Pathogenomics of Listeria spp

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International Journal of Medical Microbiology 297 (2007) 541–557 www.elsevier.de/ijmm

Pathogenomics of Listeria spp. Torsten Haina, Som S. Chatterjeea, Rohit Ghaia, Carsten Tobias Kuennea, Andre´ Billiona, Christiane Steinwega, Eugen Domanna, Uwe Ka¨rstb, Lothar Ja¨nschb, Ju¨rgen Wehlandb, Wolfgang Eisenreichc, Adelbert Bacherc, Biju Josephd, Jennifer Scha¨ra, Ju¨rgen Kreftd, Jochen Klumppe, Martin J. Loessnere, Julia Dorschtf, Klaus Neuhausf, Thilo M. Fuchsf, Siegfried Schererf, Michel Doumithg, Christine Jacquetg, Paul Marting, Pascale Cossarth, Christophe Rusniocki, Philippe Glaseri, Carmen Buchrieseri, Werner Goebeld, Trinad Chakrabortya, a

Institute for Medical Microbiology, Justus-Liebig-University, Frankfurter Strasse 107, D-35392 Giessen, Germany Helmholtz Centre for Infection Research, Department of Cell Biology and Research Group Cellular Proteomics, Inhoffenstraße 7, D-38124 Braunschweig, Germany c Lehrstuhl fu¨r Organische Chemie und Biochemie, Lichtenbergstrasse 4, D-85747 Garching, Germany d Theodor-Boveri-Institut (Biozentrum), Lehrstuhl fu¨r Mikrobiologie, Universita¨t Wu¨rzburg, D-97074 Wu¨rzburg, Germany e Food Microbiology Laboratory, Institute of Food Science and Nutrition, ETH Zu¨rich, Schmelzbergstrasse 7, CH-8092 Zu¨rich, Switzerland f Abteilung Mikrobiologie, Zentralinstitut fu¨r Erna¨hrung und Lebensmittelforschung, Technische Universita¨t Mu¨nchen, Weihenstephaner Berg 3, D-85354 Freising, Germany g Laboratoire des Listeria, Centre National de Re´fe´rence des Listeria, World Health Organization Collaborating Center for Foodborne Listeriosis, F-75015 Paris, France h Institut Pasteur, Unite´ des Interactions Bacte´ries-cellules, Inserm U604, INRA USC2020, F-75015 Paris, France i Institut Pasteur, Unite´ de Ge´nomique des Microorganismes Pathoge`nes, CNRS URA 2171 F-75015 Paris, France b

Received 8 January 2007; received in revised form 15 March 2007; accepted 16 March 2007

Abstract This review provides an overview of recent progress in the exploration of genomic, transcriptomic, and proteomic data in Listeria spp. to understand genome evolution and diversity, as well as physiological aspects of metabolism utilized by the bacteria when growing in diverse and varied environments. r 2007 Elsevier GmbH. All rights reserved. Keywords: Listeria; Genomics; Transcriptomics; Proteomics; Bacteriophages; Metabolism

Introduction Corresponding author. Tel.: +49 64 1994 1250;

fax: +49 64 1994 1259. E-mail address: [email protected] (T. Chakraborty). 1438-4221/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2007.03.016

The genus Listeria comprises a group of Gram-positive bacteria of the phylum Firmicutes with low G+C content, also including the genera Bacillus, Clostridium, Enterococcus, Streptococcus, and Staphylococcus. Listeria

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spp. are facultative anaerobic, non-sporulating rods which have no capsule and are motile at 10–25 1C (Collins et al., 1991; Rocourt, 1999; Sallen et al., 1996). Listeria have been isolated from a variety of origins including soil, water, plants, feces, decaying vegetables, meat, seafood, dairy products, and asymptomatic human and animal carriers (Seeliger and Jones, 1986; Weis and Seeliger, 1975). The natural habitat of Listeria is decaying plant material, where they live as saprophytes. Listeria are able to multiply at high salt concentrations (10% NaCl) and at a broad range of pH (pH 4.5–9) and temperature (0–45 1C) (Grau and Vanderlinde, 1990). The genus Listeria consists of six species: L. monocytogenes, L. innocua, L. welshimeri, L. ivanovii, L. seeligeri, and L. grayi. Only two of the species, L. monocytogenes and L. ivanovii are pathogenic. L. monocytogenes causes severe illnesses both in humans and in animals whereas L. ivanovii is almost only associated with infections in animals. Human listeriosis is a foodborne disease, and it has been estimated that 99% of all human listeriosis cases are caused by consumption of contaminated food products (Mead et al., 1999). The ubiquitous presence of listeriae in the environment can lead to their occurrence in food-processing environments and the food chain. Moreover, the ability of the bacterium to grow at refrigerator temperatures increases the risk of food contamination. Clinical symptoms often manifest as meningitis, meningoencephalitis, septicemia, abortion, prenatal infection, and also gastroenteritis (Vazquez-Boland et al., 2001). The occurrence of listeriosis is quite low with 2–15 cases per million population per year. However, the high mortality rate of about 20–30% in those developing listeriosis (pregnant women, elderly, and immunocompromised persons) makes L. monocytogenes a serious human pathogen (Farber and Peterkin, 1991; Mead et al., 1999). Listeriosis in animals is predominantly a foodborne disease which is often transmitted by consumption of spoiled silage causing abortions, stillbirths, and neonatal septicemia in sheep and cattle (Alexander et al., 1992; Chand and Sadana, 1999; Dennis, 1975; Gill, 1937; Ramage et al., 1999; Sergeant et al., 1991; Wesley, 1999). Listeriae like many environmental pathogens are capable of growth and survival in disparate environments ranging from the soil to the food chain finally, where they are a deadly cause of disease in susceptible animal and human hosts. Apart from understanding the molecular basis of host–pathogen interactions in disease, the bacterial species within this genus provide us with an excellent opportunity to understand (i) the evolution of virulence within the genus, (ii) mechanisms by which signals are sensed when transiting between different environments, (iii) the genes and their products involved in these transitions, (iv) physiology and metabolic requirements for growth under these conditions, and (v) properties that determine host tropism. A

detailed understanding of the factors that underlie the interactions between listeriae in its different growth environments, such as in the food chain or during infection of vertebrate hosts, are a prerequisite to understanding and predicting the global factors that have contributed to the shaping of the listerial pangenome. Research performed within the Listeria consortium within the PathoGenoMik Network is at the focus of this review. The overall aim of the consortium was the generation and development of tools for genome-based research and their application in understanding many of the questions relating to the evolution of virulence within this genus, as well as adaptive responses to growth in different environments. This review documents how data derived from whole-genome sequencing, transcriptomics, proteomics, and whole-cell metabolic studies obtained from growing the bacterium under a variety of different conditions is providing an unparalleled overview of processes involved in adaptive changes of cellular physiology as these bacteria alternate between saprophytic and parasitic lifestyles.

The listerial pangenome Currently, the complete genome sequences of L. monocytogenes EGD-e (serotype 1/2a) (Glaser et al., 2001) (http://genolist.pasteur.fr/ListiList/), L. monocytogenes F2365 (serotype 4b) (Nelson et al., 2004), and L. innocua CLIP 11262 (serotype 6a) (Glaser et al., 2001) (http:// genolist.pasteur.fr/ListiList/) are published. Additionally, the incomplete genomes of L. monocytogenes F6854 (serotype 1/2a) and L. monocytogenes H7858 (serotype 4b) (Nelson et al., 2004) are available. The genome sequences of L. welshimeri SLCC 5334 (serotype 6b), L. seeligeri SLCC 3954 (serotype 1/2b), and L. ivanovii PAM 55 (serotype 5), and an L. monocytogenes serotype 4a strain have recently been completed, and the genome sequencing of L. grayi is nearing completion (Hain et al., 2006a) (http:// www.genomesonline.org). This study will not only provide genome information of all species comprising this genus, but also information on lineage-specific gene content in the different L. monocytogenes strains including a large number of partially sequenced genomes of different L. monocytogenes serotypes by the BROAD Institute (http:// www.broad.mit.edu/seq/msc/).

General chromosome features of Listeria species All of the listerial genomes sequenced to date are circular chromosomes with sizes that vary between 2.7 and 3.0 Mb. Approximately 89% of the genome sequence of the different Listeria genomes, is coding, and 62.5% of these have an assigned function. Although strain- and serotype-specific genes were identified, all

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Listeria genomes revealed a high synteny in gene organization and content. The lack of inversions or shifting of large genome segments could be due to the low occurrence of transposons and insertion sequence elements present in the sequenced Listeria genomes (Buchrieser et al., 2003). However, localized DNA rearrangements of single gene loci are present even though it is a rather rare event. In comparison with the genome sizes of L. monocytogenes, L. innocua, L. seeligeri, and L. ivanovii (2.7–3.0 Mb), L. welshimeri has one of the smallest genomes of the genus Listeria (Hain et al., 2006b). As in the genomes of the other listerial strains L. welshimeri has a low G+C content of 36.4%, which is slightly lower compared to that of L. monocytogenes EGD-e and L. innocua, L. seeligeri, and L. ivanovii (37.0–38.0%). Interestingly, L. welshimeri harbors a prophage, which is inserted within the region between the tRNAarg and ydeI genes. In L. ivanovii the species-specific Listeria

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pathogenicity island 2 (LIPI-2) is also flanked by the genes for tRNAarg and ydeI suggesting that this region is an evolutionary ‘‘hot spot’’ of genome evolution for Listeria spp. Thus, the LIPI-2 element was probably introduced by transduction into the L. ivanovii genome implying a role for bacteriophages as vehicles for horizontal gene transfer in listeriae (Fig. 1). Comparative genomic analysis of chromosomal regions between L. welshimeri, L. innocua, and L. monocytogenes showed a strong overall conservation of synteny, which is a typical feature among all members of the genus. Additionally, a cluster of paralogous genes encoding partial components of an F0F1-ATP synthase is translocated with respect to L. monocytogenes and L. innocua in L. welshimeri. The smaller size of the L. welshimeri genome is the result of deletions in genes involved in virulence and of ‘‘fitness’’ genes required for intracellular survival, transcription factors, LPXTG- and LRR-containing

Fig. 1. Identification of putative horizontally transferred genes (HTGs) using SIGI-HMM (Waack et al., 2006). The outermost circle represents the scale in kb starting with the origin of replication at position 0. The following double circles show in gray the distribution of coding sequences (CDS) of L. monocytogenes (second circle), L. monocytogenes F2365 (third circle), L. innocua (fourth circle), and L. welshimeri (fifth circle). rRNA operons are colored in blue, a putative prophage region in black, and HTGs in red. The innermost circle displays the conservation on the level of nucleotides among the above-mentioned genomes. The figure was drawn using GenomeViz software (Ghai et al., 2004).

