Medium-chain-length polyhydroxyalkanoates synthesis by Pseudomonas putida KT2440 relA/spoT mutant: bioprocess characterization and transcriptome analysis
Mozejko‑Ciesielska et al. AMB Expr
Medium‑chain‑length polyhydroxyalkanoates synthesis by Pseudomonas putida KT2440 relA/ spoT mutant: bioprocess characterization and transcriptome analysis
Justyna Mozejko‑Ciesielska 0 3
Dorota Dabrowska 2
Agnieszka Szalewska‑Palasz 1
Slawomir Ciesielski 2
0 Department of Microbiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn , Oczapowskiego 1A, 10‐719 Olsztyn , Poland
1 Department of Molecular Biology, University of Gdansk , Gdansk , Poland
2 Department of Environmental Biotechnology, University of Warmia and Mazury in Olsztyn , Olsztyn , Poland
3 Department of Microbiology, Faculty of Biology and Biotechnology, Uni‐ versity of Warmia and Mazury in Olsztyn , Oczapowskiego 1A, 10‐719 Olsztyn , Poland
Pseudomonas putida KT2440 is a model bacteria used commonly for medium‑ chain‑ length polyhydroxyalkanoates (mcl‑ PHAs) production using various substrates. However, despite many studies conducted on P. putida KT2440 strain, the molecular mechanisms of leading to mcl‑ PHAs synthesis in reaction to environmental stimuli are still not clear. The rearrangement of the metabolism in response to environmental stress could be controlled by stringent response that modulates the transcription of many genes in order to promote survival under nutritional deprivation conditions. Therefore, in this work we investigated the relation between mcl‑ PHAs synthesis and stringent response. For this study, a relA/spoT mutant of P. putida KT2440, unable to induce the stringent response, was used. Additionally, the transcriptome of this mutant was analyzed using RNA‑ seq in order to examine rearrangements of the metabolism during cultivation. The results show that the relA/spoT mutant of P. putida KT2440 is able to accumulate mcl‑ PHAs in both optimal and nitrogen limiting conditions. Nitrogen starvation did not change the efficiency of mcl‑ PHAs synthesis in this mutant. The transition from exponential growth to stationary phase caused significant upregulation of genes involved in transport system and nitrogen metabolism. Transcriptional regulators, including rpoS, rpoN and rpoD, did not show changes in transcript abundance when entering the stationary phase, suggesting their limited role in mcl‑ PHAs accumulation during stationary phase.
Global regulation; Pseudomonas putida KT2440; Polyhydroxyalkanoates; Stringent response; Transcriptomics
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To survive in the harsh conditions, microorganisms need
to be able to adapt to a competitive and changing
environment. In response to changes in environmental
conditions, the physiological status of microorganisms can
change due to the actions of transcriptional regulatory
systems. Generally, transcription regulation occurs at two
different levels. Firstly, regulation can drive the
expression of relevant pathway genes in reaction to a specific
inducer. Secondly, global regulatory systems can adjust
the expression of the pathway gene cluster in response
to the general physiological status of the microorganism.
The main regulators at this higher level are alternative
RNA polymerase sigma subunits (Dı́az and Prieto 2000).
One of the survival strategies, that microorganisms
living in stressful environments have developed, is
synthesis and accumulation of polyhydroxyalkanoates (PHAs)
in aerobic and anaerobic conditions (Poirier et al. 1995).
PHAs play an important role in central metabolism
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because they serve as a reservoir of carbon and reducing
equivalents. The most common PHA is
poly(3-hydroxybutyrate) (PHB), composed of monomers
containing four carbon atoms, whereas medium-chain-length
PHAs, containing from 6 to 14 carbon atoms in single
monomer, are less abundant. The accumulation of PHAs
may increase the survival capabilities of these bacteria
in extreme environments or when nutrient availability
is poor (Ayub et al. 2009). Because of the complexity of
PHAs metabolism knowledge on the re-configuration
of the bacteria whole metabolism under the conditions
that lead to mcl-PHAs synthesis is still not clear. In
Pseudomonas species the pha cluster is very well conserved
and is organized into two operons phaC1ZC2D and
phaFI. Two polymerases (PhaC1 and PhaC2), a
depolymerase (PhaZ), a transcriptional activator (PhaD) and
proteins involved in granule formations (PhaF and PhaI)
are essential to accumulate and synthesize these
biopolyesters. It has been suggested that PhaG encodes
transacylase, being not co-localized with the pha gene cluster, is
also involved in mcl-PHAs synthesis from non-related
carbon sources (Hoffmann and Rehm 2004). It is known
that PHAs are accumulated by pseudomonads mainly in
response to unbalanced growth conditions such as a lack
of nitrogen, which links PHAs accumulation to the
stringent response (López et al. 2015).
The stringent response modifies the physiology of the
bacterium to such an extent that it can survive difficult
environmental circumstances during its lifecycle. This
response is mediated by the alarmons-unusual
nucleotides, guanosine tetraphosphate (ppGpp) and guanosine
pentaphosphate (pppGpp), often referred collectively to
(p)ppGpp, which primarily affects the transcriptional
program of the bacterial cell (Potrykus and Cashel 2008).
In model bacteria, Escherichia coli and many beta- and
gammaproteobacteria, including P. putida, two enzymes
modulate the levels of ppGpp (Mittenhuber 2001;
Atkinson et al. 2011): the ppGpp synthetase RelA, responsive
to amino acid starvation and ppGpp
synthetase/hydrolase SpoT, producing (p)ppGpp under other nutrients
limitations and stresses (Potrykus and Cashel 2008).
