Francisella isolates from patients suffering from tularemia in Germany are generally strains of the species F. tularensis subsp. holarctica. To our knowledge, no other Francisella species are known for Germany. Recently, a new Francisella species could be isolated from a water reservoir of a cooling tower in Germany.
We identified a Francisella sp. (isolate W12-1067) whose 16S rDNA is 99% identical to the respective nucleotide sequence of the recently published strain F. guangzhouensis. The overall sequence identity of the fopA, gyrA, rpoA, groEL, sdhA and dnaK genes is only 89%, indicating that strain W12-1067 is not identical to F. guangzhouensis. W12-1067 was isolated from a water reservoir of a cooling tower of a hospital in Germany. The growth optimum of the isolate is approximately 30°C, it can grow in the presence of 4–5% NaCl (halotolerant) and is able to grow without additional cysteine within the medium. The strain was able to replicate within a mouse-derived macrophage-like cell line. The whole genome of the strain was sequenced (~1.7 mbp, 32.2% G + C content) and the draft genome was annotated. Various virulence genes common to the genus Francisella are present, but the Francisella pathogenicity island (FPI) is missing. However, another putative type-VI secretion system is present within the genome of strain W12-1067.
Isolate W12-1067 is closely related to the recently described F. guangzhouensis species and it replicates within eukaryotic host cells. Since W12-1067 exhibits a putative new type-VI secretion system and F. tularensis subsp. holarctica was found not to be the sole species in Germany, the new isolate is an interesting species to be analyzed in more detail. Further research is needed to investigate the epidemiology, ecology and pathogenicity of Francisella species present in Germany.
Francisella isolateW12-1067Cooling towerGenome sequencePathogenicity island FPIEnvironmentalLegionellaGermany
Francisella tularensis is an facultative intracellular pathogen that causes tularemia in humans and a wide range of animals . Strains of F. tularensis subsp. (Ft.) tularensis can be lethal to humans and are mostly associated with cases of tularemia in the U.S.A. Doses as low as 10–20 bacteria can be infective . Transmission mostly occurs via aerosol, alimentary ingestion or skin inoculation. In addition, F. tularensis is suspected as a potential bacterial biological weapon . The species Ft. novicida is almost avirulent for humans in contrast to mice, and is thought to be an opportunistic pathogen [3, 4]. Ft. novicida is assumed to constitute an environmental lineage along with F. philomiragia. In rare cases the latter has also been associated with human disease in immunocompromised individuals [4, 5].
Human infections caused by F. tularensis are rare in Germany, but seroprevalence studies in wild animals revealed a high seroprevalence of F. tularensis in wildlife in eastern Germany [6–8]. In Germany, Ft. holarctica is generally identified in affected animals or humans as well as in known vectors (like ticks and other arthropods) [1, 9–11]. Other as yet known species of the genus Francisella are F. hispaniensis[12, 13], F. halioticida, F. piscida, F. noatunensis, F. asiatica, F. noatunensis subsp. orientalis and F. philomiragia subsp. noatunensis[18, 19]. Very recently, a new Francisella species (F. guangzhouensis) was isolated from a cooling tower in China, which had not been reported before .
However, to our knowledge, no species other than Ft. holarctica has been identified in Germany until now. Therefore, our new isolate W12-1067 is the first aquatic isolate identified in Germany which does not belong to the species F. tularensis and is closest related to F. guangzhouensis.
Strains, media and growth conditions
Strains used in this study were Ft subsp. holarctica LVS (ATCC 29684), Ft. novicida U112 (ATCC 15482), F. philomiragia (ATCC 25015), Legionella pneumophila Paris (CIP 107629) and the new environmental Francisella isolate W12-1067. Francisella strains were cultivated in medium T  (1% brain heart infusion broth [Difco Laboratories, Inc., Sparks, MD, USA], 1% bacto tryptone [Difco], 1% technical casamino acids [Difco], 0.005 g of MgSO4, 0.01% FeSO4, 0.12% sodium citrate, 0.02% KCl, 0.04% K2HPO4, 0.06% L-cysteine and 1.5% glucose) or on enriched cystine-heart agar (CHA [Difco], 1% brain heart infusion broth, 1% proteose-peptone, 1% D-glucose, 0.5% NaCl, 0.1% L-cystine, 1.5% agar and 1% hemoglobin). W12-1067 and L. pneumophila Paris were cultivated in ACES-buffered yeast extract (AYE) broth [1% N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 1% yeast extract, 0.04% L-cysteine and 0.025% ferric pyrophosphate, adjusted to pH 6.8 with 3 M potassium hydroxide (KOH) and sterile filtrated], on ACES-buffered charcoal–yeast extract (BCYE) agar  or on GVPC agar plates (Heipha Dr. Müller GmbH, Eppelheim, Germany, BCYE agar supplemented with 80,000 IE polymyxin B, 1 mg/l of vancomycin and 80 mg/l of cycloheximide). Isolate W12-1067 was initially cultivated on GVPC agar plates. The U937 human macrophage-like cell line ATCC CRL-1593.2 and the mouse macrophage cell line J774A.1 were cultivated in RPMI 1640 + 10% FCS medium (PAA/GE Healthcare Europe GmbH, Freiburg, Germany) at 37°C and 5% CO2.
