Transcriptome Analysis of the Intracellular Facultative Pathogen Piscirickettsia salmonis: Expression of Putative Groups of Genes Associated with Virulence and Iron Metabolism

The intracellular facultative bacteria Piscirickettsia salmonis is one of the most important pathogens of the Chilean aquaculture. However, there is a lack of information regarding the whole genomic transcriptional response according to different extracellular environments. We used next generation sequencing (NGS) of RNA (RNA-seq) to study the whole transcriptome of an isolate of P. salmonis (FAVET-INBIOGEN) using a cell line culture and a modified cell-free liquid medium, with or without iron supplementation. This was done in order to obtain information about the factors there are involved in virulence and iron acquisition. First, the isolate was grown in the Sf21 cell line; then, the bacteria were cultured into a cell-free liquid medium supplemented or not with iron. We identified in the transcriptome, genes associated with type IV secretion systems, genes related to flagellar structure assembly, several proteases and sigma factors, and genes related to the development of drug resistance. Additionally, we identified for the first time several iron-metabolism associated genes including at least two iron uptake pathways (ferrous iron and ferric iron uptake) that are actually expressed in the different conditions analyzed. We further describe putative genes that are related with the use and storage of iron in the bacteria, which have not been previously described. Several sets of genes related to virulence were expressed in both the cell line and cell-free culture media (for example those related to flagellar structure; such as basal body, MS-ring, C-ring, proximal and distal rod, and filament), which may play roles in other basic processes rather than been restricted to virulence.


Introduction
In Chile, Piscirickettsiosis is the most important bacterial disease in salmonid fish and causes high mortalities and significant economic losses during the sea water phase (approximately U$ 100 million per year) (reviewed in [1]). The etiologic agent is P. salmonis, a facultative PLOS ONE | DOI: 10.1371/journal.pone.0168855 December 29, 2016 1 / 17 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 iron-activated genes and 50 iron-repressed genes that were involved in iron acquisition or oxidative stress defense, revealing that iron is an important regulator of flagellar biogenesis [24]. This demonstrate the usefulness of this approach for discovery new metabolic pathways in prokaryotes.
In this paper, we are aimed at gain knowledge on the pathways related to several mechanisms used for iron metabolism and virulence in P. salmonis. We present for the first time information concerning the transcriptomic response of P. salmonis via NGS technology and the pathways involved in the growth of this bacterium in different culture conditions. In particular, considering media lacking iron, which is expected to be an important factor involved in bacterial growth [25]. We perform a series of experiments using different cultures (a cell line and a liquid medium supplemented or not with iron) to describe the full transcriptomic response of P. salmonis under these conditions.

Experimental design
Sf21 cells from 4 culture flasks containing monolayers infected with P. salmonis that reached 95-100% CE were centrifuged. Then, an aliquot of 2 ml from the supernatant named "CC medium" (cell culture medium) was centrifuged at 4000 x g for 10 min at 4˚C. The pellet was used for total RNA extraction. The rest of the supernatant was also centrifuged, and the pellet was cultured in liquid medium supplemented or not with FeSO 4 named "CFC-I medium" (cell-free culture medium with iron supplementation) and "CFC-N medium" (cell-free culture medium without iron supplementation), respectively. The infected media were incubated at 25˚C and 100 rpm. After 24 h, a pool of bacterial pelleted from each condition (CFC-I and CFC-N), was obtained for total RNA extraction.

RNA isolation procedures
Total RNA was extracted using TRIzol (Invitrogen) following the manufacturer's instructions. The RNA pellets dissolved in diethylpyrocarbonate (DEPC)-treated water were quantified using the Qubit 1 fluorometer (Invitrogen) and stored at -80˚C. The quality of RNA extraction was analyzed according to the RNA quality number (RQN) using the Fragment Analyzer 1 .

Library preparation and transcriptome sequencing
To sequence the transcriptome, RNA samples were treated with DNase I (Fermentas) and purified with the RNeasy MinElute Cleanup Kit (Qiagen) to remove traces of DNase I. Depletion of rRNA was accomplished using the Ribo-Zero™ Kit (Bacteria)-Low Input. Purification of the rRNA-depleted samples was accomplished with the RNeasy MinElute Cleanup Kit (Qiagen). RNA-seq libraries of the samples were prepared using the ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Epicentre 1 ) according to the manufacturer's protocol. The assessment of the library quantity and quality were completed using qPCR (Illumina) and the Fragment Analyzer 1 , respectively. The samples were sequenced using the v2 Reagent Kit (300 cycles PE kit) with NGS equipment (Illumina Miseq 1 ).