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proteins as well as 55 genes involved in carbohydrate transport and metabolism. In total, 482 genes are absent from L. welshimeri as compared to L. monocytogenes. Of these, 249 deletions are commonly absent in both L. welshimeri and L. innocua, suggesting similar genome evolutionary paths from an ancestor. Further analysis of the gene content of L. monocytogenes strains EGD-e (serotype 1/2a) and F2365 (serotype 4b), L. innocua and L. welshimeri revealed 157 genes commonly absent in the sequenced L. monocytogenes F2365 serotype 4b, L. innocua, and L. welshimeri strains, but present in the L. monocytogenes EGD-e serotype 1/2a genome. We also identified 311 genes specific to L. welshimeri that are absent in the other two species, indicating gene expansion in L. welshimeri including horizontal gene transfer. This includes several insertions of unique genes which confer new adaptive properties for niche-specific environmental survival of L. welshimeri e.g. degradation of D-xylose or degradation of proteins by species-specific proteases, the NatAB ABC transport system that catalyses ATP-dependent extrusion of electrogenic Na+ or a complex of genes coding for type I C restriction–modification (R–M) system, which is often considered to confer phage resistance, genetic recombination, or horizontal gene transfer. The species L. welshimeri appears to have been derived from early evolutionary events and an ancestor more compact than L. monocytogenes that led to the emergence of nonpathogenic Listeria. Phylogenomic analysis based on whole-genome sequences reveals the same phylogenetic relationship as determined by 16S-rRNA sequencing of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, and L. grayi which branch the genus in three main groups: the first group consists of the closely related species L. monocytogenes, L. innocua, and L. welshimeri whereas L. welshimeri reveals the deepest branching of this group. L. seeligeri and L. ivanovii exhibit the second group and L. grayi seems to be very distant from both groups.

Listeria bacteriophage genomics Many bacteriophages specific for Listeria have been isolated (reviewed in (Loessner and Calendar, 2005)), but despite the publication of the complete genome sequences of L. monocytogenes EGD-e and L. innocua CLIP11262 (Glaser et al., 2001), the potential influence of temperate Listeria bacteriophages on the host cell phenotype is still not understood. Only little information was previously available on Listeria bacteriophage genomes, gene expression, and the possible function of gene products (Loessner et al., 2000; Zimmer et al., 2003). In the framework of the PathoGenoMik project, the DNA genomes of different Listeria bacteriophages

infecting L. monocytogenes (A006, A500, and P35), L. innocua, and L. ivanovii (B054, B025) were sequenced and analyzed. Also included was the broad-host-range phage A511, which can infect approximately 95% of all L. monocytogenes strains belonging to serovars 1/2 and 4 (Loessner and Busse, 1990; Wendlinger et al., 1996). The Listeria phages investigated feature genomes between 35.8 and 134.5 kb in size, with G+C contents between 35.5 and 40.8 mol% (Dorscht et al., unpublished, manuscript submitted). Except for P35, the group of phages belonging to the Siphoviridae family (P35, A006, and A500) revealed an overall similar genome organization, where open reading frames (ORFs) are organized into functional clusters in a life-cycle-specific manner, also reflected by the direction of transcription (Loessner et al., 2000; Zimmer et al., 2003). Identification of the attB integration sites in the bacterial genome revealed that both A500 and A006 specifically target the 30 ends of tRNA genes (Dorscht et al., unpublished, manuscript submitted). We also found that both A118 and A500 utilize programmed translational frameshifting to generate major capsid and tail proteins with C-termini of different length, a decoding strategy first discovered in Listeria phage PSA (Loessner and Calendar, 2005; Zimmer et al., 2003). Phage B054 is a Myovirus, and its larger genome features 80 predicted ORFs. In fact, most of the additional genes appear to specify products required for building the more complex contractile tail. On an overall basis, many of the B054 genes specify proteins with high similarity to phage-encoded proteins in the genome of L. innocua Clip11262 (Glaser et al., 2001; Nelson et al., 2004), pointing to a close relationship of B054 to the putative prophage f11262.4 harbored by this strain (Nelson et al., 2004). Interestingly, B054 integrates into the 30 end of gene EF-ts, predicted to encode a translation elongation factor (Dorscht et al., unpublished, manuscripts submitted), and represents the first reported case of phage integration into such a gene. B025 features an invariable unit genome with overlapping 30 ends, consisting of complementary singlestranded overhangs of 10 nucleotides. The integration locus of B025 into a tRNAarg gene (Dorscht et al., unpublished, manuscript submitted) is identical to the attB of phage PSA (Lauer et al., 2002). In contrast to the conserved genome arrangement observed for B054 and the L. innocua prophage f11262.4, the genome of B025 apparently features a rather extensive mosaicism: the majority of the 65 predicted ORFs encodes proteins with high similarity to L. monocytogenes phages A118 and PSA, and to a (defective) prophage (prophage f11262.6; (Nelson et al., 2004)) contained in the bacterial chromosome of L. innocua Clip11262 (Glaser et al., 2001). P35 (infects L. monocytogenes serovar 1/2 strains) appears to be significantly different from the other

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Listeria phages investigated so far. A surprising finding was that the P35 genome completely lacks any genes associated with establishing or controlling a lysogenic status. Loss of the ability to integrate into the host genome has the advantage of avoiding homoimmunity suppression, and thereby enables the phage to infect a wider range of host strains, and explains the broad host spectrum of P35. The broad-host-range phage A511 (Zink and Loessner, 1992) belongs to the Myoviridae family (Zink and Loessner, 1992) and consists of a non-flexible, contractile tail, and an isometric capsid. In contrast to the other Listeria bacteriophages described, A511 belongs to a poorly characterized group of virulent (i.e. strictly lytic) phages (Loessner and Calendar, 2005; Loessner and Rees, 2005). A511 features a 134.5-kb genome with a G+C content of 36 mol%, specifying 190 ORFs and 16 tRNAs. In summary, phages A006 and A500 (and A118) form a closely related group. B025 and PSA are also related, whereas B054 is more distant to these two groups, and P35 is clearly different from all other phages. Phage A511 has a relatively large genome highly similar to that of P100 (Carlton et al., 2005), is unrelated to the smaller Listeria phages A006, A500, P35, B025, and B054, and features intergeneric relationships to non-Listeria phages K and LP65 (Chibani-Chennoufi et al., 2004; O’Flaherty et al., 2004).

Population structure and lineages in L. monocytogenes Epidemiological data indicate that not all strains of L. monocytogenes are equally capable of causing disease in humans but that differences in virulence among strains may exist. Although 13 serovars are described for the species L. monocytogenes, about 98% of the strains isolated from patients are of serovars 1/2a, 1/2b, 1/2c, and 4b (Jacquet et al., 2002). Furthermore, all major outbreaks of listeriosis and most of the sporadic cases are due to strains of serovar 4b suggesting that strains of this serovar may possess unique virulence properties. Heterogeneity in virulence has also been observed in the mouse infection model (Brosch et al., 1993). These phenotypic differences among Listeria strains may be in part due to genetic differences among L. monocytogenes isolates. To investigate the genetic diversity of L. monocytogenes and Listeria in general, a focused ‘‘Listeria biodiversity array’’ containing probes representative of specific sequences of two L. monocytogenes strains of serovar 1/2a (EGD-e) and 4b (CLIP80459) and of one apathogenic Listeria innocua strain (CLIP11262) was developed. The gene content of 113 Listeria strains of different epidemiological characteristics and virulence potentials was then characterized with this array

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(Doumith et al., 2004b). Hybridization results identified distinct patterns of presence or absence of genes among the L. monocytogenes and Listeria sp. strains tested. These gene content patterns correlated with the previously defined three lineages (lineage I serovars 1/2a, 1/ 2c, 3a, and 3c; lineage II serovars 4b, 4d, 4e, 1/2b, 3b, and 7; and lineage III serovars 4a, and 4c). This result together with the fact that many studies have found an association between L. monocytogenes serovars and various phenotypic characteristics leading to the definition of three lineages among L. monocytogenes strains suggests that these lineages mirror the evolution within the species due to an early divergence from ancestral L. monocytogenes. Analysis of the gene content patterns allowed also to subdivide the three lineages into five groups that correlated with serovars: I.1 (serovars 1/2a and 3a), I.2 (serovars 1/2c and 3c), II.1 (serovars 4b, 4d and 4e), II.2 (serovars 1/2b, 3b, and 7), and group III (serovars 4a and 4c). For each of the first four groups, containing the four major serovars of L. monocytogenes implicated in human disease lineage-specific genes were identified. Based on the macroarray comparison results, a multiplex PCR assay was then developed targeting genes in each of the groups of marker genes allowing to distinguish the four major L. monocytogenes serovars (1/2a, 1/2b, 1/2c, and 4b). This multiplex PCR is highly specific for the identification of pathogenic L. monocytogenes strains allowing ‘‘molecular serotyping’’ within some hours instead of several days (Doumith et al., 2004a). The specificity and reliability of this multiplex PCR test has recently been validated through a multicenter study (Doumith et al., 2005). Similar approaches using shotgun microarrays and DNA–DNA hybridization corroborated these results by also identifying genes specific for L. monocytogenes of the different lineages (Call et al., 2003; Zhang et al., 2003). Analysis of the distribution of the known virulence genes (inlAB, prfA, plcA, hly, mpl, actA, plcB, uhpT, and bsh) among the 113 strains tested revealed that the known virulence factors are present in all L. monocytogenes strains. However, the distribution of 55 genes coding for putative surface proteins of the internalin/ LPXTG/GW motif-containing family belonging to three sequenced Listeria genomes (L. monocytogenes EGD-e sv1/2a, L. monocytogenes CLIP80459) revealed a pronounced heterogeneity, suggesting that some of them may play a role in virulence differences. Based on these results, two internalin protein-encoding genes inlI (lmo0333) and inlJ (lmo2821) were further characterized leading to the identification of a new virulence factor (Sabet et al., 2005). The inlJ deletion mutant is significantly attenuated in virulence after intravenous infection of mice or oral inoculation of transgenic hEcad mice. inlJ encodes an LRR protein that is structurally related to the listerial invasion factor InlA. The

