The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence
- Eva Trost1, 2,
- Lisa Ott3,
- Jessica Schneider1, 2, 4,
- Jasmin Schröder1,
- Sebastian Jaenicke4,
- Alexander Goesmann4,
- Peter Husemann5,
- Jens Stoye5,
- Fernanda Alves Dorella6,
- Flavia Souza Rocha6,
- Siomar de Castro Soares6,
- Vívian D'Afonseca6,
- Anderson Miyoshi6,
- Jeronimo Ruiz7,
- Artur Silva8,
- Vasco Azevedo6,
- Andreas Burkovski3,
- Nicole Guiso9,
- Olivier F Join-Lambert10,
- Samer Kayal11 and
- Andreas Tauch1Email author
© Trost et al; licensee BioMed Central Ltd. 2010
Received: 8 September 2010
Accepted: 30 December 2010
Published: 30 December 2010
Corynebacterium pseudotuberculosis is generally regarded as an important animal pathogen that rarely infects humans. Clinical strains are occasionally recovered from human cases of lymphadenitis, such as C. pseudotuberculosis FRC41 that was isolated from the inguinal lymph node of a 12-year-old girl with necrotizing lymphadenitis. To detect potential virulence factors and corresponding gene-regulatory networks in this human isolate, the genome sequence of C. pseudotuberculosis FCR41 was determined by pyrosequencing and functionally annotated.
Sequencing and assembly of the C. pseudotuberculosis FRC41 genome yielded a circular chromosome with a size of 2,337,913 bp and a mean G+C content of 52.2%. Specific gene sets associated with iron and zinc homeostasis were detected among the 2,110 predicted protein-coding regions and integrated into a gene-regulatory network that is linked with both the central metabolism and the oxidative stress response of FRC41. Two gene clusters encode proteins involved in the sortase-mediated polymerization of adhesive pili that can probably mediate the adherence to host tissue to facilitate additional ligand-receptor interactions and the delivery of virulence factors. The prominent virulence factors phospholipase D (Pld) and corynebacterial protease CP40 are encoded in the genome of this human isolate. The genome annotation revealed additional serine proteases, neuraminidase H, nitric oxide reductase, an invasion-associated protein, and acyl-CoA carboxylase subunits involved in mycolic acid biosynthesis as potential virulence factors. The cAMP-sensing transcription regulator GlxR plays a key role in controlling the expression of several genes contributing to virulence.
The functional data deduced from the genome sequencing and the extended knowledge of virulence factors indicate that the human isolate C. pseudotuberculosis FRC41 is equipped with a distinct gene set promoting its survival under unfavorable environmental conditions encountered in the mammalian host.
Corynebacterium pseudotuberculosis is generally regarded as an important animal pathogen and the etiological agent of a disease that is commonly called caseous lymphadenitis [1, 2]. This bacterium is predominantly isolated from sheep and goats (biovar ovis), but has been recognized also in other animals, including horses and cattle (biovar equi) . The importance of caseous lymphadenitis varies greatly around the world, but this disease is found in all major sheep and goat production areas . C. pseudotuberculosis is a significant cause of morbidity in sheep and goats, and caseous lymphadenitis in these animals resulted in economic losses, for instance in wool, milk and meat production [2, 3]. C. pseudotuberculosis is a facultative intracellular pathogen that is able to survive and grow in macrophages, thus escaping the immune response of the host [1, 4]. A close phylogenetic relationship between C. pseudotuberculosis and Corynebacterium ulcerans was proposed as both species are unique among the corynebacteria in producing phospholipase D. The sphingomyelin-degrading enzyme is regarded as the major virulence factor for C. pseudotuberculosis[5, 6]. This exotoxin promotes the hydrolysis of ester bonds in sphingomyelin in mammalian cell membranes and contributes to the spread of the bacterium from the initial site of infection to secondary sites within the host.