Stringent response contributes to stress adaptation,
antibiotic tolerance, expression of virulence traits and
acquisition of persistent phenotypes in pathogenic bacteria.
The regulation of the mode of action of the RelA/SpoT
enzymes has been extensively studied, but the regulatory
mechanisms that manage transcription of their genes are
still not fully understood (Brown et al. 2014).
Recently, Brigham and co-workers (2012) revealed that
the polyhydroxybutyrate production cycle in Ralstonia
eutropha H16 is regulated by the stringent response. The
results indicated that R. eutropha mutant unable to
produce ppGpp did not accumulate PHB unless the stringent
response was chemically induced. Previously, Ruiz et al.
(2001) had shown that alarmones accumulation and PHB
degradation are associated in Pseudomonas oleovorans:
as PHB were degraded, ATP and ppGpp levels increased.
They suggested that the stringent response influenced
PHB utilization by activation of RpoS synthesis. Because
of these previous studies and the importance of the
stringent response in reprogramming bacterial transcription,
we hypothesized that P. putida deficient in the stringent
response may be impaired in mcl-PHAs synthesis.
Thus, to examine the role of the stringent response in
mcl-PHAs synthesis, P. putida KT2440, a model
organism for mcl-PHAs production, was used. A set of
experiments were performed to determine the possibility of
mcl-PHAs accumulation by P. putida KT2440 mutant
deficient in stringent response and to investigate the
molecular background of this process. Firstly, in
shaking flasks experiments, the mcl-PHAs accumulation
bioprocess stimulated by nitrogen starvation in P. putida
KT2440 relA/spoT mutant and wild type of P. putida
KT2440 was compared. Additionally, P. putida KT2440
rpoN mutant was used in this comparison to examine
the role of RpoN in mcl-PHAs synthesis under
nitrogen deprivation conditions. Secondly, the cultivation of
P. putida KT2440 relA/spoT mutant in a bioreactor was
conducted to monitor cell growth and biopolymers
accumulation over time. Moreover, the transcriptome of P.
putida KT2440 relA/spoT mutant cultivated in the
bioreactor was analyzed in order to show rearrangements of
the metabolism during fermentation towards mcl-PHAs
synthesis.
Materials and methods
Bacterial strain and growth conditions
Cells of P. putida KT2440 (ATCC® 47054™), P. putida
KT2440 rpoN mutant (Köhler et al. 1989), and P. putida
KT2440 relA/spoT (Sze et al. 2002) mutant were taken
from a deep-frozen stock and grown overnight in Luria–
Bertani broth (1% w/v tryptone, 0.5% w/v yeast extract,
1% NaCl) with shaking at 30 °C with 200 rpm for 24 h
before inoculation. All studied strains were cultivated
under nitrogen-limiting and non-limiting conditions. For
all cultivations, the nitrogen-limited medium contained
the following components per liter: 2 g Na2HPO4⋅12H2O,
14.9 g KCl, 46.72 g NaCl, 14.5 g Tris, 2.05 g MgCl2, 3.53 g
Na2SO4, 1 g (NH4)2 SO4, 1 g MgSO4⋅7H2O, and 2.5 mL of
trace element solution. In the non-limited experiments,
the level of (NH4)2 SO4 was adjusted to 10 g/L. Each
liter of trace element solution contained per liter: 20 g
FeCl3⋅6H2O, 10 g CaCl2⋅H2O, 0,03 g CuSO4⋅5H2O, 0,05 g
MnCl2⋅4H2O, 0,1 g ZnSO4⋅7H2O dissolved in 0.5 N HCl.
All cultures were supplemented with oleic acid (10 mL/L)
as the only carbon source in the production media. The
250-mL Erlenmeyer flasks containing 100 mL of a
mineral medium were incubated for 48 h at 30 °C in a rotary
shaker at 200 rpm. The shaking flasks cultivations were
performed in six replicates for each condition and for
each strain.
The fermentation study of P. putida KT2440 relA/spoT
mutant was carried out in a 5 L working volume in a
bioreactor (BioFlo 110, New Brunswick Scientific) at 30 °C
with an aeration rate of 4 L/min. Parameters like
dissolved oxygen, pH value, biomass, mcl-PHA, nitrogen
and carbon concentrations were controlled during the
experiments.
pH-value was maintained at seven through the
modulated addition of concentrated 1 N NaOH and 1 N HCl.
The dissolved oxygen was monitored during the whole
cycle with O2 electrode (InPro 6800, Mettler Toledo
GmbH, Switzerland). Total fermentation time was 48 h.
Analytical methods
The samples from shake flasks experiment were taken
after 48 h of cultivation in order to measure cell dry
weight and PHA concentration. The cell density of the
cultures in the bioreactor was monitored by
measuring the absorbance at 600 nm (OD600) using a
spectrophotometer. During the cultivation in the bioreactor the
samples were taken at 8, 17, 24, 32, 41 and 48 h for
measurements of cell dry weight, mcl-PHAs accumulation,
ammonium/carbon concentration and for determination
of monomers composition and their concentrations. To
measure cell dry weight (CDW), the cells in 100 mL
culture broth were harvested by centrifugation at 11.200×g
for 10 min, washed twice with hexane to remove unused
oleic acid and once with distilled water. The collected
cells were then weighed after lyophilization. The
lyophilization process was performed by Lyovac GT2 System
(SRK Systemtechnik GmbH) for 24 h. Ammonium and
total organic carbon (TOC) concentration was measured
spectrophotometrically using the Hach Lange DR 2800
spectrophotometer (Hach Lange, Düsseldorf DE) and
the LCK303 kit for ammonium and LCK380 kit for TOC
according to the manufacturer’s instructions.
