Stress Response and Adaptation of Listeria monocytogenes 08-5923 Exposed to a Sublethal Dose of Carnocyclin A

Abstract

Carnocyclin A (CCLA) is an antimicrobial peptide produced by Carnobacterium maltaromaticum ATCC PTA-5313, which can be used to control the growth of Listeria monocytogenes in ready-to-eat meat products. The aim of this research was to elucidate the cellular responses of L. monocytogenes 08-5923 exposed to a sublethal dose of CCLA. Microarray, quantitative reverse transcription-PCR, tandem mass spectrometry, and electron microscopy were used to investigate the alteration in gene expression, protein production, and morphological changes in cells of Listeria following treatment with CCLA. The genes involved in metabolism (baiE, trn, and pykA), cell wall synthesis (murZ and dacB2), and cell division (clpE and divIVA) were upregulated following a 15-min exposure to CCLA as a result of stress responses. Genes involved in cell division, cell wall synthesis, flagellar synthesis, and metabolism were downregulated after 4 h as a result of adaptation. Analysis of total soluble proteins confirmed the downregulation of pykA and gnd after 4 h of exposure to CCLA. The absence of flagella was observed in L. monocytogenes following 30 h of exposure to CCLA. A sublethal dose of CCLA induced adaptation in L. monocytogenes 08-5923 by inhibition of expression of genes and proteins critical for synthesis of cell wall structures and maintaining metabolic functions. Both the mannose- and cellobiose-specific phosphotransferase systems could be targets for CCLA.

INTRODUCTION

Listeria monocytogenes has been responsible for numerous food-borne illness outbreaks as a result of consumption of contaminated ready-to-eat (RTE) meat products (1). A listeriosis outbreak from a single manufacturer in Canada in 2008 resulted in 22 deaths and 57 confirmed positive cases (http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/listeria-monocytogenes-eng.php).

To control the growth of L. monocytogenes, bacteriocin-containing antimicrobials such as Micocin (Griffith Laboratories, Canada) can be used in RTE meat products. One active compound in Micocin, carnocyclin A (CCLA), is a 5.9-kDa cyclic, class IIc bacteriocin (2) that has strong antilisterial activity. CCLA forms ion channels in the membrane and results in dissipation of membrane potential, which leads to cell death (3). Neither the stress response of Listeria to CCLA nor the development of resistance in Listeria have been elucidated. Previous research focused on class IIa bacteriocins, which cause cell surface alterations in Listeria (4) and, in particular, result in cell lysis by targeting the mannose-specific phosphotransferase (PTS) system (5, 6). The disruption of the mannose-specific PTS system plays a role in bacteriocin resistance (7, 8). Alternative sigma factors, such as σ^B (sigB) (9–11) and σ⁵⁴ (rpoN) (12), are also involved in resistance to class IIa bacteriocins. These alternative sigma factors are also involved in other environmental stresses, such as cold shock, acid, and osmotolerance in Listeria (13, 14).

It has been postulated that CCLA can trigger alteration in the expression levels of genes that are also regulated by σ^B/σ⁵⁴. The differential expression of genes and proteins and the morphology of L. monocytogenes 08-5923, one of the two strains that were involved in the 2008 listeriosis outbreak in Canada (GenBank accession number NC_013768) (15), in the presence and absence of CCLA was analyzed.

MATERIALS AND METHODS

Bacterial strains and cultures.

L. monocytogenes 08-5923 was grown in tryptic soy broth (TSB; Becton, Dickinson, Ontario, Canada) at 21°C for 24 h prior to use. Carnobacterium maltaromaticum ATCC PTA-5313 (formerly Carnobacterium maltaromaticum UAL307) was grown in all-purpose Tween (APT; BD-Canada) broth at 21°C.

CCLA isolation and purification.

CCLA was isolated from an overnight culture of C. maltaromaticum ATCC PTA-5313 grown at 21°C and purified according to previous methods (2).

RNA isolation.

