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Exploration of Salt Adaptation Mechanisms in Desulfovibrio vulgaris Hildenborough 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

1University of Oklahoma, Norman, OK2Oak Ridge National Laboratory, Oak Ridge, TN. 3Temple University, Philadelphia, PA. 4Lawrence Berkeley National Laboratory, Berkeley, CA.5University of Missouri, Columbia, MO.6Miami University, Oxford, OH7Virtual Institute for Microbial Stress and Survival, Berkeley, CA

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, heat-shock 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 down-expressed. 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.

Abstract

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.

Materials and Methods

Results

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).

K138

References

Email: zhili.he@ou.eduPhone: 405-325-3958Web site: http://ieg.ou.edu/

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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).

4. Zhou J, Bruns MA, and Tiedje JM (1996). Appl. Environ. Microbiol. 62:461-468.

Acknowledgements

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.

ORFs (Numbered from 1 to 12)

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Microarray datReal time PCR datay = 1.0y = -1.0

1. DVU22982. DVU22813. DVU26494. DVU23565. DVU16026. DVU33717. DVU22868. DVU15749. DVU302810.DVU126711.DVU443212.DVU2048

Microarray data (log2 ratio)

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RT-PCR verification of microarray data

12 genes expressed in different levels were chosen for real-time PCR.

The correlation between microarray data and RT-PCR results were very good with r2 = 0.96 (n = 12).

Gene expression of D. vulgaris related to operons for salt adaptation

Comparison of expression levels of function-known genes under salt adaptation and salt shock

Cell growth at the LS medium containing different concentrations of NaCl

Table 2. Top 5 up- or down-regulated function-known genes under salt shock and salt adaptation conditionsSalt shock (250 mM for 120 min) Salt adaptation (cells grew at the LS medium containing 500 mM NaCl)DVU no. Gene log2(ratio) Z-score Predicted function DVU no. Gene log2(ratio) Z-score Predicted functionDVU2817 acrA 4.14 7.88 multidrug resistance protein DVU3384 zraP 2.87 4.52 zinc resistance-associated proteinDVU2073 cheY-2 3.44 5.67 Chemotaxis protein CheY DVU2441 hspC 2.2 2.67 heat shock protein, Hsp20 familyDVU0834 rnhB 2.72 5.3 conserved domain protein DVU0142 trpS 1.89 2.73 tryptophanl-tRNA synthetaseDVU1079 trmE 2.59 4.41 tRNA modification GTPase DVU0593 lysE 1.88 2.53 L-lysine exporterDVU0536 hmcA 2.37 4.23 HmcA DVU2981 leuA 1.56 2.23 2-isopropylmalate synthaseDVU3030 ackA -2.3 -4.11 acetate kinase DVU2445 tonB -2.27 -4.07 TonB dependent receptor proteinDVUA0100flrC -1.98 -3.66 sigma-54 transcriptional regulator DVU2572 feoA -2.21 -3.02 ferrous iron transport protein ADVU3187 hup-4 -1.97 -3.75 DNA-binding protein HU DVU2444 flaB3 -1.93 -2.3 flagellinDVU3026 divK -1.87 -3.65 DNA-binding response regulator DVU1307 rpsS -1.62 -2.15 ribosomal protein S19DVU0881 fusA -1.83 -3.24 translational elongation factor G DVU1769 hydA -1.58 -2.04 periplasmic [Fe] hydrogenase, large subunit

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• 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.

• 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.

