Bioresource Technology 223 (2017) 105–114

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Genome-scale metabolic reconstruction for the insidious bacterium

in aquaculture Piscirickettsia salmonis
Pablo Fuentealba a,b, Camila Aros b, Yesenia Latorre b, Irene Martínez b, Sergio Marshall c, Pau Ferrer d,
Joan Albiol d, Claudia Altamirano b,e,⇑
a
Doctorado en Biotecnología, Pontificia Universidad Católica de Valparaíso – Universidad Federico Santa María, Valparaíso, Chile
b
Laboratorio of Cultivos Celulares, Escuela de Ingeniería Bioquímica, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile
c
Laboratorio of Genética e Inmunología Molecular, Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Curauma, Chile
d
Laboratorio de Biología de Sistemas, Departamento Ingeniería Química, Universidad Autónoma de Barcelona, Barcelona, Spain
e
CREAS CONICYT Regional GORE Valparaíso R0GI1004, Av. Universidad, Curauma, Chile

h i g h l i g h t s

First genome scale metabolic model was reconstructed for Piscirickettsia salmonis.

P. salmonis catabolised amino acid as carbon and energy source.

The metabolic reconstruction of P. salmonis comprising 445 reactions.

The model reproduces the growth rate measure in defined liquid broth.

The model is a metabolism analysis tool of P. salmonis.

a r t i c l e i n f o a b s t r a c t

Article history: Piscirickettsia salmonis is a fish bacterium that causes the disease piscirickettsiosis in salmonids. This
Received 2 June 2016 pathology is partially controlled by vaccines. The lack of knowledge has hindered its culture on laboratory
Received in revised form 4 October 2016 and industrial scale. The study describes the metabolic phenotype of P. salmonis in culture. This study
Accepted 11 October 2016
presents the first genome-scale model (iPF215) of the LF-89 strain of P. salmonis, describing the central
Available online 15 October 2016
metabolic pathway, biosynthesis and molecule degradation and transport mechanisms. The model was
adjusted with experiment data, allowing the identification of the capacities that were not predicted by
Keywords:
the automatic annotation of the genome sequences. The iPF215 model is comprised of 417 metabolites,
Piscirickettsia salmonis
Genome-scale metabolic model
445 reactions and 215 genes, was used to reproduce the growth of P. salmonis (lmax 0.052 ± 0.005 h1).
Metabolic reconstruction The metabolic reconstruction of the P. salmonis LF-89 strain obtained in this research provides a baseline
Nutritional requirement that describes the metabolic capacities of the bacterium and is the basis for developing improvements to
Defined medium its cultivation for vaccine formulation.
Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction in the aquaculture industry, equivalent to US$ 120 million in losses

1.1. Piscirickettsia salmonis and its impact
Piscirickettsiosis or salmon rickettsial septicaemia (SRS) is a dis-
ease that affects salmonids and is caused by Piscirickettsia salmonis
a facultative gram-negative bacterium (Henríquez et al., 2013;
Mauel et al., 2008; Mikalsen et al., 2008; Rozas and Enríquez,
2014). In Chile, the disease causes annual mortality rates of 35%
⇑ Corresponding author at: Escuela de Ingeniería Bioquímica de la Pontificia
Universidad Católica de Valparaíso, Avenida Brasil 2085, Valparaíso, Chile.
E-mail address: claudia.altamirano@pucv.cl (C. Altamirano).
(Figueroa et al., 2011). Attempts to control the disease use different
prophylactic mechanisms, including vaccines, whose effectiveness
has been questioned (Marshall and Tobar, 2014). The vaccine pro-
duction method involves propagation of P. salmonis using culture
technology from animal cell lines. This technology is used because
the culture media reported in the literature do not result in high
cell concentrations, are difficult to scale up and have deficient
traceability, even though they have lower operational cost. The cul-
ture media reported in the literature are mainly based on complex
substrates, hindering the possibility of further study about the
nutritional requirements of P. salmonis. However, our group has
developed a chemically defined culture medium (reference PCT/

