Comparative and functional genomics of Listeria spp.

Journal of Biotechnology 126 (2006) 37–51

Review

Comparative and functional genomics of Listeria spp. Torsten Hain 1 , Christiane Steinweg 1 , Trinad Chakraborty ∗ Institute for Medical Microbiology, Justus-Liebig-University, Frankfurter Strasse 107, D-35392 Giessen, Germany Received 18 October 2005; received in revised form 8 February 2006; accepted 29 March 2006

Abstract The genus Listeria comprises a group of non-sporulating, Gram-positive, soil bacteria belonging to the low G + C group of microorganisms. The genus consists of only six species, L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua, L. welshimeri, and L. grayi. L. monocytogenes and L. ivanovii are the only known pathogens of this group. Comparative whole-genome sequencing of representative strains comprising the entire genus is currently being performed and nearing completion. In the genus Listeria, genome reduction has led to the generation of non-pathogenic species from pathogenic progenitor strains. Indeed, many of the regions absent in the non-pathogenic species represent commonly deleted genes. Speciation and diversity of strains has been achieved by horizontal gene transfer of DNA encoding novel genes probably required for niche specific survival. The sequencing of several listerial genomes has also been accompanied by studies using global strategies involving wholegenome transcriptional profiling and proteomics to examine the adaptative changes of L. monocytogenes to growth in different environments and to catalogue the genes mediating these responses. We review this data and present information on the expression profile of L. monocytogenes EGD-e inside the vacuolar and the cytosolic environments of the host cell using whole-genome microarray analysis. Of the 484 genes regulated during intracellular growth 41 genes are species-specific, being absent from the genome of the non-pathogenic L. innocua CLIP 11262 strain. There were 25 genes that are strain-specific i.e. absent from the genome of the L. monocytogenes F2365 serotype 4b strain suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes in host cells. © 2006 Elsevier B.V. All rights reserved. Keywords: Listeria; Genome; Sequencing; Intracellular; Transcriptome; Proteome

Contents 1. 2. 3. ∗ 1

Comparative genomics of Listeria spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Status of the comparative listerial genome sequencing project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sequencing and annotation strategy of the L. welshimeri and the L. seeligeri genome . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. Tel.: +49 641 99 41250; fax: +49 641 99 41259. E-mail address: [email protected] (T. Chakraborty). Both coauthors contributed equally to the work.

0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.03.047

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T. Hain et al. / Journal of Biotechnology 126 (2006) 37–51

General chromosome features of Listeria species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Origin of pathogenicity of the genus Listeria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA-based microarrays for identification and discrimination of Listeria species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transcriptome analysis of L. monocytogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The intracellular gene expression profile of L. monocytogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Global proteomic analysis of L. monocytogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Comparative genomics of Listeria spp. The genus Listeria comprises a group of Grampositive bacteria with low GC-content, closely related to the genera Bacillus, Clostridium, Enterococcus, Streptococcus and Staphylococcus. Listeria spp. are facultative, anaerobic, non-sporulating rods which have no capsule and are motile at 10–25 ◦ C (Collins et al., 1991; Rocourt, 1999; Sallen et al., 1996). Listeria have been isolated from a variety of origins including soil, water, plants, faeces, decaying vegetables, meat, seafood, diary 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 ◦ C) (Grau and Vanderlinde, 1990). The genus Listeria consists of six different 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 overwhelmingly 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 ubiquity of Listeriae enables them to enter the food-processing environments and the food chain. Moreover, the ability of the bacterium to grow at refrigerating temperatures increases the risk of food contamination. Clinical symptoms often manifest as meningitis, meningoencephalitis, septicaemia, abortion, prenatal infection and also gastroenteritis (Vazquez-Boland et al., 2001b). The occurrence of listeriosis is quite low

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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). Phylogenetic analysis based on the 16S and 23SrRNA coding genes as well as the prs, ldh, vclA, vclB and the iap gene indicated that the five out of six Listeria species are divided in two lineages. L. monocytogenes and L. innocua form one group while the second group includes L. welshimeri, L. ivanovii, and L. seeligeri. In the latter group, L. welshimeri appears to be the most distant species while L. grayi forms the deepest branch within the genus (Fig. 1) (Schmid et al., 2005). Within L. monocytogenes three different evolutionary branches have been distinguished that correlate with the serotypes (Bibb et al., 1989; Brosch et

Fig. 1. Phylogenetic tree of the genus Listeria based on 16S and 23 S rRNA, iap, prs, vclB and ldh (Schmid et al., 2005). Non-pathogenic and pathogenic strains are marked in blue and red, respectively.