Although infections due to C. pseudotuberculosis are predominantly observed in sheep and goats, infections due to this pathogen also occur in humans [7, 8]. The number of human infections is rare, but it might be underestimated as corynebacteria are often considered as skin contaminants in clinical specimens . Published cases of human infections by C. pseudotuberculosis usually presented as suppurative lymphadenitis [7, 8], with the exception of one case of eosinophilic pneumonia . Most patients revealed a classical risk exposure of close contact with animals, in particular with sheep. C. pseudotuberculosis infects humans via superficial wounds, forming abscesses in the regional draining lymph nodes after an incubation period ranging from weeks to months. In most of the published cases, antibiotic treatment alone was unsuccessful and the antimicrobial therapy was therefore supplemented by surgical interventions [7, 8]. The general problem in achieving an effective treatment of C. pseudotuberculosis infections in humans and animals is probably related to the facultative intracellular lifestyle of this bacterium, as it can survive and multiply in macrophages . The cell death and subsequent release of the pathogen lead to necrotic lesions and the formation of thick collagen capsules that cannot be penetrated by antibiotics .
In the present study, we characterize the genome of C. pseudotuberculosis FRC41 that was isolated from the inguinal lymph node of a 12-year-old French girl with necrotizing lymphadenitis . This patient had no underlying disease or predisposing conditions. The diagnosis of necrotizing lymphadenitis was supported by 16 S rDNA gene sequencing, a taxonomic classification of the bacterial isolate and Western blot assays revealing the presence of phospholipase D in the patient's serum. The patient relapsed many times despite a surgical drainage and an adapted antimicrobial treatment, although C. pseudotuberculosis FRC41 turned out to be highly susceptible to a wide range of antibiotics in vitro. However, the patient recovered after a broad spectrum intravenous antimicrobial therapy with imipenem-cilastatin, rifampin and ofloxacin for four months, followed by an oral therapy with rifampin and ofloxacin for six months . Here, we report the functional annotation of the complete genome sequence of C. pseudotuberculosis FRC41, the detection of potential virulence factors and the deduced gene-regulatory networks controlling their expression.
Results and discussion
Pyrosequencing and annotation of the C. pseudotuberculosis FRC41 genome
The annotation of the C. pseudotuberculosis FRC41 genome sequence was performed with the GenDB software system  and resulted in the detection of 2,110 protein-coding regions. Furthermore, 49 tRNA genes were predicted by the tRNAscan-SE program  and four rrn operons were detected on the leading strands of the chromosome (Figure 1B). A plot of the calculated G/C skew [(G-C)/(G+C)] indicated a bi-directional replication mechanism of the C. pseudotuberculosis chromosome (Figure 1A). According to the presence and distribution of six conserved DnaA boxes, the oriC is located downstream of the dnaA coding region . The G/C skew and the biased distribution of architecture imparting sequences (AIMS) on the leading and lagging strands indicated the presence of a dif region  at the expected position of 180° from oriC, dividing the chromosome of C. pseudotuberculosis FRC41 into two replichores of similar size (Figure 1B). Synteny analysis by reciprocal best matches with BLASTP  revealed a highly conserved order of orthologous genes between the chromosomes of C. pseudotuberculosis FRC41 and C. diphtheriae NCTC 13129 (Figure 1C), which is consistent with the close phylogenetic relationship of both species  and the observation that genetic rearrangements are rare in the genomes of species belonging to the main lineage of the genus Corynebacterium[20, 21]. The calculated reciprocal best BLASTP hits  were used also to compare the predicted proteome of C. pseudotuberculosis FRC41 with the complete set of proteins encoded in the genome of C. diphtheriae NCTC 13129 . This comparative content analysis at the proteome level revealed that 1610 proteins (76.3%) of C. pseudotuberculosis FRC41 share a homologue in the genome of C. diphtheriae NCTC 13129 (data not shown). The characteristic features of C. pseudotuberculosis FRC41 are apparently based on a distinct gene set, defining its lifestyle and pathogenicity, such as the pld gene encoding phospholipase D . In the following sections, we describe a collection of relevant genes contributing to the lifestyle and pathogenicity of C. pseudotuberculosis FRC41 and deduce their integration into a transcriptional gene-regulatory network.