Twenty-five ml of log-phase L. monocytogenes 08-5923 (optical density at 600 nm [OD₆₀₀] of 0.2) was grown in TSB or in TSB with 7 μg/ml CCLA (1/10 the MIC on L. monocytogenes 08-5923) for 4 h at 21°C (final OD₆₀₀, 0.5) to obtain cells that were at the mid-log phase of growth. RNAprotect bacterial reagent (Qiagen Inc., Ontario, Canada) was added to the cell culture according to the manufacturer's instructions. Total RNA was isolated using the RNeasy minikit (Qiagen Inc.) and treated with DNase I (New England BioLabs Ltd., Ontario, Canada) on column according to the manufacturers' protocol. RNA quantity was measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Ontario, Canada), and the samples were stored at −80°C. RNA quality and quantity were assessed on an Agilent 2100 bioanalyzer (Agilent Technologies Inc., Ontario, Canada) using an Agilent Nano 6000 assay, and an RNA with an integrity number (RIN) of >7.0 was used for subsequent microarray experiments. For quantitative reverse transcriptase real-time PCR (qRT-PCR), total RNA was isolated as described above from cells in lag phase (15 min), mid-log phase (4 h), and early stationary phase (30 h).

Gene expression microarrays and data analysis.

The L. monocytogenes EGD-e 385 K gene expression microarray from Roche NimbleGen, including all of the 2,857 annotated open reading frames (ORFs) of the genome, was used. The cDNA was synthesized from RNA extracted from three independent biological repetitions (SOP#M007 and SOP#M008; J. Craig Venter Institute [http://pfgrc.jcvi.org/index.php/microarray/protocols]) and labeled with Cy3 monoreactive dye (GE Healthcare Life Sciences, Ontario, Canada). Hybridization of the labeled cDNA probes using the NimbleGen hybridization station and washing were carried out according to the NimbleGen protocol. Microarray raw data were preprocessed and normalized by NimbleScan (version 2.6; Roche NimbleGen).

GeneSifter software (trial version; Geospiza, Inc.) (16) was used for the analysis of normalized results. The statistical analyses were the averages from three independent samples (CCLA-treated L. monocytogenes 08-5923 versus untreated) with the cutoff being a 2-fold change in expression level and P values of <0.05 to determine differentially expressed (DE) genes. Genes that showed 2-fold or greater changes in expression levels were selected for subsequent qRT-PCR.

Total cytosoluble protein identification.

L. monocytogenes 08-5923 was grown in TSB or in TSB with CCLA at 21°C for 4 h. Cells were pelleted by centrifugation at 10,600 × g for 1 min and washed once with 1× SigmaFAST protease inhibitor cocktail tablet (Sigma-Aldrich Ltd., Ontario, Canada) reconstituted in double-distilled water (ddH₂O). Cell pellets were resuspended in the fresh cocktail, mixed 1:1 (vol/vol) with 0.1-mm Zirconia-silica beads (BioSpec Inc., OK), and lysed by bead beating for 45 s a total of 3 times. The cell envelope was removed by centrifugation at 10,600 × g for 2 min. The concentration of the total soluble protein was determined by Bio-Rad protein assay by following the manufacturer's instructions (Bio-Rad Laboratories Ltd., Ontario, Canada).

The total soluble proteins were separated by SDS-PAGE with 16% polyacrylamide (17). Five μg of proteins from the untreated and treated Listeria samples were loaded onto each lane and subjected to electrophoresis at 130 V for 80 min. The gel was fixed (45 min in a solution containing 50% methanol and 10% acetic acid), and protein bands were visualized by EZBlue gel staining (Sigma-Aldrich Ltd.). The density of protein bands was measured by AlphaEase FluorChem densitometry (Alpha Innotech; Fisher Scientific) to identify differentially expressed proteins.

The differentially expressed proteins in treated and untreated L. monocytogenes 08-5923 samples were analyzed by the Mass Spectrometry Facility, Department of Chemistry, University of Alberta. Protein bands that were differentially expressed were excised from the SDS-PAGE gel, digested in gel by trypsin (Sigma-Aldrich Ltd.), and analyzed by ultraperformance liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer (UPLC-ESI-Q-TOF-MS) (Q-TOF Premier; Waters, MA). Proteins were identified from mass spectrometry data using the Mascot search engine (Matrix Science).

qRT-PCR and data analysis.