Mcl-PHAs were extracted from lyophilized cells using
the chloroform/methanol procedure for quantitative
and qualitative analysis of biopolymers. The monomeric
composition of the purified mcl-PHAs was determined
using a methanolysis protocol as described previously
(Mozejko and Ciesielski 2014). The concentrations of
methyl esters were estimated by a gas chromatography
(GC) equipped with a capillary column Varian VF-5 ms
with a film thickness of 0.25 μm (Varian, Lake Forest,
USA). Pure standards of methyl 3-hydroxy-hexanoate,
-octanoate, -nonanoate, -decanoate, -undecanoate,
-dodecanoate, -tetradecanoate, -hexadecanoate were
used to generate calibration curves for the methanolysis
assay. All samples were analyzed in triplicates.
Cell dry weight and PHA concentration analysis in
biomass from shake flasks and fermentor were performed
in this same way. Student t test was used to find
statistically significant differences between biomass and PHA
concentration.
RNA isolation
One aliquot of 20 mL from each of cultures were
collected and centrifuged at 4000×g to pellet the cells and
then transferred to a Falcon tube containing RNAlater
solution (Sigma). Total RNA extraction was performed
using a commercial RNA extraction kit (A&A
Biotechnology) according to the manufacturer’s protocol.
Isolated RNA samples were treated with On-Column DNase
I Digest Set (Sigma) to remove traces of DNA. Each time
the absence of contaminating DNA was proven by PCR
reaction. The RNA quantity, quality was checked using
capillary electrophoresis (Agilent 2100 Bioanalyzer,
California, USA). The RNA integrity number (RIN) of every
RNA sample used for sequencing was more than 8.0.
Reverse transcription PCR analysis
Reverse transcription was performed using a SuperScript
Vilo™ cDNA Synthesis Kit (Invitrogen) according to the
manufacturer’s instruction. The cDNA reaction for each
sample contained 1 μg of total RNA. Samples, without
reverse transcriptase (RT) were used as a negative
control. The synthesized first strand cDNA was suspended in
sterile water and stored at −20 °C. Real-time PCR
reaction was performed using SYBR Green technology in an
ABI 7500 real-time PCR system (Applied Biosystems,
USA) in MicroAmpTM optical 96-well reaction plates
(Applied Biosystems, USA). The primer pairs used for
real-time amplification are given in Table 1. The reactions
were run using the thermal cycling parameters as follows:
95 °C for 3 min, then 40 cycles of 95 °C for 15 s, and 60 °C
for 1 min. After performing a run, a final standard
melting curve stage was included. In each run, negative
controls (no cDNA) for each primer set were included. For
quantification of the fluorescence values, a calibration
curve was made using dilution series from 5 × 10−7 to
5 ng of P. putida KT2440 genomic DNA sample.
Normalized expression levels of the examined transcripts were
estimated relative to the 16S rRNA gene, as its
expression is known to remain relatively constant throughout
growth phase of P. putida. Then, the concentration of P.
putida KT2440 DNA was converted to a genome
equivalent for calculation of copy numbers in the real-time
PCR assays (Cottyn et al. 2011). For the convenience,
the genome size of P. putida KT2440 (6.18 × 106 bp)
available at NCBI (National Center for Biotechnology
Table 1 Details of the PCR primers used in this study
Information) was used to estimate the mean mass of the
P. putida KT2440 genome accordingly to the equation:
data were deposited to the NCBI Sequence Read Archive
(SRA) database with BioProject accession PRJNA374570.
m = (n × mw)/AN
where n is the genome size in base pairs, mw is the
average molecular weight per base pairs (660 g/mol), and AN
is the Avogadro constant (6.023 × 1023 molecules/mol).
Library construction, illumina sequencing and data
analysis
RNAseq template libraries were constructed with 1 μg
of the enriched mRNA samples using Truseq RNA
Sample Preparation Kit (Illumina, California, USA)
according to the manufacturer’s instructions. Deep sequencing
was performed by Illumina HiSeq 2500 according to the
manufacturer’s description with a read length of 1 × 50
nucleotides. Sequence reads were pre-processed to trim
low-quality reads and filter reads shorter than 20 bp
using FASTX Tool Kit. Genome sequences and
annotation data of P. putida KT2440 were downloaded from
NCBI (downloaded on 10 November, 2016). Reads that
mapped to non-coding RNA sequences and reads that
did not map to unique positions were excluded from
further analysis. Remaining reads were mapped to P. putida
KT2440 genome using Bowtie with the default
parameters. The reads per gene values of all genes were
calculated from the SAM output files. Testing for differential
expression was performed with DESeq and R software
package that uses a statistical model based on the
negative bionomial distribution (Anders and Huber 2010).
Statistical analysis was performed and genes with a false
discovery rate (FDR) p value correction <0.05 were
determined as differentially regulated genes. The raw RNAseq
Results
PHAs synthesis in shake flask cultures experiment
In order to examine the relationship between the stringent
response and mcl-PHAs synthesis, P. putida KT2440 and
its mutant with non-functional relA/spoT genes were
cultivated in shaking flasks. Additionally, an RpoN-deficient
mutant of P. putida KT2440 was used to reveal the role of
RpoN in the regulatory network that controls mcl-PHAs
synthesis in culture supplemented with oleic acid. All
cultivations were carried out in six replicates under
optimal growth conditions and under nitrogen limitation.