The transcriptional regulatory repertoire of C. pseudotuberculosis FRC41
Iron regulation in physiology and virulence of C. pseudotuberculosis
The dtxR gene of C. pseudotuberculosis FRC41 encodes a homologue of the diphtheria toxin repressor DtxR that is activated by iron and controls a complex gene-regulatory network involved in iron homeostasis in corynebacteria [30, 31]. The detection of a putative DtxR-binding site in the fagA-fagD intergenic region supports the previously observed differential expression pattern of the fagABC operon in vitro. We combined the functional annotation of the C. pseudotuberculosis FRC41 genome sequence with a bioinformatic motif search for DtxR-binding sites using a hidden Markov model and a position weight matrix with input data from the DtxR regulon of C. glutamicum. In this way, additional genes were assigned to the DtxR regulon of C. pseudotuberculosis FRC41, including several gene clusters involved in the utilization of various host compounds as iron sources (Figure 3). One gene region revealed similarity to the hemin utilization system HmuTUV from C. diphtheriae. The corresponding hemin binding protein HtaA is probably associated with the cell envelope and involved in the utilization of heme iron . Two gene clusters assigned to the DtxR regulon of C. pseudotuberculosis FRC41 include htaA-like genes (htaD and htaF) that are associated with genes encoding membrane proteins (htaE and htaG), suggesting a role of these clusters in the acquisition of iron from the host (Figure 3). The hmuO gene of the DtxR regulon encodes heme oxygenase that releases iron from the protoporphyrin ring of heme and facilitates the acquisition of iron from heme and hemoglobin . Because of the potential toxicity of both, iron and heme, the expression of hmuO in C. diphtheriae is under complex control, comprising the iron-responsive repressor DtxR and the heme-dependent activators ChrA and HrrA that are part of the two-component signal transduction systems ChrA-ChrS and HrrA-HrrS [35, 36]. A BLAST search across the C. pseudotuberculosis FRC41 genome revealed two response regulators that share similarity with ChrA and HrrA. As the hrrA gene of C. pseudotuberculosis FRC41 is part of the DtxR regulon, a complex hierarchical control of hmuO gene expression might be established in this bacterium (Figure 3).
Iron acquisition can moreover involve the synthesis and secretion of high-affinity iron chelators, termed siderophores, which are synthesized by nonribosomal peptide synthetases or by biosynthesis pathways independent of these multimodular enzymes [37, 38]. The genome of C. pseudotuberculosis FRC41 contains two DtxR-regulated gene clusters that are probably associated with pathways for siderophore biosynthesis independent of nonribosomal peptide synthetases (Figure 3). The ciu locus comprises the ciuABCD (ABC-type transporter), ciuE (siderophore biosynthesis-related protein) and ciuF (putative efflux protein) genes. The predicted product of the ciuE gene is similar to aerobactin biosynthesis enzymes . The expression of the ciuA gene encoding the lipoprotein receptor of the ABC transport system has been detected in vivo by a reporter transposon system . The ciu gene region detected in the genome of C. pseudotuberculosis FRC41 is similar to the ciu gene locus from C. diphtheriae NCTC 13129, with the exception that it lacks the ciuG gene, encoding a protein of unknown function .
The second DtxR-regulated gene cluster related to siderophore biosynthesis and excretion includes four genes probably constituting the biosynthesis pathway (ogs, ocd, odc and tsb), a gene encoding an efflux protein (mdtK) and four genes encoding an ABC-type transporter (stsABCD) (Figure 3). Ornithine cyclodeaminase (ocd) and ornithine decarboxylase (odc) as well as monooxygenase (ogs) and synthetase (tsb) functions encoded in this gene cluster are components of widely distributed routes for siderophore biosynthesis . Additional DtxR-binding sites were detected in front of ftn (ferritin) and fas (fatty acid synthase) and the sdhCAB (succinate dehydrogenase) operon that are also part of the DtxR regulon in C. glutamicum[31, 42]. Ferritins act primarily in iron storage and are thus central to the natural regulation of iron in the bacterial cell .