http://dx.doi.org/10.1016/j.biortech.2016.10.024
0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
106 P. Fuentealba et al. / Bioresource Technology 223 (2017) 105–114
CL2014/000065), facilitating the characterisation of the kinetics of
P. salmonis in pure culture with the objective of understanding the
metabolic processes that sustain its growth.
1.2. Genome-scale metabolic reconstruction
Advances made in sequencing techniques and bioinformatics
tools for annotating genomes have facilitated the construction pro-
cess of genome-scale metabolic models to improve the under-
standing of organisms from a systemic perspective. This type of
model has led to improvements in fermentation processes by pre-
dicting the impact of genetic modifications on intracellular flux
distribution (Hendry et al., 2016). Different methods can be used
to calculate the metabolic fluxes, where the most widespread is
the classical metabolic flux analysis (MFA), which describes the
flux distribution of known cell phenotypes (Iwatani et al., 2008).
Another option is flux balance analysis (FBA) which is an MFA
approach that describes the fluxes of the metabolic network with-
out constraining the model with experimental data (Feist and
Palsson, 2010). This directed approach to improve cell performance
is in contrast to the traditional random mutation strategy that aims
to induce the appropriate mutation followed by its identification.
The validation of a metabolic model is a slow and costly process.
However this is compensated by the capacity and efficiency with
which problems are resolved (Blazeck and Alper, 2010). This has
led to the technological development of multiple fermentation pro-
cesses, mainly involving the synthesis of organic compounds, such
as polyketides, antibiotics, pigments, polymers, biodiesel, etc. (Liu
et al., 2010). This tool has also improved the understanding of bac-
terial pathogens, leading to the proposal of new control strategies
(Chaudhury et al., 2013; Juneja et al., 2016). This is the case with
the bacterium Francisella tularensis, which is related to P. salmonis.
The studies by Raghunathan et al. (2010) and Chaudhury et al.
(2013) present the metabolic reconstruction of F. tularensis, which
led to the characterisation of the capacities that result in
pathogenicity and the subsequent identification of new targets
for drugs. No metabolic reconstructions have been reported for
any other bacteria related to P. salmonis, such as Legionella tularen-
sis and Coxiella burnetii.
However, significant advancements have been made in the
metabolic reconstructions of other organisms, such as Escherichia
coli and Saccharomyces cerevisiae which have more robust meta-
bolic models (Kim et al., 2012). These have led to the successful
experiments reported by researchers such as Park et al. and Lee
et al., who used this method to improve the production of valine
and threonine, respectively, in E. coli, through gene overexpression
and deletion, reaching industrial production levels (Lee et al., 2007;
Park et al., 2007). Another similar example is that of Alper et al.,
who used an in silico analysis to predict a knockout series to
improve production of lycopene by 37% (Alper et al., 2005). In
another case involves the production of Interleukin-2 (IL-2) in
E. coli, in which the supplementation of amino acids needed to
increase IL-2 production was predicted (Sadeghyzadeh et al.,
2009).
The present study aims to identify the metabolic capacities of P.
salmonis LF-89 through the construction of a genome-scale meta-
bolic model that simulates the behaviour of the bacterium in terms
of growth, nutrient demand and the generation of extracellular
metabolites, under defined culture conditions. This is possible
because in recent years many efforts have been made to sequence
the genome of P. salmonis, resulting in the construction of different
assemblies for the LF-89 and other strains: EM-90, A1-15972, B1-
32597 and AUSTRAL-005. The genome-scale metabolic reconstruc-
tion process integrates current knowledge of P. salmonis, undoubt-
edly enhancing understanding about the metabolic phenotype
arising during cultivation. The genome-scale metabolic model is
then used to identify and determine the capacities that support
the growth of the bacterium, and its predictive capacity is evalu-
ated by comparison with experimental data. This information
could be used for the development of vaccine formulations against
SRS.
2. Materials and methods
2.1. Metabolic reconstruction
Three genome assemblies and a full genome of P. salmonis LF-89
strain were used. These are available on the NCBI database. The dif-
ferent genome sequences were housed in the SEED database
(http://theseed.org/wiki/Main_Page) and were annotated automat-
ically using the RAST tool (Rapid Annotaion using Subsystem Tech-
nology) (Henry et al., 2010). The metabolic reconstruction was
carried out based on the predicted metabolic genes, whose E-
values obtained when aligning the sequences with the BLASTX tool