After 48 h of growth, final cell dry weight (CDW) ranged
from 0.63 to 0.98 g/L (Fig. 1). Under nitrogen limitation,
the wild-form and rpoN mutant accumulated the largest
amounts of mcl-PHAs (15.9 and 17.7% mcl-PHAs of CDW,
respectively). Under optimal conditions, these two strains
accumulated significantly less mcl-PHAs (3.8 and 11.1%
mcl-PHAs of CDW, respectively; p < 0.05). The relA/spoT
mutant synthesized similar amounts of mcl-PHAs in both
optimal and nitrogen limiting conditions (10.6 and 11.2%
mcl-PHAs of CDW, respectively). In both conditions the
PHA concentration in relA/spoT mutant cells was
significantly lower than in wild-form cells (p < 0.05).
PHAs synthesis during bioreactor cultivation
To characterize the process of mcl-PHAs synthesis by P.
putida KT2440 relA/spoT mutant, fed-batch culture was
carried out for 48 h in a 5-L bioreactor. The first evidence
of mcl-PHAs synthesis was noted at 24 h, but up to 32 h
of cultivation, mcl-PHAs concentration remained below
1 2 3 4 5 6
biomass PHA concentration
Fig. 1 Mcl‑PHAs content and biomass concentration of
Pseudomonas putida KT2440 wild‑type (1 and 2), P. putida KT2440 relA/spoT
mutant (3 and 4) and P. putida KT2440 rpoN mutant (5 and 6). The
shake flasks cultivation was performed under nitrogen limitation (1, 3,
and 5) and optimal (2, 4, and 6) conditions. Each data represents the
mean ± standard deviation
4.5% CDW. After that, mcl-PHAs concentration started
to increase rapidly, reaching 12.3% CDW at 48 h, when
cell dry weight (1.74 g/L) also reached its maximum
value. During fermentation, total nitrogen and
phosphorus concentration decreased with time, whereas total
carbon concentration was similar throughout cultivation
(0.8 g/L). As can be seen in Fig. 2, ammonium was
completely consumed during the first 8 h of cultivation
The major repeat units of the mcl-PHAs produced by
P. putida KT2440 relA/spoT mutant on oleic acid were
3-hydroxyoctanoate and 3-hydroxyhexanoate, whereas
3-hydroxyhexanoate and 3-hydroxydodecanoate were
found in smaller amounts (Table 2). The composition of
the PHAs synthesized by this mutant was similar to
composition of mcl-PHAs produced by other Pseudomonas
species cultivated on fatty acids (Ciesielski and Mozejko
2015).
Fig. 2 Growth and mcl‑PHAs accumulation during 48 h cultivation of
P. putida KT2440 relA/spoT mutant in bioreactor
Analysis of mcl‑PHAs related genes using reverse
transcription real‑time PCR
The transcriptional expression levels of phaC1, phaZ,
phaC2, phaD, phaI, phaF, and phaG genes were
examined. The transcription of all these genes was investigated
in flask cultures at 48 h of cultivation. The results in Fig. 3
show that the mRNA copy numbers varied between
analyzed strains and conditions. Nitrogen limiting
conditions did not increase transcription of phaC1, phaC2, and
phaZ genes in any of the analyzed strains. Under both
conditions, expression of the phaZ gene was significantly
higher in the wild-type than in the mutant. Under
nitrogen limiting conditions, the number of phaD gene
transcripts was significantly higher in the wild-form than in
the mutant (20.0 vs. 3.6 million copies). Although phaD
is considered a possible regulator of the PC1 promoter,
these results did not show that the changes in phaD had
any effect on phaC1, phaC2, and phaZ expression.
In all strains, phaI and phaF expression was much
higher than expression of other genes directly involved in
PHAs synthesis, and the transcript numbers of phaI and
phaF were higher in nitrogen limiting conditions.
Similar profile was observed for phaG, although its
expression level was rather comparable to phaZ gene. Whereas
the expression of phaI, phaF, and phaG in the relA/spoT
mutant was significantly higher than their expression in
the other strains, the expression of phaC1, phaC2, phaZ,
and phaD did not differ significantly between strains.
Additionally, the expression of phaG was investigated
at six time-points during relA/spoT mutant cultivation
in the bioreactor (Fig. 4). The phaG transcript number
increased from the beginning of cultivation until 41 h
and then decreased. The changes in phaG gene
transcription between the exponential growth phase and the
stationary phase that were obtained using real-time PCR
and RNA-seq were the same.
Transcriptional analysis using RNA‑seq
To investigate how P. putida KT2440 relA/spoT responds
at the molecular level to a decrease in nutrient
availability and metabolically adapts to deteriorating
conditions, samples were withdrawn for RNA-seq at the
end of the exponential phase (24 h) and in the
middle of the stationary phase (41 h). The sequence reads
matched to 5517 coding genes in the P. putida KT2440
genome (Nelson et al. 2002), indicating that the
sequencing was deep enough to cover almost all kinds of
transcripts in the cells. From the sample withdrawn at 24 h,
18,290,865 sequences of cDNA from mRNA transcripts
were obtained; from the sample taken at 41 h, 10,847,701
sequences were obtained.
