Transcript Poster - Institute for Environmental Genomics

Exploration of Salt Adaptation Mechanisms in Desulfovibrio vulgaris Hildenborough
K138
Email: [email protected]
Phone: 405-325-3958
Web site: http://ieg.ou.edu/
Zhili He1,2,7, Qiang He2,3,7, Eric J. Alm4,7, Judy D. Wall5,7, Matthew W. Fields6,7, Terry C. Hazen4,7, Adam P. Arkin4,7, and Jizhong Zhou1,2,7
http://vimss.lbl.gov/
Cell culture and treatment: D. vulgaris cells were grown at the LS medium with or
without yeast extract. To test the effects of amino acids on D. vulgaris growth, yeast extract was
removed. NaCl was added into the LS medium to make desired concentrations when the LS
medium was made.
D. vulgaris oligonucleotide array: 70mer oligonucleotide arrays that containing all
ORFs were constructed as described (He et al., in press).
Target preparation, labeling and array hybridization: Total cellular RNA was
isolated and purified using TRIzolTM Reagent, and then labeled with Cy5 dye. Genomic DNA
was isolated and purified from D. vulgaris as described previously (Zhou et al., 1996), and then
labeled with Cy3 dye. The labeled RNA and genomic DNA were co-hybridized to the array at
45oC with 50% formamide for 16 hrs in the dark. Image and data analysis were the same as
described previously (Chhabra et al., 2006; Mukhopadhyay et al., in press).
Results
Cell growth at the LS medium containing different concentrations of NaCl
Fig. 1
 50 and 100 mM NaCl did not affect
the cell growth, and cells reached the
stationary stage approximately 28 h after
inoculation.
 The cell growth was inhibited by 250
and 500 mM NaCl in two ways: the
growth rate and the final biomass
(measured by OD).
 D. vulgaris could not grow in the LS
medium in presence of 1 M NaCl.
 100, 250 and 500 mM were chosen for
further experiments.
1.4
1.2
1.4
Fig. 4
A.
0 mM NaCl
500 mM NaCl
0.8
0.6
0.6
0.4
0.4
OD600
-0.11
0.22
0.17
0.2
0.2
0.0
0.0
10
20
30
0.05
2-isopropylmalate synthase
3-isopropylmalate dehydratase, large subunit
3-isopropylmalate dehydratase, small subunit
conserved hypothetical protein
3-isopropylmalate dehydrogenase
0.14
0.62
0.17
0.23
0.45
0.71
0.60
1.02
0.77
1.63
1.51
1.21
cytochrome c3
formate dehydrogenase formation protein FdhE
formate dehydrogenase, beta subunit
formate dehydrogenase, alpha subunit
0.73
0.46
0.54
0.64
0.68
0.65
1.43
1.23
0.56
0.53
-0.24
0.75
2.24
1.90
1.57
1.20
0.66
1.32
heat shock protein, Hsp20 family
heat shock protein, Hsp20 family
heat shock protein HtpG
phage shock protein C (regulating Leu synthesis)
hypothetical protein
phage shock protein A
-0.78
-0.98
-0.78
0.52
0.54
0.12
-0.42
-0.19
0.37
-0.15
0.07
-0.78
-1.17
-1.20
-1.13
-1.01
-0.50
-0.41
-0.23
-0.32
-0.69
-0.94
-0.49
-1.11
-1.27
-1.31
-1.46
-1.19
-0.06
-0.62
0.14
-0.69
-0.62
-0.74
-0.20
phospho-2-dehydro-3-deoxyheptonate aldolase
3-dehydroquinate synthase
chorismate mutase/prephenate dehydratase
3-phosphoshikimate 1-carboxyvinyltransferase
prephenate dehydrogenase
anthranilate synthase, component I
anthranilate synthases component II
anthranilate phosphoribosyltransferase
indole-3-glycerol phosphate synthase
N-(5-phosphoribosyl)anthranilate isomerase
tryptophan synthase, beta subunit
tryptophan synthase, alpha subunit
-0.50
-0.61
-0.86
-0.70
-2.26
-1.15
-0.09
-0.46
-0.57
-0.15
-0.25
-0.24
0.27
-0.14
0.02
-0.32
-0.40
-0.62
-0.93
-0.50
0.37
tonB dependent receptor domain protein
ABC transporter, periplasmic substrate-binding protein
ABC transporter, permease protein
ABC transporter, permease protein
ABC transporter, ATP-binding protein
tolQ protein, biopolymer transport proteins
ferrous iron transport protein B
ferrous iron transport protein A
hypothetical protein
ferrous ion transport protein
pssA
leuA
leuC
leuD
DVU2984
leuB
1.2
DVU2809
DVU2810
DVU2811
hspC
pspC
leuB
DVU0460 DVU0461 DVU0462
DVU2382
fdnG-3
B.