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) on NCBI were significant.
Annotations for related bacteria were also reviewed, along with
the predicted metabolic context for the annotation. The predicted
transporters annotated automatically were evaluated using the
BLAST tool from the TCDB database (http://www.tcdb.org). The
information on the correctly predicted enzymes and transporters
was stored on a local database. The provisional version of the
metabolic reconstruction was inspected manually and corrected
to replicate the phenotype capacities; this process was based on
Henry et al. (2010) and Thiele and Palsson (2010).
2.2. Metabolic modelling
Model compliance with the mass balances was evaluated using
FBA. This also provided the metabolic fluxes (v) through the con-
structed network. This network was represented as a stoichiomet-
ric matrix (S), in which the rows are the metabolites (m) and the
columns are the reactions (n). It was assumed that the intracellular
metabolites are in a pseudo-steady state, and thus the mass bal-
ance can be expressed as:
Sv ¼0
The FBA was carried out using COBRA ToolBox v2.0 for Matlab.
The MFA was carried out using the stoichiometrically balanced
model, defining the biomass equation as objective function for
maximisation. Some measured fluxes were used as model con-
straints to predict the substrate uptake, metabolite production
and growth rates. The resulting linear FBA problem was solved in
the COBRA Toolbox using the free access solver GLPK.
2.3. Biomass equation
The biomass equation defines all the components required for
the synthesis of a new cell. The composition of molecules and
macromolecules that define the biomass composition are based
on the model of the related bacterium F. tularensis (Raghunathan
et al., 2010). The stoichiometry coefficients of the molecules and
macromolecules were estimated from the elemental composition
of the biomass of P. salmonis obtained with a combustion elemen-
tal analyser. The different coefficients were calculated using
numerical fitting, using constraints that define the expected range
of each compound in the bacterial biomass composition.
2.4. Strain and culture conditions
The bacterium P. salmonis LF-89 (ATCC VR-1361) was kept in
cryovials at 80 °C (Henríquez et al., 2013), and stocks were
 P. Fuentealba et al. / Bioresource Technology 223 (2017) 105–114
activated in complex medium (Henríquez et al., 2013). The P. salmonis
culture experiments were carried out in the defined medium
described in patent application PCT/CL2014/000065. The culture
medium is composed of 6.3 g/L K2HPO4; 9 g/L NaCl; 0.1 g/L
MgSO4*7H2O; 0.08 g/L CaCl2*2H2O; 0.032 g/L ferric citrate, MEM
vitamin solution from Sigma-AldrichÒ 2X, 0.56 g/L histidine, 1 g/L
cysteine, 0.41 g/L phenylalanine, 0.52 g/L valine, 0.48 g/L leucine,
0.48 g/L isoleucine, 0.66 g/L methionine, 0.65 g/L threonine,
0.54 g/L lysine, 0.52 g/L proline, 0.65 glutamic acid and 0.65 g/L
arginine. Culture samples were used to characterise the growth,
production and consumption kinetics of P. salmonis, data that
was used to model the metabolism of the bacterium.
2.5. Characterisation of culture kinetics
The specific growth rate was determined fitting the function
representing the growth kinetics of P. salmonis in the defined med-
ium, whose expression is derived and normalised by the biomass
production at the corresponding time. The growth of P. salmonis
in the culture was determined using turbidimetry at 600 nm and
this parameter was then converted to its equivalent dry cell weight
using a calibration curve.
The amino acid consumption rates and NH+4 production rates
were determined from concentrations measured by HPLC. The
samples were filtered with a 0.22 lm PDVF membrane, derivatised
using the AccQ*Fluor Reagent Kit and ran in a Waters AccQ*Tag
column using acetate buffer as the mobile phase (19.05 g of
sodium trihydrated acetate (NaAc*3H2O), 2.37 mL of triethylamine,
1 mg EDTA in 800 mL of miliQ water, pH 5.02 with H3PO4, diluted
to 1 L). The chromatography was carried out by elution gradient
(acetonitile:water (60:40) and acetate buffer, pH 5.02) at 37 °C,
with a pressure of 1200 psi and a flow rate of 1 mL/min. The spe-
cies were identified by fluorescence by exciting the sample to
250 nm and emitting at 395 nm. The consumption and production
profiles were fitted to functions whose derivation and normalisa-
tion by biomass production in the respective time gave the specific
consumption and production rates of the respective compounds.
The production of organic acids was measured by HPLC with a
Bio-Rad Aminex HPX-87H column (300  7.8 mm). The samples
were filtered with a 0.22 lm PDVF membrane. 5 mM sulphuric
acid was used as the mobile phase. The chromatography was car-
ried out at 35 °C with a flow of 0.6 mL/min and a pressure of
750 psi. The species were measured at 215 nm with UV detector.
The total protein production rate was calculated from the total
protein concentration measured with the Bicinchoninic Acid Kit for