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al., 1994; Graves et al., 1994; Piffaretti et al., 1989). Thus, serovars 1/2a, 1/2c and 3c strains belong to lineage I; serovars 4b, 1/2b and 3b strains to lineage II; and serovars 4a and 4c strains to lineage III. Lineage I serovars are associated with sporadic cases in humans and animals while lineage II serovars are responsible for epidemic outbreaks of foodborne infections as well as sporadic cases in humans and animals. Serovars of lineage III have been almost solely isolated from animal hosts (Jeffers et al., 2001). A hallmark of the pathogenic Listeria species is their ability to breach endothelial and epithelial barriers of the infected host including the intestinal, blood–brain and fetoplacental barrier. Both L. monocytogenes and L. ivanovii are facultative intracellular parasites that are able to invade and replicate in phagocytic and nonphagocytic cells (Vazquez-Boland et al., 2001b). The infectious life cycle of L. monocytogenes in tissue culture cells has been studied in detail (Cossart et al., 2003). The entry of the bacterium into the eukaryotic cell and the incorporation into a membranebound vacuole is triggered by two proteins, internalins InlA and InlB, which are expressed on the surface of the bacterium (Gaillard et al., 1991; Lecuit et al., 1997). Remarkably, L. monocytogenes reveals host cell tropism due to the specificity of InlA for human ECadherin (Lecuit et al., 1999; Schubert et al., 2002; Pizarro-Cerda et al., 2004) and for InlB to the receptor for the globular head of the complement factor C1q (gC1q-R), with the hepatocyte growth factor receptor (c-Met) and with glycosaminoglycans including heparan sulphate (Pizarro-Cerda et al., 2004). The most inventive feature of L. monocytogenes is its ability to survive and multiply within eukaryotic host cells and to induce cell-to-cell spread. The genes responsible for the intracellular life cycle of the bacterium are located within a locus of the chromosome between prs and ldh which is referred as LIPI-1 (Listeria pathogenicity island 1) (VazquezBoland et al., 2001b). Following the lysis of this vacuole, which is mediated by two secreted proteins, the pore-forming hemolytic toxin listeriolysin (LLO) and phosphatidylinositol-specific phospholipase C (PlcA) (Cossart et al., 1989; Dramsi and Cossart, 2002; Marquis et al., 1995; Portnoy et al., 1988) the bacterium replicates in the cytosol. The bacterial surface protein ActA leads to actin-based intra- and intercellular movements and finally cell-to-cell spread where

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the bacterium upon contact with the plasma membrane induces pseudopod-like protrusions that invaginate into a neighbouring cell and can be ingested (Mounier et al., 1990; Tilney and Portnoy, 1989). The lysis of the two plasma membranes is mediated by LLO together with the phophatidylcholine-specific phospholipase C (PlcB) which enables the bacterium to infect the second cell (Vazquez-Boland et al., 2001b) (Fig. 2). Another species-specific Listeria pathogenicity island 2 (LIPI2) was described for L. ivanovii, which contains apart from the membrane-damaging virulence factor SmcL,

Fig. 2. Intracellular life cycle of L. monocytogenes. (A) Schematic representation of the virulence factors involved in the cellular infection process adapted from Vazquez-Boland et al. (2001b). (B) Immunofluorescence microscopic image of infected P388D1 macrophages with wild type EGD-e 8 h post-infection (magnification, ×63) as described by Chatterjee et al. (2006). FITC-phalloidin was used for actin tail staining and L. monocytogenes was detected using ␣-ActA monoclonal antibody N81 (Niebuhr et al., 1993) followed by a secondary Cy3 conjugated goat ␣-mouse IgG polyclonal antibody (Dianova).

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10 genes encoding for internalin (Inl) proteins located on a 22 kb chromosomal locus (Gonzalez-Zorn et al., 2000; Vazquez-Boland et al., 2001a,b; DominguezBernal et al., 2006). The relatively small number of species comprising the genus Listeria and the clear differences that separate pathogenic from non-pathogenic species make these bacteria attractive as models to examine for the evolution of pathogenicity in this genus. Also, the spectrum of ecological niches occupied by these bacteria, i.e., from the abiotic environment to the intracellular compartments of the infected host cell, raises questions as to how these features have evolved within these species. With these considerations in mind, we initiated a comparative genome sequencing project to investigate the differences in gene composition and organization between the genomes of pathogenic and apathogenic Listeria species which contribute to the virulence, tropism and survival of these bacteria in widely diverse environmental conditions. 2. Status of the comparative listerial genome sequencing project Currently, the complete genome sequences of L. monocytogenes EGD-e (serotype 1/2a) (Glaser et al., 2001), L. monocytogenes F2365 (serotype 4b) (Nelson et al., 2004) and L. innocua CLIP 11262 (serotype 6a) (Glaser et al., 2001) 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) have recently been completed and the genomes of a L. monocytogenes serotype 4a strain and L. grayi are currently being sequenced (http://www.genomesonline.org). When completed, this study will not only comprise data from all species but will also provide information on lineage-specific gene content in the different L. monocytogenes strains. 3. Sequencing and annotation strategy of the L. welshimeri and the L. seeligeri genome In order to sequence the genomes of L. welshimeri SLCC 5334 and L. seeligeri SLCC 3954 we constructed