Growth without additional cysteine was done on BCYE agar plates without additional cysteine. Physiological characteristics of analyzed strains were determined by using API ZYM (bioMérieux Deutschland GmbH, Nürtingen, Germany).
Chitinase activity tests were done on 1.5% agarose plates containing 0.1% deacetylated glycol chitin. Chitinase activity experiments were done as described earlier , with the modification that the deacetylated glycol chitin was suspended in 0.01 M sodium phosphate (pH 5.5) by heating. In short, bacteria were grown in medium T overnight. The supernatant was precipitated by isopropanol and the protein pellet was resuspended in PBS (concentrated 40-fold). 50 μl were inoculated into agar plates (as described earlier ) and incubated for two days at 37°C. Degrading activity was visualized by incubation with 0.01% Calcofluor Brightener 28 (Sigma-Aldrich Chemie GmbH, Munich, Germany) for 10 min, washing two times with water and then incubation at room temperature (RT) overnight.
The NaCl sensitivity assays were done in 96-well plates in a total volume of 200 μl of medium T and ~2 × 107 bacterial cells. Plates were incubated 2–3 days at 37°C and 5% CO2, and then the optical density (OD) at 600 nm was measured using an Infinite 200 reader (Tecan Deutschland GmbH, Crailsheim, Germany).
Intracellular multiplication of Francisellain host cells
To determine which amoeba strain may be suitable to be used for replication assays, isolate W12-1067 (~1010 cells) was suspended in 1 ml of dH2O and 100 μl were plated onto NN-agar plates (14 g/l of agar in dH2O). The amoeba strain (15 μl, A. castellanii ATCC 30010, A. castellanii ATCC 30234, A. castellanii 50739, A. lenticulata 45 ATCC 50703, A. lenticulata 118 ATCC 50706, Hartmannella vermiformis OS101, Hartmannella vermiformis ATCC 50256 and Naegleria gruberi ATCC 30244, respectively) was dropped onto the centre of the plates and incubated at RT or at 30°C for 7 days. The plates were inspected daily for movement and replication of the amoeba. All amoeba tested were motile and not killed by isolate W12-1067. Therefore, no further experiments were performed using amoebae as host cells.
For differentiation into macrophage-like cells, U937 cells were adjusted to 3 × 105 cells/ml and transferred into 100 ml of fresh RPMI medium containing 10% fetal calf serum (10% FCS), and PMA (phorbol-12-myristate-13-acetate, 1 mg/ml in dH2O [P-8139; Sigma-Aldrich Chemie]) was added at a concentration of 1:20,000. After incubation for 36 h at 37°C and 5% CO2, the supernatant was discarded and adherent cells were washed once with 10 ml of 0.2% EDTA in PBS. Cells were mechanically detached from the flask bottom with RPMI + 10% FCS, transferred into 50 ml tubes and centrifuged at 800 g for 10 min. All cells were counted after trypan blue staining in a Neubauer counting chamber and adjusted to 5 × 105 cells/ml with RPMI + 10% FCS. To each well of a 24-well plate 1 ml of the cell suspension was added and incubated for adhesion during 2 h at 37°C and 5% CO2. Macrophage-like mouse cell line J774A.1 was also grown in fresh RPMI medium containing 10% fetal calf serum and treated as described above, but without the differentiation step (no PMA treatment).
For both cell lines, stationary phase bacteria grown for 3 days on CHA or BCYE agar were diluted in plain RPMI medium and the infection was done with a multiplicity of infection (MOI) of 1, 10 or 100 (time point 0 h) for 2 h at 37°C and 5% CO2. Cells were washed three times with RPMI and incubated with 50 μg/ml of Gentamycin for 1 h to kill extracellular bacteria. Cells were washed again three times with RPMI and covered with 1 ml of RPMI + 10% FCS. For colony-forming unit (CFU) determination at various time points of infection, coincubations of cells and bacteria were lysed by addition of 10 μl of 10% Saponin (S4521, Sigma-Aldrich Chemie) for 5 min, and serial dilutions were plated on BCYE agar. In a control experiment we showed that Saponin treatment did not affect the number of remaining CFU of strain W12-1067 (data not shown).