Protein Determination from Sigma-Aldrich. The calibrated curve
was prepared with BSA (bovine serum albumin) in five concentra-
tions: 200, 400, 600, 800 and 1000 lg/mL. The samples of defined
medium post-culture were centrifuged at 10000 RPM for 10 min
and then filtered with a pore size of 0.22 lm. The cell free medium
was concentrated using Amicon Ultra-15 3 KDa filters from Merck
Millipore to eliminate interference produced by the cysteine in the
MDm. Each reaction was incubated for 30 min at 37 °C and its
absorbance measured using a spectrophotometer at 562 nm.
3. Results and discussion
3.1. Metabolic reconstruction
3.1.1. Genome annotation of P. salmonis LF-89 strain
The full genome of LF-89 has been sequenced (Pulgar et al.,
2015), having available different draft sequences (Annexe 1). The
most refined sequence gives a genome size of 3.18 Mb [GenBank:
CP011849.2], predicting 3143 genes and 2863 proteins. Three plas-
mids are also recognised, measuring 180 kb [GenBank: CP011850],
107
33 Kb (GenBank: CP011851] and 51 kb [GenBank: CP011852].
Intracellular bacteria related such as F. tularensis and L. pneu-
mophila, which vary from 0.9 to 2 Mb and 3.35 to 3.6 Mb, respec-
tively, depending on the strain (Jules and Buchrieser, 2007;
Larsson et al., 2009; Steinert et al., 2007). Bacteria comparable to
P. salmonis have smaller genomes, implying a limited capacity to
colonise and proliferate in several different environments (Singh
et al., 2013; Zaneveld et al., 2011). The reduced genomes in bacte-
ria makes them more dependent on their surroundings, which can
lead to transformation of intracellular facultative bacteria into
obligatory, as is the case with C. burnetii (Singh et al., 2013).
The genome assembly of LF-89 available in GenBank [GenBank:
CP011849, LELB00000000.1, ASSK00000000.2, AMFF00000000.2]
were used to made one refining genome annotation. For that, first
these sequences were annotated automatically using the RAST tool
of the ModelSeed database. This information enable to make one
draft metabolic reconstruction that then was corrected manually,
with bibliographic information, comparisons with related species
and metabolic modelling.
The ModelSeed database groups genetic potential identified
around a set of abstract functional roles, under the concept of a
subsystem (Aziz et al., 2008). These subsystems, shown in Fig. 1A,
are organised in decreasing order by the gene percentage predicted
for each. These subsystems group together predicted genes related
to cell processes from molecule and macromolecule synthesis
(DNA, RNA, lipids, proteins, amino acids), central metabolism, cell
structures, compound transit, response to environmental stimuli
(stress response, chemotaxis and mortality), cell cycle, respiration,
regulation mechanisms and virulence, among others. The genetic
potential grouped into subsystems of cofactors, vitamins and pros-
thetic groups, amino acid metabolism and protein metabolism
accounted of the highest number of genes, taking 31.3% of the total
of predicted genes (Fig. 1A).
3.1.2. General metabolic capabilities
In P. salmonis, predictions were made for the genes required for
glycolysis and most of those for gluconeogenesis and non-
oxidative pentose phosphate pathway. Also, the predictions have
been made for most of the genes that code the enzymes involved
in the Krebs cycle, anaplerotic pathways, pathway for nucleotides,
some amino acid and polysaccharides.
Genome annotation of P. salmonis allowed to predict the capac-
ity to metabolise galactose, whose carbon can be incorporated in
glycolysis as glucose 6-phosphate. Further, glycerol genes were
predicted which code for enzymes that allow incorporation this
substrate at the glycolysis as glycerone phosphate.
Greater metabolic potential is predicted for amino acid assimi-
lation (Fig. 2). It is predicted that threonine is transformed sequen-
tially to glycine and serine, then synthesising pyruvate. Pyruvate
synthesis is also predicted when alanine. The catabolic pathways
predicted for proline, arginine, glutamine and histidine converge
in the synthesis of glutamic acid, which would be incorporated into
the Krebs cycle as 2-oxoglutarate. Asparagine is likely deamined to
aspartic acid, which is incorporated into the Krebs cycle as oxaloac-
etate. It can be stated that P. salmonis may incorporate carbon from
amino acids along the glycolytic pathway at the pyruvate level and
in the Krebs cycle at the 2-oxoglutarate and oxaloacetate level.
More details of metabolic capacities of P. salmonis LF-89 predicted
and evidenced in Annexe 2 of Supplementary material.
3.1.3. Predicted transport mechanisms
By annotating the different genome assemblies and the full gen-
ome of P. salmonis automatically, it was found that the genetic
potential related to transport mechanisms is around 7% (Fig. 1A).
Of this proportion of the predicted genes, 40% correspond to the
incorporation of cations, 23% are related to Type II, IV and VI secre-
108 P. Fuentealba et al. / Bioresource Technology 223 (2017) 105–114