Furthermore, the ripA gene encoding a DNA-binding transcription regulator of the AraC protein family was assigned to the DtxR regulon of C. pseudotuberculosis FRC41 (Figure 3). The expression of the orthologous ripA gene from C. glutamicum is also controlled directly by DtxR . Under iron limitation, the RipA protein acts as a repressor of genes coding for iron proteins in C. glutamicum. Candidate RipA-binding sites were detected by bioinformatic pattern searches in the genome sequence of C. pseudotuberculosis FRC41 in front of the acn (aconitase) gene and upstream of the sdhCAB (succinate dehydrogenase) and cydABDC operons (cytochrome bd-type menaquinol oxidase and ABC-type transporter), thereby linking the availability of iron with the expression of genes in the citrate cycle and the respiratory energy metabolism of C. pseudotuberculosis. Due to its high oxygen affinity, the cytochrome bd oxidase is used in many bacteria under microaerobic growth conditions . Additional RipA-binding sites were detected in front of the katA (catalase) gene and in the dps-fpg1 (DNA protection during starvation protein, formamidopyrimidine-DNA glycosylase) intergenic region, integrating protective functions into the RipA regulon (Figure 3). Dps-like proteins effectively protect DNA against oxidizing agents by nonspecific DNA-binding and physical sequestration that limits DNA accessibility to detrimental factors . They also act as iron-binding and storage proteins and catalyze the oxidation of ferrous iron to ferric iron by H2O2, which prevents the formation of hydroxyl radicals by the Fenton reaction . The lsr2 gene of C. pseudotuberculosis FRC41 encodes the multifunctional histone-like protein and transcription regulator Lsr2 (Figure 2) that shares a number of physical properties with Dps-like proteins and the ability to bind DNA sequences with little specificity . Like Dps, the Lsr2 protein may physically protect corynebacterial DNA against reactive oxygen intermediates . Formamidopyrimidine-DNA glycosylase is a primary participant in the repair of DNA lesions caused by oxidative damage . A second gene (fpg2) encoding formamidopyrimidine-DNA glycosylase was detected in the genome of C. pseudotuberculosis FRC41, but the upstream region of fpg2 apparently lacks a RipA-binding site. Catalase is another important protective component in the bacterial oxidative stress response which is involved in the detoxification of H2O2. Hence, the gene composition of the DtxR-RipA gene-regulatory network revealed the interdependence of iron metabolism and oxidative stress response and the regulatory connection between distinct physiological functionalities of the corynebacterial cell, including an important role in virulence of C. pseudotuberculosis.
Manganese and zinc regulation in physiology and virulence of C. pseudotuberculosis
As manganese and zinc ions are apparently involved as cofactors in the oxidative stress response of pathogens, the corresponding regulons involved in metal ion uptake were identified in the genome sequence of C. pseudotuberculosis FRC41 (Figure 4B). The uptake of manganese in C. pseudotuberculosis FRC41 is mediated by a typical manganese ABC-type transport system (mntABCD) that is negatively controlled at the transcriptional level by the metalloregulator MntR (Figure 4B). The manganese ABC transporter is composed of: a lipoprotein receptor (MntA), anchored to the cell membrane and functioning as an extracellular cation-binding protein; a cytoplasmic ATP-binding protein (MntB); and two integral membrane proteins (MntC and MntD) that mediate the cation flux . A homologous gene cluster and its manganese-dependent transcriptional control by the metalloregulator MntR have been examined in the genome of C. diphtheriae. The uptake of zinc ions in C. pseudotuberculosis FRC41 is probably mediated by two ABC transport systems (znuB1C1A1 and znuB2A2C2), as both gene regions are specified by the presence of Zur-binding sites (Figure 4C). The Zur protein is a metalloregulator of the ferric uptake regulator (FUR) family of DNA-binding transcription regulators [65, 66]. The znu ABC transporter genes are key components of actinobacterial Zur regulons and their expression is generally repressed by Zur in a zinc-dependent manner . Zinc resistance might be facilitated in C. pseudotuberculosis FRC41 by the ArsR-type transcription regulator Znr that probably controls the expression of the czcE gene encoding a cobalt/zinc/cadmium efflux system [23, 67, 68]. The yciC gene coding for a putative P-loop GTPase of the COG0523 protein family is also part of the Zur regulon in C. pseudotuberculosis FRC41 (Figure 4C). The YciC protein may function as a metallochaperone/insertase to enable the in vivo assembly of zinc-containing proteins under environmental conditions of zinc deficiency .