Electron microscopy (EM)
J774A.1 cells were infected with Francisella strain W12-1067 (MOI of 10) at 37°C as described above. Cells were fixed 96 h post infection with 2.5% glutaraldehyde in 0.05 M HEPES buffer. Bacteria cultivated in medium T were fixed with 4% paraformaldehyde 5% glutaraldehyde in 0.05 M HEPES buffer for 2 h at RT. All samples were post-fixed with osmium tetroxide (1% in distilled water) and uranyl acetate (2% in distilled water), dehydrated stepwise in a graded ethanol series and embedded in LR White resin (Science Services GmbH, Munich, Germany) which was polymerized at 60°C overnight. Thin sections were prepared with an ultramicrotome (UC-T; Leica, Vienna, Austria) and counterstained with uranyl acetate and lead citrate.
Samples were examined using a transmission electron microscope (EM 902; Carl Zeiss Microscopy GmbH, Jena, Germany) at 80 kV, and the images were recorded using a slow-scan charge-coupled-device camera (Pro Scan elektronische Systeme GmbH, Lagerlechfeld, Germany).
Genome sequencing, ORF finding and annotation
Genome sequencing of chromosomal DNA of isolate W12-1067 was performed by Eurofins MWG Operon (Eurofins Medigenomix GmbH, Ebersberg, Germany): (i) Short insert shotgun library (FLX + library): 1 μg of DNA was fragmented using a Covaris E210 instrument (Covaris Inc., Woburn, MA) according to manufacturer’s instructions. End-repair, dA-tailing and ligation of barcoded adapter were performed following New England Biolabs’ instructions (New England Biolabs GmbH, Frankfurt/Main, Germany). Emulsion-based clonal amplification (emPCR amplification) was performed following Roche’s instructions (Roche Diagnostics GmbH, Mannheim, Germany). FLX + Sequencing: Sequencing was performed on an FLX + platform according the manufacturer’s instructions using 1/8 plate. The sequencing process was controlled by the Roche 454 software gsRunProcessor v2.8 (shotgun signal processing pipeline). (ii) 8 kb mate-pair-like library (Long-Jumping-Distance library): Creation of the 8 kb mate-pair-like library was done at Eurofins MWG Operon (Ebersberg, Germany) using their proprietary protocol. (iii) Illumina Sequencing: For sequencing, the library was loaded on an Illumina MiSeq machine. Cluster generation and paired-end sequencing was performed using the manufacturer’s instructions. MiSeq Control Software 2.2.0 was used for sequencing. For processing of raw data RTA version 1.17.28 and CASAVA 1.8.2 were used to generate FASTQ-files. (iv) Data analysis: A two-step hybrid de novo assembly was conducted using the sequencing data of the two libraries. First, the FLX + data (long reds, single-end) has been assembled separately using the Roche 454 software Newbler (v2.6). The resulting contigs as well as the Illumina long-jumping-distance pairs (Illumina mate-pair-like) were then assembled together with a hardware-accelerated assembly pipeline based on the Convey hardware and software tools (http://www.conveycomputer.com) that mimic a standard de novo assembly using the Velvet assembler (Eurofins proprietary assembly pipeline) [25, 26]. The draft genome (all contigs) was annotated by using the RAST server, freely available at http://www.patricbrc.org.
16S rDNA gene and the multi-gene locus (in frame gene sequence) of genes fopA, gyrA, rpoA, groEL, sdhA and dnaK of strain W12-1067 and available homologous sequences from Francisella species and L. pneumophila Paris (obtained from GenBank) were used for nucleotide comparison. The multi-gene locus of L. pneumophila Paris exhibited no fopA gene because no homolog is present within the genome. Phylogenetic analysis (phylogenetic tree) was generated by using the ClustalW program (ClustalW 2.1; http://www.patricbrc.org).
This whole genome shotgun project has been deposited at DDB/EMBL/GenBank under accession AWHF00000000. The version described in this paper is version [AWHF01000000].
Results and discussion
During 2012 health authorities of the city of Heilbronn (Germany) observed some coincident spots of Legionnaires’ disease (LD). Therefore, different putative sources were screened for the presence of Legionella species using GVPC (glycine-vancomycin-polymyxin-cycloheximide) agar plates. The investigation of a water reservoir of a cooling tower led to the isolation of strain W12-1067. The isolate W12-1067 was first thought to belong to the genus Legionella because of its habitus and growth on GVPC agar plates, but PCR analysis did not support this finding. Preliminary 16S rDNA PCR analysis performed by the German Consultant Laboratory for Legionella (Dresden, Germany) revealed that this strain may belong to the genus Francisella. The identified isolate was not involved in the LD outbreak. However, the strain was send for further analysis to the Centre for Biological Threats and Special Pathogens at the Robert Koch Institute (Berlin, Germany). Here, W12-1067 was identified to be the first German isolate of the genus Francisella which did not belong to the species F. tularensis.
Analysis of strain W12-1067
First we performed 16S rDNA PCR and sequenced the PCR product. The phylogenetic analysis of the 16S rDNA revealed that isolate W12-1067 is a close representative of the recently identified new environmental Francisella species F. guangzhouensis. The phylogenetic tree of 16S rDNA of different Francisella strains is given in Figure 1A, corroborating the close relationship of isolate W12-1067 with F. guangzhouensis and other Chinese cooling tower isolates of this species (99% identity). The other Francisella strains analyzed revealed DNA identities of 16S rDNA sequences of 94–95% with 16S rDNA of isolate W12-1067 and 83% identity with the 16S rRNA gene of L. pneumophila Paris.