Fig. 1. Proportion of the genetic potential predicted in the P. salmonis genome. (A) Genetic potential grouped in subsystem. ‘‘Others” includes predicted genes in subsystems
of the metabolism of phosphorus, potassium, sulphur, nitrogen, aromatic compounds, iron acquisition, dormancy and sporulation. (B) Transporters predicted in P. salmonis.

tion systems and 19% to the import and export of peptidoglycan,
siderophores and different organic molecules (Fig. 1B). The remain-
ing proportion corresponds to transporters of peptides and amino
acids, representing 16%, and of saccharides and alcohols, which
only constitutes 2% of the total (Fig. 1B). It can be seen that this
bacteria has greater capacity to assimilate amino acids from the
surrounding environment than saccharide or any alcohol. This is
shown in Table 1, which presents the different peptide and amino
acid transporters, totalling 19 predicted genes versus 3 gene pre-
dictions for the potential incorporation of a saccharide, glycerol
or glycerol 3P. The predicted saccharide transporter is attributed
the capacity to potentially incorporate arabinose, galactose and
xylose. In F. tularensis, a greater diversity of transporters for amino
acids and peptides has been reported regarding carbon sources,
such as saccharides and alcohols (Meibom and Charbit, 2010).
The L. pneumophila genome codes around 12 ABC transport sys-
tems, permeases for amino acids and proteases, which are highly
induced during growth in macrophages (Manske and Hilbi, 2014;
Price et al., 2014). For the other aspects of its genome, a possible
transporter for D-xylose (galactose, arabinose) proton symporter
and under certain culture conditions evidence has been found of
the metabolisation of glycerol marked with C14 during its growth
(Manske and Hilbi, 2014). Comparing the substrate incorporation
capacities of P. salmonis and the aforementioned bacteria, it can
 P. Fuentealba et al. / Bioresource Technology 223 (2017) 105–114 109