small (1.5–3 kb) and medium (5 kb) insert plasmid libraries for a shotgun sequencing approach as well as Fosmid and BAC libraries with large fragments of around 40 and 50 kb for filling gaps and contig ordering. Clones from all libraries were sequenced from both ends. The obtained sequences were evaluated and then assembled using the Phred/Phrap/Consed software (Ewing et al., 1998; Ewing and Green, 1998; Gordon et al., 1998). Sequence gaps in both genomes were first closed by primer walking on plasmid, BAC and Fosmid clones using the program Autofinish (Gordon et al., 2001). The remaining physical gaps were closed by sequencing of PCR products. For ordering the contigs we aligned the contigs with the L. monocytogenes EGD-e genome as a reference by using the program Nucmer (Delcher et al., 2002). Repetitive elements, like rrn-operons which occurred in both genomes were assembled independently. To re-sequence low quality regions we applied primer walking, resequencing of reads and sequencing of PCR products. The GenDB 2.0 (Meyer et al., 2003) annotation system was used for a preliminary automatic annotation. All orthologs between the L. welshimeri and the L. seeligeri genome and the genomes of L. monocytogenes EGD-e, L. innocua CLIP 11262 and L. monocytogenes F2365 were predicted. The remaining genes were manually annotated.

4. 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 in length. Listeriae are members of the low G + C group of bacteria that includes Bacillus, Lactococcus, Staphylococcus and Streptococcus and have a G + C− content of between 36.4% and 41.5%. The genome sequence encodes approximately 2800 putative protein coding genes, of which ∼65% genes have an assigned function. Although both strainand serotype-specific genes were identified, all Listeria genomes revealed a highly conserved 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 (IS) elements in all sequenced Listeria genomes. However, DNA rearrangements of single

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gene loci are present in Listeria genomes even though it is a rather rare event.

5. Origin of pathogenicity of the genus Listeria To look for clues to the evolution of the genus Listeria we compared the currently sequenced genomes of the apathogenic species L. welshimeri and L. seeligeri with respect to the L. monocytogenes EGD-e genome. A global analysis of chromosomal regions present in L. welshimeri and L. seeligeri in comparison to L. monocytogenes EGD-e revealed genome reduction resulting from deletions within the L. welshimeri and L. seeligeri genome, respectively. Another well-investigated hot spot for evolution is the chromosomal locus between prs and ldh of pathogenic Listeriae harbouring the LIPI-1 element. It has previously been established that six of the virulence factors required for key steps in the intracellular life cycle (prfA, plcA, hly, mpl, actA and plcB) are clustered on the 9-kb LIPI-1 chromosomal fragment. The overall organization of LIPI-1 is conserved between the two pathogenic species L. monocytogenes and L. ivanovii but all of these genes are missing in the non-pathogenic species with one exception, L. seeligeri (Chakraborty et al., 2000; Kreft et al., 1999; Vazquez-Boland et al., 2001a). In this species, an ORF is inserted in the opposite orientation between plcA and prfA which disrupts the positive autoregulatory loop required for PrfA-dependent virulence gene activation (Karunasagar et al., 1997; Kreft et al., 1999) (Fig. 3). Similar insertions are found between several genes of the LIPI-1 cluster of L. seeligeri and suggest that insertional inactivation of genes within the LIPI-1 locus may have preceeded gene deletions leading to the total loss of this gene cluster in the nonpathogenic species. Thus, the genus Listeria probably evolved by genome reduction leading to the generation of non-pathogenic species from a progenitor strain already harboring the virulence genes. Indeed many of the regions absent in the non-pathogenic species represent commonly deleted genes. This hypothesis also explains the existence of a natural atypical Listeria innocua strain containing the virulence gene cluster which could be a relic of the common ancestor of L. monocytogenes and L. innocua (Johnson et al., 2004).