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consensus sequence of LRR defined a novel subfamily of cysteine-containing proteins belonging to the internalin family (Sabet et al., 2005). In contrast to strains, which appear to have a higher potential to cause disease, also strains that seem to have lower virulence are present in the species L. monocytogenes. Most interestingly, the gene content comparison using the DNA array showed that in L. monocytogenes of serovar 4a and 4c, belonging to lineage III and mainly described as animal pathogens (Jeffers et al., 2001; Wiedmann et al., 1997), 13 of the 25 L. monocytogenes specific surface proteins, including all known internalins except inlAB, were missing (Doumith et al., 2004b). Surface proteins are known to be important for host–pathogen interactions. The fact that many surface proteins are missing from strains of this lineage might indicate adaptation to a different niche, the animal production environment, than for lineage II and I isolates. This is in line with a recent study where L. monocytogenes populations present at farms, in foodprocessing plants, at retail, and in the human population were examined. Analyses of over 400 strains identified one dominant strain in pork products among L. monocytogenes serovar 1/2a strains isolated from foodprocessing plants. Unexpectedly, this strain was not identified among the human isolates tested. DNA array characterization of these pork product-associated strains identified a specific genetic profile and the absence of five genes predicted to encode internalins and other cell surface proteins, similar to the lineage III isolates (Hong et al., 2007). Thus surface protein diversity may explain/contribute to the adaptation of L. monocytogenes to different environments.

Transcriptional profiling of intracellularly growing L. monocytogenes L. monocytogenes is a facultative intracellular pathogen, which can breach the vacuolar compartment to gain access to the cytosol of the host cell. Identification of its genes that are expressed during invasive infection is important for understanding the infection processes. Whole-genome-based adaptive changes during bacterial intracellular growth were examined in two different cell types, P388D1 macrophages and the epithelia cell line Caco-2, by interrogating for differences between RNAs isolated from infected cells to that isolated from bacteria growing in broth cultures. In the first study using infected P388D1 macrophages, wild-type L. monocytogenes strain EGD-e and its isogenic double deletion mutant DhlyDplcA (which remains entrapped in the vacuolar compartment of the host cell) were examined. Survival properties of DhlyDplcA and EGD-e strains inside P388D1 cells showed characteristics typical for adaptation and growth, respectively (Chatterjee et al.,

2006). Thus to model adaptation and growth of listeriae within different intracellular compartments, bacterial cells were collected from infected P388D1 cells at 1 h (for DhlyDplcA) and at 4 and 8 h (for EGD-e) post-infection and were subjected to whole-genome microarray-based expression profiling. A total of 484 genes comprising 17% of the total genome were differentially regulated to enable survival in the altered environment. Of these 484 genes, 301 were up-regulated and 182 genes were down-regulated during intracellular growth. Sixty-six genes were specifically upregulated for adaptation in the vacuolar and 115 genes for growth in the cytosolic compartment of P388D1 cells. A total of 41 genes were species specific, being absent from the genome of the non-pathogenic L. innocua CLIP 11262 strain; and 25 genes were strain specific, being absent from the genome of the previously sequenced L. monocytogenes F2365 serotype 4b strain. The expression profile of intracellular L. monocytogenes following invasion of epithelial cells was also investigated using whole-genome DNA microarrays (Joseph et al., 2006). Approximately 19% of the genes were found to be differentially expressed by at least 1.6fold after 6 h of infection relative to their level of transcription when grown in brain–heart infusion (BHI) medium. Among them were 279 up-regulated genes and 272 down-regulated genes including species-specific genes, regulatory genes, and those of yet unknown function. The differential regulation of some of these genes was confirmed by real-time RT-PCR analysis. Strong transcriptional induction was observed for the PrfA-controlled virulence genes confirming their indispensability for intracellular survival and replication. To validate the biological relevance of the intracellular gene expression profile, a random mutant library of L. monocytogenes was screened for intracellular growth deficiencies. Interfacing and extrapolation of the results of mutant screening and microarray data revealed that approximately 36% of all up-regulated genes are indeed required for listerial proliferation in the cytosol of epithelial cells. Apart from the strong intracellular induction of all previously known PrfA-regulated genes, genes comprising the class I stress response [dnaJ, dnaK, grpE, and hrcA (lmo1472–1475) and groEL and groES (lmo2068–2069)], all of which are classical chaperone proteins, members of the CtsR-regulated class III stress-responsive genes [clpP, clpC, clpE, clpB (lmo2468, lmo0232, lmo0997, and lmo2206)], which are ATP-dependent proteases, and 26 members of the class II stress-response family (mediated by sigma factor B) were strongly induced during survival in both the vacuolar and cytosolic compartments of P388D1 cells. In addition, induction of listerial SOS response genes [lexA (lmo1302), recA (lmo1398), radA (lmo0233), lmo2676 (coding for an UV-damage repair protein), and polIV (lmo1975)] was observed in the

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cytosol of the host cells. Furthermore, the expression of several bacterial cell division genes [divIVA (lmo2020), ftsZA (lmo2032–2033), ftsXE (lmo2506–2507), ftsL (lmo2040), ftsH (lmo0220), minC (lmo1545), minD (lmo1544), and smc (lmo1804)], replication genes [chromosomal replication initiation gene dnaA (lmo0001) and DNA primase gene dnaG (lmo1455)], and major autolysins [iap (lmo0582), spl (lmo2505), and murA (lmo2691)] were down-regulated during transit in the phagolysosome and during intracytosolic growth. Cumulatively, these data suggest that L. monocytogenes may be experiencing stress during intracellular survival. Approximately 30 up-regulated genes coding for ABC transporters, phosphotransferase systems (PTS), other transport systems, and genes known to be under carbon catabolite repression (CCR) control, were identified indicating that intracellularly replicating listeriae are mainly CCR-derepressed. Among the transcriptionally induced genes are those that are involved in the pentose phosphate cycle, providing evidence that this is a major catabolic pathway for the synthesis of necessary catabolic intermediates. The induced transcription of the genes encoding glycerol kinases (lmo1034, lmo1538) and the reduced intracellular growth of respective mutants suggest that glycerol plays a role as carbon source for listerial growth in the cytosol of the host cells apart from glucose and phosphorylated glucose. This hypothesis is further strengthened by the strong up-regulation of plcB (9.8-fold) encoding a broad-spectrum phospholipase which could provide glycerol from host-derived phospholipids. Indeed replication efficiency of a plcB mutant is significantly reduced within the cytosol of the host cell (Goetz et al., 2001). Together with the up-regulation of the PrfA-dependent hpt gene encoding a hexose phosphate transporter and the down-regulation of the glycolysis genes, these data further strengthen the assumption that the intracellular level of free glucose is low and that glucose is not the predominant carbohydrate source for listeriae growing inside host cells. Another interesting finding was the induction of 10 genes located within a chromosomal region that ranges from lmo1142 (pduS) to lmo1208 (cbiP). This 55-kb gene cluster encodes the factors for coenzyme B12 synthesis and for the B12-dependent degradation of 1,2-propanediol and ethanolamine (Buchrieser et al., 2003). Together with the reduced replication efficiency of an insertional knockout of eutB in the cytosol of epithelial cells, the results suggest that ethanolamine, probably derived from phosphatidyl ethanolamine by the activity of PlcB, is used as an alternative nitrogen source for replication of L. monocytogenes as shown for Salmonella typhimurium (Heithoff et al., 1999). L. monocytogenes is entirely dependent on reduced nitrogen sources for its nitrogen supply. Within the cytosol of the host cell, the amount of ammonia is low as excess of this substrate is lethal to mammalian cells. This

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is in line with the up-regulation of genes involved in the glutamine–glutamate synthesis pathway such as glnA (lmo1299) and gltAB (lmo1733-lmo1734). Among the differentially regulated genes involved in anabolic pathways, we observed the up-regulation of genes for synthesis of amino acids which L. monocytogenes – but not the host cells – is able to synthesize, namely tryptophan, isoleucine, leucine, valine, and arginine. The importance of isoleucine and valine biosynthesis for growth in epithelial cells was confirmed by a threefold attenuation of an insertional ilvD mutant. The upregulation of key genes such as glnA in cytosolically grown L. monocytogenes suggests that the intracellular availability of even free non-essential amino acids like serine and glutamine, is limited. In this context, the strong up-regulation of clpB encoding a subunit of the ATP-dependent Clp protease could be of special importance. It has already been shown that this protease is required for efficient intracellular growth of L. monocytogenes, possibly by degradation of unnecessary proteins, thus providing peptides as amino acid sources for intracellularly growing L. monocytogenes.