Moreover, the genome-wide motif search for Zur-binding sites in C. pseudotuberculosis FRC41 detected three genes (cpfrc_00168, cpfrc_00313, cpfrc_02059) encoding proteins with a carboxy-terminal sorting (LPxTG) signal that is generally used by Gram-positive bacteria to anchor proteins to the cell wall . The Cpfrc_00168 protein contains two CnaB-like domains that may be involved in the positioning of a ligand binding domain away from the corynebacterial cell surface and is encoded adjacent to a putative sialoprotein-binding protein. The secreted proteins Cpfrc_00313 and Cpfrc_02059 contain actinobacterial surface-anchored protein domains for their covalent attachment to the cell wall . The Cpfrc_00313 protein is encoded next to the components of a transporter and may act together with the Cpfrc_00314 protein as a substrate receptor for this system. Hence, the reconstruction of regulons participating in metal ion uptake of C. pseudotuberculosis FRC41 led to the detection of genes that may fulfill novel functions in sensing the presence of zinc in the environment.
Genes encoding adhesive pili in C. pseudotuberculosis FRC41
Candidate virulence factors of C. pseudotuberculosis FRC41 and their integration into a gene-regulatory network
Candidate determinants contributing to virulence of C. pseudotuberculosis FRC41
Predicted protein function
phospholipase D (sphingomyelin-degrading enzyme)
corynebacterial protease CP40 (serine protease)
secreted subtilisin-like serine protease
secreted subtilisin-like serine protease
secreted trypsin-like serine protease
neuraminidase H (sialidase)
resuscitation-promoting factor interacting protein (D,L-endopeptidase)
resuscitation-promoting factor A (muralytic enzyme)
resuscitation-promoting factor B (muralytic enzyme)
nitric oxide reductase
nonribosomal peptide synthetase 1
nonribosomal peptide synthetase 2
acetyl-CoA carboxylase β-subunit involved in fatty acid synthesis
acyl-CoA carboxylase β-subunit involved in mycolic acid synthesis
acyl-CoA carboxylase β-subunit involved in mycolic acid synthesis
Likewise, a DNA-binding site for GlxR was detected in front of the rpfI gene encoding an invasion-associated protein that is involved in cell surface organization and adhesion of corynebacteria . The homologue of RpfI in M. tuberculosis (named RipA) revealed endopeptidase activity and interacts with the resuscitation-promoting factor RpfB, representing a peptidoglycan glycosidase [86, 87]. Two genes (rpfA and rpfB) encoding resuscitation-promoting factors are present in the genome of C. pseudotuberculosis FRC41 (Table 1). Important roles in pathogenesis for peptidoglycan hydrolytic enzymes have been proposed  and an analogous system combining the activities of a muramidase and an endopeptidase contributed to the virulence of Listeria monocytogenes. As previously demonstrated in C. glutamicum[74, 90], the expression of rpfI, rpfA and rpfB in C. pseudotuberculosis FRC41 is probably under complex control by three regulatory proteins, GlxR, RamB and RamA (Figure 6).
Another potential virulence factor of C. pseudotuberculosis FRC41 is represented by the nor gene encoding nitric oxide reductase (Table 1). This enzyme is generally involved in the detoxification of nitric oxide and consequently necessary for the long-term persistence of pathogens in macrophages . The toxic properties of nitric oxide are used by the host immune system to kill or slow down the growth of pathogenic bacteria . Interestingly, the expression of the nor gene was not induced upon the infection of macrophages by animal C. pseudotuberculosis. As the expression of nor is typically activated by a transcription regulator in response to the presence of nitric oxide , the regulatory pattern of nor transcription and its contribution to the protection against nitric oxide remains unclear.