The RNA-seq analysis revealed that, 104 genes were
singnificantly differentially expressed between 24 and
Table 2 Monomeric composition of mcl-PHAs synthesized by Pseudomonas putida KT2440 relA/spoT mutant
mcl‑PHAs composition (mol%)
3HB 3‑hydroxybutyrate, 3HHx 3‑hydroxyhexanoate, 3HO 3‑hydroxyoctanoate, 3HN 3‑hydroxynonanoate, 3HD 3‑hydroxydecanoate, 3HUD 3‑hydroxyundecanoate,
3HDD 3‑hydroxydodecanoate, 3HTD 3‑hydroxytetradecanoate, 3HHxD 3‑hydroxyhexadecanoate, n.d. not detected
41 h of the cultivation. Most of these differentially
expressed genes were up-regulated (78 genes), with fold
changes ranging from 8.6 to 106.3 (Additional file 1: Table
S1). These genes were classified into categories according
to the UniProt annotation pipeline (Fig. 5). With regard
to the genes directly involved in mcl-PHAs synthesis or
degradation, their expression did not differ significantly
between the exponential and the stationary phase.
With the exception of genes coding for proteins
involved in amino acid biosynthesis and metabolism,
many more genes were upregulated during the
stationary phase than were downregulated. Almost half of all
the genes showing significant differences in transcription
(42 genes) were classified as coding for
unknown/hypothetical proteins due to a lack of corresponding genes in
databases. The next two largest groups of differentially
transcribed genes were associated with cell membrane,
cell wall proteins and transport and binding protein.
Although there were some downregulated genes in these
two groups, as in the group of unknown/hypothetical
proteins, in all three groups the number of
downregulated genes was less than the number of those that were
upregulated. This pattern was also observed in the genes
involved in secondary metabolites biosynthesis,
transport, and catabolism, and in those responsible for
nitrogen compounds catabolism.
More specifically, the genes that were highly
upregulated in the stationary phase are mostly involved in
the expression of branched-chain amino acid ABC
transporters (e.g. urtA, urtC, urtD). Other
upregulated genes code for proteins that participate in
nitrogen metabolism. Among these genes were the nitrite
reductase small (nirD) and large (nirB) units, and the
nitrite transporter (nasA). Other upregulated genes
code for the urease subunits gamma (ureA) and alpha
(ureC) and for urease accessory proteins (ureD, ureE,
ureF, ureJ). Moreover, genes that were upregulated in
the stationary phase are involved in fatty acid
metabolism: long-chain-fatty-acid-CoA-ligase (PP_2709)
and short-chain-dehydrogenase (PP_2711).
Furthermore, STRING analysis indicated that some of the
significantly upregulated genes that code for
hypothetical proteins (PP_2708, PP_2710, PP_2711) are most
likely also involved in fatty acids metabolism. Genes
involved in the β-oxidation cycle (fadA, fadB, fadAx,
fadBx) did not show significant changes in their
transcription. Most of the downregulated genes belonged
to the group of hypothetical proteins, with one
exception: transcription of the gene that codes for the amino
acid transporter LysE was about 50 times lower in the
stationary phase.
Although some genes directly involved in mcl-PHAs
synthesis and degradation changed during a shift
from exponential growth to stationary phase, these
changes were not statistically significant. However, to
show even small changes of this genes transcription,
the values of RPKM (Reads Per Kilobase per Million)
were used to calculate fold-change values (Table 3).
Genes coding for phaC1, phaZ, phaC2, phaF, and
phaI showed upregulation in the range from 1.3 to
1.8. PhaG gene coding for (R)-3-hydroxydecanoyl
ACP:CoA transacylase showed almost six-fold increase
in the stationary phase. Only gene, that showed small
downregulation was phaD, its calculated fold-change
was only at the level of 1.1.
Similarly, genes playing the regulatory functions were
also investigated accurately (Table 4). All genes coding
for RNA polymerase subunits and sigma factors
displayed small downregulation. The transcription of Lrp
(leucine-responsive regulatory protein) increased about
1.3 in stationary phase, similarly Anr (anaerobic
regulatory protein), that showed 1.2 upregulation. Catabolic
repression control protein (Crc) changed slightly in the
stationary phase (fold-change = 1.1).
Most of the transcriptional regulators showed only
small changes in expression between exponential growth
and stationary phase (Table 4). The first exception was
transcriptional regulator from Fis family (ntrC), the
main nitrogen stress factor, that showed almost ninefold
upregulation. The second exception was transcriptional
regulator from TetR family that was activated in
stationary phase (fold change 13.2).
Fig. 3 The result of quantitative real‑time reverse transcription PCR analysis of phaC1, phaZ, phaC2, phaD, phaI, phaF, and phaG genes. Samples
were taken at 48 h of cultivation. Each data represents the mean ± standard deviation
Discussion
The stringent response is a global regulatory system,
which mediates major changes in gene expression in
response to growth-limiting stress conditions. Because,
polyhydroxyalkanoates accumulation is a central
feature of survival physiology when cells are stressed, it was
hypothesized that these two mechanisms are linked. A
set of cultivations performed both in flask cultures and
in a bioreactor showed that a P. putida KT2400 mutant
with non functional relA/spoT genes is able to produce
and accumulate mcl-PHAs. The result of this
examination is surprising in the light of observations made by
Brigham et al. (2012), who showed that
poly(3-hydroxybutyrate) (PHB) is regulated by the stringent response
8 17 24 32 41 48
Time (h)
Fig. 4 The result of quantitative real‑time reverse transcription PCR
analysis of phaG gene. Samples were taken during cultivation of P.
