al., 2005), which poses a major threat for the food industry. Some studies have shown that L. monocytogenes biofilms are more resistant to disinfectants than are planktonic-grown cells (Berrang et al., 2008; Folsom & Frank, 2006; Pan et al., 2006). Several mechanisms have been proposed to explain the increased resistance of biofilms against disinfectants, such as the restricted penetration of the biofilm, the slow growth rate of biofilm cells and the induction of resistance mechanisms in the biofilm (Lewis, 2001). It has been suggested that biofilms contain many microniches in which bacteria are stressed and consequently induce stress resistance mechanisms (Costerton et al., 1995). However, it has also been shown previously that detached biofilms are not more resistant to disinfectants than planktonic cells (Kastbjerg & Gram, 2009; Stopforth et al., 2002), which might indicate that resistance is dependent on the restricted penetration of the biofilm.

    For L. monocytogenes biofilms, two specific morphotypes have been described thus far. Static biofilms generally consist of small rod-shaped cells, which form microcolonies or homogeneous layers (Kalmokoff et al., 2001; Rodriguez et al., 2008). In contrast, continuous-flow biofilms consist of a network of elongated cells that form knitted chains and surround ball-shaped microcolonies (Rieu et al., 2008). An important role for motility was shown in these types of biofilms. Although loss of motility initially decreased surface attachment, in later stages the formation of hyperbiofilms was observed (Todhanakasem & Young, 2008). Furthermore, continuous-flow biofilm formation is dependent on the activation of the SOS response (van der Veen & Abee, 2010). So far, little is known about the function and expression of other stress response mechanisms during static and continuous-flow biofilm formation and their impact on the resistance of biofilms to disinfectants.

    In this study, we investigated the role of the class I heat-shock response in L. monocytogenes biofilm formation and disinfectant resistance. The class I heat-shock response contains molecular chaperones that are activated under conditions that result in the accumulation of damaged, denatured or aggregated proteins and provides protection or reactivates these proteins (Narberhaus, 1999). We selected to study hrcA, which encodes the regulator of the class I heat-shock response, and dnaK, which encodes a molecular chaperone that is important for protein protection and disaggregation of denatured proteins (Diamant et al., 2000). Maintenance of protein quality is essential during exposure to stress conditions (Liberek et al., 2008) and dnaK may therefore be a very important factor during biofilm formation and disinfection resistance. The class I heat-shock response is controlled by the autoregulatory HrcA transcriptional repressor, which was first shown in Bacillus subtilis to recognize the CIRCE operator sequence (TTAGCACTC-N9-GAGTGCTAA) located in front of these genes (Schulz & Schumann, 1996). In L. monocytogenes, CIRCE operator sequences were later also identified in front of the class I heat-shock genes (Gahan et al., 2001; Hanawa et al., 2000), and HrcA-dependent regulation of these genes has been confirmed with transcription and mutant analyses (Hu et al., 2007). hrcA and dnaK are located within the hrcA–grpE–dnaK operon, which contains both a SigB and a SigA promoter (Hu et al., 2007). A second SigB promoter is located upstream of the dnaK coding region. Comparison of the transcription profiles of a wild-type strain and its isogenic ΔhrcA mutant showed that HrcA is also indirectly involved in the induction and repression of 56 other genes encoding proteins with functions in resistance to heat, cold, acid and oxidative stress (Hu et al., 2007). In the current study, we investigated the role of HrcA and its regulon member DnaK in L. monocytogenes biofilm formation and disinfection resistance.

    METHODS

    Strains, media and plasmids.