The metabolism of extra- and intracellularly growing L. monocytogenes Most of the pregenomic knowledge concerning L. monocytogenes physiology was based on growth studies performed under extracellular conditions and basically showed that L. monocytogenes is a heterotrophic, facultative anaerobically growing Gram-positive bacterium that cannot be readily cultured in a basal mineral salt medium with glucose as sole carbon source. L. monocytogenes was therefore routinely cultured in the rich BHI medium, and most molecular studies on virulence genes and their regulation were indeed carried out in this medium. Several defined minimal media were designed in the past (Premaratne et al., 1991; Tsai and Hodgson, 2003) which contain a more or less complex mixture of amino acids, the vitamins biotin, riboflavin, thiamine, and lipoate as essential additives (Tsai and Hodgson, 2003). These minimal media support a (often rather slow) growth of L. monocytogenes. Earlier studies (Tsai and Hodgson, 2003) and our own data showed that the L. monocytogenes EGD-e strain (in liquid culture medium with glucose as carbon source and glutamine as nitrogen source) is strictly dependent on cysteine, biotin, riboflavin, thiamine, and lipoate. Addition of methionine and other amino acids, especially the branched-chain amino acids Ile, Leu, and Val, greatly enhances growth of L. monocytogenes in this medium. The genome sequence of L. monocytogenes (Glaser et al., 2001) indicates that the gene sets for most catabolic and anabolic pathways of L. monocytogenes

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are similar to those of the extensively studied Bacillus subtilis (which like L. monocytogenes belongs to the group of low G+C Gram-positive bacteria). The observed growth dependency of L. monocytogenes for the mentioned vitamins, cysteine and glutamine can be readily explained by the genome sequence since the genes for the biosyntheses of these vitamins and for sulfate and nitrate reductases are missing, and the lack of these reductases renders L. monocytogenes dependent on reduced nitrogen and sulfur sources, i.e. glutamine and cysteine. Glutamine can be partially replaced by ammonium ions (due to the presence of a gene for glutamate dehydrogenase) but not by nitrate (Tsai and Hodgson, 2003). However, the need for the additional amino acids for efficient growth under extracellular conditions cannot be explained by the genome data as all genes for these amino acid biosynthesis pathways have been identified.

Extracellular metabolism and its impact on virulence To obtain a clearer view of the basic metabolism of L. monocytogenes, 13C isotopologue studies were performed with bacteria grown in a defined minimal medium with uniformly 13C-labeled glucose as carbon source (Eisenreich et al., 2006). The major conclusions derived from these studies are summarized in Fig. 2. The major flux of glucose occurs under aerobic conditions via the pentose phosphate pathway (PPP) down to 2oxoglutarate. The subsequent step in the citrate cycle

leading to succinyl-CoA is interrupted because the gene coding for 2-oxoglutarate dehydrogenase is absent. Oxaloacetate and the other C4-dicarboxylic acids are synthesized by carboxylation of pyruvate via pyruvate carboxylase and the reverse (reductive) reactions in the citrate cycle, respectively. Clearly, the need for CO2 in the synthesis of the central metabolite oxaloacetate favors glucose degradation via PPP over glycolysis since PPP but not glycolysis delivers CO2. The exclusive synthesis of oxaloacetate by pyruvate carboxylation probably slows down biosynthesis of amino acids requiring C4 intermediates, especially the branchedchain amino acids. This metabolic limitation may explain that these amino acids are required for efficient growth in minimal media. Glucose uptake by L. monocytogenes is completely dependent on phosphoenolpyruvate PTS-mediated transport (Mertins et al., 2007) and occurs – as also indicated in Fig. 2 – by co-transport via several PTS. L. monocytogenes possesses the genetic information for about 40 PTS, but a complete pts-G gene which determines the major glucose-specific PTS in many bacteria, is absent in L. monocytogenes. These conclusions are based on yet unpublished physiological studies with mutants defective in specific PTS (Ecke et al., unpublished data). L. monocytogenes is unable to utilize C2 carbon sources (and hence fatty acids) due to a missing glyoxylate cycle but can efficiently grow in defined minimal media with glycerol as sole carbon source (Tsai and Hodgson, 2003; and our own unpublished data). The presence of two glycerol uptake facilitators, two PrfA (virulence regulator ) - low activity in MM

Growth in MM (minimal medium) with glucose Other PTS

PTS-Gluc/Man (Lmo0096-98; lmo0781)

PTS-Bgl

Hpr-His-P hprK

Glucose-6- P (external) Ser, Gly

hpt

Hpr

Hpr-Ser-P

CcpA

Glucose-6-P

Uptake

Gluconate-6-P eno

Glycolysis PEP

Ala(Val, IIe, Leu) pycA Oxaloacetate Asp, Thr,Lys, Cys, Met

PentosePhosphate Cycle

Xylulose-5-P

Pyruvate CO2

- CO2

Erythrose-4-P Heptulose-7-P

Carbon Catabolite Repression

Tyr, Phe,Trp

pdh Citrate cycle

Citrate

Acetyl-CoA 2- Oxoglutarate

Glu, Arg, Pro

Fig. 2. Carbon flux analysis of Listeria monocytogenes. Strength of the arrows indicates the preferred reactions. De novo synthesized amino acids are in red, poorly or not synthesized ones in black. Note that the synthesis of oxaloacetate is a bottleneck, probably the reason for the preference for the pentose-phosphate pathway. PrfA activity is low during balanced growth in minimal medium and increases in the later growth phase.

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glycerol kinases, a glycerol-3-phosphate dehydrogenase as well as two dihydroxyacetone kinases (DhaK) belonging to category C (Barabote and Saier, Jr., 2005) in the genome indicates that L. monocytogenes possesses a highly developed glycerol metabolism. The mode of C metabolism has a strong impact on the activity of PrfA, the central transcriptional regulator of virulence genes and hence on virulence of this pathogen. When L. monocytogenes grows in minimal medium in the presence of PTS sugars, like glucose, mannose, or cellobiose, strong CCR is observed and PrfA activity is low, especially in the presence of cellobiose (Mertins et al., 2007). In contrast, all PrfA-regulated genes are highly induced in the presence of the non-PTS carbon source glycerol, underlining the role of PTS sugars for the inhibition of PrfA activity. However, neither CcpA (Milenbachs et al., 1997) nor Hpr-Ser-P (Mertins et al., 2007), the two major components of CCR control in Gram-positive bacteria, act as modulators of PrfA activity in L. monocytogenes. These results rather suggest that component(s) of subsequent steps dependent on the common basic PTS pathway is (are) involved in the modulation of PrfA activity. This conclusion is further supported by recent studies (Eisenreich et al., 2006; Marr et al., 2006) which showed that over-expression of PrfA inhibits glucose uptake and affects the expression of genes that depend on the efficiency of glucose uptake (Stulke and Hillen, 2000). More recently, evidence has been provided to implicate phosphorylated Hpr as the inhibitor of PrfA in the presence of PTS sugars (Herro et al., 2005).

Intracellular metabolism Little is known about the acquisition of nutrients for efficient intracellular replication of L. monocytogenes and the specific adaptation mechanisms to the conditions encountered in the cytosol of host cells. The study of the metabolism of L. monocytogenes growing within mammalian cells by metabolite flux measurements is not yet possible due to the low sensitivity of nuclear magnetic resonance technique used for the elucidation of the metabolome of extracellular growing L. monocytogenes. However, some interesting conclusions concerning the intracellular metabolism can be drawn from the expression profile of intracellularly replicating L. monocytogenes (see above) and the interfacing of these transcriptome data with the results obtained by screening of a random L. monocytogenes mutant library for mutants impaired in intracellular replication (Joseph et al., 2006). Many genes and operons known to be under CCR control in L. monocytogenes (Mertins et al., 2007) are up-regulated, suggesting that PTS-mediated glucose uptake is not the major route by which the necessary carbon sources for intracellularly growing L. monocyto-

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genes are provided. The down-regulation of the genes encoding the major glycolysis enzymes is in line with this assumption. The up-regulation of the genes for both glycerol kinases and for glycerol-3-phosphate dehydrogenase rather suggests that glycerol may play a major role in the C metabolism at least under the applied conditions. The impaired intracellular replication of a mutant deficient in glycerol kinase and glycerol-3phosphate dehydrogenase supports this view. The strong upregulation of the L. monocytogenes specific PrfA-regulated hpt (uhpT) gene, encoding a transporter for phosphorylated hexoses (Chico-Calero et al., 2002; Milohanic et al., 2003), suggests that phosphorylated glucose may be an alternative carbon source for L. monocytogenes during intracellular replication. Together with the transcriptome data discussed above, it appears that intracellularly replicating listeriae avoid competing for the major carbon and nitrogen sources of the host cell, i.e. glucose and glutamine, respectively, and rather take advantage of alternative carbon and nitrogen sources deriving from excess or storage components, like phospholipids, glycogen, arginine, and others.

Listerial thiol metabolism Glutathione (gamma-glutamyl-cysteine-glycine, GSH) is a peptide thiol involved in several important cellular processes. It has a role in thiol redox homeostasis, protection against reactive oxygen intermediates and also in reduction of ribonucleotides, hence in DNA synthesis. Normally GSH is formed by the consecutive action of two peptide bond-forming enzymes, gammaglutamyl-cysteine ligase (GshA) and glutathione synthetase (GshB), an enzyme with an ATP-grasp fold. In the genomes of L. monocytogenes and L. innocua, GshB seemed to be missing. However, the large ORF Lmo2770 encodes an N-terminal GshA-like and a Cterminal ATP-grasp domain. By mutant construction and biochemical analysis it was demonstrated that Lmo2770 encodes a novel multidomain enzyme (GshF) for the synthesis of GSH (Gopal et al., 2005). GshF proved to be involved in oxidative stress defence and intracellular multiplication of the bacteria. This gene most presumably arose by fusion of two ancestral genes. Orthologous genes are found in a number of bacteria which phylogenetically are only distantly related to each other, pointing to spread of the gene by horizontal gene transfer.