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We also identified a number of novel genes both in the L. welshimeri and L. seeligeri genome which are probably required for niche specific survival and leading to the speciation and diversity of this Listeria species. Thus, the genomes of L. monocytogenes EGD-e and L. innocua CLIP 11262 contain 270 (9.5%) L. monocytogenes EGD-e- and 149 (5%) L. innocuaspecific genes, respectively. These encode for surface and transport proteins, as well as for regulatory factors. Similarly, whole-genome comparisons of L. monocytogenes strains F2365 (4b), F6854 (1/2a), H7858 (4b) and EGD-e (1/2a) revealed a total of 51, 97, 69 and 61 strain-specific genes, respectively. Several of the strainspecific genes encode putative surface-associated proteins and pathways for the transport and metabolism of carbohydrates (Nelson et al., 2004). While surface proteins are important for the interaction of the bacterium with its environment, regulatory and transport proteins reflect its ability to adapt and to colonize a variety of different ecosystems (Buchrieser et al., 2003; Glaser et al., 2001). The observation that during the evolution of the genus Listeria both reduction and gain of genes has played an important role leads to the hypothesis that as a consequence of deletion of the virulence gene cluster the primordial ancestor ceased to be only an intracellular pathogen and adapted to an extracellular way of life by the uptake of novel genes which enabled it to colonize different ecological niches. The importance of uptake of DNA encoding novel genes which are required for niche specific survival is reflected by the relative small number of only four nonpathogenic Listeria species which survived gene loss and subsequent selection. Bacteriophages may have played an important role in gene acquisition because in all sequenced Listeria genomes at least one prophage or remnants of bacteriophage genomes have been identified. Currently, two temperate bacteriophages, A118 and PSA, were isolated from L. monocytogenes serovar 1/2a and L. monocytogenes serovar 4b strains, respectively and their nucleotide sequences completely determined (Loessner et al., 2000; Zimmer et al., 2003). Comparative genome analysis of both phages showed very few similarities between each other, which may reflect that the host cell specificity of these two listerial phages is also manifested by differences among diverse lineages of L. monocytogenes (Zimmer et al., 2003).

42 T. Hain et al. / Journal of Biotechnology 126 (2006) 37–51 Fig. 3. Virulence gene cluster of L. monocytogenes EGD-e and its orthologs among other Listeriae using Geco (unpublished software). Listerial species and strains used in this analysis: L. monocytogenes EGD-e (serotype 1/2a) and L. innocua CLIP 11262 (serotype 6a) (Glaser et al., 2001), L. monocytogenes F2365 (serotype 4b) and the incomplete genomes of L. monocytogenes F6854 (serotype 1/2a) and L. monocytogenes H7858 (serotype 4b) (Nelson et al., 2004), genome sequences of L. welshimeri SLCC 5334 (serotype 6b), L. seeligeri SLCC 3954 (serotype 1/2b) and L. ivanovii PAM 55 (serotype 5) have recently been completed and the genomes of a L. monocytogenes serotype 4a strain and L. grayi are currently being sequenced (http://www.genomesonline.org). Genes that are >50% identical by blastp analysis with >75% coverage in protein length are marked using identical colours.

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In contrast, the transmission of transposons and plasmids may not have played a major role in the evolution of the genus Listeria since only the L. innocua CLIP 11262 and the L. monocytogenes H7858 genome carry a single plasmid. Furthermore, the insertion of a Tn916-like transposon has solely been described for L. monocytogenes EGD-e (Glaser et al., 2001; Nelson et al., 2004).

6. DNA-based microarrays for identification and discrimination of Listeria species The availability of the published genome sequence for L. monocytogenes (Glaser et al., 2001) allowed the generation of a whole-genome microarray to examine relationships between listerial strains at both species and serovar level as well as to probe for regulatory networks enabling listerial growth and survival under a wide variety of growth conditions. Several microarray-based strategies were developed to differentiate between the six listerial species (Volokhov et al., 2002) and for discrimination among L. monocytogenes serovars (Borucki et al., 2003; Rudi et al., 2003) and phylogenetic lineages (Call et al., 2003; Zhang et al., 2003). Microarray based assays were also applied to investigate the genome evolution within the genus Listeria (Doumith et al., 2004) and for identification of natural atypical L. innocua strains harboring genes of the LIPI-1 (Johnson et al., 2004). The accurate detection of foodborne pathogens such as L. monocytogenes is an important step forward to ensure food safety and proper control of industrial processes in food technology. The Center for Food Safety and Applied Nutrition (FDA, USA) recently developed a multipathogen oligonucleotide array (FDA-1) to identify foodborne pathogenic bacteria that includes a multi gene probe set for Listeriae for environmental and biodefense application (Sergeev et al., 2004).