putida KT2440 relA/spoT mutant in bioreactor
in R. eutropha H16. In their study, a R. eutropha spoT2
mutant accumulated no detectable PHB under
conditions of nitrogen starvation, confirming the hypothesis
that guanosine tetraphosphate (ppGpp) plays an
important role in the production of PHB. A possible
relationship between the stringent response and PHB utilization
was shown previously by Ruiz et al. (2001) in P.
oleovorans GPo1. ATP and ppGpp levels increase
concomitantly with PHAs degradation in P. oleovorans cells. It
was postulated that ppGpp is an activator of RpoS
synthesis that controls the genes involved in PHB
metabolism (Ruiz et al. 2001; Brigham et al. 2012). Here, we
show that nitrogen limitation positively influences
mclPHAs synthesis both in the wild strain and the rpoN
mutant of P. putida, but in RpoN-independent manner as
it was shown by Hoffmann and Rehm (2004). Although
this observation suggests that this process is dependent
mainly on nitrogen availability, nitrogen limitation did
not change the efficiency of mcl-PHAs synthesis in the
relA/spoT mutant. Under the stress conditions, in the
wild type cells, elevated amount of ppGpp would
destabilize RNA polymerase complexes with housekeeping
sigma factor promoting transcription from stress related
promoters that depend on alternative sigma factors with
lower affinity to core RNA polymerase. In this situation,
as a result of ppGpp-deficiency due to the mutations,
the transcription from the stress-responsive promoters
can be impaired, partially due to the insufficient pool of
free RNA polymerase core molecules that could bind to
other σ factors related to stress tolerance, such as RpoN
or RpoS (Potrykus and Cashel 2008).
In the flask culture experiment, the transcription of
the genes directly involved in mcl-PHAs synthesis and
degradation differed depending on the strain of
bacteria and on the availability of nitrogen. The transcription
of phaC1 and phaC2 were similar in all strains in both
conditions. This observation is in contrary to results of
Hoffmann and Rehm (2005) who showed that phaC1
gene expression was slightly induced in P. putida KT2440
under nitrogen starvation when sodium gluconate was
used. Transcription of phaZ was significantly
upregulated in P. putida KT2440 wild-type under both optimal
and nitrogen limiting conditions. It could be suggested
that both mutants do not effectively activate processes
leading to recovery of energy from PHAs. The number
of transcripts of phaC1, phaZ, phaC2 and phaD differed
significantly from those of phaF and phaI, which
indicates that these operons are differentially regulated, as
in de Eugenio et al. (2010). According to previous
studies (Klinke et al. 2000; de Eugenio et al. 2010), the phaD
gene acts as an activator of the phaC1 and phaI
promoters. In our study, however, phaC1 was not induced by
high expression of phaD gene in wild type of P. putida
KT2440 under nitrogen limiting conditions. On the
contrary, obtained results support the possible regulation
of the phaI promoter by phaD, which controls the phaI
and phaF gene (Prieto et al. 1999). This was reflected in
Fig. 5 Gene ontology analysis of the significantly differentially expressed genes between exponential growth and stationary phase of P. putida
KT2440 relA/spoT mutant cultivation
Table 3 Differences in pha genes transcription between exponential growth (24 h) and stationary phase (41 h) expressed
in RPKM
Table 4 Differences in regulatory genes transcription between exponential growth (24 h) and stationary phase (41 h)
expressed in RPKM
the higher expression of the phaI and phaF genes by this
strain under nitrogen limitation than under optimal
conditions. Because the expression of phaD gene was highest
in the wild type strain cultivated under nitrogen limiting
conditions, it could be suggested that this gene induction
is dependent on nitrogen availability. In rpoN mutant the
difference in phaD gene expression was smaller between
conditions, therefore phaI and phaF genes expression
difference between conditions was also smaller. Because the
expression level of phaD gene was much lower in rpoN
mutant than in wild type under nitrogen limiting
conditions it could be speculated that a regulation of this gene
could be RpoN dependent. Accordingly to Hoffmann
and Rehm (2005), RpoN might be a negative regulator
of phaF transcription, particularly when excess nitrogen
is available, however it was not observed in our study
because the expression of phaF gene was at the same level
both in wild type and rpoN mutant. In a case of relA/spoT
mutant, phaD gene expression was independent on the
used conditions. It is worth emphasizing that, in the
relA/spoT mutant, the phaI/phaF genes expression was at
comparable level in both conditions. Expression of these
genes in relA/spoT mutant was significantly higher than
in wild-type strain and rpoN mutant, which could suggest
that this operon is regulated by the stringent response in
negative manner.
RNA-seq analysis revealed that genes directly involved
in mcl-PHAs synthesis and degradation in relA/spoT
mutant during cultivation in bioreactor did not show
statistically significant changes in transcription between
exponential growth and stationary phase. Similarly to
the results obtained by Poblete-Castro et al. (2012) and
Fu et al. (2015), the highest upregulation was noticed for
phaI and phaF genes. In the mentioned reports, as well
as in this study, upregulation of phaI was higher, that
confirm superiority of phaI in relation to phaF gene (de
Eugenio et al. 2010). The higher induction of phaI/phaF
operon in comparison to phaC1/phaZ/phaC2/phaD
operon, as well as its significantly higher transcription
in flasks culture confirms both independent regulation
of phaI/phaF genes, and possible negative influence of
stringent response on these genes expression. The
highest upregulation (5.9 fold-change) showed phaG gene
linking fatty acid de novo biosynthesis with PHAs
biosynthesis when non-related carbon sources are utilized
(Hoffmann and Rehm 2004). The flasks culture showed
that its transcription was nitrogen dependent but
RpoNindependent. The same observation was previously made
by Hoffman and Rehm (2004), when sodium gluconate
was used as a carbon source. However, in this work oleic
acid was used as the only carbon source, therefore high
induction of this gene is surprising. In order to prove
this observation phaG gene transcription was
examined at six time-points during relA/spoT mutant
cultivation in bioreactor using reverse transcription real-time
PCR. Obtained results showed gradual increase of this
gene transcription until 41 h of cultivation, confirming
the results from RNA-seq. It seems that this gene must
have (an) additional function(s) in P. putida KT2440
metabolism.