2.7.1.6-5.4.2.2 Galactose

Glycogen D-glucose6P
RNA
2.2.1.1 D xylulose 5P
5.3.1.9

erythrose 4P
D-fructose6P
UTP
2.5.1.54-4.2.1.20
3.1.3.11 2.7.1.1 5.1.3.1-5.3.1.6 CTP
Glycerol GTP
L-tryptophan
D-fructose1.6P
6.3.5.5-2.7.4.6
ATP
2.2.1.2-2.2.1.1
2.7.1.30-1.1.1.94 6.3.5.5-6.3.4.2
4.1.2.13

glycerone P D ribose 5P 2.4.2.14-2.7.4.6

2.4.2.14-2.7.1.40
5.3.1.1 glyceraldehyde 3P 2.7.6.1

L-serine PRPP
1.2.1.59-4.2.1.11

2.4.2.14-2.7.4.6+
2.1.2.1+ phodphoenolpyruvate
4.1.1.31 2.4.2.14-2.7.4.6

2.7.9.2 2.7.1.40 6.3.5.5-2.7.4.6

dATP
glycine 6.3.5.5-2.7.4.6+
pyruvate 2.6.1.21-5.1.1.1+ L-alanine dGTP
acetaldehyde
1.2.4.1-2.3.1.12 dCTP
4.1.2.5

L-proline dTTP
6.2.1.1-1.2.1.3 acetyl-CoA
L-threonine
1.5.1.2-1.5.1.12
1.1.1.38 2.3.3.1-1.1.1.42
DNA
oxaloacetate 2 oxoglutarate 1.4.1.2 L-glutamate 6.3.1.2 L-glutamine

1.1.1.37 2.3.1.109-3.5.1.96 L-arginine

2.6.1.1

formamide
1.4.3.2
4.3.1.3-3.5.3.8
L-glutamate
1.2.4.2-6.2.1.5
Polyamines
malate
L-histidine

L-aspartate succinate

6.3.5.4

1.3.5.1
L-asparagine 4.2.1.2

fumarate

L-phenylanine 1.14.16.1+ L-tyrosine

Fig. 2. Representation of the metabolic network of P. salmonis LF-89 reconstructed from the automatic annotations of reported genome sequences and experimental evidence.
(Blue) amino acids for which P. salmonis is auxotrophic. (Light green) molecules that are synthesised. (Dark green) macromolecules comprising the biomass of the metabolic
model. (Yellow) metabolite that is not catabolised. (Peach) alternative substrates with predicted capacity for assimilation. (Red arrow) manually annotated reaction. (Black
arrow) predicted reactions. (EC Codes) predicted enzymes in the genome assemblies of P. salmonis that catalyse the specified reaction, labelled individually (one EC code), at
intervals with the initial and final enzyme of the compacted pathway (two EC codes) or labelled with the presence of other reactions involved in the pathway without a
sequential correspondence (EC+). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

be seen that a wide range of transport systems for amino acids pre- and this was attained in zone 2. The specific growth rates for P. sal-
dominate, which is in contrast to the rare capacity to incorporate monis are in agreement with the information reported for other
another substrate type. The predicted capacity to transport saccha- bacteria. In the case of F. tularensis, specific growth rates of
rides and alcohols in these bacteria is similar to that of P. salmonis. 0.022 h1 have been reported in Chamberlain medium (formulated
solely with amino acids) (Raghunathan et al., 2010). As shown
3.2. Characterisation of P. salmonis culture kinetics in defined medium below, the change in lmax during the growth of P. salmonis is in
agreement with the simultaneous consumption of three amino
P. salmonis was cultured in flasks in defined medium, reaching a acids that act as the carbon and energy sources, though different
maximum dry cell weight (DCW) concentration of 690 ± 50 mg/L at preferences are reflected in the consumption rates (Annexe 3).
68 h, after which the culture entered a steady state (Fig. 3). During Studies of L. pneumophila reveal a predomination towards catabo-
the growth phase, variations were identified in the growth kinetics lism and capture of amino acids during the different growth phases
(Fig. 3), showing three zones: from 4 to 36 h (zone 1), from 37 to (Jules and Buchrieser, 2007; Steinert et al., 2007).
56 h (zone 2) and from 57 to 68 h (zone 3). The maximum specific With regard to the consumption rates of the amino acids in the
growth rate (lmax) of P. salmonis was 0.052 ± 0.005 h1 (Annexe 3) defined medium, it was found that there are amino acids with high
110 P. Fuentealba et al. / Bioresource Technology 223 (2017) 105–114

Table 1
Principal substrate transporters derived from the automatic annotation of reported genome assemblies of P. salmonis.