Mapping of reads and description of transcriptomes
The reads obtained from the CFC and CC media samples were mapped to the annotated genome of the FAVET-INBIOGEN (Whole Genome Shotgun (WGS) project accession number LELB01000000) isolate using the CLC Genomics Workbench. The P. salmonis transcriptomes were characterized according to the functional classification of the Rast server [27] and Kyoto Encyclopedia of Genes and Genomes (KEEG) through the automatic metabolic pathway annotation server KAAS [28]. According to Humphrys et al [29], we defined two different cut-offs to asses a minimal gene expression following sequencing. The minimal gene expression was considering for a gene with a minimum of a RPKM (Reads Per Kilobase of transcript per Million mapped reads) ! 0.1 and a minimum of 10 mapped reads. Additionally, to assess the relative abundance of genes between both grown conditions, we used the test of proportion through Kal's test statistic using CLC Genomic Workbench [30], The genes were considered as differentially expressed with FDR (false discovery rate) 0.01.

Results
Comparisons between transcriptomes obtained under different culture conditions P. salmonis from the CC medium had 2188 genes with a minimal level expression (RPKM ! 0.1 and a minimum of 10 mapped reads). In the CFC-I medium, P. salmonis expressed 2264 genes and 2296 in the CFC-N medium with a minimal level expression.
For the 6 genes used, there was a high correlation between Ct and RPKM (Read per kilobase per million mapped reads) values for each gene in both samples (Fig 1).
A total of 1205 genes were differentially expressed, where 876 genes were up regulated and 328 genes were down regulated in CFC-I culture medium compare to CC culture medium with FDR 0.01. The number of differentially expressed genes related to virulence is presented in Table 1. When we compared CFC-I with CFC-N, this analysis revealed 323 genes differentially expressed (FDR 0.01), with 145 genes up regulated when iron was supplemented in the media. A description of the number of genes related to virulence, differentially expressed presented in Table 2.

Pathways associated with virulence and iron metabolism
We sought to divide the analysis in two main mechanisms that the bacteria could use to interact with the host. In particular, we analyzed virulence factors such as flagellar structure, type IV secretion system, sigma factors, proteases, chaperones and mobile proteins elements, as described previously. We also analyzed the pathways related with uptake, utilization and storage of the iron (see below).