DVU2383
aroA
DVU2987
DVU0464
DVU2384
trpE
pspF
pspA
trpG
DVU2385
feoA
trpD
oppC
trpC
trpF-1
DVU2387
trpB-2
trpA
tolQ-1
DVU2573
feoA
More confidence may be expected to incorporate
operons or pathways for gene expression analysis.
cytochrome c3
formate dehydrogenase formation protein FdhE
formate dehydrogenase, beta subunit
formate dehydrogenase, alpha subunit
ferrous iron transport protein B
ferrous iron transport protein A
hypothetical protein
ferrous ion transport protein
glycine/betaine/L-proline ABC transporter, permease protein
glycine/betaine/L-proline ABC transporter, ATP binding protein
6
r2 = 0.96 (n = 12)
2
0
-2
-4
-4
2
-2
0
0.2
0.0
20
40
60
80
100
120
140
0
20
2
40
60
-2
Microarray dat
Real time PCR data
y = 1.0
y = -1.0
-4
-6
-8
0
1
2
3
4
5
6
7
8
9
ORFs (Numbered from 1 to 12)
10
11
12
13
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
100
120
140
Hours after inoculation (h)
• With yeast extract, D. vulgaris
growth was inhibited ~ 35% by 500
mM NaCl, and its growth was
inhibited ~ 80% by 500 mM NaCl
without yeast extract (Fig. 4).
• The results suggest that yeast extract
significantly affects the growth of D.
vulgaris in the presence of NaCl,
which may be because certain
substances in yeast extract help D.
vulgaris cells adapt to high salinity
environments.
• Leu, Trp and Leu+Trp significantly
relieved the inhibition of D. vulgaris
grown at the LS medium without
yeast extract and with 500 mM NaCl
(Fig. 5), and other amino acids,
products or precursors of Trp did not
relieve such an inhibition (not shown).
• The results are consistent with
mciroarray data, and suggest that
tryptophan and leucine may play
important roles in D. vulgaris
adaptation to salt stress.
Summary
1. Genes for leucine biosynthesis, heat-shock proteins, formate dehydrogenases, sensory
box histidine kinases/response regulators, and peptidases were highly up-expressed in
NaCl-adapted cells.
2. Predicted genes involved in tryptophan biosynthesis, ribosomal protein synthesis,
energy metabolism, and iron transport were down-expressed.
4
6
8
5. Microarray data are consistent with RT-PCR results and physiological studies.
6. Future studies will focus on the analysis of metabolites accumulated in the cell.
References
1. Chhabra SR, He Q, Huang KH, Gaucher SP, Alm EJ, He Z, Hadi MZ, Hazen TC, Wall JD, Zhou, J, Arkin AP and
Singh AK (2006). J. Bacteriol. 188: 1817-1828.
2. He Q, Huang KH, He Z, Alm EJ, Fields MW, Hazen TC, Arkin AP, Wall JD, and Zhou J. Appl. Environ. Microbiol.
(in press).
3. Mukhopadhyay A, He Z, Yen HC, Alm EJ, He Q, Huang K, Baidoo EE, Chen W, Borglin SC, Redding A, Holman
HY, Sun J, Joyner DC, Keller M, Zhou J, Arkin AP, Hazen TC, Wall JD, and Keasling JD. J. Bacteriol. (in press).
 12 genes expressed in
different levels were chosen for
real-time PCR.
4. Zhou J, Bruns MA, and Tiedje JM (1996). Appl. Environ. Microbiol. 62:461-468.
 The correlation between
microarray data and RT-PCR
results were very good with r2 =
0.96 (n = 12).
This research was funded by the U.S.
Department of Energy (Office of
Biological and Environmental
Research, Office of Science) grants
from the Genomes To Life Program.
Acknowledgements
Microarray data (log2 ratio)
0
80
4. Yeast extract, leu, Trp, or/and Leu+Trp significantly relieved the inhibition of D.
vulgaris grown under 500 mM NaCl conditions, which suggests that an accumulation
of metabolites (e.g. leucine and tryptophan) and availability of nutrients may increase
the adaptability of D. vulgaris to high salt conditions.