7. Transcriptome analysis of L. monocytogenes Whole-genome transcriptional profiling of pathogenic bacteria also permits new insights into adaptive responses activated by the bacteria when growing in the natural environment or in the niche of

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the human host. In the past three years genome-wide transcriptome studies have been used to catalogue and analyze genes belonging to different regulons (PrfA, σ B , σ 54 and VirR) and to examine overlapping regulatory interactions. The first regulon that was mapped was the PrfAregulon (Milohanic et al., 2003). PrfA (positive regulatory factor A) is well known as the major regulator of the genes of the virulence gene cluster (prfA, plcA, hly, mpl, actA and plcB), internalins (inlA, inlB and inlC) and a hexose phosphate transporter (hpt). A whole-genome macroarray analysis comparing gene expression in the wild-type of L. monocytogenes EGDe to that of an isogenic PrfA-deletion mutant indicates that the PrfA-regulon consists of 73 differentially regulated genes that could be broadly divided into three groups. The first group consisted of the 10 known genes of the PrfA-regulon including genes of the virulence gene cluster (prfA, plcA, hly, mpl, actA and plcB), internalins (inlA, inlB and irpA) and the intracellular activated hexose phosphate transporter as well as two new genes lmo0788 and lmo2219 which harbour a PrfAbinding sequence in their upstream region. lmo0788 encodes for an unknown protein which shares similarities with BadF/BadG/BcrA/BcrD proteins that may be involved in ATP hydrolysis while lmo2219 codes for Parvulin-like peptidyl-prolyl isomerase and is similar to the PrsA homolog of B. subtilis. PrsA plays a major role in protein secretion as molecular chaperone by helping the post-translocational extracellular folding of several secreted proteins (Kontinen and Sarvas, 1993). The second group of PrfA-regulon genes were down regulated indicating that PrfA itself can act as an activator as well as a repressor protein. The members of this group (8 genes) appear to be involved in sugar transport and utilization. Seven of these genes are organized in an operon (lmo0178–0184). The third group comprises the largest class of genes up regulated by PrfA. Among these 53 genes only two genes, lmo2067 encoding for a bile salt hydrolase (BSH) and the gene for lmo0596 coding for an unknown protein, contain a PrfA consensus binding site in their flanking upstream region. Surprisingly, binding motifs for the alternative sigma factor, SigB (σ B ), were observed in the upstream region for 22 genes of this group. The existence of σ B binding motifs in genes up regulated by PrfA support the hypothesis that PrfA can directly or indirectly interact with other alternative sigma fac-

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tors such as σ B to induce expression of different sets of listerial genes. In addition, it has been shown that the promoter prfAp2 of the prfA gene is also a functional σ B -dependent promoter, and a SigB deletion mutant was impaired in the mice model of infection (Nadon et al., 2002). Notably, SigB contributed to invasion of L. monocytogenes by controlling expression of inlA and inlB (Kim et al., 2005). The alternative sigma factor, σ B is a well-known regulator of the general stress response in Gram-positive bacteria such as Listeria, Staphylococcus and Bacillus (Becker et al., 1998; Haldenwang, 1995; Wu et al., 1996). The activity of σ B in B. subtilis is known to be post-translationally regulated by a finely tuned network composed of two sequentially linked partnerswitching modules (Helmann, 1999). The sigB operon consists of sigB itself and seven regulators of sigB genes (rsb). Genes such as rsbW, rsbX regulate σ B negatively while rsbV is a positive regulator of σ B . Under normal conditions σ B is inactivated by antiSigB protein RsbW by direct binding. For an activation of the sigB regulon, sequestration of the RsbW protein (anti-σ B ) by RsbV (anti-anti-σ B ) has to occur which results in the release of the previously complexed σ B protein, and transcribes genes with σ B -dependent specificities (Abee and Wouters, 1999; Haldenwang, 1995; Helmann, 1999). In general induction of the sigB operon is influenced by different environmental stimuli such as starvation in stationary phase, heat, freeze and thaw cycles, acid and alkaline shock (Boylan et al., 1993; Gaidenko and Price, 1998; Volker et al., 1999). In B. subtilis, more than 100 stress-responsive genes have been assigned to be under the control of the σ B transcription factor (Petersohn et al., 2001; Price et al., 2001). Recently, based on a specialized 208-gene microarray 55 σ B -regulated genes were identified in L. monocytogenes (Kazmierczak et al., 2003). First, using Hidden Markov model-based searches for σ B consensus binding sites, 170 putative σ B consensus sequences within the genome of L. monocytogenes were identified. The specialized 208-gene microarray was subsequently created using 166 genes harboring σ B consensus sequences and also contained virulence and other genes involved in the general stress response. For 54 of the 55 σ B controlled genes upstream σ B consensus sequences were detected. These 55 genes controlled