Virulence-associated genes
Flagellar structures. Although this pathogen has been described as a non-motile bacterium, a total of 46 genes involved in flagellum biosynthesis, assembly, structural organization, function, and the flagella motor genes were expressed in P. salmonis under different conditions     (FDR 0.01) (S1 Table). When we compared CFC-I with CFC-N, we did not find any genes differential expressed between treatments. All these flagella related genes have been involved in protein export (especially of virulence factors) in non-motile bacteria. Several proteins that form the flagellar metabolic pathway may export other proteins to the extracellular space, especially those linked to virulence [31,32]. Maezawa et al. [33] reported the existence of hundreds of flagellar-expressed hooks and basal body structures in a bacterium that lacked the filament portion of the flagellum, supporting previous suggestions of the possible specialization of these genes in protein export from the bacterium to the host. Abby and Rocha [34] suggested that non-flagellar type III secretion systems (injectisomes) evolved from the bacterial flagellum. Additionally, P. salmonis can produce extracellular products (EPCs), at least some of which are thermo-labile exotoxins that probably play a role in the pathogenesis of Piscirickettsiosis [35]. Overall, the results showed that P. salmonis expressed several genes that were associated with the flagellar structure; However, the fliC gene that encodes the filament of the flagellum was not found in the annotated genome. Moreover, the subunits flaA and flaB which also encode the filament of the flagella were expressed. This results show that P. salmonis is expressing another host-cell adaptation system (i.e., the type III secretion system), which likely delivery virulence factors such as flagellar component and exotoxins into eukaryotic cells. Further analysis of this secretion system are required in order to understand the interaction of these factors in the host.
The type IV secretion system. Data from our experiment showed that the FAVET-IN-BIOGEN isolate expressed 24 genes annotated in the icm/dot system. Among this group, eight genes were differentially expressed in CC medium (FDR 0.01) compared with CFC-I (S1 Table), and two were differentially expressed between CFC-I and CFC-N (S2 Table). The group of genes that form the icm (intracellular multiplication)/dot (defective organelle trafficking) system have been described as a type IV secretion system (T4SS) that is responsible for intracellular survival and multiplication. T4SS has been described in L. pneumophila and C. burnetii as one of the most important virulence factors of these pathogens [36-39], which are both pathogens, are phylogenetically related to P. salmonis.
Proteases. Bacterial pathogens express a group of proteases that contribute to virulence either directly due to host interactions or indirectly (e.g., allowing replication in the host environment) [40]. Because P. salmonis is a facultative intracellular bacterium, we would expect to find expression of alternative proteases related to these processes that have not been previously described in this bacterium. Indeed, the transcriptome of the FAVET-INBIOGEN isolate revealed 20 genes (e.g., clpP, clpA, clpS and clpX) encoding proteases and chaperons that were expressed under all culture conditions and would contribute to bacterial adaptation to the hostile conditions produced by host cells [41]. Moreover, clpB, whose expression has been described previously in P. salmonis, was differentially expressed in the CC medium compared with CFC-I medium (FDR 0.01) (S1 Table), but it was not differentially expressed between CFC-N and CFC-I.
Sigma factors. Bacterial pathogens have alternative sigma factors that regulate the expression of virulence genes and virulence-associated genes in response to a particular stimuli, which can be critical for host establishment [42]. Alternative sigma factors are a class of proteins that constitute essential dissociable subunits of the prokaryotic RNA polymerase [43]. The association of appropriate alternative sigma factors with the core RNA polymerase provides a mechanism for cellular responses mediated through the redirection of transcription initiation [44].
The transcriptomic responses of the FAVET-INBIOGEN isolate under all culture conditions showed 10 genes related with sigma factors. Seven were differentially expressed between CC medium and CFC-I (S1 Table), and two between CFC-I and CFC-N (S2 Table). Among those expressed genes; rpoE, encoding the sigma factor E (σ E ) that controls specific functions involved in the interaction of the bacterium with the host (e.g., synthesis, assembly and homeostasis of lipopolysaccharides and outer membrane porins including several proteases) [45]; rpoD, which has been described as a housekeeping gene in Gram-negative bacteria; rpoH, which is associated with the heat-shock response; rpoS, which contributes to bacterial survival in stressful environments [44]; and rpoN, which has been reported to be involved in flagellar regulation [46].
Chaperones. Molecular chaperones are essential for the survival of all three kingdoms of life and have been shown to play key roles in the integration of cellular and organismal responses to environmental fluctuation. Heat shock proteins (Hsp) comprise a subgroup of molecular chaperones that are induced in response to adverse environmental conditions. These proteins have been reported for several bacteria during infection processes, and thus form a first line of attack that helps consolidate pathogen virulence [47]. Similar to many other pathogenic bacteria, we expect that P. salmonis would express several groups of chaperones that were important for the invasion process in the host. Indeed, numerous genes encoding Hsp33, Hsp40, Hsp60, Hsp70 and Hsp90 family proteins were annotated in the transcriptomes of P. salmonis. The genes encoding the heat shock protein GrpE, GroES, GroEL, Hsp90 and Hsp70, which were more expressed in the CC medium, and the gene encoding the Hsp33, hsp40 and "Protein export cytoplasm chaperone" proteins were more expressed in the CFC-I medium (FDR 0.01) (S1 Table). When we compared CFC-I with CFC-N only 2 genes were not consistently differentially expressed, hsp40 which was more expressed in CFC-I and hsp90 which was more expressed in CFC-N (FDR 0.01) (S2 Table).
Mobile protein elements. The analysis of the transcriptomes in this study revealed more than forty annotations defined as "mobile protein elements", which are defined as DNA segments encoding enzymes and other proteins that allow movement of the genomic DNA [48]. Sixteen of them were differentially expressed between CC medium and CFC-I (S1 Table), and seven between CFC with and without iron supplementation (S2 Table) (FDR 0.01).