We then sequenced the whole genome of W12-1067, resulting in an annotated draft genome of this strain. We used the sequences of the genes fopA, gyrA, rpoA, groEL, sdhA and dnaK (see Table 1) to build a multi-gene locus (9918 bp for strain W12-1067) for each strain analyzed. With these 6-loci concatenated sequences we performed a further phylogenetic analysis (Figure 1B). The overall DNA identity was only 89%, indicating that W12-1067 is not identical to strain F. guangzhouensis, but that F. guangzhouensis is the closest relative identified to date. The overall DNA identity of the gene cluster of isolate W12-1067 to other Francisella strains analyzed was 80–81% and that to L. pneumophila Paris 67% (Figure 1B).
Genes used for phylogenetic tree analysis
% DNA similarity to
Small subunit ribosomal RNA
Large subunit ribosomal RNA
Francisella outer membrane protein A
DNA gyrase subunit B
DNA-directed RNA polymerase A
Heat shock protein 60, chaperone
Succinate dehydrogenase subunit A
Heat shock protein K, chaperone
Type strain of F. guangzhouensis sp. nov (Qu et al. ).
Similar to strain W12-1067, F. guangzhouensis strains had been isolated from water of air conditioning systems of cooling towers in China, during a routine investigation to detect Legionella. The growth optimum of this species ranged between 25 and 28°C and it showed growth on BCYE-alpha (minus cysteine) Legionella-agar plates. Furthermore, no virulence to mice was found for this strain [20, 28]. No further information about virulence properties of this species was available yet.
We investigated the growth of strain W12-1067 on different agar plates and within different liquid media. Francisella sp. strain W12-1067 grew well on BCYE, GVPC and CHA plates. The isolate grew faster in medium T than in AYE medium, and it did not grow in the cell culture medium RPMI (data not shown). Whereas L-cysteine within BCYE agar plates is necessary for the growth of L. pneumophila Paris, it stimulates the growth of F. philomiragia, and the growth of strain W12-1067 was nearly similar with or without additional cysteine (see Additional file 1: Figure S1A). Growth in medium T revealed that the growth optimum of strain W12-1067 is about 30°C (Figure 2A), but growth of strains F. philomiragia and Ft. novicida in general was faster and reached a higher cell density compared to that of the growth of strain W12-1067 (Figure 2B–D). Growth of strain W12-1067 in media with NaCl was reduced from a concentration of 4–5% NaCl, which was comparable to growth of strains F. philomiragia and Ft. novicida (up to 6–7% NaCl), but more resistant than the non-halotolerant strain Ft. holarctica LVS (see Additional file 1: Figure S1B). Using the API ZYM assay kit (bioMérieux), W12-1067 showed a profile similar to F. guangzhouensis (data not shown). In contrast to F. guangzhouensis, isolate W12-1067 was negative for alkaline phosphatase activity and showed only very low activity of the acid phosphatase, which is in good agreement with only one putative phosphatase encoding gene (peg_768) present within the genome sequence (see below).
These experiments were followed by co-culture studies using macrophage-like cell lines of human (U937) or mice (J774A.1) origin as host cells. We found that strain W12-1067 was able to persist within U937 cells (data not shown), and it replicated intracellularly in J774A.1 cells (Figure 3). Replication within J774A.1 was slower than that of L. pneumophila Paris. However, W12-1067 multiplied about 16-fold within 4 days of co-incubation, with a 24 h lag-phase at the beginning of the infection (Figure 3), indicating that the new isolate is able to infect and multiply within eukaryotic cells. Cells of strain W12-1067 grown in medium T at 37°C possessed a rod-shaped, slightly pleomorphic morphology (Figure 4A). EM analysis of the infection assay revealed that strain W12-1067 was localized intracellularly within a vacuole 96 h after infection (Figure 4B–D). The data indicate that W12-1067 is able to replicate intracellularly, but from EM analysis it was not clear yet whether the bacteria were able to escape from the vacuole. Experiments are under way to investigate this question further.