qRT-PCR experiments were performed to validate the differential expression of genes at different times of exposure to CCLA. The experimental design included three biological replicates and two technical replicates within each biological replicate. Genes that were ≥2-fold up- or downregulated (P < 0.05) from the microarray experiments, as well as genes encoding the differentially expressed proteins, were quantified using SYBR green (Invitrogen, Ontario, Canada). Primers (Tables 1 and 2) were designed (using Primer3 [http://frodo.wi.mit.edu/primer3/]) and tested for specificity with genomic DNA prior to the analysis. Total RNA was isolated and reverse transcribed into cDNA using SuperScript III reverse transcriptase (Promega, WI) according to the manufacturer's protocol. Quantifications of the transcripts were carried out (7500 Fast real-time PCR system; Applied Biosystems, CA) using a QuantiFast SYBR green PCR kit (Qiagen Inc.). Relative quantification values were obtained using a comparative threshold cycle method (ΔΔC_T) (18). The housekeeping gene rpoB was selected as the reference. The C_T slope method was used to validate the internal control (rpoB). rpoB was amplified from 10-fold serial dilutions of genomic DNA prepared from L. monocytogenes. The C_T slope was linear, and the qRT-PCR efficiency of rpoB was 100.9%. A one-tailed unpaired t test was performed for determination of the significance (P < 0.05) in differential expression of genes.

TABLE 1.

Primers used in qRT-PCR for confirmation of the genes found differentially expressed in microarray experiments

Gene	Primer sequence (5′–3′)
	Forward	Reverse
ftsE	TATGCGATGGAAGTGGTTGA	CTCACCACCGGAAAGTTCAT
ftsX	AATGGTTGGATGACCTTTGC	CGTTGCAAGCTTGTTCATGT
rpoB	GAAATCTGGGTTCGTCGTGT	GCTACGTTTGGACGTTGGTT
fliF	TCTACATGAACACGCCCAAA	AAAATGTTGCCGCTTTTGTC
clpE	AGCAAACTTTGGGTCGAATG	GTTCACGGTTTGCTTGGTTT
baiE	ACAAGCAAAGGCAGACATCC	CAATAAAGCCATGCCCAGTT
deoR	CAGGCCTTGAGCAAAATGAT	ATAATACGCGCGATTGGAAG
hisC	TGCGAAACGTTTTGAACAAG	CAGCACCAGAACGCGTAATA
flgD	AAATGGCGCAACTTTCCTTA	CGCCGTTTAGTGAAACACCT
yitT	TGGATTTTCCAGCGAATACC	ATCACTACCGCCAGTTGTCC
tgl	GACAAGCCGAGTTTCAAAGC	CGGCATCTGTGACAGTTTGT
lmo0867	CAATCGGCTATCTCGCTTTT	TGCGATAATTGCCATCGTTA
ywzB	ACTTTTTGGGCACTTCAAGC	CCTAACACAATCGAAATAATCACAA
lmo1796	CACCCGGAAGACGTTGTTAT	ACGAATCGCTTTCGCAATAC
ftsW	GGGATCGCTAGTCTGATTGC	TACCAAGCATCATCGACAGC
lmo2311	GCTCAATGCGTTTATGACGA	CGCTGGCAAATAATCTCGTT
trn	CCGAAAAGAGTCGCGATAAT	TGCTGCATAGGTGGATGCTA
hrcA	GGGATATTCCCGATGGTCTT	TCTTGTGGGCCCTAGGAGTA
abc	AATGAGAGCGCCAATTATGG	TGCGCGATACCTTCTACAAA
fmt	AGAAGCCGACCTGCTTGTAA	GTCAAGCAGCGCGTAGTGTA
lmo2317	CCCTTTTCAACCGTGATGTT	GCTCACGAACCCTTTCCATA
mfs	GGGGAAAAAGCTGGAATTGT	TTGCAATCGTTGGCATGTAT
malP	CAATTGGATGGAGCGAAAAT	CGCCATTACGGTGAATTTCT
mptC	ATTCCAGCTGCAGCACTTTT	CAACGGCAACTACCATTCCT
mbl	GCAATTCGTGAAGTTGCTGA	ACCGGAAGGCTCAAAAATCT
gidA	GGCGTGTGTAAAGGCGTTAT	GGTTATTTGGACCGCTTGAA
rodA	CTTCCGCTTGGTCTTGTAGC	CAATGAGCGCAATCGAACTA
divIVA	TCCGTGAGCGTTTACGTATG	CGCAAGTTCTGTCGCATCTA
murZ	GCGATTTATTTACGGGCAGA	TTGCACGAACAGCTGCTAAC
fliM	GTGTGACATCACCGAACGAC	TCTTCCACCGAGAAAAATGG
motB	TTCATGGAAGCGATCATTCA	CGCACCATGATTTCTACACG
mptAB	ACGTTTCGGCAATACCAAAG	ACAGAGTGAGCCATGGAACC
murE	TTATGGATCTCCGACCCAAG	ACGATACATCGTGCCAATCA
pdp	AGTGAAAATCGCACCGAAAG	GCAATAATTCCGAGGCATGT
agrB	CTCCGGCAGACACAGAAAGT	TGCGAATGGTATTAGCAACG
bglK	CCGAAAAATGGCTTGGTAAA	CCGCGAACTAATTCACCATT
cheA	CGACGTTCCGAATTGAGATT	CGTGAACGTGTTGAATGTCC
celC	TTGCAGCTATTCGTGACACC	CATACCAGATCCGCCGTAGT
dacB2	ACCAAAAAGCGAGAGAAGCA	GGCATTTTCTTGCAACCATT
celA	ATTTCGAGGAAGCAGAAGCA	TTCCACTTTTTCACCGGAAG
celB	TGGATATCGTGATGCTTGGA	CGGCTTTCCCGTTTAACATA