by σ B were involved in the stress response (gadB, ctc, lmo1433 coding for a glutathione reductase and opuCoperon) and virulence (inlA, inlB and bsh). Thus, σ B regulates genes to adapt to environmental changes as well as genes involved in the virulence process of the pathogenic bacterium. The alternative sigma factor σ 54 is known to regulate several classes of genes involved in carbon and nitrogen metabolism, flagella biosynthesis and virulence (Studholme and Buck, 2000), in Gram-positives and Gram-negatives. The role of the alternative sigma factor σ 54 was examined by comparing the wild-type L. monocytogenes EGD-e and an isogenic σ 54 mutant (Arous et al., 2004). In this global study analyzing the regulatory function of σ 54 , 77 genes were identified by macroarray transcriptional profiling and nine proteins using two-dimensional gel electrophoresis. Most of the genes were directly involved in carbohydrate metabolism, particularly in pyruvate- and amino acidmetabolism and included several amino acid transporters. However, only the mannose specific PTS system mptACD was directly regulated by σ 54 revealing an indirect role of σ 54 in inducing the remaining genes. σ 54 was also involved in the sensitivity to antibacterial peptides such as subclass IIa bacteriocins (Robichon et al., 1997). A mpt mutant also showed reduced sensitivity to subclass IIa bacteriocins. The permease has been suggested to act as docking molecule for the membrane pore-forming bacteriocins (Dalet et al., 2001; Gravesen et al., 2002). Mouse infection studies have not revealed a role for the σ 54 mutant in virulence. It has been suggested that the bacterium compensates for the loss of the sigma factor activity by using alternative regulatory or metabolic systems. Recently, a novel virulence associated gene VirR was detected by signature tagged mutagenesis (STM) as being critical for survival and growth of L. monocytogenes EGD-e in mice (Mandin et al., 2005). VirR encodes for a response regulator and belongs to a two component system including the histidine kinase VirS. The VirR regulon was identified by comparison of the wild-type with its isogenic deletion mutant virR. This indicated that 12 genes were under control of the response regulator. Among these genes the dlt operon as well as a homolog of the mprF gene of S. aureus was observed. The gene products of the dlt operon are responsible for catalysing the incorpo-

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ration of d-alanine residues into the cell wall associated lipoteichoic acids (LTAs) (Abachin et al., 2002). MprF mediates the biosynthesis of lysylphosphatidylglycerol, which represents an important determinant in the resistance of S. aureus to defensins (Peschel et al., 1999; Staubitz et al., 2004). Both proteins seem to be involved in the alteration of the charge of bacterial cell surface. The authors speculate that impaired activation of the dlt operon or mprF controlled by VirR results in early elimination of Listeriae throughout the immune system of the host, because unmodified cell surface structure such as LTA’s or peptidoglycan will be easily recognized by Toll like receptors that activate components of the innate immune system (Takeda and Akira, 2005). The VirR regulon therefore comprises in addition to the PrfA-regulon, a second regulon that affects the virulence of the pathogenic bacterium.

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COG analysis compiling all three above-mentioned comparisons revealed that a large number of genes for carbohydrate and amino acid transport and metabolism are up regulated indicating a switch to alternative carbon sources as energy source. Genes belonging to the categories of cell cycle control and nucleotide transport and metabolism were down regulated indicating lowered replication and cell division activities within the host cell environment (Fig. 4). Comparative genome analysis of the intracellular gene repertoire of L. monocytogenes EGD-e (serovar 1/2a) with another strain of L. monocytogenes (serovar 4b) and the apathogenic strain L. innocua showed 41 species-specific and 25 strain-specific genes. These results reveal that the gene pool for intracellular replication among strains of the same species varies and suggest that different intracellularly induced gene sets may be responsible for sporadic or epidemic infections of this foodborne pathogen.