Subproteomes of Listeria Genome sequencing and transcriptomics allow for the identification of genes and their transcription into RNA, but they do not necessarily specify actual protein

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both the importance of this subproteome and demonstrating the reliability of the techniques used. The comparison between wild-type L. monocytogenes and the non-pathogenic species L. innocua was performed to reveal proteins probably involved in pathogenicity and/ or the adaptation to their respective lifestyles. In addition to the eight known virulence factors, all specific for L. monocytogenes, eight additional proteins were identified that exhibit the typical key feature defining the known listerial virulence factors. This result clearly demonstrates that the major difference between the pathogenic and non-pathogenic species, noted in the comparative genome analysis, manifests itself strongest in the secretome (Trost et al., 2005). Our bioinformatic analysis was recently extended by Desvaux and Hebraud (2006). Looking at the cell wall, the issue of covalently linked proteins was first addressed. A newly developed gel-less strategy with greatly increased sensitivity compared to 2D-PAGE was developed and used in a comparison of a sortaseA-deletion mutant with the wild-type strain, demonstrating that SrtA is required for the cell wall anchoring of InlA. This work was extended to cover SrtB and further SrtA substrates (Bierne et al., 2002). Because the majority of surface proteins are not covalently linked to the cell wall, this initial investigation was followed by an analysis of non-covalently cell wall-associated proteins. For this purpose, a new method for the isolation of defined surface proteome fractions based on serial extraction of proteins with different salts was developed and particular attention paid to ensure the integrity of the cell membranes during the treatment. Fifty-five cell wall-associated surface

synthesis. A proteomic approach yields both complementary expression data and additional information, e.g., on protein transport, cellular localization, protein–protein interactions, and post-translational modifications. As almost all listerial virulence factors are either secreted or surface associated, the three accessible surface subproteomes, the secretory, cell wall-associated, and membrane-associated proteins that are involved in host–pathogen interactions were examined in detail (Figs. 3 and 4). This had the additional advantage of reducing the complexity of the samples, thus achieving higher selectivity and sensitivity. Both classical two-dimensional electrophoresis coupled with protein identification by MALDI-MS, mixed 1D-PAGE/liquid chromatography-tandem mass spectrometry (LC-MS/MS), and newly developed gelless procedures based on nano LC-MS/MS peptide sequenching were employed, enabling differential semiquantitative protein expression analyses. Comparative analyses between the EGD-e wild-type strain and isogenic mutants or L. innocua revealed several proteins that seem to be specific for the life cycle of L. monocytogenes and might be involved in pathogenic functions. Extracellular proteins of bacterial pathogens are crucial for host–pathogen interaction as well as highly variable and adaptive. Using predictive bioinformatic and experimental proteomic approaches, the original signal peptide (SP) prediction (Glaser et al., 2001) was reanalyzed and greatly improved and led to the identification of 105 proteins in the culture supernatant of L. monocytogenes. Among these, all currently known virulence factors with an SP were detected, showing

all L. monocytogenes CDSs all identified lipoproteins all identified TM proteins all identified ribosomal proteins all identified LPxTG-proteins all identified GW-proteins all identified hyd. anchor-proteins all identified P60-proteins all othe rident. virulence factors all other identified proteins

200000 150000 100000

Mol. Weight [Da]

70000 50000 30000 20000 15000 10000 7000 5000 3000 3

4

5

6

7

8

9

10

11

12

13

pI

Fig. 3. Experimental coverage of the predicted Listeria monocytogenes proteome.

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Fig. 4. Listeria monocytogenes surface proteins and host–pathogen interactions between InlA and E-cadherin, and InlB and the hepatocyte growth factor receptor (HGFR).

proteins were identified by N-terminal sequencing and mass spectrometry. About 16% of these proteins are of unknown function and three proteins have no orthologs in the non-pathogenic L. innocua. Remarkably, a relatively high number of proteins with a function in the cytoplasmic compartment were identified in this surface proteome. These proteins had neither predicted or detectable signal peptides nor could any modification be observed except for removal of the N-terminal methionine. One of these proteins, enolase, was shown to be present in the cell wall of the pathogen by immuno-electron microscopy and, along with heat shock factor DnaK, elongation factor TU, and glyceraldehyde-3-phosphate dehydrogenase, it was found to be able to bind human plasminogen with high specificity in overlay blots and surface plasmon resonance experiments. The data suggest a possible ‘‘moonlighting’’ role of these proteins as receptors for human plasminogen on the bacterial cell surface, where the recruited host protease might assist in the invasion process (Schaumburg et al., 2004). The analysis of membrane proteins is particularly difficult mainly due to their hydrophobicity and the complexity of this subproteome that comprises about one third of all proteins. Lipoproteins, anchored to the membrane by a diacylglyceryl moiety, however, could be rendered accessible by a deletion of the prolipoprotein diacylglyceryl transferase (Lgt) that is not lethal in Gram-positive bacteria. Without membrane anchor, the proteins were released into the culture supernatant so that a comparison of the wild-type and mutant secretomes allowed for the experimental verification of

26 of the 68 predicted lipoproteins in L. monocytogenes. The additional analysis of Dlgt/DprfA double mutants led to the identification of three lipoproteins the extracellular levels of which are regulated by PrfA, and one, OppA, that is post-translationally modified depending on PrfA. Furthermore, in contrast to earlier studies in Escherichia coli we unambiguously demonstrated that lipidation by Lgt is not a prerequisite for activity of the lipoprotein-specific signal peptidase II (Lsp) in Listeria (Baumgartner et al., 2007). Integral membrane proteins were addressed by a novel protocol for the efficient preparation of membrane fractions that overcomes difficulties associated with ribosomes. By combining 1D-SDS-PAGE LC-MS/MS, 301 different proteins were identified, including 70 proteins that exhibited 2–15 transmembrane domains. In these experiments, a remarkably high ratio of proteins was detected in gel sections that did not match their expected migration behavior during SDS-PAGE. LaneSpector, a general visualization tool was developed to compare systematically apparent and calculated protein masses. The detailed presentation of the LaneSpector plot promotes the validation of the analytical process and helps to reveal relevant biological processes such as proteolysis or other post-translational modifications (Wehmhoner et al., 2005). Starting from these premises the L. monocytogenes wild type, its PrfA deletion mutant, and L. innocua were reanalyzed. These studies led to the detection of Lmo1695. Lmo1695 belongs to the recently identified new VirR-dependent virulence regulon identified for L. monocytogenes (Mandin et al., 2005). We could demonstrate that Lmo1695 is required

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for lysinylation of phospholipids indicating that this protein has the same function as MprF of Staphylococcus aureus, known to be involved in bacterial antimicrobial cationic peptide (CAMP) resistance. In contrast to staphylococcal MprF, Lmo1695 is not only involved in lysyl phosphoglycerol synthesis, but also essential for lysinylation of diphosphoglycerol, a phospholipid widely distributed in bacterial membranes. A Dlmo1695 mutant lacking lysinylated phospholipids was particularly susceptible to CAMPs of human and bacterial origin and was attenuated for infection of epithelial cell lines. Moreover, these results demonstrated an adaptation of L. monocytogenes in response to being challenged with CAMPs, which may regulate bacterial growth and stress response mechanisms during the infection (Thedieck et al., 2006).

Influence of a food-borne anti-listerial, multispecies microbial consortium on the transcriptome of L. monocytogenes One important source of food poisoning L. monocytogenes is red-smear cheese, which is characterized by a red-brown-orange colored, complex multi-species microbial consortium (smear) on its surface (Maoz et al., 2003; Wenning et al., 2006). These cheeses provide excellent growth conditions for yeasts and bacteria, including L. monocytogenes, and have often been implicated in severe outbreaks of listeriosis. L. monocytogenes and other species of Listeria still appear frequently in red-smear cheese, even when pasteurized milk has been used for cheese making. Rudol and Scherer (2001) reported an incidence of L. monocytogenes of more than 6% in various soft cheeses, with cell counts of up to 106 cm2 of cheese. To control L. monocytogenes in red-smear cheese, antagonistic substances such as bacteriocins or bacteriocin-producing starter bacteria have been used (Carnio et al., 2000; Loessner et al., 2003). Even though bacteriocins have ‘‘high potentials’’ in helping to achieve food preservation in a more ‘‘natural’’ way, many questions remain. A better understanding of the development of bacteriocin-resistant pathogens in food, and strategies to mitigate their emergence, as well as investigations on the molecular mechanisms involved are necessary (for reviews see (Carnio et al., 2000; Cotter et al., 2005; Drider et al., 2006)).

Anti-listerial action of complex microbial consortia Some complex red-smear cheese microbial multispecies consortia exclude L. monocytogenes through yet unknown interactions (Eppert et al., 1997; Maoz

et al., 2003). However, exclusion does not appear to be just a passive mechanism, e.g. through nutrient depletion, but rather an active attack by bactericidal substances. Maoz et al. (2003) described a red-smear cheese consortium which is inhibitory for L. monocytogenes, but the inhibitory activity seems not to be caused by a single bacteriocin-producing bacterium. In order to characterize this inhibitory action, microarray experiments have been performed. This technique should be an ideal entrance to characterize the mode of action of antagonistic substances. Regulation responses of the genes should give hints towards the molecular mechanisms involved in anti-listerial activities. The inhibitory consortium was grown on agar plates. Filter membranes were placed on this consortium and L. monocytogenes were spread on top. After 1–4 h, L. monocytogenes was harvested, RNA was isolated, reverse transcribed and labeled, and used for microarray hybridization. L. monocytogenes cells were highly stressed, and after 4 h, a two-log reduction in colonyforming units prohibited the isolation of enough RNA for later time points. Controls were prepared from mixed bacterial cultures similar in species composition as the red-smear consortium, but without any antilisterial activity. Furthermore, known bacteriocins (e.g. nisin) were applied to L. monocytogenes for comparison (Gabert et al., unpublished, manuscript in preparation). Another control study was undertaken to account for the low pH response of L. monocytogenes, since fresh cheeses have low pH values and it was necessary to distinguish between a general pH stress response and a specific response to anti-listerial effects (Satorhelyi et al., unpublished, manuscript in preparation).