Superfamily(sf)/family(f)/gen Family Description

ABC-sf/OppC 3.A.1.5.1 Transporter of oligopeptide of 2–5 amino acids with high affinity by tripeptides,
ABC-sf/OppA 3.A.1.5.1 belonging to the system OppA,B,C,D,F (Doeven et al., 2008)
ABC-sf/OppB 3.A.1.5.1
ABC-sf/OppD 3.A.1.5.1
ABC-sf/OppF 3.A.1.5.1
MFS-sf/POT-f/TppB (YdgR- DtpA) 2.A.17.1.2 Transport by symport of di/tripeptides (Bippes et al., 2013; Weitz et al., 2007)
MFS-sf/POT-f/DtpB(YhiP) 2.A.17.1.3 Transporte by symport of di/tripeptides (Harder et al., 2008)
MFS-sf/POT-f/YbgH(DtpD) 2.A.17.1.4 Permease of di/tripeptides peptide (Casagrande et al., 2009)
MFS-sf/POT-f/IraB 2.A.17.1.1 Transport by symport of di/tripeptides
ABC-sf/MetQ 3.A.1.24.1 Transporter of methionine constituted by the subunits of methionine bond,
ABC-sf/MetN 3.A.1.24.1 permease system and bond to ATP (Kadaba et al., 2008)
ABC-sf/MetI 3.A.1.24.1
APC-sf/SSS-f/PutP 2.A.21.2.1 Transport by symport of proline:Na+ (Rivera-Ordaz et al., 2013)
MHS-f/ProP 2.A.1.6.4 Transport by symport of proline/glycine:H+/Na+ (Culham et al., 1993)
DAACS-f/GltP-GltS 2.A.23.1.2 Glutamate/aspartate:Na++H+ symport
APC-sf/HAAAP-f/TyrP 2.A.42.1.1 Transporter of tyrosine
MFS-sf/ PhtJ 2.A.1.53.2 Transporter of valine (Chen et al., 2008)
MFS-sf/ PhtA 2.A.1.53.1 Transporter of threonine (Sauer et al., 2005)
APC-sf/AGCS-f/AgcS 2.A.25.1.3 Transporte by symport of alanine:Na+ (Moore and Leigh, 2005)
MFS-sf/ MFS-f/AraE 2.A.1.1.2 Transporte by symport of arabinose (xylose; galactose):H+
MFS-sf/ GlpT 2.A.1.4.3 Glycerol-P:Pi antiport (Law et al., 2008)
MIP-sf/MIP-f/GlpF 1.A.8.2.1 Glycerol facilitator

800 4
Zone 1 Zone 2 Zone 3

600 3
DCW ln(X/X0)
DCW (mg/L)

400 2

200 1
DCW (mg/L)

0 0 DCW ln(X/X0)
0 20 40 60 80
Time (h)

Fig. 3. Time course evolution of P. salmonis cultivated in the defined medium. (Circle) Growth profile of P. salmonis at initial pH of 6 (initial inoculum of 90 mg/L). (Square)
Linearised growth profile to identify fluctuations in specific growth rate of P. salmonis during culture in the defined medium. ln (X/X0), where X is the biomass over time and
X0 is the biomass at time 0. DCW: Dry cell weight. Zone 1: growth phase between the 4–36 h of culture. Zone 2: growth phase between the 37–56 h of culture. Zone 3: growth
phase between the 57–68 h of culture.

(Fig. 4A) and low (Fig. 4B) demand. The specific consumption rates
for each one were calculated for the different zones identified
during cell growth. The amino acids that were consumed
preferentially were glutamic acid, threonine and arginine. The
maximum specific consumption rate for glutamic acid
(0.36 ± 0.03 mmol/(g DCW*h)) was attained in zone 1, and then fell
to 0.35 ± 0.02 mmol/(g DCW*h) in zone 2, and the acid was no
longer detected in zone 3. The maximum specific consumption rate
for threonine was obtained in zone 2 (0.54 ± 0.03 mmol/
(g DCW*h)), and for arginine it was obtained in zone 3
(0.31 ± 0.02 mmol/(g DCW*h)).
The other nine amino acids in the defined medium showed low
demand, with maximum specific consumption rates below those of
glutamic acid, threonine and arginine. Of this group, proline
showed in the zone 1, an specific consumption rate throughout
the culture of 0.084 ± 0.004 mmol/(g DCW*h), which decreases
progressively in zone 2 and 3. Lysine specific consumption was
stable in zone 1 and 2, presenting a consumption rate average of
0.067 ± 0.006 mmol/(g DCW*h). The other amino acids in the
defined medium (histidine, cysteine, valine, methionine, leucine,
isoleucine and phenylalanine) showed specific consumption rates
below 0.04 mmol/(g DCW*h) (Annexe 3). The measured agrees
with data reported for Streptomyces tsukubaensis, for which the
maximum amino acid consumption rate was in the order of
0.05 mmol/(g DCW*h) for a specific growth rate of 0.049 h1
(Huang et al., 2013). This is in agreement with the topology of
the metabolic network, in which the amino acids are fundamen-
tally required for protein biosynthesis and in some other cases
for amino acids.
As a result of the catabolism of amino acids, a significant
amount of NH+4 accumulates in the extracellular medium, reaching
a concentration of 18.65 mM at 68 h (Fig. 4C). The specific produc-
tion rate of this metabolite fluctuates in the different growth zones,
reaching a value of 1.69 ± 0.06 mmol/(g DCW*h) in zone 3. This
fluctuation is attributed to the change in carbon sources catabo-
lised by P. salmonis, as the glutamic acid and threonine lead to
 P. Fuentealba et al. / Bioresource Technology 223 (2017) 105–114 111