Global transcriptomics of the relA/spoT mutant
revealed that only some regulatory genes showed
upregulation although these changes were not statistically
significant. During the stationary phase the highest
activation was shown by genes coding for NtrC
transcriptional regulator (fold-change 8.8) and an undetermined
gene coding for a transcriptional regulator from TetR
family (fold-change 13.2). NtrC, a global regulator,
activates the transcription of many genes for the uptake
and catabolism of various nitrogen sources and should
be activated only during nitrogen limitation
(Chubukov et al. 2014). NtrC activated relA gene during
nitrogen starvation, therefore it is accepted that NtrC couples
nitrogen starvation stress and stringent response (Brown
et al. 2014). One of the other genes regulated by NtrC is
glnK. The PII-like protein that is produced by this gene
regulates multiple cellular functions related to
nitrogen metabolism, including ammonium transport and
assimilation via glutamine synthetase, nitrogen fixation
and nitrogen-responsive transcriptional regulation, by
means of protein–protein interactions (García-González
et al. 2009). Our results showed its upregulation, which
is in accordance with the results of Poblete-Castro et al.
(2012). In their work glnK showed a large change in both
mRNA abundance and protein level when P. putida was
subjected to dual limitation of nitrogen and carbon.
One of the regulators specific to the stationary phase is
highly conserved bacterial protein Lrp
(leucine-responsive regulatory protein), which can act both as a
transcriptional repressor and activator (Pletnev et al. 2015)
influencing on more than 400 genes in E. coli. Among
them there are genes responsible for amino acid
synthesis, catabolism and the utilization of various carbon
sources (Tani et al. 2002). Most probably, Lrp is
upregulated by ppGpp in P. putida KT2440 like in E. coli, which
would explain the fact that the increase in abundance of
Lrp transcripts during the transition to the stationary
phase was not significant.
The RpoS factor, encoded by rpoS, activates the
transcription of genes involved in the bacterial general stress
response. Therefore, it was expected that the number of
rpoS gene transcripts will increase entering stationary
phase. However, the number of rpoS gene transcripts
did not increase in the relA/spoT mutant; the same was
observed with the rpoN and rpoD genes. The observed
relation between rpoS expression and the stringent
response is in agreement with previous reports. For
instance, high ppGpp levels resulted in increased rpoS
transcription in E. coli (Gentry et al. 1993), and the P.
aeruginosa relA mutant strain, which synthesizes less
ppGpp than the wild type, had reduced but not abolished
RpoS protein levels (Erickson et al. 2004). In E. coli, poor
induction of the RpoS regulon in the ppGpp-null mutant
likely results from lower induction of the rpoS gene, weak
stabilization of RpoS by IraP, and poor competition of
core RNAP for RpoS (Traxler et al. 2008).
The Crc (catabolite repression control) protein is a key
regulator involved in the repression by catabolites in
Pseudomonas at translational level. Generally, the action
of Crc is limited to the exponential growth phase and it is
not observed when the cultures entered into the
stationary phase (La Rosa et al. 2014). In our study the number
of mRNA transcripts of Crc decreased in the stationary
phase, but they were still present, most likely reducing
mcl-PHAs synthesis by repressing phaC1 translation.
The gene whose expression was upregulated the most
was a transcriptional regulator from the TetR family
(PP_2475) (fold change 13.2). Follonier et al. (2013) found
that when P. putida KT2440 was grown in medium
supplemented with octanoate, the same gene was
upregulated to a much lesser extent under elevated total
pressure and elevated oxygen pressure (fold change of
1.59 and 1.7, respectively). A similar upregulation (1.56
fold-change) of a TetR family transcriptional regulator
was shown by Fu et al. (2015) for P. putida LS46 grown
on waste fatty acid but not on glycerol. The authors
postulated that this factor is responsible for short chain fatty
acid degradation.
In this study 104 genes were significantly differentially
expressed between exponential growth and stationary
phase. Most of them were responsible for the
expression of the branched-chain amino acid ABC
transporters and proteins engaged in nitrogen metabolism. Similar
observation was made by Poblete-Castro et al. (2012)
when wild-type of P. putida KT2440 was grown under
nitrogen-limitation. Authors observed that expression of
the branched-chain amino acid ABC transporter was up
to 16-fold higher as a result of stress. Other upregulated
genes were responsible for the urea assimilation system
(e.g. UreE, UreJ, and UreA). The activation of urease may
increase competitive fitness of bacteria under
nitrogenlimiting conditions, since urease catalyzes the hydrolysis
of urea to yield ammonia. According to Poblete-Castro
et al. (2012) the same system was activated both under
nitrogen limitation and under carbon–nitrogen dual
limitation. However, our examination showed that
upregulation of the genes responsible for branched-chain amino
acid ABC transporters and nitrogen metabolism was
much stronger than in the above mentioned paper. The
transcription of genes coding for branched-chain amino
acid ABC transporters was even more than 100 times
higher in the stationary phase (utrA). Similarly, nitrogen
metabolism related genes showed more than 100 times
higher activity in the stationary phase (e.g. nirD). The
differences in gene transcription between exponential
growth and the stationary phase in the relA/spoT mutant
were unexpectedly high in comparison to values obtained
by other authors for P. putida KT2440 (Poblete-Castro
et al. 2012; Follonier et al. 2013). It is postulated that most
of the highly upregulated genes are stringent response
dependent. This group comprises genes coding for
branched-chain amino acid ABC transporters and
nitrogen metabolism. Especially, glnK gene coding for nitrogen
regulatory protein P-II showed unusually high number of
transcripts in the stationary phase (RPKM = 47668.9).