Contact with anti-listerial microbial consortia causes extensive transcriptome changes It was found that nearly 400 genes are up- or downregulated upon contact with the anti-listerial consortium. From these, 117 genes were strongly induced (43 times for at least two consecutive time points). The overlap between this response and the low pH response is surprisingly small: of the strongly induced genes, only 13 were also induced by low pH and some of those can be considered general stress proteins (e.g. rpsF, ltrC, usp family protein lmo2673). Among the strongly induced genes, a major group can be identified which is involved in energy supply (e.g. catabolic enzymes, sugar activation, co-enzyme metabolism, respiration, and others). In accordance to these findings, uptake systems (mainly for sugars, but also for oligopeptides and iron) are induced. This finding would fit well to the pore-forming activity of some bacteriocins, depleting the cell of the membrane potential (Cotter et al., 2005). Under these conditions, Listeria cells are severely stressed, which is demonstrated

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by the induction of general stress proteins (rpsF, rsbX, rbfA, compatible solute transporter lmo1427, grpE, repressor of HrcA-lmo1475, ltrC, usp family protein lmo2673). The cells react by inducing genes involved in cell wall synthesis (lmo0265, ddlA, lmo1112, regulator of cell wall synthesis-lmo1800) and by changing the lipid composition (branched-chain lipid synthase-lmo1754). A further large group of highly induced genes includes those involved in DNA repair and maintenance (recF, inosine monophosphate dehydrogenase-lmo0132, pyrAE, recS). Interestingly, genes involved in bacteriocin production are induced which could be evidence for a counterattack mounted by L. monocytogenes (mccE homolog-lmo2141, bacteriocin production-lmo2776). In addition, several detoxification systems for small compounds for, e.g., export of harmful low-molecular-weight substances are induced (lmo0134, lmo0749, lmo0979, lmo1117, lmo1505, and lmo2723). Other genes induced are active in iron uptake, potassium balance, pH maintenance, and sulfur metabolism. A further prominent group of induced genes comprises several regulators. Most interestingly is the observation that bvrBlmo2787 and bvrA-lmo2788 specifically repress a PrfA response, with PrfA being the principal regulator of virulence genes (Brehm et al., 1999).

Bioinformatic tools for Listeria research Large-scale genomic analyses necessitate the development of bioinformatics tools for predictions, comparisons, and visualization. To visualize the circular architecture of a microbial genome, the software package GenomeViz was implemented (Ghai et al., 2004). This allows one to plot data from several different experiments or predictions on a single interactive circular map making it possible, for example, to compare predictions of horizontally transferred genes of several genomes in a single plot. Any kind of quantitative (e.g. microarrays) or qualitative (e.g. COGs) data may be plotted and visualized. This helps provide broad overviews of interesting genomic features across several genomes. A large number of prokaryotic genomes are provided with the software for immediate visualization. In addition, programs are available for common file reformatting tasks for GenomeViz (Ghai et al., 2004; http://www.uniklinikum-giessen.de/genome/ genomeviz/intro.html). To visualize conservation of genes across related genomes GECO, a browser-based program was developed (Kuenne et al., 2007). GECO makes it possible to ‘‘walk’’ along a reference genome and compare regions between related genomes. It is easy to visualize regions of gene insertions, deletions, or frameshifts that may be relevant to development of pathogenicity. Orthologs in different genomes are plotted with same colors to enable easy

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identification of conservation and divergence. Multiple genomes must be visualized for understanding and identifying important regions so the software currently provides visualization for all available microbial genomes (400) (Kuenne et al., 2007; http://bioinfo.mikrobio. med.uni-giessen.de/databases/geco.php). Bacterial surface proteins are crucial to interaction with the environment, especially the host immune response, and represent highly relevant candidates for vaccine design and pharmacological intervention. A software pipeline, AUGUR, to automate analysis of all major surface protein types in Gram-positive bacteria has been developed (Billion et al., 2006). The pipeline integrates several motif prediction tools, performs ortholog analyses on all genomes, and provides querying and visualization of the results. Both predefined as well as customized hidden Markov models and neural networks are used in predicting signal peptides, lipoproteins, LPXTG motifs, GW modules, LRR domains, LysM and NlpC/P60 domains, and transmembrane helices. This provides a comprehensive coverage of the surface proteome across all gram-positive genomes. Comparative visualization of distribution of any motif in any set of genomes is possible in AUGUR. Experimentally verified results are being added to the database with the aim to continuously improve the prediction results for this important subproteome (Billion et al., 2006; http://bioinfo.mikrobio.med.unigiessen.de/databases/augur.php). No large-scale proteomics project is possible without appropriate bioinformatic tools. Therefore, LEGER (Dieterich et al., 2006a; http://leger2.helmholtz-hzi.de/ cgi-bin/expLeger.pl), a program to support functional genome analyses by combining information obtained by applying bioinformatic methods and from public databases to improve the original annotations, was developed. LEGER is the first comprehensive Listeria information system focusing on the functional assignment of genes and proteins including sub-cellular localization, alleviates the functional exploration of complex data, and presents results of systematic postgenome studies, thus facilitating comparative analyses combining computational and experimental results. LEGER was then extended by the VIS-O-BAC visualization tools (Dieterich et al., 2006b; http://leger2. helmholtz-hzi.de/cgi-bin/vis-o-bac.pl) that permit the visualization of regulatory information derived from typical ‘‘omic’’ approaches with regard to three biologically relevant aspects, the genome structure (operon organization), the organization of genes in pathways (KEGG) and the genes to GO terms. These graphical visualizations clearly accelerate both the validation of regulatory information and the detection of affected biological processes. VIS-O-BAC currently supports the exploration of both transcriptomics and proteomics data set for 25 bacterial species. The web service

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MineBlast (Dieterich et al., 2005; http://leger2. helmholtz-hzi.de/cgi-bin/MineBlast.pl) was added for literature searches and presentation based on data mining results from PubMed, starting from protein sequences of interest. A test with 74 selected protein sequences of L. monocytogenes EGD-e that were originally annotated without functional assignment (Glaser et al., 2001) revealed for 50% (37 proteins) of all subjected sequences additional functional information that could be extracted from UniProt and PubMed. For 22 proteins, a highly reliable functional annotation was retrieved from homologs, and in five of these cases, precise gene names can now be added to the corresponding database entries. The results of two genome-wide analyses of L. monocytogenes EGD-e and L. innocua are included in the latest LEGER database release.

Outlook and perspectives The studies described above represent the firstgeneration exploration of genomic data in Listeria spp. to understand genome evolution and diversity, to examine expression transcriptomics, proteomics, and analyze physiological aspects of metabolism utilized by the bacteria when growing in diverse and varied environments. This information is also being used to explore strategies used by the bacteria when growing in microbial consortia on ripening smear cheese. Clearly, much more remains to be developed and the potential of these technologies is only now being realized. Thus questions regarding RNA stability, the role of small RNAs in regulating growth and virulence, and the transcriptional circuits with their overlapping functions remain to be studied. Factors that determine protein localization, as for example in the polarized ActAmediated actin polymerization during intracellular growth, as well as those regulating post-translational modifications are poorly understood. Specific expression patterns, i.e. transcriptome- and proteome-based signatures in response to physiological and antimicrobial stress could be used to identify new targets for preventing contamination of the food chain. This spectrum of technologies could also be used to study cellular physiology under in vitro conditions, as described above, as a prerequisite to understanding the pathophysiology of L. monocytogenes during host infection. As we understand how the transition from the environment to the food chain and finally the infected host cell is sensed and mediated by the bacterium, this information can be applied to understand if and how other pathogens of the low-G+C family of organisms, such as Staphylococcus, Streptococcus, Clostridia, and Bacillus, apply these strategies,

and may open new avenues for control and prevention of disease.

Acknowledgments The work reported herein was funded by the Bundesministerium fu¨r Bildung und Forschung (BMBF) within the PathoGenoMik Network of the National Bacterial Genomics Initiative. Work at the Institut Pasteur received financial support from the Re´seau National des Genopoles.

References Alexander, A.V., Walker, R.L., Johnson, B.J., Charlton, B.R., Woods, L.W., 1992. Bovine abortions attributable to Listeria ivanovii: four cases (1988–1990). J. Am. Vet. Med. Assoc. 200, 711–714. Barabote, R.D., Saier Jr., M.H., 2005. Comparative genomic analyses of the bacterial phosphotransferase system. Microbiol. Mol. Biol. Rev. 69, 608–634. Baumgartner, M., Karst, U., Gerstel, B., Loessner, M., Wehland, J., Jansch, L., 2007. Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes. J. Bacteriol. 189, 313–324. Bierne, H., Mazmanian, S.K., Trost, M., Pucciarelli, M.G., Liu, G., Dehoux, P., Jansch, L., Garcia-del Portillo, F., Schneewind, O., Cossart, P., 2002. Inactivation of the srtA gene in Listeria monocytogenes inhibits anchoring of surface proteins and affects virulence. Mol. Microbiol. 43, 869–881. Billion, A., Ghai, R., Chakraborty, T., Hain, T., 2006. Augur – a computational pipeline for whole genome microbial surface protein prediction and classification. Bioinformatics 22, 2819–2820. Brehm, K., Ripio, M.T., Kreft, J., Vazquez-Boland, J.A., 1999. The bvr locus of Listeria monocytogenes mediates virulence gene repression by beta-glucosides. J. Bacteriol. 181, 5024–5032. Brosch, R., Catimel, B., Milon, G., Buchrieser, C., Vindel, E., Rocourt, J., 1993. Virulence heterogeneity of Listeria monocytogenes strains from various sources (food, human, animal) in immunocompetent mice and its association with typing characteristics. J. Food Prot. 56, 296–301. Buchrieser, C., Rusniok, C., Kunst, F., Cossart, P., Glaser, P., 2003. Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: clues for evolution and pathogenicity. FEMS Immunol. Med. Microbiol. 35, 207–213. Call, D.R., Borucki, M.K., Besser, T.E., 2003. Mixed-genome microarrays reveal multiple serotype and lineage-specific differences among strains of Listeria monocytogenes. J. Clin. Microbiol. 41, 632–639. Carlton, R.M., Noordman, W.H., Biswas, B., de Meester, E.D., Loessner, M.J., 2005. Bacteriophage P100 for control of Listeria monocytogenes in foods: genome sequence, bioinformatic analyses, oral toxicity study, and application. Regul. Toxicol. Pharmacol. 43, 301–312.