Iron metabolism-associated genes
As mentioned before, few studies are available in the literature regarding iron metabolism in P. salmonis. Thus, to characterize the mechanisms underlying iron homeostasis in this bacterium is important, in order to understand more deeply how the bacteria interact with the host. In all the media used, a total of 41 genes related to iron homeostasis were expressed and sorted into four functional groups: uptake/transport, intracellular utilization, storage and.iron regulation (S1-S3 Figs).
Iron uptake. The transcriptomic response of P. salmonis was associated with three pathways involved with the transport for siderophores. Both transcriptomes contained homologues of vibrioferrin (pvsA, pvsB, pvsC, pvsD, pvsE, and pvuA), a member of the carboxylate class of siderophores that has been described in marine a bacteria [49,50]. Among them, pvsB, pvsC, pvsD, pvsE were differentially expressed in CC medium versus the CFC-I medium (FDR 0.01) (S1 Table). Additionally, a homologue of fhuC, which is involved in the iron uptake by siderophores of the hydroxamate class, was also represented in both transcriptomes, but more expressed in CC medium (FDR 0.01) (S1 Table). A homologue of the feoB gene and other of the feoA gene that are take up Fe 2+ directly were expressed in both cultures media. In addition, a homologous gene fecD that is involved in iron uptake by siderophores of the dicitrate class was also contained in all transcriptomes analyzed of the bacteria (associated with CC, CFC-I, CFC-N), but without showing statistical differences between both grown conditions.
When we compared CFC-I with CFC-N two genes were differentially expressed (feoA and pvuA) which were more expressed in CFC-N (FDR 0.01) (S2 Table).
Iron utilization. Iron-sulphur [Fe-S] clusters and ferredoxins (fdx) play an important role in cellular processes, such as bacterial respiration, cofactor biosynthesis and gene expression [51]. As expected, the [Fe-S] cluster assembly pathway was represented in both growth conditions assayed for P. salmonis by genes encoding for the following proteins: a cysteine desulphurase that catalyzes the release of sulphur from L-cysteine, which is one of the essential components of the CFC medium; the SufE protein that accepts the sulphur molecule that is necessary for [Fe-S] cluster assembly [52]; an ATPase SufC protein and two assembly proteins (SufB and SufD) [53]; a gene encoding a likely iron binding protein from the SufA family; and a gene associated with the iron binding protein IscA, which is involved in [Fe-S] cluster assembly. All these genes were over represented in CFC-I medium in relation to CC medium (FDR 0.01) (S1 Table). More importantly, we detected the expression of iscR, which is a necessary regulator of [Fe-S] cluster assembly [54,55] and that was 6-fold more represented in CFC-I medium than CC medium (FDR 0.01) (S1 Table).
Comparing CFC-I with CFC-N there were four genes differentially expressed (pvuA, 4Fe-4S ferredoxin-iron sulfur binding, IscR and probable iron binding protein from the HesB_IscA_SufA family) (FDR 0.01), which three of them more expressed in CFC-I (S2 Table).
Iron storage. As expected, all the transcriptomes contained the bfr gene that encodes bacterioferritin, which was 1.4 fold more expressed in CFC-I medium than CC medium (FDR 0.01) (S1 Table), and 1.06 fold more expressed in CFC-I than CFC-N medium (S2 Table). This protein is involved in intracellular iron storage and has been described as the most important storage protein for iron in bacteria [56,57].
Iron regulation. Similar to many other prokaryotes, iron homeostasis in P. salmonis appear to be regulated by the fur gene. The fur gene that encodes the main transcriptional regulator of iron genes [58,59] was almost 3-fold more represented in CFC-I medium when compared with the CC medium (FDR 0.01) (S1 Table). However, the "RNA-binding protein hfq" gene was also differentially expressed in all comparison (S1 and S2 Tables). This gene has been described to encode a chaperone of a small RNA named RhyB that was also responsible for iron homeostasis, albeit at a different level than fur [60,61]. A further analysis of the small RNA will be required in order to understand more deeply this mechanism.
Oxidative stress. High concentrations of iron result in an oxidant environment that catalyzes reactive species of oxygen, mainly through the Fenton reaction. These molecules are toxic causing cell death [62,63]. Several expressed genes were observed under both conditions, e.g: alkyl hydroperoxidase D (ahpD), alkyl hydroperoxide reductase C (ahpC), catalase (kat) and superoxide dismutase (sodB), which are associated with protection of the cell against oxidative stress and were up regulated in the CFC-I medium compared with CC medium (FDR 0,01) (S1 Table). Other seven genes were differentially expressed between CFC-I and CFC-N, four of them were more expressed in CFC with iron supplementation (FDR 0.01) (S2 Table).