    L. monocytogenes strains EGD-e and mutants thereof (Table 1⇓) were grown in brain heart infusion (BHI) broth (Becton Dickinson). The temperature-sensitive suicide plasmid pSvS26 was constructed by cloning the XhoI–ScaI-digested origin of replication fragment from vector pGH9 : ISS1 in the NheI–ScaI-digested plasmid pNZ5319. The ΔhrcA and ΔdnaK deletion mutants were constructed with plasmids pSvS38 and pSvS34, which contain the flanking regions of these genes, and the primers hrcA-1 to hrcA-4 for ΔhrcA and dnaK-1 to dnaK-4 for ΔdnaK (Table 2⇓) following the protocol described previously (Lambert et al., 2007). This resulted in a 1014 bp internal in-frame deletion for hrcA and a 1818 bp internal in-frame deletion for dnaK. Both deletions were verified by sequencing. Genomic expressed promoter reporter strains were constructed using the site-specific integration plasmids pIMK-Pr.hrcA-EGFP and pIMK-Pr.dnaK-EGFP. These plasmids are derivatives of plasmid pIMK2-EGFP. The promoter regions of hrcA, containing both SigA and SigB promoters, and of dnaK, including the specific SigB promoter located in front of dnaK, were amplified using primers hrcA-5, hrcA-7, dnaK-5 and dnaK-7 and cloned into pIMK2-EGFP as a SacI–NcoI fragment, which replaced the constitutive active Phelp promoter in front of enhanced GFP (EGFP). The hrcA and dnaK genomic complementation mutants were constructed with the plasmids pIMK-hrcA and pIMK-dnaK. These plasmids were made by replacing the Phelp promoter and EGFP gene of plasmid pIMK2-EGFP with the genes hrcA and dnaK. These genes, including promoter regions and a terminator for dnaK, were amplified using the primers hrcA-5, hrcA-6, dnaK-5 and dnaK-6 and cloned as SacI–SmaI and SacI–SalI fragments, respectively, in pIMK2-EGFP. The plasmid pIMK-hrcA still contained the terminator present behind the EGFP, while for plasmid pIMK-dnaK, this terminator was replaced with the native terminator of dnaK.

    Table 1.

    Bacterial strains and plasmids used in this study

    Table 2.

    PCR primers used in this study

    Biofilm formation

    Static biofilm formation.

    Overnight-grown cultures (18 h, 20 °C, BHI) were used for inoculation (1 %) of 12-well polystyrene microtitre plates (Greiner Bio-One) containing 3 ml BHI broth. After 48 h static incubation at 20 °C, the medium was removed and biofilms were washed three times with PBS (Merck). Biofilms were dispersed in 1 ml PBS by pipetting rigorously. Dispersal of the biofilms into single cells was microscopically verified. The empty wells were stained with 0.1 % crystal violet (Merck) to verify complete removal of the biofilm. Dispersed biofilm cells were serially diluted in PBS and plated on BHI agar. After 2 days incubation at 30 °C, colonies were enumerated. Three independent biological experiments using two replicates each were performed for static biofilm formation experiments.

    Continuous-flow biofilm formation.

    Overnight-grown cultures (18 h, 20 °C, BHI) were diluted in BHI (1 %) and used for inoculation of a flow cell (BST FC 281; Biosurface Technologies). Bacteria were left to adhere for 1 h and BHI broth (20 °C) was pumped through the flow cell with a flow of 10 ml h−1. Biofilms were harvested after 48 h and dispersed in 10 ml PBS. Dispersal of the biofilms into single cells was microscopically verified. Dispersed biofilm cells were serially diluted in PBS and plated on BHI ager. Agar plates were incubated for 2 days at 30 °C and colonies were enumerated. Three independent biological experiments using two replicates each were performed for continuous-flow biofilm formation experiments.

    Quantitative real-time PCR.

    RNAprotect (Qiagen) was used according to the manufacturer's protocol to quench planktonic cells and biofilms. RNA extraction and cDNA synthesis was performed according to the protocols described previously (van der Veen et al., 2010). Quantitative real-time PCR was performed on a ABI Prism 7000 Sequence Detection System (Applied Biosystems) using 2× Sybr Green PCR Master Mix (Applied Biosystems) and 200 nM primers (Table 2⇑) in a total volume of 20 μl. Expression levels were normalized using the average expression of the housekeeping genes tpi, rpoB and 16S rRNA.

    Microscopy.

    A BX41 microscope and MNIBA3 filter (Olympus) were used for phase-contrast and fluorescence microscopy, respectively.

    Disinfection treatments.

    One millilitre of dispersed static and continuous-flow biofilms (48 h, 20 °C, BHI) and 1 ml of planktonic-grown cells (24 h, 20 °C, BHI) were centrifuged for 2 min at 5000 g and resuspended in 1 ml sterile water (20 °C). Benzalkonium chloride (Merck) or peracetic acid (Sigma-Aldrich) was added to these cell suspensions to a final concentration of 20 μg ml−1. Samples were taken in a time series up to 15 min and diluted (1/10) in neutralizing liquid [3 g lecithin l−1 (VWR International), 3 % (v/v) Tween-80 (Merck), 5 g sodium thiosulphate l−1 (VWR International), 1 g l-histidine l−1 (Sigma-Aldrich), 0.34 g potassium dihydrogen phosphate l−1 (Merck)]. After subsequent serial dilution in PBS, samples were plated on BHI agar. After 3–5 days incubation at 30 °C, colonies were enumerated. Three independent biological replicates were performed for all disinfection experiments.