Whole genome sequencing and sequence analysis of strain W12-1067
The genomic DNA was sequenced using 454 shotgun reads and paired-end Illumina reads (for details, see Methods), followed by de novo assembly of the sequence, which was done by Eurofins (Ebersberg, Germany), resulting in 73 contigs and a chromosomal size of approximately 1,704,745 bp. The draft genome was annotated by using the RAST server (freely available at http://rast.nmpdr.otg). All genes encoded by the draft genome sequence of isolate W12-1067 (GenBank AWHF01000000) are given in the Additional file 2: Table S1. The draft genome exhibits 1,541 protein-coding genes (peg), three copies of the 16S-23S-5S rRNA gene locus (plus one additional 5S rRNA gene) and 37 tRNA genes (Table 2). The G + C content is 32.2% and therefore not significantly different from various other Francisella strains already sequenced [12, 29–37]. However, the number of protein-coding genes and of tRNA genes seemed to be lower compared with other Francisella strains (Table 2), but this is not surprising since the W12-1067 genome is of draft quality and the others are finished. The genes encoding the tRNAs are present in four (tRNA-Leu), three (tRNA-Ala, -Ile, -Met, -Arg, -Gly, -Ser) and two (tRNA-Val, -Thr) copies. Like in other Francisella genomes, W12-1067 genome exhibits only one copy of the other tRNA genes (Asn, Asp, Cys, Gln, Glu, His, Lys, Phe, Pro, Trp and Tyr). The tRNA genes for Ala and Ile are only present within the ribosomal RNA locus, which is also true for the other Francisella strains and for most other bacteria [12, 31, 38, 39]. Genome sequence comparison using the MAUVE program (http://www.DNASTAR.com) demonstrated that the genome of strain W12-1067 is poorly similar to the genomes of Ft. holarctica, Ft. novicida, F. philomiragia or F. hispaniensis AS02-814 (Ft. novicida-like 3523), indicating the evolutionary distance between these strains (data not shown). The MAUVE alignment of the draft genome sequence of strain W12-1067 with the genome sequence of Ft. novicida U112 is shown in the Additional file 3: Figure S2. The genome sequence of strain F. guangzhouensis 08HL01032T was not yet available for a comparative analysis, but a G + C content of about 32.5% of its draft genome was reported .
Genome features of variousFrancisellagenome sequences
Chromosome size (bp)
Nr. of protein-coding genes
GC content (%)
5S rRNA genes
3 + 1
3 + 1
3 + 1
3 + 1
16S RNA genes
23S RNA genes
GenBank accession number
*Draft genome (73 contigs); Fnov-U112, F. tularensis subsp. novicida U112; Fphi-25017, F. philomiragia strain ATCC25017; F-TX077308, Francisella spp. strain TX077308.
In F. tularensis strains six different insertion sequences (IS) elements (ISFtu1–ISFtu6) have been described. IS elements are important elements of F. tularensis genomes and are thought to be generally stable among different isolates [36, 40]. Within the genome of Francisella isolate W12-1067, we identified several mobile elements and transposases (Table 3). For the element ISFw3 (1,177 bp in length) we identified 18 copies within the genome sequence. The element encodes a putative integrase of 359 amino acids with 70% amino acid identity to the protein encoded by the IS481 element of Wolbachia endosymbiont of Drosophila simulans. All other elements are only present as a single copy. There is only one element (ISFw7) with significant similarity to one of the ISFtu elements of F. tularensis. ISFw7 encodes a putative transposase exhibiting 78% amino acid identity to ISFtu1 of Ft. holarctica strains. Of the 14 mobile elements identified, six (ISFw4, 5, 7, 9, 10 and peg_1255) exhibit homologs within Francisella strains (Table 3).
Mobile elements and transposases ofFrancisellastrain W12-1067
A number of genes involved in Francisella pathogenesis have been identified in various different studies, including in vivo negative selection screens of transposon mutant libraries of Francisella strains [41–44], reviewed in  and . We therefore looked for homologs of these virulence genes within the genome sequence of Francisella strain W12-1067. Some of them are shown in Table 4, and others, like LPS, capsule, type IV pili or FPI-associated genes, are described below. Various known virulence factors of Francisella exhibit homologs in strain W12-1067. For the most important virulence factors of F. tularensis, encoded by the genes of the Francisella Pathogenicity Island (FPI), no close homologs could be identified within the genome. However, we identified an FPI-like island at the genome sequence which will be discussed later.
(Putative) virulence factors of strain W12-1067
Macrophage growth locus protein
Ferrous iron transport protein
Twitching motility protein
Peptidyl-prolyl cis-trans isomerase
Toxin secretion ABC transporter
CBS domain, putative hemolysin
ClpB chaperone domain
Chitinase 1 (372 aa)
Chitinase 2 (590 aa)
Chitinase_glyco-hydro_19 domain, PP location, CBM
Chitinase 3 (437 aa)
Chitinase_glyco-hydro_19 domain, SP
Chitinase 4 (979 aa)
GH18_chitinase-like superfamily, SP, two internal repeats
Interestingly, the genome of isolate W12-1067 exhibit six different chitinases of different sizes and putative location (cytoplasmic, periplasmic or extracellular) (Table 4), which was in good agreement with the phenotypically identified chitinase activity within the supernatant of Francisella W12-1067 cells (see Additional file 1: Figure S1C). Four chitinases (peg_818, 1009, 1044 and 1477) exhibit a signal peptide and therefore could be secreted by the general secretion system (Sec). Two of the chitinases (peg_0816 and peg_0818) are separated by a gene encoding a putative DNA-invertase (peg_0817). We could not identify a homolog of this protein in the available genome sequences of Francisella strains. We were unable to determine whether the invertase is involved in DNA inversion in Francisella and whether this may influence the expression of the nearby chitinase genes. Chitinase peg_0816 is 76% identical to chitinase peg_0818. Both chitinases exhibit a homolog in F. philomiragia 25017 (Fphi_0512) and 25015, but not in the other Francisella strains, whereas homologs of the chitinases 1, 4 and 5 could be found in the genomes of Ft. tularensis, F. holarctica and F. philomiragia.