TABLE 2.

Primers used in qRT-PCR for confirmation of the downregulation of proteins

Gene name	Primer sequence (5′–3′)
	Forward	Reverse
alaS	GGTAGCTGCTGGAAACGAAG	TTTCGCTCTCGATTGTTCCT
adhE	CAAGCTGGCTTCAAAGTTCC	ACGATCGAATGCTTCTGCTT
pflA	CCCACTTAACCCAGAAGCAG	TACCACCGTTGATAGCACCA
pnpA	TATTCACTCGTGGCCAAACA	GCCAGTTTCCCCAACACTAA
pykA	AAGTGCTGCAGTTGTTGTGG	TTGCATCTTTAGCACCAACG
gnd	GGAAGAGAATGCGGACAAAA	ATCTGTAGCATCGCCAGCTT
fri	GGCGAACAAATGGATGAAGT	GCTTCTTCTACGCTCGCATT
rpsK	TACTCGTAAACGCCGTGTGA	TAGGGAACCTGCACTTGACC
rpsH	AGTAAGCCAGGTTTGCGTGT	TTTAGCACGGGCTTCTTTGT
rplU	TGAAACAGGTGGGAAACAAA	TAGCGGAATCTCCACCTACG
rplL	AATTTGGCGTAACTGCTGCT	ACCAGTGATTTCACGAACCA
hup	GCAGCGAAAGCAGTAGAAGC	GTACGAGGGTTACGGCCTTT
rpsT	TTGATGAAGCAGCTGCAAAC	CGAGCAGCATTGTTTTTGTG
csp	GAAAAAGGCTTCGGTTTCATC	ATTCAACGCTTTGACCTTCG

Cell morphology examination.

Culture of L. monocytogenes 08-5923 was fixed with 10% (vol/vol) formaldehyde and pelleted by centrifugation at 11,000 × g for 15 s. The cell pellet was resuspended in 0.1 M phosphate buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde, stained with 2% sodium phosphotungstate (PTA), and examined using a transmission electron microscope (TEM; Philips Morgagni 268; FEI, OR) operating at 80 kV.

Microarray data accession number.

The complete microarray data set generated in this study is deposited for public access in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE56558.

RESULTS

Functional classification of DE genes.

cDNA microarray experiments were performed to compare the expression levels of genes in untreated and treated (CCLA for 4 h) L. monocytogenes. Global differential gene expression changes in response to CCLA were determined using a high-density NimbleGen microarray for L. monocytogenes of 2,857 genes. Forty genes were found to be differentially expressed with a minimum threshold of a 2-fold difference (P < 0.05) (Table 3). These genes were grouped into several functional categories, including cell division, cell wall synthesis, motility, transport, and translation (Table 3).

TABLE 3.