    Data analyses.

    The reparameterized Gompertz model (Zwietering et al., 1990) was used to fit the inactivation curves of the disinfectant treatments with the following equation:

    Figure image not available in archive
    where A is the difference between the surviving population and the initial population (log10 c.f.u. per well), k is the maximum specific inactivation rate (log10 min−1) and ts is the duration of the shoulder (min). The inactivation curves were fitted in Microsoft Excel by minimizing the residual sum of squares using the Excel Solver add-in.

    Student's t-test (P<0.05) was used to identify significant differences in resistance against inactivation treatments, biofilm formation and gene expression.

    RESULTS

    Expression of hrcA and dnaK during biofilm formation

    To investigate whether hrcA and dnaK are activated during L. monocytogenes biofilm formation, quantitative real-time PCR experiments were performed (Fig. 1⇓). Compared with planktonic cells, expression of dnaK in static biofilms was induced (1.5-fold), while no difference in hrcA expression was observed. However, expression of hrcA and dnaK was highly induced in continuous-flow biofilms. The expression of hrcA was 3.3-fold higher in continuous-flow biofilms compared with planktonic-grown cells and the expression of dnaK was 4-fold higher. Promoter activities of hrcA and dnaK in biofilms were further analysed with promoter reporters. Continuous-flow biofilm formation resulted in high expression of EGFP for both promoter reporters (Fig. 2⇓), while no expression for the hrcA promoter or very faint expression for the dnaK promoter was observed during static biofilm formation (data not shown). These results show that hrcA and dnaK are activated during continuous-flow biofilm formation in particular.

    Figure image not available in archive
    Fig. 1.

    Expression of hrcA and dnaK during biofilm formation. Differential expression between 48 h planktonic cultures (black), 48 h static biofilms (light grey) and 48 h continuous-flow biofilms (dark grey) grown at 20 °C in BHI is shown relative to expression in planktonic cultures, which was set at 1. The bars represent the mean±sd of three independent biological experiments. *Significant difference compared with planktonic cultures (P<0.05, t-test).

    Figure image not available in archive
    Fig. 2.

    Activation of the hrcA (a) and dnaK (b) promoter during continuous-flow biofilm formation. Micrographs show fluorescence (1) and phase-contrast (2) pictures of cells expressing EGFP from the hrcA and dnaK promoter after 48 h biofilm formation in BHI at 20 °C.

    The impact of HrcA and DnaK on biofilm formation

    To assess the role of HrcA and DnaK in static and continuous-flow biofilm formation, differences in biofilm formation between the wild-type strain and in-frame deletion and complementation mutants were determined (Fig. 3⇓). The wild-type and mutant strains showed no differences in planktonic growth (data not shown). The ΔhrcA mutant showed increased biofilm formation compared with the wild-type strain under static-biofilm-forming conditions, indicating that constitutive activation of the class I heat-shock response in this mutant is beneficial for static biofilm formation. The importance of the class I heat-shock response in static biofilm formation was furthermore emphasized by the deficiency in static biofilm formation by the ΔdnaK mutant. Static biofilm formation was restored to wild-type levels in the hrcA and dnaK complementation mutants. Compared with the wild-type strain, a deficiency in continuous-flow biofilm formation was also observed for the ΔdnaK mutant. Continuous-flow biofilm formation was restored to wild-type levels in the dnaK complementation mutant, which shows that the presence of DnaK is important for continuous-flow biofilm formation. However, continuous-flow biofilm formation was also reduced with the-ΔhrcA mutant. Furthermore, continuous-flow biofilm formation was not completely restored to wild-type levels using the hrcA complementation mutant, although a significantly higher amount of biofilm was produced with this strain compared with the ΔhrcA mutant (P<0.05, t-test). These results might indicate that a constitutive highly expressed class I heat-shock response is not beneficial for continuous-flow biofilm formation. On the other hand, HrcA was previously shown to activate a range of stress proteins (Hu et al., 2007), and it remains to be elucidated whether these contribute to continuous-flow biofilm formation.

    Figure image not available in archive
    Fig. 3.