8
4
B. NaCl adaptation
were grown in the LS medium containing
500 mM NaCl (added before inoculation).
Samples were taken at OD ~= 0.40 for
microarray hybridization.
RT-PCR verification of microarray data
6
50
 Salt adaptation: D. vulgaris cells
 Differences and similarities in gene
expression were seen between salt
adaptation and salt shock (Table 3).
glycine/betaine/L-proline ABC transporter, periplasmic-binding protein
8
40
3. Genes involved in glycine/betaine/L-proline ABC transport, Na+/H+ transport, K+
uptake and transport, and proline biosynthesis and transport were not significantly
changed.
 Salt shock: D. vulgaris cells were
grown in the LS medium to the mid-log
(OD ~= 0.40), and NaCl was added to the
culture. Samples were taken for microarray
hybridization after 30-min NaCl (250 mM)
treatment.
phospho-2-dehydro-3-deoxyheptonate aldolase
3-dehydroquinate synthase
chorismate mutase/prephenate dehydratase
3-phosphoshikimate 1-carboxyvinyltransferase
prephenate dehydrogenase
10
30
0 mM NaCl
500 mM NaCl
0
DVU2442
feoB
pleD
2-isopropylmalate synthase
3-isopropylmalate dehydratase, large subunit
3-isopropylmalate dehydratase, small subunit
conserved hypothetical protein
3-isopropylmalate dehydrogenase
4
20
0.4
Hours after inoculation
Predicted function
Fig. 3
10
1.0
0.0
Table 3. Comparison of genes differentially expressed under salt shock and salt adaptation conditions
DVU No.
Gene name Salt adaptation Salt shock
Leucine biosynthesis
DVU2981
leuA
1.48
2.03
DVU2982
leuC
1.26
1.26
DVU2983
leuD
1.04
0.62
DVU2984
1.29
0.65
DVU2985
leuB
1.26
0.64
Formate dehydrogenase
DVU2809
0.77
-1.04
DVU2810
1.63
0.22
DVU2811
1.51
-0.64
DVU2812
fdnG-3
1.21
-1.10
Tryptophan biosynthesis
DVU0460
-1.11
1.68
DVU0461
-1.27
1.48
DVU0462
-1.31
1.58
DVU0463
aroA
-1.46
1.24
DVU0464
-1.19
1.30
Fur regulation
DVU2571
-1.07
2.16
DVU2572
-2.08
2.87
DVU2573
-1.84
1.42
DVU2574
-1.27
-0.13
Glycine/betaine/L-proline ABC transporters
DVU2297
0.55
2.33
DVU2298
opuBB
0.48
2.61
DVU2299
proV
1.03
4.36
0
0.2
Salt adaptation (cells grew at the LS medium containing 500 mM NaCl)
DVU no. Gene
log2(ratio) Z-score
Predicted function
DVU3384 zraP
2.87
4.52
zinc resistance-associated protein
DVU2441 hspC
2.2
2.67
heat shock protein, Hsp20 family
DVU0142 trpS
1.89
2.73
tryptophanl-tRNA synthetase
DVU0593 lysE
1.88
2.53
L-lysine exporter
DVU2981 leuA
1.56
2.23
2-isopropylmalate synthase
DVU2445 tonB
-2.27
-4.07
TonB dependent receptor protein
DVU2572 feoA
-2.21
-3.02
ferrous iron transport protein A
DVU2444 flaB3
-1.93
-2.3
flagellin
DVU1307 rpsS
-1.62
-2.15
ribosomal protein S19
DVU1769 hydA
-1.58
-2.04
periplasmic [Fe] hydrogenase, large subunit
tRNA modification GTPase
HmcA
acetate kinase
sigma-54 transcriptional regulator
DNA-binding protein HU
DNA-binding response regulator
translational elongation factor G
70
0.1
Table 2. Top 5 up- or down-regulated function-known genes under salt shock and salt adaptation conditions
conserved domain protein
60
0.6
Comparison of expression levels of function-known genes under salt
adaptation and salt shock
Predicted function
multidrug resistance protein
Chemotaxis protein CheY
50
0.4
1.48
1.26
1.04
1.29
1.26
Salt shock (250 mM for 120 min)
DVU no. Gene
log2(ratio) Z-score
DVU2817 acrA
4.14
7.88
DVU2073 cheY-2
3.44
5.67
DVU0834 rnhB
2.72
5.3
DVU1079 trmE
2.59
4.41
DVU0536 hmcA
2.37
4.23
DVU3030 ack A
-2.3
-4.11
DVUA0100flrC
-1.98
-3.66
DVU3187 hup-4
-1.97
-3.75
DVU3026 divK
-1.87
-3.65
DVU0881 fusA
-1.83
-3.24
40
0.3
Fig. 2 Operon structure
0.11
0.39
0.45
-0.11
0.43
-0.94
-1.07
-2.08
-1.84