Identification of differentially expressed genes in CCLA-treated L. monocytogenes 08-5923^a

Gene name and function	Gene annotation	Product	Fold change
Cell division
clpE	lmo0997	ATP-dependent protease	+3.69
ftsW	lmo1071	Cell division protein FtsW	−2.14
divIVA	lmo2020	Cell division initiation protein	−2.01
rodA	lmo2428	Similar to cell division protein RodA	−2.64
ftsX	lmo2506	Cell division transport system permease protein	−2.08
ftsE	lmo2507	Cell division transport system ATP-binding protein	−2.32
mbl	lmo2525	Rod shape-determining protein MreB	−2.03
Cell wall synthesis
pdp	lmo0835	Peptidoglycan binding protein	−2.21
murE	lmo2038	UDP-N-acetylmuramoylalanyl-d-glutamate-2,6-diaminopimelate ligase	−2.02
murZ	lmo2552	UDP-N-acetylglucosamine 1-carboxyvinyltransferase	−2.41
dacB2	lmo1855	d-Alanyl-d-alanine carboxypeptidase	−2.41
Membrane functions
abc	lmo0767	ABC transporter, permease protein	+2.13
mfs	lmo1250	Major facilitator superfamily	+2.05
ywzB	lmo2527	Similar to B. subtilis YwzB protein	+2.37
mptAB	lmo0096	PTS system, mannose-specific IIA and IIB component	+2.14
mptC	lmo0097	PTS system, mannose-specific IIC component	−2.29
celA	lmo2765	PTS system, cellobiose-specific IIA component	+2.35
celB	lmo2762	PTS system, cellobiose-specific IIB component	+2.10
celC	lmo2763	PTS system, cellobiose-specific IIC component	+2.81
Gene regulation
deoR	lmo2107	Similar to deoR family transcriptional regulator	+2.28
hrcA	lmo1475	Heat-inducible transcription repressor	+2.43
agrB	lmo0048	Similar to Staphylococcus two-component sensor histidine kinase AgrB	+3.18
Nucleotide and protein synthesis
fmt	lmo1823	Methionyl-tRNA formyltransferase	−2.07
hisC	lmo1925	Histidinol-phosphate aminotransferase	−2.26
gidA	lmo2810	tRNA uridine 5-carboxymethylaminomethyl modification enzyme GidA	−2.61
Metabolism
tgl	lmo0717	Transglycosylase	−2.01
baiE	lmo0754	Bile acid 7-alpha dehydratase	−2.74
trn	lmo1903	Thioredoxin	−2.21
yitT	lmo1909	YitT family protein	−2.05
malP	lmo2121	Maltose phosphorylase	+2.01
bglK	lmo2764	Beta-glucoside kinase	+2.85
Motility
flgD	lmo0696	Flagellar basal body rod modification protein	−2.70
fliF	lmo0713	Flagellar MS-ring protein	−2.20
motB	lmo0686	Chemotaxis protein	−2.23
fliM	lmo0699	Flagellar motor switch protein	−2.26
cheA	lmo0692	Two-component sensor histidine kinase	−2.82
Unknown
lmo0867	lmo0867	Hypothetical protein	−2.39
lmo1796	lmo1796	Hypothetical protein	−2.33
lmo2311	lmo2311	Hypothetical protein	+2.25
lmo2317	lmo2317	Hypothetical protein	+2.07

^aIdentification of differentially expressed genes in CCLA-treated (4 h) L. monocytogenes 08-5923 is represented as fold change (n = 3; P < 0.05). +, upregulation; −, downregulation.

Differential expression of total soluble proteins.

To gain further insight, the protein expression patterns of whole-cell lysate from L. monocytogenes with and without exposure to CCLA (4 h) were compared using SDS-PAGE. Out of 11 protein bands that were visibly different in density (Fig. 1) between the lysate from the CCLA-treated and untreated cells, there were 14 proteins identified by UPLC-ESI-Q-TOF-MS. The proteins were involved in glycolysis (PykA), pyruvate metabolism (AdhE and PflA), stress response (Csp protein), DNA replication (Hup), translation (Rps and Rpl proteins), and purine/pyrimidine biosynthesis (PnpA) (Table 4). The downregulation of PykA in treated L. monocytogenes confirmed the observed downregulation of the gene encoding this protein as shown in Fig. 2G.

FIG 1.

Differential production of proteins in L. monocytogenes 08-5923; treated (CCLA for 4 h) and control samples are shown on an SDS-PAGE gel. Proteins expressed differentially are indicated by arrows. CoA, coenzyme A.

TABLE 4.