    Comparative analysis of static and continuous-flow biofilm formation between wild-type, ΔhrcA, hrcA-c, ΔdnaK and dnaK-c strains. Biofilm formation in BHI at 20 °C after 48 h under static (light grey) and continuous-flow (dark grey) conditions is shown. The bars represent the mean±sd of three independent biological experiments. *Significant difference compared with wild-type (P<0.05, t-test); #significant difference compared with the complementation strain (P<0.05, t-test).

    The role of HrcA and DnaK in resistance against disinfectants

    The disinfectants benzalkonium chloride (Fig. 4⇓) and peracetic acid (Fig. 5⇓) were used to investigate the role of HrcA and DnaK in disinfection resistance of planktonic cells and static and continuous-flow biofilm cells. Biofilms were detached and dispersed to exclude the possible influence of biofilm matrix components on the resistance of biofilm cells, thereby focusing on the intrinsic resistance of cells grown in biofilms. Parameter estimates were determined after modelling the inactivation curves of wild-type and mutant strains (Table 3⇓). Differences in disinfectant resistance are reflected in the surviving population (A), the maximum specific inactivation rate (k) or the duration of the shoulder (ts). Differences in disinfectant resistance between wild-type and mutant strains and between planktonic and biofilm cells are considered significant when any of the three parameters A, k or ts of the inactivation curve is significantly different. Compared with planktonic-grown cells, increased resistance of dispersed static biofilm cells was observed for the wild-type strain after exposure to benzalkonium chloride (Fig. 4⇓ and Table 3⇓) and peracetic acid (Fig. 5⇓ and Table 3⇓). In contrast, resistance of dispersed continuous-flow biofilm cells was only increased after peracetic acid treatments. Furthermore, a role for HrcA and DnaK in the resistance against disinfectants was identified. Planktonic cells and dispersed static and continuous-flow biofilm cells of the ΔhrcA mutant showed lower resistance against benzalkonium chloride compared with the wild-type strain (Fig. 4⇓ and Table 3⇓). Resistance of planktonic cells and dispersed static and continuous-flow biofilm cells of the ΔhrcA mutant was also lower after peracetic acid treatment (Fig. 5⇓ and Table 3⇓). Furthermore, planktonic cells and dispersed static and continuous-flow biofilm cells of the ΔdnaK mutant were less resistant to benzalkonium chloride (Fig. 4⇓ and Table 3⇓) and peracetic acid (Fig. 5⇓ and Table 3⇓) treatment compared with the wild-type strain. Complementation of the ΔhrcA and ΔdnaK mutant completely or partly restored the disinfectant sensitive phenotype of the mutants (Fig. 4⇓, Fig. 5⇓ and Table 3⇓). These results revealed that HrcA and DnaK play an important role in the resistance of L. monocytogenes planktonic and biofilm cells against disinfectants.

    Figure image not available in archive
    Fig. 4.

    Disinfection treatment of planktonic and biofilm cells with benzalkonium chloride. Inactivation of 24 h planktonic cells (a), cells from a 48 h dispersed static biofilm (b) and cells from a 48 h dispersed continuous-flow biofilm (c) treated with 20 μg benzalkonium chloride ml−1 for 15 min at 20 °C of wild-type (⧫), ΔhrcA (▪), hrcA-c (□), ΔdnaK (▴) and dnaK-c (▵) strains is shown. The graphs represent the mean±sd of three independent biological experiments. Data points below the detection limit [log10(Nt/N0)≈−6.5] are not shown.

    Figure image not available in archive
    Fig. 5.

    Disinfection treatment of planktonic and biofilm cells with peracetic acid. Inactivation of 24 h planktonic cells (a), cells from a 48 h dispersed static biofilm (b) and cells from a 48 h dispersed continuous-flow biofilm (c) treated with 20 μg peracetic acid ml−1 for 15 min at 20 °C of wild-type (⧫), ΔhrcA (▪), hrcA-c (□), ΔdnaK (▴) and dnaK-c (▵) strains is shown. The graphs represent the mean±sd of three independent biological experiments. Data points below the detection limit (log10(Nt/N0)≈−6.5) are not shown.

    Table 3.

    Parameter estimates of the inactivation curves after treatment with disinfectants

    Values shown are the mean±sd.