Chitinases (FTN_0627 and FTN_1744) have been detected as essential virulence factors of Ft. novicida and for biofilm formation [42, 44, 47]. In L. pneumophila a chitinase was also found to be involved in the infectivity of Legionella for mice . It would be interesting to further analyze the role of the different chitinases in strain Francisella W12-1067.
Furthermore, we identified three hypothetical proteins (peg_523, 567 and 1109) containing ankyrin-repeat domains. These proteins did not exhibit significant homology to any known protein (Table 4). For L. pneumophila it was shown that ankyrin-repeat-containing proteins are involved in the pathogen–host interplay during intracellular replication [49, 50].
Furthermore, we looked for genes encoding putative antibiotic resistance proteins. We identified ten putative multidrug resistance proteins (peg_126, 152, 681, 682, 683, 764, 1134, 1430, 1444 and 1445). In addition, we identified a Chloramphenicol acetyltransferase (peg_183), a Chloramphenicol phosphotransferase (peg_1413) and a Streptomycin-6 kinase (peg_1416) without a homolog in any of the available Francisella genomes. We performed growth inhibition experiments with the isolate W12-1067 and found that levels of resistance to erythromycin, chloramphenicol and streptomycin were comparable to those of F. philomiragia (data not shown). We also identified a putative Acriflavin resistance protein (peg_810) exhibiting 85% amino acid identity to the AcrB protein (Fphi_1007) of F. philomiragia 25017.
Since we identified putative signal peptides at the N-terminus of some of the chitinases and chitinase activity within the supernatant, we searched the genome sequence for genes of the general Sec system. The identified proteins are given in the Additional file 4: Table S2. We identified all proteins necessary for a putative functional Sec system plus two signal sequence recognition proteins (SRP) and three different signal peptidases. Therefore, strain W12-1067 seems to encode a functional Sec system for the transport of proteins across the inner membrane. We could not detect genes encoding homologs of a type-II secretion system (T2SS), but proteins (HlyB, HlyD, TolC2) of a putative T1SS (Table S2). We were also able to identify a putative functional Tol-Pal system generally involved in vitamin B12 or colicin translocation, but also in capsule synthesis and outer membrane vesicle formation [51, 52]. The presence of a putative T6-like SS will be discussed in the next section.
A new putative homolog of the Francisella pathogenicity island (FPI) and regulatory proteins
T6SS are widely distributed amongst diverse Gram-negative species. It is a complex molecular machine which injects effector proteins to target cells or bacteria . T6SS are involved in virulence and in eukaryotic cell targeting. They are also reported to have antibacterial activity [54, 55]. Most systems are able to function “anti-eukaryotically” or “anti-bacterially”, and one system has been reported to be able to do both [53, 56, 57]. It was also proposed that T6SS may target and defend predatory eukaryotes in the environment . For an overview see .
Francisella strains exhibit an FPI encoding a functional T6SS, needed for preventing phagolysosomal fusion and escape into the cytosol, which is therefore essential for intracellular replication and pathogenicity of Francisella[59–63]. The island is ~33 kb in length, encodes for 15–19 open reading frames (ORFs) and is present in Ft. tularensis strains, Ft. novicida and F. philomiragia (Figure 5A). In strains of F. tularensis this island is duplicated [36, 64–66]. However, it is still unclear yet whether both copies are needed for full virulence of these strains. Genes of the locus were named igl (intracellular growth locus, ) and pdp (pathogenicity determinant proteins, ). The complete locus was identified in 2004, and it seems to be acquired via horizontal gene transfer because of a lower G + C content compared with the core genome [60, 64, 65].