The previous search for macrophage-induced genes of animal C. pseudotuberculosis by means of a cloned promoter library provided two gene tags showing significant induction rates in macrophages . The nucleotide sequence of the respective gene tags revealed similarity to nonribosomal peptide synthetases (44-fold induction) and to the β-subunit of acyl-CoA carboxylases (24-fold induction), respectively. The genome sequence of C. pseudotuberculosis FRC41 encodes two nonribosomal peptide synthetases, NrpS1 and NrpS2 (Table 1). These modular enzymes are used by microorganisms to participate in the synthesis of many secondary metabolites, including for instance siderophores and antibiotics . As both nrpS genes were not assigned to the iron-responsive DtxR regulon of C. pseudotuberculosis FRC41 and siderophore biosynthesis is carried out by alternative pathways independent of nonribosomal peptide synthetases, a physiological role in iron metabolism of the two proteins cannot be deduced from the current data. However, the strong upregulation of gene expression in macrophages points toward a protective or toxic function during the infection of at least one nonribosomal peptide synthetase . A role in virulence of a secondary metabolite produced by a nonribosomal peptide synthetase has been demonstrated in Streptomyces acidiscabies. This phytopathogen produces thaxtomin A which is necessary for the infection of potato tubers .
Three genes coding for β-subunits of acyl-CoA carboxylases are present in the genome of C. pseudotuberculosis FRC41 (Table 1). These genes are located in highly conserved regions of corynebacterial genomes and are essential for either fatty acid synthesis (dtsR1) or mycolic acid synthesis (dtsR2 and accD3) . The acetyl-CoA carboxylase of C. glutamicum consists of the biotinylated α-subunit AccBC, the β-subunit AccD1 (DtsR1) and the small AccE protein. The acyl-CoA carboxylase involved in mycolic acid synthesis of C. glutamicum consists of the two β-subunits AccD2 and AccD3 (DtsR2 and AccD3) in addition to AccBC and AccE [95, 96]. The expression of the respective genes in C. pseudotuberculosis FRC41 is probably controlled by the global transcription regulator GlxR (Figure 6), providing hints that this regulatory protein has a key function in connecting both the central metabolism and the expression of candidate virulence factors in this pathogenic bacterium. Mycolic acids, which are long-chain α-alkyl-β-hydroxy fatty acids, are important components of the corynebacterial cell wall  and probably associated with the pathogenicity of C. pseudotuberculosis. They can provide a thick layer at the outer surface of the cell that protects the bacterium from antibiotics and the host's immune system . Moreover, the noncovalently bound trehalose dimycolate is a well-established immunostimulatory compound with toxic properties . Variations in the amount of mycolic acids and differences in other cell surface properties provide a basis for explaining the aggregation capacity of C. pseudotuberculosis in suspension, the strong aggregation within macrophages, the formation of thick capsules after the release of the pathogen from macrophages , and the occurrence of virulent and attenuated strains .