    DISCUSSION

    So far, not much is known about the activation and requirement of stress response genes in L. monocytogenes biofilm formation and subsequently in the resistance of biofilms against disinfectants. Therefore, we chose to investigate the involvement of the class I heat-shock response, which is one of the most important stress resistance mechanisms of L. monocytogenes, in biofilm formation and disinfectant resistance by expression and mutant analyses. We selected HrcA and DnaK, which are the repressor and a chaperone protein, respectively, of the class I heat-shock response. The class I heat-shock response is, together with the class III heat-shock response (Nair et al., 2000), responsible for protein maintenance and turnover in many bacteria. A role for class I heat-shock response genes in biofilm formation has not been established in L. monocytogenes thus far, although a role in resistance to heat, cold, acid and ethanol has been established (Gahan et al., 2001; Hanawa et al., 1999; Liu et al., 2002). Our results show that hrcA and dnaK are specifically induced during continuous-flow biofilm formation. Furthermore, mutant analyses indicate that HrcA and DnaK are involved in both static and continuous-flow biofilm formation. The impact of the class I or class III heat-shock response on the biofilm-forming behaviour of Streptococcus mutans has been shown previously. Forced downregulation of dnaK in S. mutans resulted in a reduced static-biofilm-forming capacity (Lemos et al., 2007). In this organism, the capacity to form biofilms was also reduced in a clpP mutant, ClpP being an ATP-dependent protease of the class III heat-shock response (Lemos & Burne, 2002). A mutant of clpP in Pseudomonas fluorescens also showed a reduced biofilm-forming capacity (O'Toole & Kolter, 1998). Our results show that DnaK is important for both static and continuous-flow biofilm formation in L. monocytogenes, indicating that active protein quality maintenance influences the biofilm-forming capacity. This was furthermore highlighted with the induced static-biofilm-forming capacity of the hrcA mutant, which shows induced dnaK expression. However, the hrcA mutant showed reduced biofilm formation during continuous-flow biofilm formation. For L. monocytogenes, it was shown previously that continuous-flow biofilm formation was dependent on the activation of the SOS response factor YneA, which results in the formation of knitted chains composed of elongated cells (van der Veen & Abee, 2010). It might be possible that the induction of the class I heat-shock response in this mutant, or the differential expression of numerous other indirectly regulated genes (Hu et al., 2007), affects continuous-flow biofilm formation by preventing the activation of the SOS response.

    Biofilms generally show higher resistance against disinfectants than planktonic-grown cells. However, the genetic mechanisms responsible for this increased resistance are not known. Therefore, we investigated the role of HrcA and DnaK in the resistance of static and continuous-flow biofilms against the disinfectants benzalkonium chloride and peracetic acid, which are two of the most commonly used disinfectants in the food industry (McDonnell & Russell, 1999). Previously, it has been shown that L. monocytogenes biofilms are more resistant against benzalkonium chloride and peracetic acid than planktonic-grown cells (Romanova et al., 2007; Stopforth et al., 2002), although detached and suspended biofilm cells appeared to be equally sensitive to peracetic acid. Benzalkonium chloride and peracetic acid use different modes of action to kill bacteria: benzalkonium chloride disrupts the cell membrane, which results in leakage of intracellular material, while peracetic acid is an oxidizing agent that is expected to attack essential cellular components such as DNA and proteins (McDonnell & Russell, 1999). Resistance to benzalkonium chloride has previously been related to the fatty acid composition of the cell membrane and the induction of an efflux pump (Romanova et al., 2006; To et al., 2002), while no peracetic acid resistance mechanism has been reported thus far. The reduced disinfectant resistance of biofilm cells and planktonic cells of the ΔdnaK mutant indicates that protein maintenance is an important factor contributing to resistance. This result is in line with the hypothesis that peracetic acid disrupts cellular proteins.

    Therefore, it somewhat surprising that the ΔhrcA mutant, which shows induced expression of dnaK and other class I heat-shock genes that are involved in protein maintenance, is more sensitive to disinfectants than the wild-type strain. However, it has been shown previously that HrcA is also indirectly involved in the up- and downregulation of 56 other genes, including genes encoding proteins with functions in oxidative, acid, heat and cold stress resistance (Hu et al., 2007), and this may offer an explanation for the observed disinfection sensitivity of the ΔhrcA mutant.

    In conclusion, our study showed that HrcA and DnaK are important for L. monocytogenes static and continuous-flow biofilm formation and disinfection resistance. The specific HrcA-regulated genes that are involved in disinfectant resistance remain to be elucidated in future studies.

    Acknowledgments

    We thank Saskia van Schalkwijk for construction of the ΔhrcA and ΔdnaK mutants.

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