This hypothesis could be supported by results obtained
for Corynebacterium glutamicum (Brockmann-Gretza
and Kalinowski 2006). In this study, the C. glutamicum
rel mutant was used to reveal genes controlled by the
stringent response. According to the results obtained by
Brockmann-Gretza and Kalinowski (2006), many genes
coding for branched-chain amino acid ABC transporters
and nitrogen metabolism were negatively controlled by
the stringent response. High expression of phaI and phaF
genes observed during the experiment in flasks suggests
that these PHA granule associated proteins can be also
negatively regulated by ppGpp. Other genes are
positively affected by the stringent response. Among them
are genes involved in global regulation, with rpoS being
the most important (Brockmann-Gretza and Kalinowski
2006). RpoS controls the general stress response
therefore it plays an important role in adaptation to nutritional
and environmental stress. In contrary to P. putida
deficient in stringent response examined in this study, RpoS
expression in the wild type strain is increasing during
transition from exponential growth to stationary phase
when ppGpp are at the sufficient level. Similar behaviour
was observed in the case of genes encoding Lpr, PsrA and
GacA transcriptional regulators, that are likely positively
affected by stringent response. It is known that psrA and
gacA are involved in rpoS activation (Venturi 2003), thus
RpoS is additionally restricted. The stringent response
deficiency makes cellular metabolism disordered by
transcriptional regulators deactivation, and therefore cells are
much more unbalanced in a stressful environment.
In conclusion, we showed that the P. putida KT2440
relA/spoT mutant is able to accumulate mcl-PHAs,
therefore it could be postulated that the stringent response is
not necessary for polyhydroxyalkanoates synthesis using
oleic acid as an external carbon source. The
composition of mcl-PHAs monomers was similar to that of
mclPHAs synthesized by other Pseudomonas species using
fatty acids. Comparative analysis showed that nitrogen
limitation did not influence mcl-PHAs synthesis,
suggesting that the global regulator did not act properly in
the studied mutant. Expression of phaI/phaF genes in
the relA/spoT mutant was significantly higher than in the
wild type strain and the rpoN mutant, which suggests
that this operon is negatively regulated by the stringent
response. The small changes in mcl-PHAs related genes
between the exponential growth and stationary phases
can be due to the fact that they are activated more or less
directly by the RpoS global regulator. Surprisingly, the
substantial activation of the phaG gene was observed.
RNA-seq analysis showed that the transition from
exponential growth to the stationary phase caused significant
changes in genes encoding the branched-chain amino
acid ABC transporters and proteins engaged in
nitrogen metabolism. Most of these genes were upregulated,
confirming that the stringent response affected their
transcription negatively in P. putida KT2440.
Transcriptional regulators, including rpoS, rpoN and rpoD, did not
show changes when entering the stationary phase,
therefore it could be suggested that they are influenced
positively by stringent response. Although global regulation
was non-functioning in the P. putida KT2440 relA/spoT
mutant, mcl-PHAs synthesis was made possible by the
activity of other transcriptional factors that remain to be
determined.
Additional file 1: TableS1. Significantly differentially expressed genes in
the stationary phase (41 h) with adjusted p‑ value (Adj. p‑ value) lower than
0.05. The genes are sorted according to fold‑ change.
Abbreviations
PHA: polyhydroxyalkanoates; mcl‑PHA: medium‑ chain‑length polyhydroxyal‑
kanoates; PHB: poly(3‑hydroxybutyrate); P. putida: Pseudomonas putida; ppGpp:
guanosine tetraphosphate; (p)ppGpp: guanosine pentaphosphate; ATP:
adenosine triphosphate; CDW: cell dry weight; GC: gas chromatography; TOC:
total organic carbon; RIN: RNA integrity number; RT: reverse transcriptase;
NCBI: National Center for Biotechnology Information; FDR: false discovery rate;
RPKM: Reads Per Kilobase per Million; 3HB: 3‑hydroxybutyrate; 3HHx: 3‑hydrox ‑
yhexanoate; 3HO: 3‑hydroxyoctanoate; 3HN: 3‑hydroxynonanoate; 3HD:
3‑hydroxydecanoate; 3HUD: 3‑hydroxyundecanoate; 3HDD: 3‑hydroxydode ‑
canoate; 3HTD: 3‑hydroxytetradecanoate; 3HHxD: 3‑hydroxyhexadecanoate;
n.d.: not detected; C. glutamicum: Corynebacterium glutamicum.
Authors’ contributions
JMC participated in the design of the study and carried out the molecular
studies, and RNAseq data analysis as well as drafted manuscript. DD per‑
formed the experiments. ASzP helped to draft manuscript and gave some
interpretation on data. SC participated in the design of the study, supervised
the work and helped to draft the manuscript. All authors read and approved
the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the
article (and its supporting material).
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