The complete genome sequence of C. pseudotuberculosis FRC41 of human origin provides detailed insights into the gene repertoire contributing to the virulence of this bacterium that was isolated from a rare case of necrotizing lymphadenitis . By combining in silico data obtained from the genome annotation with previous experimental knowledge, occasional observations on genes that affect the virulence of C. pseudotuberculosis were integrated into a global view on the pathogenicity of this species. The reconstruction of the DtxR regulon for instance provides a comprehensive set of genes involved in the acquisition of iron and extends the initial observation that a fagB mutant was not impaired in iron uptake . A systematic mutational characterization of the newly detected iron transporters and siderophore biosynthesis gene clusters may help to dissect the contribution of each system to the virulence of C. pseudotuberculosis FRC41. It is very likely that the ciu siderophore biosynthesis and transport system may complement the uptake of iron by the FagABCD transporter under certain environmental conditions, as the ciuA gene was shown earlier to be expressed in C. pseudotuberculosis. Likewise, a reporter transposon system indicated that a gene coding for a fimbrial subunit is expressed during growth of C. pseudotuberculosis in standard medium . As the genome sequence of C. pseudotuberculosis FRC41 revealed two gene clusters encoding adhesive pili, it is interesting to examine how the respective pilin monomers or pilus polymers contribute to the adherence of C. pseudotuberculosis to host tissue and how their expression is controlled. We detected a DNA-binding site for the cAMP-sensing transcription regulator GlxR in the spaA-srtB intergenic region, whereas the iron-responsive regulator DtxR was proposed to control the assembly of pilin subunits in C. diphtheriae NCTC 13129 . Another previous study provided two DNA sequence tags for macrophage-induced genes  that were linked to the nonribosomal peptide synthetase genes nrpS1 and nrpS2 and to the acyl-CoA carboxylase subunit genes dtsR1, dtsR2 and accD3 by means of the genome sequence of C. pseudotuberculosis FRC41. The detection of the latter genes is consistent with the observation that the cell surface of C. pseudotuberculosis is an important factor contributing to virulence . Future work should compare the knowledge deduced from the genome sequence of C. pseudotuberculosis FRC41 with the genetic information generated from other human isolates or animal pathogens of the different biovars , or even extend the genome comparison to the pangenomic level. This global comparative approach with a larger set of sequenced genomes may provide comprehensive insights into the distinctive features of each biovar or strain. As the currently available commercial vaccines are unable to fully protect susceptible animals [102, 103], the extended knowledge of potential virulence factors and novel antigens of C. pseudotuberculosis might be helpful for the design of more effective vaccines and molecular diagnostics to control caseous lymphadenitis in sheep and goats to reduce thereby the occupational risk exposure for humans.
Bacterial strain and growth conditions
C. pseudotuberculosis FRC41 was isolated from the inguinal lymph node of a 12-year-old French girl with necrotizing lymphadenitis . The taxonomic identification of this clinical isolate was recently confirmed using multiplex PCR and the nucleotide sequence of the pld gene (N. Guiso, unpublished data). C. pseudotuberculosis FRC41 was routinely grown in brain-heart-infusion (BHI) broth or on Columbia agar with sheep blood at 37°C.
Preparation of chromosomal DNA for genome sequencing
The preparation of chromosomal DNA from C. pseudotuberculosis FRC41 was performed as follows: 50-ml aliquots of cultures grown for 48-72 h were centrifuged at 4°C and 2,000 × g for 20 min. The cell pellets were resuspended in 0.6 ml Tris/NaCl buffer [10 mM Tris (pH 7.0), 10 mM EDTA (pH 8.0), 300 mM NaCl] and transferred to VK01 Precellys lysing tubes. The cells were lysed by means of a Precellys 24-Dual Tissue Homogenizer, using two cycles of 6,500 rpm for 15 sec with an interval of 30 sec. The chromosomal DNA was purified by phenol/chloroform/isoamyl alcohol (25:24:1) extraction and precipitated with ethanol. The DNA concentration was determined with a Tecan Infinite 200 Microplate Reader.
Sequencing of the C. pseudotuberculosis FRC41 genome
A total of 5 μg of purified chromosomal DNA was used for constructing a single-stranded template DNA library. The DNA concentration of the library was measured by using the Agilent RNA 6000 Nano Kit. The preparation of the single-stranded template DNA library and DNA sequencing were performed according to manufacturer's protocols (Roche Applied Science). The sequencing of C. pseudotuberculosis DNA was carried out with the Genome Sequencer FLX Instrument and Titanium chemistry (Roche Applied Science). The sequence data were assembled with the GS de novo Assembler Software (Version 2.3). According to the 454 Newbler Metrics file, 286,938 reads representing 94,447,635 bases were assembled. Using the default cutoff of 500 bases for the size classification of the contigs, ten large contigs (≥ 500 bases) and one small contig (313 bases) were obtained to give a total size of 2,319,243 bases. The gap closure process was supported by the related reference contig arrangement tool r2cat, using the C. diphtheriae NCTC 13129 genome sequence as a reference . The remaining gaps in the genome sequence were closed by PCR with Phusion hot start high-fidelity DNA polymerase (Finnzymes) and genomic template DNA. All primers used in this study were synthesized by Metabion. The PCR assays were carried out with a TProfessional PCR thermocycler (Biometra) according to standard protocols (Finnzymes). The amplified DNA fragments linking the individual contigs were sequenced by IIT Biotech. All DNA sequences were uploaded into the Consed program  to generate the complete genome sequence of C. pseudotuberculosis FRC41.