8. The intracellular gene expression profile of L. monocytogenes To investigate the intracellular transcriptional response of L. monocytogenes EGD-e in different compartments i.e. the vacuole and the host cell cytoplasm, we used two different listerial strains, wild-type EGD-e and the isogenic double mutant hlyplcA to infect P388D1 murine macrophage cells. To determine the intraphagosomal transcriptome profile of L. monocytogenes, total bacterial RNA isolated from hlyplcA cultured in BHI was compared to that isolated from hlyplcA 1 h post-infection. The expression profile of bacteria isolated from the cytosol of infected macrophages was detected by comparing whole RNA isolated from EGD-e cultured in BHI to that isolated from bacteria at 4 and 8 h post-infection. In this analysis, we identified 484 differential regulated genes, of which 301 genes were up regulated and 182 genes were down regulated (Chatterjee et al., 2006). Several different regulons were induced during the intracellular life cycle of the bacterium. We observed genes of the PrfA, CtsR, HrcA, σ B , σ 54 , OhrR and VirR regulon. Interestingly, the majority of genes regulated by VirR were also intracellularly up regulated excepting lmo1695 and lmo1696 confirming the potential role of the VirR regulon for intracellular survival and pathogenicity of the bacterium. A comprehensive

9. Global proteomic analysis of L. monocytogenes Whole cell proteomics is an important tool in bridging the divide between whole-genome transcription studies and to an understanding of cellular physiology in microorganisms. Two-dimensional (2-D) gel electrophoresis was used to analyze the response of L. monocytogenes under acidic conditions (Phan-Thanh, 2002) or following the application of osmotic shock conditions (Duche et al., 2002). Two of the 59 proteins identified following altered salinity of the growth medium were identified as Ctc and GbuA, both are known to be involved in the osmotic stress tolerance of the bacterium (Gardan et al., 2003; WemekampKamphuis et al., 2002). In addition, both proteins are also σ B -dependent (Cetin et al., 2004; Kazmierczak et al., 2003). To investigate the role of σ B in the acid response, the proteome expression profile of L. monocytognes EGD-e was compared to its isogenic deletion mutant sigB which was examined following acid exposure. Nine proteins were identified including 6-phosphofructokinase (Pfk), UDPglucose-4-epimerase (GalE), ClpP protease and an unknown protein Lmo1580, which were assigned as putative σ B -regulated proteins involved in acid adaptation (Wemekamp-Kamphuis et al., 2004).

46 T. Hain et al. / Journal of Biotechnology 126 (2006) 37–51 Fig. 4. Functional COG analysis using Augur (unpublished software) of intracellular differential regulated listerial genes after different infection times of P388D1 macrophages including the double mutant hlyplcA in BHI versus hlyplcA after 1 h post-infection, EGD-e in BHI versus EGD-e after 4 h post-infection and EGD-e in BHI versus EGD-e after 8 h post-infection. Up regulated genes are marked as red bars and down regulated genes are indicated as green bars for each COG category (http://www.ncbi.nlm.nih.gov/COG/). The data corresponding to the intracellular expression profile have been obtained from Chatterjee et al. (2006).

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Induction of biofilm formation caused by foodborne pathogenic bacteria such as L. monocytogenes represents a serious problem for food safety in foodprocessing industries. Thus, proteomic comparisons of the global protein expression profiling from biofilmand planktonic-grown cells of L. monocytogenes are highly relevant (Hefford et al., 2005; Helloin et al., 2003; Tremoulet et al., 2002). Hefford and colleagues identified nineteen proteins, which were up regulated in listerial biofilms. These genes are involved in the overall stress response, envelope and protein synthesis, biosynthesis, energy generation, and regulatory functions (Hefford et al., 2005). Comparison of the proteomes between L. monocytogenes EGD-e cells in exponential or stationary phase of growth at 37 ◦ C identified 38 differentially regulated proteins. These proteins were associated with translation, cellular metabolism and stress adaptation (Folio et al., 2004). Global changes of protein expression profile of L. monocytogenes ScottA grown in batch cultures from exponential growth to stationary phase, showed that more than 50% of all proteins identified in this study changed in their expression level in the transition process (Weeks et al., 2004). 2-D electrophoresis analysis was also used to create a partial proteome reference maps for L. monocytogenes EGD-e of exponentially grown cells under standardized conditions. This reference map was used to compare the wildtype EGD-e with four different L. monocytogenes food isolates of the serovars 1/2a and 1/2b indicating variations of the proteome reference maps among these serovars (Ramnath et al., 2003). The bacterial cell envelope serves as an interactive compartment to sense for different environmental conditions, e.g., by using two component systems, influx and efflux systems or by expressing a number of extracellular proteins which both are associated with the cell envelope and are secreted to the external environment. In addition, extracellular proteins play a major role in infection diseases caused by pathogenic bacteria. To exploit the whole proteome information of L. monocytogenes, the cell-membrane as well as the cellwall proteome and the secretome of L. monocytogenes were determined (Wehmhoner et al., 2005; Calvo et al., 2005; Schaumburg et al., 2004; Trost et al., 2005). 301 membrane proteins were analyzed by SDS-PAGE and liquid chromatography-tandem mass spectrometry to characterize the membrane subproteome (Wehmhoner