ARTICLE IN PRESS T. Hain et al. / International Journal of Medical Microbiology 297 (2007) 541–557

Carnio, M.C., Holtzel, A., Rudolf, M., Henle, T., Jung, G., Scherer, S., 2000. The macrocyclic peptide antibiotic micrococcin P(1) is secreted by the food-borne bacterium Staphylococcus equorum WS 2733 and inhibits Listeria monocytogenes on soft cheese. Appl. Environ. Microbiol. 66, 2378–2384. Chand, P., Sadana, J.R., 1999. Outbreak of Listeria ivanovii abortion in sheep in India. Vet. Rec. 145, 83–84. Chatterjee, S.S., Hossain, H., Otten, S., Kuenne, C., Kuchmina, K., Machata, S., Domann, E., Chakraborty, T., Hain, T., 2006. Intracellular gene expression profile of Listeria monocytogenes. Infect. Immun. 74, 1323–1338. Chibani-Chennoufi, S., Dillmann, M.L., Marvin-Guy, L., Rami-Shojaei, S., Brussow, H., 2004. Lactobacillus plantarum bacteriophage LP65: a new member of the SPO1-like genus of the family Myoviridae. J. Bacteriol. 186, 7069–7083. Chico-Calero, I., Suarez, M., Gonzalez-Zorn, B., Scortti, M., Slaghuis, J., Goebel, W., Vazquez-Boland, J.A., 2002. Hpt, a bacterial homolog of the microsomal glucose-6-phosphate translocase, mediates rapid intracellular proliferation in Listeria. Proc. Natl. Acad. Sci. USA 99, 431–436. Collins, M.D., Wallbanks, S., Lane, D.J., Shah, J., Nietupski, R., Smida, J., Dorsch, M., Stackebrandt, E., 1991. Phylogenetic analysis of the genus Listeria based on reverse transcriptase sequencing of 16S rRNA. Int. J. Syst. Bacteriol. 41, 240–246. Cotter, P.D., Hill, C., Ross, R.P., 2005. Bacteriocins: developing innate immunity for food. Nat. Rev. Microbiol. 3, 777–788. Dennis, S.M., 1975. Perinatal lamb mortality in Western Australia. 6. Listeric infection. Aust. Vet. J. 51, 75–79. Desvaux, M., Hebraud, M., 2006. The protein secretion systems in Listeria: inside out bacterial virulence. FEMS Microbiol. Rev. 30, 774–805. Dieterich, G., Karst, U., Wehland, J., Jansch, L., 2005. MineBlast: a literature presentation service supporting protein annotation by data mining of BLAST results. Bioinformatics 21, 3450–3451. Dieterich, G., Karst, U., Fischer, E., Wehland, J., Jansch, L., 2006a. LEGER: knowledge database and visualization tool for comparative genomics of pathogenic and non-pathogenic Listeria species. Nucleic Acids Res. 34, D402–D406. Dieterich, G., Karst, U., Wehland, J., Jansch, L., 2006b. VISO-BAC: exploratory visualization of functional genome studies from bacteria. Bioinformatics 22, 630–631. Doumith, M., Buchrieser, C., Glaser, P., Jacquet, C., Martin, P., 2004a. Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. J. Clin. Microbiol. 42, 3819–3822. Doumith, M., Cazalet, C., Simoes, N., Frangeul, L., Jacquet, C., Kunst, F., Martin, P., Cossart, P., Glaser, P., Buchrieser, C., 2004b. New aspects regarding evolution and virulence of Listeria monocytogenes revealed by comparative genomics and DNA arrays. Infect. Immun. 72, 1072–1083. Doumith, M., Jacquet, C., Gerner-Smidt, P., Graves, L.M., Loncarevic, S., Mathisen, T., Morvan, A., Salcedo, C., Torpdahl, M., Vazquez, J.A., Martin, P., 2005. Multicenter validation of a multiplex PCR assay for differentiating the

555

major Listeria monocytogenes serovars 1/2a, 1/2b, 1/2c, and 4b: toward an international standard. J. Food Prot. 68, 2648–2650. Drider, D., Fimland, G., Hechard, Y., McMullen, L.M., Prevost, H., 2006. The continuing story of class IIa bacteriocins. Microbiol. Mol. Biol. Rev. 70, 564–582. Eisenreich, W., Slaghuis, J., Laupitz, R., Bussemer, J., Stritzker, J., Schwarz, C., Schwarz, R., Dandekar, T., Goebel, W., Bacher, A., 2006. 13C isotopologue perturbation studies of Listeria monocytogenes carbon metabolism and its modulation by the virulence regulator PrfA. Proc. Natl. Acad. Sci. USA 103, 2040–2045. Eppert, I., Valdes-Stauber, N., Gotz, H., Busse, M., Scherer, S., 1997. Growth reduction of Listeria spp. caused by undefined industrial red-smear cheese cultures and bacteriocin-producing Brevibacterium lines as evaluated in situ on soft cheese. Appl. Environ. Microbiol. 63, 4812–4817. Farber, J.M., Peterkin, P.I., 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55, 476–511. Ghai, R., Hain, T., Chakraborty, T., 2004. GenomeViz: visualizing microbial genomes. BMC Bioinform. 5, 198. Gill, D.A., 1937. Ovine bacterial encephalitits (circling disease) and the bacterial genus Listerella. Aust. Vet. J. 13, 46–56. Glaser, P., Frangeul, L., Buchrieser, C., Rusniok, C., Amend, A., Baquero, F., Berche, P., Bloecker, H., Brandt, P., Chakraborty, T., Charbit, A., Chetouani, F., Couve, E., de Daruvar, A., Dehoux, P., Domann, E., Dominguez-Bernal, G., Duchaud, E., Durant, L., Dussurget, O., Entian, K.D., Fsihi, H., Garcia-del Portillo, F., Garrido, P., Gautier, L., Goebel, W., Gomez-Lopez, N., Hain, T., Hauf, J., Jackson, D., Jones, L.M., Kaerst, U., Kreft, J., Kuhn, M., Kunst, F., Kurapkat, G., Madueno, E., Maitournam, A., Vicente, J.M., Ng, E., Nedjari, H., Nordsiek, G., Novella, S., de Pablos, B., Perez-Diaz, J.C., Purcell, R., Remmel, B., Rose, M., Schlueter, T., Simoes, N., Tierrez, A., Vazquez-Boland, J.A., Voss, H., Wehland, J., Cossart, P., 2001. Comparative genomics of Listeria species. Science 294, 849–852. Goetz, M., Bubert, A., Wang, G., Chico-Calero, I., VazquezBoland, J.A., Beck, M., Slaghuis, J., Szalay, A.A., Goebel, W., 2001. Microinjection and growth of bacteria in the cytosol of mammalian host cells. Proc. Natl. Acad. Sci. USA 98, 12221–12226. Gopal, S., Borovok, I., Ofer, A., Yanku, M., Cohen, G., Goebel, W., Kreft, J., Aharonowitz, Y., 2005. A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis. J. Bacteriol. 187, 3839–3847. Grau, F.H., Vanderlinde, P.B., 1990. Growth of Listeria monocytogenes on vacuum packaged beef. J. Food Prot. 53, 739–741. Hain, T., Steinweg, C., Chakraborty, T., 2006a. Comparative and functional genomics of Listeria spp. J. Biotechnol. 126, 37–51. Hain, T., Steinweg, C., Kuenne, C.T., Billion, A., Ghai, R., Chatterjee, S.S., Domann, E., Karst, U., Goesmann, A., Bekel, T., Bartels, D., Kaiser, O., Meyer, F., Puhler, A., Weisshaar, B., Wehland, J., Liang, C., Dandekar, T., Lampidis, R., Kreft, J., Goebel, W., Chakraborty, T., 2006b. Whole-genome sequence of Listeria welshimeri reveals common steps in genome reduction with Listeria

ARTICLE IN PRESS 556

T. Hain et al. / International Journal of Medical Microbiology 297 (2007) 541–557

innocua as compared to Listeria monocytogenes. J. Bacteriol. 188, 7405–7415. Heithoff, D.M., Conner, C.P., Hentschel, U., Govantes, F., Hanna, P.C., Mahan, M.J., 1999. Coordinate intracellular expression of Salmonella genes induced during infection. J. Bacteriol. 181, 799–807. Herro, R., Poncet, S., Cossart, P., Buchrieser, C., Gouin, E., Glaser, P., Deutscher, J., 2005. How seryl-phosphorylated HPr inhibits PrfA, a transcription activator of Listeria monocytogenes virulence genes. J. Mol. Microbiol. Biotechnol. 9, 224–234. Hong, E., Doumith, M., Duperrier, S., Giovannacci, I., Morvan, A., Glaser, P., Buchrieser, C., Jacquet, C., Martin, P., 2007. Genetic diversity of Listeria monocytogenes recovered from infected persons and pork, seafood and dairy products on retail sale in France during 2000 and 2001. Int. J. Food Microbiol. 114, 187–194. Jacquet, C., Gouin, E., Jeannel, D., Cossart, P., Rocourt, J., 2002. Expression of ActA, Ami, InlB, and listeriolysin O in Listeria monocytogenes of human and food origin. Appl. Environ. Microbiol. 68, 616–622. Jeffers, G.T., Bruce, J.L., McDonough, P.L., Scarlett, J., Boor, K.J., Wiedmann, M., 2001. Comparative genetic characterization of Listeria monocytogenes isolates from human and animal listeriosis cases. Microbiology 147, 1095–1104. Joseph, B., Przybilla, K., Stuhler, C., Schauer, K., Slaghuis, J., Fuchs, T.M., Goebel, W., 2006. Identification of Listeria monocytogenes genes contributing to intracellular replication by expression profiling and mutant screening. J. Bacteriol. 188, 556–568. Kuenne, C.T., Ghai, R., Chakraborty, T., Hain, T., 2007. GECO – linear visualization for comparative genomics. Bioinformatics 23, 125–126. Lauer, P., Chow, M.Y., Loessner, M.J., Portnoy, D.A., Calendar, R., 2002. Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors. J. Bacteriol. 184, 4177–4186. Loessner, M.J., Busse, M., 1990. Bacteriophage typing of Listeria species. Appl. Environ. Microbiol. 56, 1912–1918. Loessner, M.J., Calendar, R., 2005. The Listeria Bacteriophages. Oxford University Press, New York, pp. 593–601. Loessner, M.J., Rees, C.E.D., 2005. Listeria Phages: Basics and Applications. ASM Press, Washington, DC. Loessner, M.J., Inman, R.B., Lauer, P., Calendar, R., 2000. Complete nucleotide sequence, molecular analysis and genome structure of bacteriophage A118 of Listeria monocytogenes: implications for phage evolution. Mol. Microbiol. 35, 324–340. Loessner, M.J., Guenther, S., Steffan, S., Scherer, S., 2003. A pediocin-producing Lactobacillus plantarum strain inhibits Listeria monocytogenes in a multispecies cheese surface microbial ripening consortium. Appl. Environ. Microbiol. 69, 1854–1857. Mandin, P., Fsihi, H., Dussurget, O., Vergassola, M., Milohanic, E., Toledo-Arana, A., Lasa, I., Johansson, J., Cossart, P., 2005. VirR, a response regulator critical for Listeria monocytogenes virulence. Mol. Microbiol. 57, 1367–1380. Maoz, A., Mayr, R., Scherer, S., 2003. Temporal stability and biodiversity of two complex antilisterial cheese-ripening