Bioinformatic analysis of the complete genome sequence
The assembled sequence of C. pseudotuberculosis FRC41 was uploaded into the bacterial genome annotation system GenDB . The annotation of the complete genome sequence was performed as described previously , followed by manual curation. Analyses of the predicted gene content and the metabolic properties of C. pseudotuberculosis FRC41 were accomplished by the computer programs EDGAR  and CARMEN . The synteny between the genomes of C. pseudotuberculosis FRC41 and C. diphtheriae NCTC 13129 was calculated by the EDGAR software . The origin of chromosomal DNA replication was predicted with the Ori-Finder tool . The genome sequence of C. pseudotuberculosis FRC41 has been deposited in the GenBank database with accession number CP002097.
Bioinformatic analysis of the regulatory repertoire
The detection of the transcriptional regulatory repertoire of C. pseudotuberculosis FRC41 was performed by a combined bioinformatic approach using several tools and programs . Proteins containing putative DNA-binding domains were detected by means of the HMM library and genome assignments server Superfamily version 1.75 . To identify among the set of potential DNA-binding proteins those representing transcription regulators, hidden Markov model (HMM) profiles of regulatory protein families were downloaded from the Pfam database version 24.0  and used for searches against the predicted C. pseudotuberculosis FRC41 proteins by applying the HMMsearch module of the profile hidden Markov model software HMMER . Moreover, the helix-turn-helix (HTH) recognition tool  integrated in the GenDB platform was applied to scan the putative DNA-binding transcription regulators for the presence of HTH motifs. The classification of C. pseudotuberculosis FRC41 proteins into Clusters of Orthologous Groups of proteins  during genome annotation provided further indications on the role of the predicted proteins in transcriptional regulation. The genome-wide search was extended by using data on known transcription regulators from C. glutamicum. A validation step was included by performing BLASTP  searches against the NCBI protein database and evaluating results generated with the Conserved Domain Search program . During the final step of data analysis, the DNA-binding transcription regulators of C. pseudotuberculosis FRC41 were grouped into regulatory protein families [23, 26].
The detection of DNA-binding sites in the genome sequence of C. pseudotuberculosis FRC41 followed a combined workflow, using both position weight matrices and hidden Markov models . The programs PoSSuMsearch  and HMMsearch  were applied to scan the complete genome sequence of C. pseudotuberculosis FRC41. As HMMsearch does not support searching in both directions of a double strand, scanning of the reverse complementary DNA sequence was implemented. PoSSuMsearch was configured for lazy probability evaluation . The comprehensive set of validated DNA-binding sites sequences from C. glutamicum was downloaded from the reference database CoryneRegNet  and used as input for PoSSuMsearch and HMMsearch. The respective gene-regulatory network transfer between C. glutamicum and other corynebacteria on the genome-scale was described previously . E-value cut-offs were used as described for the genome-wide pattern recognition approach in C. glutamicum. Sequence logos of the detected DNA-binding sites were generated with WebLogo version 2.8.2 .
ET and JSchn acknowledge the receipt of scholarships which are embedded into the Cluster Industrial Biotechnology CLIB2021 and financed by Bielefeld University and the Ministry of Innovation, Science, Research and Technology of North Rhine-Westphalia. LO and AB were supported by the Deutsche Forschungsgemeinschaft in frame of SFB 796 (project B5). The valuable contribution of the CeBiTec sequencing team (A. Albersmeier, K. Brinkrolf, S. Schatschneider, R. Szczepanowski, P. Viehoever, A. Winkler) to this genome project is gratefullly acknowledged. The authors thank the sequencing team from IIT Biotech for the help during the gap closure process.
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