We were not able to identify a close homolog of the FPI within the genome of strain W12-1067. However, we identified two FPI-like clusters which seem to encode a further putative T6SS (Figure 5). Clusters I (Figure 5B) and II (Figure 5C) each code for 16 ORFs. Homologs of iglABC, dotU and pdbB are present, and the whole island I is similar to an FPI-like element (FTN_0038-0054) recently identified within the genome of Ft. novicida, but not yet discussed further [60, 64]. Within the genome sequence, island I is also localized between the pyrD and tyrA genes and island II between rpsU and glmS (Figure 5B and C). All genes, features of the ORFs and closest homologs of both systems identified within the genome of W12-1067 are given in Table 5. In a recently published model of an FPI-encoded T6SS-like apparatus IglAB (TssBC), IglC (TssD), DotU (TssL), PdpB (TssM) and VgrG (TssI) were identified as core proteins of the T6SS . We identified similar proteins within the identified loci of strain W12-1067. In strain F-TX077308, the FPI-like gene cluster is localized at a different site within its genome sequence (Figure 5B). Altogether these findings led us to hypothesize that these loci may encode a further T6-like SS in Francisella strain W12-1067. As mentioned above, infection assays and EM analysis indicateed that strain W12-1067 is able to replicate intracellularly (Figures 3 and 4), which also suggest that there should be a functional T6SS in this strain. However, since the EM analysis did not yet clearly demonstrate whether the strain is released into the cytosol, further experimental data are needed to verify this hypothesis.
Genes of the putative T6SSs I and II ofFrancisellaisolate W12-1067
Name (T6SS I)
Peg Nr. (aa)
Closest homolog (% aa identity)
DUF2345, VgrG-like, Rhs family
SP, lipid attachment site (IglE-like)
SP, TM domain (PdpB-like/TssM)
homologous to IglA/TssB
homologous to IglB/TssC
helical bundle domain (putative TssD)
Cl, T6SS-associated like protein
C-terminal TM domain
DUF2077 (putative DotU/TssL)
Hyp. protein (33%) Flavobacterium
IglD (49%) F-TX077308
Name (T6SS II)
Closest homolog (% aa identity)
Cl, homologous to IglA/TssB, DUF770
Cl, homologous to IglB/TssC, COG3517
2 TM domains
Hyp. protein (49%) Prevotella histicola
1369 (928) 
Cytoplasmic membrane, SP
DUF2077 (putative DotU/TssL)
DotU, (29%) Desulfonatronospira
Cl (DUF2345, vgrG)
*determined by using “psortb” (http://www.psort.org/psortb); Cl, cytoplasmic localization; TM, transmembrane domain; SP, signal peptide; (aa), number of amino acids.
Various regulatory proteins are known for Francisella, and the regulators MglAB, SspA, PmrA, FevR, MigR and Hfq were identified to be involved in the regulation of genes of the FPI [59, 61, 68–76]. In F. tularensis the genes of the FPI are upregulated during intracellular growth within macrophages [61, 68, 77–80]. In the genome of strain W12-1067 we identified only homologs of MglAB and Hfq, but also two further response regulator proteins (OmpR1 and R2) as well as two sigma factors (Sig-70 and Sig-32) and homologs of IscR, ArsR, Crp and Fur (see Additional file 5: Table S3). In addition, proteins involved in the stringent response could be identified (SspB, SpoT and RelA).
Surface structures: The wbt locus, capsule and type IV pili
LPS. The lipid A core portion of the LPS anchors the lipopolysaccharide structure to the outer membrane, whereas the O-polysaccharide chain is the predominant epitope recognized by the immune system and specifies antigenicity. Ft. tularensis subspecies-specific antisera have been generated and applied to antigen detection in F. tularensis. Furthermore, LPS is used as an antigen in seroprevalence studies and for diagnostic of human tularemia [6, 82, 83]. However, the LPS produced by F. tularensis is less endotoxic compared to other Gram-negative bacteria, such as E. coli, a phenotype also known for L. pneumophila LPS [84–86]. Genes probably involved in the biosynthesis of the O-polysaccharide chain are given in supplementary Table 4 (see Additional file 6: Table S4). Obviously, there is one cluster of genes (peg_0636-0646) for which homologs were found in Ft. novicida and F. philomiragia, whereas for genes peg_0609-0615 and peg_0628-0631 the homologs were only found in F. philomiragia or Ft. novicida U112, respectively. In addition, there is another cluster of genes (peg_0616-0627) which seems to have no homologs in F. tularensis or F. philomiragia, but homologs were identified in different bacterial species, as in Vibrio, Pseudomonas, Sulfurovum or Acinetobacter (see Additional file 6: Table S4). The LPS structure of strain W12-1067 has not yet been analyzed further.
Capsule. Electron microscopy of strain W12-1067 grown on agar plates or in medium revealed the absence of a capsule (Figure 4A and data not shown). However, we identified three ORFs encoding homologs of capBCA genes (Table 4). The capBCA locus of Francisella is similar to determinants encoding the poly-gamma-glutamic capsule in Bacillus anthracis. These genes have been shown to be essential for the virulence of F. tularensis in a murine model of tularemia [44, 87]. Further experiments will be needed to analyze the role and structure of the capsule of Francisella strain W12-1067 and to identify conditions necessary for the putative induction of capsule gene expression.