A 8 800

6 600

Amino acids (mM)

DCW (mg/L)
4 400

DCW
2 200
glu
arg
0 0 thr
0 20 40 60 80
Time (h)

B 5 800

4
600
Amino acids (mM)

DCW

DCW (mg/L)
3 His
400
Phe
Val
2
Met
200 Ile
1 Leu
Pro
0 0 Lys
0 20 40 60 80
Time (h)

25
C 800

20
600
DCW (mg/L)
NH 4+ (mM)

15
400
10

200
5
NH4+
DCW
0 0
0 20 40 60 80
Time (h)

Fig. 4. Consumption and production profile presented in the defined medium during P. salmonis culture. (A) Profile of amino acids consumed at high demand. (B) Profile of
amino acids consumed at low demand. DCW: Dry cell weight. (C) Profile of NH4+ production in P. salmonis cultures in defined medium. DCW: Dry cell weight.

the release of one amine group, while the arginine releases four
when degraded. Arginine shows the highest consumption rate in
zone 3, leading to a higher specific NH+4 production rate during this
period (Annexe 3). This is also the case in other intracellular patho-
genic bacteria that support their metabolism on the basis of amino
acid degradation, such as the related bacterium L. pneumophila
(George et al., 1980).
The accumulation of some organic acids was recorded in zone 1,
such as succinic acid and lactic acid, which showed maximum
concentrations of 0.87 mM and 0.36 mM, respectively. In zone 2,
a peak of 0.77 mM was seen in acetic acid, along with accumula-
tion of succinic acid, reaching a concentration of 1.89 mM and a
drop in lactic acid. In zone 3, a peak of 2.98 mM was seen in suc-
cinic acid, along with one of 1.01 mM in lactic acid. These were
reabsorbed by P. salmonis during the steady state phase (>60 h of
culture). Also these have been reported in the fermentative
processes of other bacterial types (Horiuchi et al., 2002) and their
production is of interest on an industrial level (Ong et al., 2006).
112 P. Fuentealba et al. / Bioresource Technology 223 (2017) 105–114
Protein secretion into the extracellular medium was also evalu-
ated by measuring total proteins in the filtrate culture broth. The
protein concentration reached 1.19 mM after 50 h of culture in
the defined medium. The molar flow in the form of proteins into
the extracellular medium was 0.04 mmol/(g DCW*h). Proteins in
the extracellular medium, may be associated with the infection
processes (Wilhelm et al., 2005).
Amino acid consumption, biomass synthesis, and NH+4, organic
acid and protein production measurements were used to calculate
the kinetics parameters that describe large part of the mass flows
during P. salmonis growth in the defined medium. These parame-
ters are the first description of the substrate demand and product
synthesis in this bacterium in pure culture.
3.3. Genome-scale metabolic model
The genome-scale metabolic reconstruction of Piscirickettsia sal-
monis LF-89 is based on the annotation of several full and partial
genome sequences reported in GenBank. The RAST tool on the
ModelSeed database can be used to automatically annotate the
reported sequences. The predicted genetic potential allowed
the definition of a base topology of the metabolic network of
P. salmonis, which was then used to construct a first metabolic
model. This was then corrected manually, which involved complet-
ing gaps in the metabolic pathways identified on the basis of thor-
ough experimental evidence. Once this was done, a metabolic
model iPF215 (Annexe 7) was obtained comprising 215 genes (It
represents 6.8% of the predicted genes in the genome
CP011849.2), 417 metabolites and 445 reactions, of which 432
are reactions that were annotated automatically and 13 were
annotated manually (Annexe 4). The manual annotations are for
reactions that complete a pathway and confer capacities observed
in culture, as well as satisfying macromolecule synthesis specifica-
tions that describe the biomass. The genome-scale metabolic
reconstruction of P. salmonis shows reduced metabolic potential
that is in agreement with models of species with similar life cycles,
such as F. tularensis and Helicobacter Pylori. In the case of F. tularen-
sis, the reported model has 605 reactions and 343 metabolites (Kim
et al., 2012). Comparing these models with E. coli, which is a widely
studied organism model, the reduced metabolic potential can be
seen. One of the latest validated models of E. coli has 2251 reac-
tions and 1136 metabolites (Senger et al., 2014).
The components used to describe the biomass of P. salmonis are
DNA, RNA, protein, polysaccharides (glucogen), glycerolipids
(phosphatidylethanolamine dioctadecanoyl), LPS, peptidoglycan
and polyamine (spermidine), as they are the constituent parts of
bacterial biomass that are commonly used in genome-scale meta-
bolic models. The stoichiometric coefficients of these in the bio-
mass (Annexe 5) were obtained by fitting the coefficients in the
literature with the elemental proportions of C, H, O and N mea-
sured in the biomass. The nucleotide proportion in the RNA and
the amino acid proportion in the protein were calculated from
the predicted genes from the annotation of the genome sequences
(Thiele and Palsson, 2010). The stoichiometry indices obtained
comply with the proposed restrictions and satisfy the element pro-
portionality of the biomass obtained in the experiments.
3.4. Metabolic simulation
The metabolic reconstruction, refinement process and data
obtained from the cultures in a chemically defined medium were
all used to construct the genome-scale metabolic model iPF215
and analyse the metabolic fluxes in order to understand how P. sal-
monis sustains its growth. This chemically defined medium is the
first effort to define the nutritional demand of P. salmonis, which
allows inferring the nutritional characteristic of the intracellular
environment that support its growth. For this reason, experimental
data available for the chemically defined medium (Section 2) was
used as environment in the simulations. The constrains used to
estimate the intracellular flows by MFA considered the specific
consumption rates of the amino acids that were identified as the
sources of carbon (threonine, glutamic acid and arginine), as well
as proline and leucine, as representatives of the less consumed
amino acids. The constraining steps of the iPF215 also consider
inactivating the entry to other carbon sources that are not present
in the defined medium. The catabolic pathway of histidine that
leads to formamide production was also constrained to zero flux,
due to the low levels of consumption of this amino acid, since is
likely used only for protein synthesis. The fermentative pathway
to ethanol production was also inactivated as production of this
metabolite was not detected in the culture medium (data not
shown). The objective function selected to simulate P. salmonis
growth was the maximisation of the biomass equation considering
the aforementioned restrictions.
The iPF215 accurately reproduced most of the dynamic charac-
teristics of the P. salmonis culture in MDm. This can be seen by
comparing the calculated values with the model with the experi-
mentally derived values (Annexe 3). The specific growth rate and
most of the specific amino acid consumption rates in the P. salmo-
nis cultures in the defined medium were accurately reproduced.
With regard to the specific growth rate, the iPF215 reproduced this
index in the different growth zones. For the case of the specific
amino acid consumption rates, the highest level of correlation
between the magnitude of predicted and calculated values was
seen for histidine, methionine and isoleucine. A higher degree of
dispersion was seen in the calculated specific consumption rates
of lysine, phenylalanine and valine with the model and the exper-
imental values. The model also suggests a specific NH+4 production
rate stable in the different growth zones for the P. salmonis culture,
while the calculations indicate that this parameter increases dur-
ing culture.
The O2 consumption and CO2 production rates calculated with
the iPF215 can be used to derive the respiratory quotient (RQ),
defined as the flow of CO2 produced divided by O2 consumed,
whose index is characteristic of a metabolism sustained by a sub-
strate type. The RQ calculated with the model values in different
phases of growth (zone 1, 2 and 3) is on average 0.93 (Annexe 6).
The calculate RQ (Annexe 6) is in agreement with the type of sub-
strate assimilated by P. salmonis, since organisms that base their
metabolism on amino acid degradation with NH+4 production have
an RQ of around 0.9 (Elliott and Davison, 1975).
The MFA performed using the iPF215 reproduced the main
experimental rates of P. salmonis observed in the defined medium.
It was also obtained, that the RQ calculated with flows CO2 and O2
of the metabolic model, are around 0.93 for the metabolism of
P. salmonis sustained on the basis of amino acid assimilation. It
is a significant accomplishment within the metabolic reconstruc-
tion of P. salmonis, as it establishes a baseline for the study of its
metabolism.
The iPF215 of P. salmonis allowed evaluating the bacterial
growth behaviour in silico. The essential genes were identified by
deleting them one by one using the OptFlux software. The
metabolic model involved 445 reactions, where 183 were found
to be essential for growth (Fig. 5). The essential reactions predicted
correspond to 122 genes. The identified essential genes could be
considered as candidates to define proliferation control strategies
in this pathogen.
P. salmonis is a pathogen that remains largely unknown on a
metabolic level and which has a significant impact on the aquacul-
ture sector. This work represent a significant advancement in the
comprehension of the bacterium from a metabolic perspective.
The iPF215 provides the first metabolic simulation to describe
 P. Fuentealba et al. / Bioresource Technology 223 (2017) 105–114 113

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