-1.27
A. Control
Without additives
2 mM Leu
2 mM Trp
2 mM Leu + 2 mM Trp
0.8
0.8
Leucine biosynthesis
DVU2981
leuA
DVU2982
leuC
DVU2983
leuD
DVU2984
DVU2985
leuB
Formate dehydrogenase
DVU2809
DVU2810
DVU2811
DVU2812
fdnG-3
Heat shock proteins
DVU2441
hspC
DVU2442
DVU2643
htpG
DVU2986
pspC
DVU2987
DVU2988
pspA
Tryptophan biosynthesis
DVU0460
DVU0461
DVU0462
DVU0463
aroA
DVU0464
DVU0465
trpE
DVU0466
trpG
DVU0467
trpD
DVU0468
trpC
DVU0469
trpF-1
DVU0470
trpB-2
DVU0471
trpA
Fur regulation
DVU2383
DVU2384
DVU2385
DVU2386
oppC
DVU2387
DVU2388
tolQ-1
DVU2571
DVU2572
DVU2573
DVU2574
Fig. 5
1.0
Gene expression of D. vulgaris related to operons for salt adaptation
Table 1. Significantly changed operons of D. vulgaris grown under 100, 250 and 500 mM NaCl
DVU No.
Gene name 100 mM 250 mM 500 mM Predicted function
1.2
1.0
0
Real time PCR data (log2 ratio)
Materials and Methods
of Oklahoma, Norman, OK
2Oak Ridge National Laboratory, Oak Ridge, TN.
3Temple University, Philadelphia, PA.
4Lawrence Berkeley National Laboratory, Berkeley, CA.
5University of Missouri, Columbia, MO.
6Miami University, Oxford, OH
7Virtual Institute for Microbial Stress and Survival, Berkeley, CA
Log2 (fold-change)
Salt adaptation mechanisms were explored in Desulfovibrio vulgaris Hildenborough
combining a global transcriptional analysis and physiological studies. D. vulgaris is a
δ-Proteobacterium, a model sulfate-reducing bacterium, and well known for its
metabolic versatility and wide distribution. D. vulgaris cells grew slower with a
longer lag and generated reduced biomass at 250 or 500 mM NaCl, and did not grow
at 1 M NaCl although growth was not significantly affected below 50 and 100 mM
NaCl conditions. Comparison of D. vulgaris grown with and without yeast extract in
the presence of 500 mM NaCl showed that D. vulgaris growth was inhibited ~ 35%
with yeast extract, and that its growth was inhibited ~ 80% without yeast extract.
Transcriptomic data revealed that predicted genes for leucine biosynthesis, heatshock proteins, formate dehydrogenases, sensory box histidine kinases/response
regulators, and peptidases were highly up-expressed in NaCl-adapted cells, and that
predicted genes involved in tryptophan biosynthesis, ribosomal protein synthesis,
energy metabolism, iron transport, and phage-related proteins were downexpressed. However, genes involved in glycine/betaine/L-proline ABC transport,
Na+/H+ transport, K+ uptake and transport, proline biosynthesis and transport, and
glycerol biosynthesis and transport were not significantly changed. This was
different from our previous observations for salt shock in D. vulgaris. External
addition of leucine or/and tryptophan into the LS medium without yeast extract
significantly relieved the inhibitation of D. vulgaris growth under 500 mM NaCl
conditions, which was consistent with the microarray data since the genes involved
in tryptophan biosynthesis are strongly regulated by feedback mechanisms. An
addition of other amino acids (e.g. glutamate and serine), precursors of tryptophan,
or products of tryptophan could not relieve inhibition. The results suggested that
the accumulation of metabolites (e.g. leucine and tryptophan) and nutrients may
increase the adaptability of D. vulgaris to high salt conditions. Further studies will
focus on the analysis of metabolites and on the elucidation of salt adaptation
mechanisms in Desulfovibrio vulgaris Hildenborough.
1University
OD600
Abstract
DVU2298
DVU2281
DVU2649
DVU2356
DVU1602
DVU3371
DVU2286
DVU1574
DVU3028
DVU1267
DVU4432
DVU2048