Type IV pili. Electron microscopy of strain W12-1067 did not show any pili on the surface of the bacteria grown in medium at 37°C (Figure 4A) or on agar plates (data not shown). However, we identified homologs of the type IV pilus (Tfp) encoding loci of F. philomiragia ATCC 25017 in the genome sequence of W12-1067 (see Additional file 7: Table S5) . We could not identify a PilA homolog, a homolog of the second PilW protein Fphi_0522 and of the two additional PilA/PilE pilus assembly proteins (Fphi_0424/0449). Tfp systems are known to be involved in bacterial pathogenesis, bacterial adhesion and twitching motility . In L. pneumophila Tfp pili are required for twitching motility, natural competence, biofilm formation and are involved in the attachment to host cells [90–93]. Tfp have been observed on the surface of Ft. novicida and Ft. holarctica[94, 95], and Tfp are involved in the pathogenicity of Francisella[96–98].
We identified three different type II toxin–antitoxin systems (peg_0599-0600, peg_0704-0705 and peg1296-1297). In type II systems, the antitoxin (small unstable protein) sequesters the toxin (stable protein) through protein complex formation (reviewed in ). Peg_0599 encodes a protein (84 amino acids, putative toxin), exhibiting a Pfam_Plasmid_Txe domain and 67% amino acid identity to the YoeB toxin of Pleurocapsa sp. PCC7319. peg_0600 encodes the respective putative antitoxin (83 amino acids), exhibiting a Pfam_PhdYeFM (type II toxin–antitoxin) and shows 60% amino acid identity to the prevent-host-death protein of Methylocystis rosea. The second system is composed of protein Peg_0704 (96 amino acids), exhibiting a HTH-XRE motif and a HigA-antidote (VapI) domain and shows 65% amino acid identity to protein LLO_065 of Legionella longbeachae NSW150. The respective putative toxin (HigB, 97 amino acids) is encoded by peg_0705, exhibiting a Pfam_plasmid killer domain, and shows 62% amino acid identity to the plasmid maintenance system killer protein of Deferribacter desulfurricans SSM1. Both described systems seemed to have no homolog in the sequences of Francisella available so far and are localized on contig_34 of the draft genome of strain W12-1067.
The third system is localized on contig_46. peg_1296 encodes for a protein (85 amino acids, putative antitoxin), exhibiting a Pfam_PhdYeFM domain (type II toxin–antitoxin system), and shows 66% amino acid identity to the plasmid-encoded (pF243) protein F243_0001 of F. philomiragia ATCC 25017 and 65% identity to the Phd protein (pFNL10_p3) of Ft. novicida. The respective putative toxin (84 amino acids) is encoded by peg_1297, exhibits a Pfam_Plasmid_Txe (YoeB) domain and shows 79% amino acid identity to F243_0002 of F. philomiragia. Plasmid pF243 is 5,072 bp long and was predicted to encode six putative ORFs . ORFs F243_0001 and F243_0002 are organized in an operon that is similar to the phd-doc post-segregation killing system operon of pFNL10 [100, 101]. This post-segregation killing mechanism relies on the difference in stability of the antitoxin and toxin. In the daughter cells the plasmid-free bacteria will be killed by the activity of the toxin [102, 103]. Chromosomally encoded toxin–antitoxin systems may stabilize chromosomal regions during evolution and seem to be involved in host regulatory networks or fitness advantages . Less is known so far about toxin–antitoxin systems in Francisella, but they have been used to construct plasmids which are stable without a selective marker gene [101, 104].
The isolation of strain W12-1067 in Germany indicates for the first time the presence of a close homolog in Europe of the new species F. guangzhouensis recently identified in China. In addition, to our knowledge this is the first report of a Francisella species other than F. tularensis isolated in Germany. Further research is needed to analyze the spectrum of Francisella species present in natural habitats in Germany.
The growth optimum of the isolate is approximately 30°C, it is able to grow without additional cysteine within the medium and the strain is halotolerant. The analysis of the genome sequence of the new isolate revealed a lot of known Francisella virulence factors, but also the absence of FPI, the major virulence factor of Francisella strains. Instead, the isolate seems to exhibit a putative new T6SS, and W12-1067 is able to replicate within eukaryotic host cells. Therefore, the isolate seems to be an interesting species to be analyzed further.
We thank U. Erikli for her careful review of the manuscript. This work was supported by the Robert Koch Institute (ZBS 2) and grant 1369-364 from the Robert Koch Institute to CL.
Cellular Interactions of Bacterial Pathogens, Centre for Biological Threats and Special Pathogens, Division 2 (ZBS 2), Robert Koch Institute
Department of Genomics and Applied Microbiology & Göttinger Genomics Laboratory, Institute of Microbiology and Genetics, Georg-August-University of Göttingen
Centre for Biological Threats and Special Pathogens, Division 4 (ZBS 4), Advanced Light and Electron Microscopy, Robert Koch Institute
Institute of Medical Microbiology and Hygiene, Consultant Laboratory for Legionella, TU Dresden
Centre for Biological Threats and Special Pathogens, Division 2 (ZBS 2), Highly Pathogenic Microorganisms, Robert Koch Institute
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