microbial consortia. Appl. Environ. Microbiol. 69, 4012–4018. Marr, A.K., Joseph, B., Mertins, S., Ecke, R., Muller-Altrock, S., Goebel, W., 2006. Overexpression of PrfA leads to growth inhibition of Listeria monocytogenes in glucosecontaining culture media by interfering with glucose uptake. J. Bacteriol. 188, 3887–3901. Mead, P.S., Slutsker, L., Dietz, V., McCaig, L.F., Bresee, J.S., Shapiro, C., Griffin, P.M., Tauxe, R.V., 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5, 607–625. Mertins, S., Joseph, B., Goetz, M., Ecke, R., Seidel, G., Sprehe, M., Hillen, W., Goebel, W., Muller-Altrock, S., 2007. Interference of components of the phosphoenolpyruvate phosphotransferase system with the central virulence gene regulator PrfA of Listeria monocytogenes. J. Bacteriol. 189, 473–490. Milenbachs, A.A., Brown, D.P., Moors, M., Youngman, P., 1997. Carbon-source regulation of virulence gene expression in Listeria monocytogenes. Mol. Microbiol. 23, 1075–1085. Milohanic, E., Glaser, P., Coppee, J.Y., Frangeul, L., Vega, Y., Vazquez-Boland, J.A., Kunst, F., Cossart, P., Buchrieser, C., 2003. Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfA. Mol. Microbiol. 47, 1613–1625. Nelson, K.E., Fouts, D.E., Mongodin, E.F., Ravel, J., DeBoy, R.T., Kolonay, J.F., Rasko, D.A., Angiuoli, S.V., Gill, S.R., Paulsen, I.T., Peterson, J., White, O., Nelson, W.C., Nierman, W., Beanan, M.J., Brinkac, L.M., Daugherty, S.C., Dodson, R.J., Durkin, A.S., Madupu, R., Haft, D.H., Selengut, J., Van Aken, S., Khouri, H., Fedorova, N., Forberger, H., Tran, B., Kathariou, S., Wonderling, L.D., Uhlich, G.A., Bayles, D.O., Luchansky, J.B., Fraser, C.M., 2004. Whole genome comparisons of serotype 4b and 1/2a strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species. Nucleic Acids Res. 32, 2386–2395. O’Flaherty, S., Coffey, A., Edwards, R., Meaney, W., Fitzgerald, G.F., Ross, R.P., 2004. Genome of staphylococcal phage K: a new lineage of Myoviridae infecting Gram-positive bacteria with a low G+C content. J. Bacteriol. 186, 2862–2871. Premaratne, R.J., Lin, W.J., Johnson, E.A., 1991. Development of an improved chemically defined minimal medium for Listeria monocytogenes. Appl. Environ. Microbiol. 57, 3046–3048. Ramage, C.P., Low, J.C., McLauchlin, J., Donachie, W., 1999. Characterisation of Listeria ivanovii isolates from the UK using pulsed-field gel electrophoresis. FEMS Microbiol. Lett. 170, 349–353. Rocourt, J., 1999. The genus Listeria and Listeria monocytogenes: phylogenetic position, taxonomy, and identification. In: Ryser, E.T., Marth, E. (Eds.), Listeria, Listeriosis and Food safety. Macel Dekker Inc., New York, pp. 1–20. Rudol, M., Scherer, S., 2001. High incidence of Listeria monocytogenes in European red-smear cheese. Int. J. Food Microbiol. 63, 91–98. Sabet, C., Lecuit, M., Cabanes, D., Cossart, P., Bierne, H., 2005. LPXTG protein InlJ, a newly identified internalin

ARTICLE IN PRESS T. Hain et al. / International Journal of Medical Microbiology 297 (2007) 541–557

involved in Listeria monocytogenes virulence. Infect. Immun. 73, 6912–6922. Sallen, B., Rajoharison, A., Desvarenne, S., Quinn, F., Mabilat, C., 1996. Comparative analysis of 16S and 23S rRNA sequences of Listeria species. Int. J. Syst. Bacteriol. 46, 669–674. Schaumburg, J., Diekmann, O., Hagendorff, P., Bergmann, S., Rohde, M., Hammerschmidt, S., Jansch, L., Wehland, J., Karst, U., 2004. The cell wall subproteome of Listeria monocytogenes. Proteomics 4, 2991–3006. Seeliger, H.P., Jones, D., 1986. Genus Listeria. In: Sneath, P.H., Mair, N.S., Sharpe, M.E., Hold, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology. Williams and Wilkins, Baltimore. Sergeant, E.S., Love, S.C., McInnes, A., 1991. Abortions in sheep due to Listeria ivanovii. Aust. Vet. J. 68, 39. Stulke, J., Hillen, W., 2000. Regulation of carbon catabolism in Bacillus species. Annu. Rev. Microbiol. 54, 849–880. Thedieck, K., Hain, T., Mohamed, W., Tindall, B.J., Nimtz, M., Chakraborty, T., Wehland, J., Jansch, L., 2006. The MprF protein is required for lysinylation of phospholipids in listerial membranes and confers resistance to cationic antimicrobial peptides (CAMPs) on Listeria monocytogenes. Mol. Microbiol. 62, 1325–1339. Trost, M., Wehmhoner, D., Karst, U., Dieterich, G., Wehland, J., Jansch, L., 2005. Comparative proteome analysis of secretory proteins from pathogenic and nonpathogenic Listeria species. Proteomics 5, 1544–1557. Tsai, H.N., Hodgson, D.A., 2003. Development of a synthetic minimal medium for Listeria monocytogenes. Appl. Environ. Microbiol. 69, 6943–6945. Vazquez-Boland, J.A., Kuhn, M., Berche, P., Chakraborty, T., Dominguez-Bernal, G., Goebel, W., Gonzalez-Zorn, B., Wehland, J., Kreft, J., 2001. Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev. 14, 584–640. Waack, S., Keller, O., Asper, R., Brodag, T., Damm, C., Fricke, W.F., Surovcik, K., Meinicke, P., Merkl, R., 2006. Score-based prediction of genomic islands in prokaryotic genomes using hidden Markov models. BMC Bioinform. 7, 142.

557

Wehmhoner, D., Dieterich, G., Fischer, E., Baumgartner, M., Wehland, J., Jansch, L., 2005. ‘‘LaneSpector,’’ a tool for membrane proteome profiling based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis/liquid chromatography-tandem mass spectrometry analysis: application to Listeria monocytogenes membrane proteins. Electrophoresis 26, 2450–2460. Weis, J., Seeliger, H.P., 1975. Incidence of Listeria monocytogenes in nature. Appl. Microbiol. 30, 29–32. Wendlinger, G., Loessner, M.J., Scherer, S., 1996. Bacteriophage receptors on Listeria monocytogenes cells are the Nacetylglucosamine and rhamnose substituents of teichoic acids or the peptidoglycan itself. Microbiology 142, 985–992. Wenning, M., Theilmann, V., Scherer, S., 2006. Rapid analysis of two food-borne microbial communities at the species level by Fourier-transform infrared microspectroscopy. Environ. Microbiol. 8, 848–857. Wesley, I.V., 1999. Listeriosis in animals. In: Ryser, E.T., Marth, E. (Eds.), Listeria, Listeriosis and Food Safety. Marcel Dekker, New York, pp. 39–73. Wiedmann, M., Bruce, J.L., Keating, C., Johnson, A.E., McDonough, P.L., Batt, C.A., 1997. Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential. Infect. Immun. 65, 2707–2716. Zhang, C., Zhang, M., Ju, J., Nietfeldt, J., Wise, J., Terry, P.M., Olson, M., Kachman, S.D., Wiedmann, M., Samadpour, M., Benson, A.K., 2003. Genome diversification in phylogenetic lineages I and II of Listeria monocytogenes: identification of segments unique to lineage II populations. J. Bacteriol. 185, 5573–5584. Zimmer, M., Sattelberger, E., Inman, R.B., Calendar, R., Loessner, M.J., 2003. Genome and proteome of Listeria monocytogenes phage PSA: an unusual case for programmed +1 translational frameshifting in structural protein synthesis. Mol. Microbiol. 50, 303–317. Zink, R., Loessner, M.J., 1992. Classification of virulent and temperate bacteriophages of Listeria spp. on the basis of morphology and protein analysis. Appl. Environ. Microbiol. 58, 296–302.