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et al., 2005). A gel independent approach using 2-D nanoliquid chromatography coupled to mass spectrometry was applied to identify novel listerial cell-wall associated proteins. Using the method, 30 proteins were identified in the cell wall proteome of L. monocytogenes (19 proteins) and the non-pathogenic strain L. innocua (11 proteins). The majority of the proteins (20) belonged to the class of LPXTG motif harboring proteins (Calvo et al., 2005). In a separate investigation of the cell wall subproteome of L. monocytogenes using 2-D electrophoresis analysis followed by N-terminal sequencing and mass spectrometry, 55 proteins were identified. For 50% of the proteins detected no specific protein secretion or anchoring motifs were discernible, but many members of the subproteome were known as proteins normally present in the bacterial cytoplasm. These so-called “moonlighting” proteins appear to have alternative functions in the cell wall (Schaumburg et al., 2004). Comparative proteome analysis indicated that 105 proteins for L. monocytogenes and 96 proteins for L. innocua respectively were secreted to the supernatant (Trost et al., 2005). Half of all identified proteins were detected in both species, but 43 proteins were specific for each species. Additionally, 16 proteins of L. monocytogenes and 7 proteins of L. innocua had no ortholog in the respective listerial species. These findings indicate that differences in the secretory protein repertoire may reflect as differences in the lifestyle of Listeriae.

10. Outlook The era of comparative genome sequencing of strains representing all species of the genus Listeria is well underway and is already providing insights into the evolution of virulence, tropism and survival strategies used by these bacterial species to persist the different ecological niches it occupies. Comparative transcriptome and proteome analysis investigating regulatory networks and subproteomes suggest common and distinct species- and strain-specific adaptive responses and imply subtle strategies used by different strains and species for survival and transmission in living and nonliving environments. This information provides access to niche specific candidate genes and proteins that will need to be evaluated by traditional physiological, biochemical and genetic approaches. These studies can

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lead not only to the development of novel ways to interrupt transmission and prevent foodborne disease but also to provide new targets for prophylactic and therapeutic strategies. Finally, as many of the low G + C bacterial genera comprise important pathogens such as Staphylococcus, Streptococcus, Clostridia, and Bacillus, information gained from the study of Listeria can add to an understanding to the evolution of virulence in this group of bacteria. Acknowledgements The work presented herein owes much to the foresight and leadership of Werner Goebel in the PathoGenoMik Network, and we are inordinately indebted to him for his support. We are grateful to R. Ghai for critical reading and for helpful discussion on the manuscript. We thank S.S. Chatterjee for providing the image of Listeria-infected cells and C. K¨unne and A. Billion for Figs. 3 and 4 using software programs Geco and Augur, respectively. We also thank C. Buchrieser and P. Glaser for information about the L. ivanovii and L. grayi genome sequencing projects. This work was supported by funds obtained from the BMBF through the Competence Network PathoGenoMik (PTJ-BIO//03u213B) to T.H. and T.C. References Abachin, E., Poyart, C., Pellegrini, E., Milohanic, E., Fiedler, F., Berche, P., Trieu-Cuot, P., 2002. Formation of d-alanyllipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes. Mol. Microbiol. 43, 1–14. Abee, T., Wouters, J.A., 1999. Microbial stress response in minimal processing. Int. J. Food Microbiol. 50, 65–91. 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. Arous, S., Buchrieser, C., Folio, P., Glaser, P., Namane, A., Hebraud, M., Hechard, Y., 2004. Global analysis of gene expression in an rpoN mutant of Listeria monocytogenes. Microbiology 150, 1581–1590. Becker, L.A., Cetin, M.S., Hutkins, R.W., Benson, A.K., 1998. Identification of the gene encoding the alternative sigma factor σ B from Listeria monocytogenes and its role in osmotolerance. J. Bacteriol. 180, 4547–4554. Bibb, W.F., Schwartz, B., Gellin, B.G., Plikaytis, B.D., Weaver, R.E., 1989. Analysis of Listeria monocytogenes by multilocus enzyme electrophoresis and application of the method to epidemiologic investigations. Int. J. Food Microbiol. 8, 233–239.

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