Genes associated with virulence processes
Our results showed that 46 genes were associated with flagellar biosynthesis, assembly, structural organization, and function (S3 Table). To date, P. salmonis has been described as a non-motile bacterium [1]. However, in this type of facultative intracellular symbiotic bacteria the presence of the flagella is unnecessary and energetically expensive unless proteins involved in the flagella pathway are also involved in other essential functions for the bacterium or the host [31].
During in vivo infection, P. salmonis replicates in the phagocyte preventing phagosome fusion with the lysosome. Therefore, the bacterium requires the expression of the necessary metabolic machinery to survive under these conditions [1,64,65] The expression of the Dot/Icm type IV secretion system (icmK, icmE, dotA and dotB) was previously described in P. salmonis [14]. Our results demonstrated that those dot/icm gene homologues were expressed both during in vitro cell line infection and in cell-free media, suggesting putative constitutive expression, confirming thereby the hypothesis previously suggested by Gomez et al. [14]. In L. pneumophila, this Type IV secretion system shows cytopathogenicity and hemolytic properties in addition to being essential for the induction of apoptosis [66,67]. To understand and analyze the functions of proteins delivered into host cells by this mechanism, it is important to improve the knowledge of the mechanisms related with P. salmonis pathogenesis.
The stressful conditions of the phagosome (i.e., oxidative burst and fusion of the phagosome with lysosomes) are capable of inducing the expression of bacterial chaperones (heat shock proteins, Hsp) and proteases, which also participate in the survival strategies of intracellular pathogens. Our results suggest similar survival strategies due to the expression of genes that are homologues of the most important classes of Hsps described in bacteria (Hps33, DnaJ, DnaK, HtpG, GroEL, GroES and GrpE proteins). All these proteins are involved in protein folding processes in E. coli, B. suis, C. jejuni and S. enterica serovar Typhimurium [47, [68][69][70]. At present, DnaK protein is the best characterized bacterial chaperone. Mutations in dnaK increase the sensitivity of normally resistant S. aureus strains to oxacillin and methicillin; moreover, mutations in dnaK or dnaJ in E. coli make the cells susceptible to fluoroquinolones [71].
The FAVET-INBIOGEN isolate also expressed the clpP and clpB genes that encoded ATPdependent proteases that were described as important virulence factors in numerous bacterial pathogens [72]. The represented clpA and clpX genes in the transcriptomes have been described in Gram-negative bacteria [40]. These genes encode two ATPase-chaperones that interact with the ClpP protease [41]. In L. pneumophila, the ClpP homologue is required for cell division and several transmission traits, including stress tolerance, cell shortening, sodium sensitivity, cytotoxicity and intracellular multiplication [73]. Moreover, the ClpB protease contributes to resistance to various stress conditions, intracellular multiplication and virulence in F. tularensis [74]. The presence of the clpB gene has been described in the P. salmonis showing significantly increased expression during infection of the SHK-1 cell line compared to the cellfree medium, indicating that clpB could also be expressed during propagation in host cells permitting the adaptation to the hostile conditions in the host [15]. We found similar results in our study, i.e. the clpB gene was significantly more expressed in the CC medium (Sf21 cell line), thereby confirming the hypothesis of Isla et al. [15]. Despite the fact that phenotypic protein expressions have not yet have been demonstrated in P. salmonis, data concerning its presence in the genome and its gene expression will allow the study of alternative mechanisms of survival in the host.