Identification of differentially expressed proteins by MS

Protein name	Product	Accession no.	Calculated pI	Nominal molecular mass (Da)	Differential expression
AlaS	Alanyl-tRNA synthetase	YP_003416804	5.13	92,938	Down
AdhE	Bifunctional acetaldehyde-coenzyme A/alcohol dehydrogenase	YP_003416936	6.48	95,135	Down
PflA	Formate C-acetyltransferase	CAK20840	5.41	83,950	Down
PnpA	Polyadenylase	YP_003416626	5.23	79,780	Down
PykA	Pyruvate kinase	YP_003416871	5.39	62,673	Down
Gnd	6-Phosphogluconate dehydrogenase	YP_003416672	5.11	52,497	Down
Fri	Nonheme iron-binding ferritin	YP_003416184	4.86	18,036	Down
RpsK	30S ribosomal protein S11	YP_003415461	11.40	13,834	Down
RpsH	30S ribosomal protein S8	YP_003415450	9.48	14,635	Down
RplU	50S ribosomal protein L21	YP_003416842	9.57	11,207	Down
RplL	50S ribosomal protein L7/L12	YP_003415499	4.54	12,462	Down
Hup	DNA-binding protein HU-beta	YP_003417289	9.65	9,876	Down
RpsT	30S ribosomal protein S20	YP_003416777	10.70	9,163	Down
Csp	Cold shock protein, CspA family	ADB71297	4.45	7,261	Down

FIG 2.

qRT-PCR measurement of DE genes in L. monocytogenes 08-5923 exposed to CCLA for 15 min (□), 4 h (■), and 30 h (▨). Untreated samples were used as a reference. Genes were grouped according to their biological function. (A) Nucleotide/protein synthesis; (B) cell wall synthesis; (C) membrane functions; (D) motility; (E) transcription regulation; (F) cell division; (G) metabolism; (H) unknown functions. Note the different y axes for panels C and D. Data are expressed as log values (fold change). Means ± standard deviations (n = 3) are given. An asterisk indicates genes significantly up- or downregulated at P < 0.05.

Confirmation of DE genes in L. monocytogenes.

To validate results from microarray experiments and to investigate the gene expression changes over time, a total of 54 genes (40 DE genes from microarray experiments and 14 DE genes from SDS-PAGE) (Fig. 2) were selected for qRT-PCR, and gene expression levels were quantified following treatment with CCLA after 15 min (lag phase), 4 h (log phase), and 30 h (early stationary phase). For a number of the genes, expression was significantly (P < 0.05) upregulated after 15 min of exposure to CCLA and downregulated after 4 h and 30 h.

The upregulation of genes encoding proteins involved in metabolism and membrane functions was observed upon 15 min of exposure to a sublethal dose of CCLA. Genes encoding proteins involved in metabolism, including bile acid 7-alpha dehydratase (BaiE), thioredoxin (Trn), and pyruvate kinase (PykA), were significantly upregulated (P < 0.05). The genes involved in cell wall synthesis (murZ and dacB2) were upregulated (P < 0.05). Genes involved in cell division, including clpE and divIVA, were upregulated, while malP was downregulated in cells exposed to CCLA for 15 min. After 4 h of exposure to CCLA, genes involved in cell division (clpE, ftsW, divIVA, rodA, ftsX, and ftsE), cell wall synthesis (mbl, murE, and murZ), flagellar synthesis (flgD, motB, and cheA), and metabolism (pykA and gnd) were significantly downregulated. Upregulation of lmo2311, which is regulated by σ⁵⁴ (19), was observed. After the cells were exposed to CCLA for 30 h, more genes were downregulated compared to those after 4 h of exposure. These genes were involved in cell division, cell wall synthesis, membrane function, transcription regulation, metabolism, and motility (Fig. 2). Both the mannose-specific PTS genes (mpt genes) and the cellobiose-specific PTS genes, such as celA, celB, and celC, were downregulated (P < 0.05).

Cell morphology.

The morphologies of untreated and treated (CCLA for 4 h or 30 h) L. monocytogenes 08-5923 samples were compared using TEM to examine whether exposure to CCLA could result in visible damage to the cell structure. No morphological changes were observed after 4 h of treatment of L. monocytogenes 08-5923 with CCLA (Fig. 3). However, after 30 h the cells had no flagella attached to the cell surface (Fig. 3). This corresponds to the downregulation of motility genes observed previously.

FIG 3.