Metabolic pathways and iron uptake
Previously, four putative genes encoding siderophore-related proteins involved in iron metabolism and transport (one hemH gene, two tonB genes, and one fur gene) were reported in the published genome of P. salmonis [75]. The results of the transcriptome analysis of our isolate revealed two pathways for iron uptake: through siderophores and iron ferrous uptake.
Several Gram-negative bacteria synthesize and excrete low-molecular-weight compounds that chelate iron with high affinity that are called siderophores [76]. Siderophores are synthesized and secreted for iron acquisition; for example, muscle tissues expressing carrier proteins in Atlantic salmon were recently demonstrated to have a significantly higher bacterial load and became a niche where P. salmonis could acquire iron [25]. The FAVET-INBIOGEN isolate expressed genes encoding a protein system homologous to a system present in V. parahaemolyticus that was involved in the biosynthesis of vibrioferrin [77]. Because P. salmonis is a Gramnegative bacterium, the expressed genes also encoded proteins of the TonB system, which is a sophisticated mechanism that supplies the required energy for ferri-siderophores uptake. The TonB system includes an integral membrane protein (ExbB), a membrane-anchored periplasmatic protein (ExbD) and the TonB protein [78,79].
The feo system is the main route described for the uptake of Fe +2 and is associated with the growth of bacteria under anaerobic or microaerobic [80,81] and to a lesser extent aerobic conditions. This system was described in E. coli K-12; however, it has also been described in Salmonella [82], Campylobacter [83], Francisella [84] and Legionella [85]. The expression of feoB serves as an alternative pathway for iron uptake by P. salmonis.
The RNA-seq data show that bacterioferritin (bfr) is a mechanism that the bacteria is using for iron storage. This is a ubiquitous system used for the other facultative intracellular bacteria, such as Salmonella and B. abortus [86,87]. When intracellular iron concentrations are very high, this family of proteins also plays an antioxidant role and sequesters iron away from oxygen free radicals [88].
To assembly the [Fe-S] cluster (in addition to serving as a source of iron), the bacterium requires a source of sulphur. The sulphur is supplied by L-cysteine, an amino acid with a thiol side chain, which is an important supplemental component of the CFC medium. After transport into the cytoplasm, the cysteine desulphurase catalyzes the release of sulphur from L-cysteine, which is necessary for [Fe-S] cluster assembly [89]. Cysteine supplementation is not required for intracellular growth in macrophages, mammalian cell lines, or natural amoebic hosts, indicating that cysteine and other essential amino acids are obtained directly from the host [90].
An environment with excess iron together with reactive oxygen species (ROS) via the Fenton reaction causes oxidative stress. This stress increases the damage associated with [Fe-S] clusters, protein carbonylation, Cys/Met-residue oxidation, membrane lipid peroxidation, and DNA damage. The main strategy employed is the production of enzymes that degrade ROS species to maintain stress levels within a range of tolerance [91]. Our bacteria expressed alkyl hydroperoxidase D (ahpD) and alkyl hydroperoxide reductase C (ahpC), which provide significant antioxidant protection and have been described in various bacteria [92][93][94]. Additionally, Chung et al. [95] demonstrated that deletion of the ahpC genes alone in V. parahaemolyticus did not significantly affect the survival of this pathogen; thus, bacteria require more than one of these antioxidant molecules. Resistance against oxidative stress through biofilm formation was studied in C. jejuni, where the over expression of ahpC was associated with a decrease in biofilm formation; conversely, an ahpC mutant increased biofilm formation [96].
Finally, one of the most important factors involved in the development of resistance to antibiotics is the ability of bacteria to share genetic resources through lateral gene transfer (LGT) [97]. Drug resistance genes are commonly associated with mobile genetic elements (the mobilome) that can be transferred between bacteria by plasmids, integrons and transposons [98].

Final remarks
Several sets of virulence-associated genes were expressed under the experimental conditions studied here; however, the response of the bacteria was similar in different media. Virulenceassociated genes were expressed in the cell line culture and also in the cell-free medium supplemented with iron, indicating that these genes could play a role in basic processes rather than specifically in pathogenesis. Furthermore, the expressed genes related to flagellar structure could play a similar role to a type III secretion system during pathogenic processes, supporting the production of extracellular products by P. salmonis [35], but still this require further analysis considering the interaction with the host.
Overall, this study provides new insights into the functional mechanisms of P. salmonis. This information will be important for improving our basic knowledge of this bacterium to gain better prevention and control strategies against Piscirickettsiosis in Chile.
Supporting Information S1 Table. Differentially expressed genes related with virulence and iron metabolism between CC medium and CFC-I medium.
(XLSX) S2 Table. Differentially expressed genes related with virulence and iron metabolism between CFC-I medium and CFC-N medium.