Transmission electron micrographs of cells of L. monocytogenes 08-5923 in the presence (treated, 4 h and 30 h) and absence (untreated) of CCLA (28,000× magnification).

DISCUSSION

Commercial preservatives that are used to control the growth of L. monocytogenes in RTE meats include Micocin, which contains carnocyclin A along with two class IIa bacteriocins, carnocyclin BM1 and piscicolin 126. Although the stress responses of L. monocytogenes to class IIa bacteriocins have been characterized, the response to class IIc cyclic bacteriocins has not been studied to date. Our study described the cellular alteration and adaptation of L. monocytogenes when exposed to a sublethal dose of a class IIc bacteriocin, which provides insight into the general stress response and resistance mechanisms to bacteriocins.

Treatment of L. monocytogenes 08-5923 with CCLA resulted in differential expression of genes that are known to be involved in cell division, cell wall synthesis, membrane function, transcriptional regulation, nucleotide and protein synthesis, metabolism, and cell motility. The PTS function, cell wall synthesis, motility, and cell division genes were affected the most. The downregulation of these genes was not because of inhibition of cell growth, as at each time point the OD₆₀₀ values of treated and untreated cells were comparable. No filamentation of cells was observed. This indicates that the downregulation of these genes was due to the adaptation to CCLA.

The upregulation of σ⁵⁴-regulated mptAB and cel detected by cDNA microarray after 4 h of exposure to CCLA indicates an immediate adaptive response of Listeria cells, as the mannose-specific (7) and cellobiose-specific PTS systems are receptors for bacteriocins (20), and the upregulation of mannose-specific PTS systems has been observed in bacteriocin-sensitive cells (21, 22). Gong et al. (3) demonstrated that CCLA forms ion-selective channels in lipid bilayers, which indicates that CCLA interaction with membranes is not receptor mediated. However, our microarray results suggested that both the mannose-specific and cellobiose-specific PTS systems are receptors for CCLA. The downregulation of the mannose-specific and cellobiose-specific PTS systems observed by qRT-PCR analysis after 30 h of exposure to CCLA demonstrates that Listeria limits the expression of genes to minimize the production of the PTS targets to CCLA. This downregulation of the PTS targets could play a role in adaptation to CCLA, as these targets have been suggested to play a role in resistance to class II bacteriocins (6, 23, 24).

The expression of genes involved in cell wall synthesis (mur) followed the same pattern as that of the PTS systems after 30 h of exposure to CCLA. Although the function of Mur proteins in the antimicrobial stress response of Listeria has not been described in the literature, Mur is the target of antibiotics in Staphylococcus aureus (25).

Absence of flagella in Listeria cells exposed to CCLA for 30 h could be a result of inhibition of flagellar synthesis, because the basal body of the flagella may not develop properly as genes encoding various components of flagella, including the MS ring (fliF), motor switch (fliM), motor rotation (motB), and rod synthesis (flgD), were downregulated. In addition, cheA, a chemotaxis gene responsible for virulence (26), was downregulated. Flagellar motility is also important for biofilm formation of L. monocytogenes (27). Others have found that stress from antimicrobials can inhibit the synthesis of flagella in E. coli (28), but the impact of bacteriocins on flagellar synthesis in L. monocytogenes has not been described in the literature. Lack of flagella can inhibit adhesion of Listeria (29) and may affect its ability to form biofilms.

The cell division gene ftsE was upregulated when L. monocytogenes was exposed to CCLA for 15 min. High pressure for 15 min also upregulates ftsE in Listeria (30). After 4 h of exposure to CCLA, ftsE was downregulated, along with ftsX and ftsW. Gene ftsE in L. monocytogenes is regulated by σ^B (31), which is a general stress response regulator. Various stresses, such as liquid smoke (32), cold (33), antibiotic (31, 34), and heat (35), also downregulate fts genes in Listeria. The up- or downregulation of fts genes may depend on the type of stress and time of exposure.

In summary, a sublethal dose of carnocyclin A targeted the PTS systems of Listeria and cells began to repair injury by upregulation of genes involved in cell wall synthesis. After 4 h, cells gradually became desensitized to CCLA and started to decrease activities such as cell division, cell wall synthesis, and motility. After 30 h, cells adapted to CCLA through a reduction of several metabolic processes and motility functions. Adaptation to bacteriocins could affect the sensitivity to antimicrobials that are used to control the growth of L. monocytogenes.