Table 3. Comparison of genes differentially expressed under salt shock and salt adaptation conditionsDVU No. Gene name Salt adaptation Salt shock Predicted functionLeucine biosynthesisDVU2981 leuA 1.48 2.03 2-isopropylmalate synthaseDVU2982 leuC 1.26 1.26 3-isopropylmalate dehydratase, large subunitDVU2983 leuD 1.04 0.62 3-isopropylmalate dehydratase, small subunitDVU2984 1.29 0.65 conserved hypothetical proteinDVU2985 leuB 1.26 0.64 3-isopropylmalate dehydrogenaseFormate dehydrogenaseDVU2809 0.77 -1.04 cytochrome c3DVU2810 1.63 0.22 formate dehydrogenase formation protein FdhEDVU2811 1.51 -0.64 formate dehydrogenase, beta subunitDVU2812 fdnG-3 1.21 -1.10 formate dehydrogenase, alpha subunitTryptophan biosynthesisDVU0460 -1.11 1.68 phospho-2-dehydro-3-deoxyheptonate aldolaseDVU0461 -1.27 1.48 3-dehydroquinate synthaseDVU0462 -1.31 1.58 chorismate mutase/prephenate dehydrataseDVU0463 aroA -1.46 1.24 3-phosphoshikimate 1-carboxyvinyltransferaseDVU0464 -1.19 1.30 prephenate dehydrogenaseFur regulationDVU2571 -1.07 2.16 ferrous iron transport protein BDVU2572 -2.08 2.87 ferrous iron transport protein ADVU2573 -1.84 1.42 hypothetical proteinDVU2574 -1.27 -0.13 ferrous ion transport proteinGlycine/betaine/L-proline ABC transportersDVU2297 0.55 2.33 glycine/betaine/L-proline ABC transporter, periplasmic-binding protein

DVU2298 opuBB 0.48 2.61 glycine/betaine/L-proline ABC transporter, permease protein

DVU2299 proV 1.03 4.36 glycine/betaine/L-proline ABC transporter, ATP binding protein

leuBleuD DVU2984pssA leuCleuA

DVU2810DVU2809 DVU2811 fdnG-3

hspC

pspC

DVU2442

pspADVU2987leuB pspF

feoA feoA

DVU2387oppCDVU2383 DVU2385DVU2384DVU2382 tolQ-1

pleD feoB DVU2573

Table 1. Significantly changed operons of D. vulgaris grown under 100, 250 and 500 mM NaClDVU No. Gene name 100 mM 250 mM 500 mM Predicted functionLeucine biosynthesisDVU2981 leuA -0.11 0.11 1.48 2-isopropylmalate synthaseDVU2982 leuC 0.22 0.39 1.26 3-isopropylmalate dehydratase, large subunitDVU2983 leuD 0.17 0.45 1.04 3-isopropylmalate dehydratase, small subunitDVU2984 -0.11 1.29 conserved hypothetical proteinDVU2985 leuB 0.05 0.43 1.26 3-isopropylmalate dehydrogenaseFormate dehydrogenaseDVU2809 0.14 0.45 0.77 cytochrome c3DVU2810 0.62 0.71 1.63 formate dehydrogenase formation protein FdhEDVU2811 0.17 0.60 1.51 formate dehydrogenase, beta subunitDVU2812 fdnG-3 0.23 1.02 1.21 formate dehydrogenase, alpha subunitHeat shock proteinsDVU2441 hspC 0.73 1.43 2.24 heat shock protein, Hsp20 familyDVU2442 0.46 1.23 1.90 heat shock protein, Hsp20 familyDVU2643 htpG 0.54 0.56 1.57 heat shock protein HtpGDVU2986 pspC 0.64 0.53 1.20 phage shock protein C (regulating Leu synthesis)DVU2987 0.68 -0.24 0.66 hypothetical proteinDVU2988 pspA 0.65 0.75 1.32 phage shock protein ATryptophan biosynthesisDVU0460 -0.78 -0.78 -1.11 phospho-2-dehydro-3-deoxyheptonate aldolaseDVU0461 -0.98 -1.17 -1.27 3-dehydroquinate synthaseDVU0462 -0.78 -1.20 -1.31 chorismate mutase/prephenate dehydrataseDVU0463 aroA 0.52 -1.13 -1.46 3-phosphoshikimate 1-carboxyvinyltransferaseDVU0464 0.54 -1.01 -1.19 prephenate dehydrogenaseDVU0465 trpE 0.12 -0.50 -0.06 anthranilate synthase, component IDVU0466 trpG -0.42 -0.41 -0.62 anthranilate synthases component IIDVU0467 trpD -0.23 0.14 anthranilate phosphoribosyltransferaseDVU0468 trpC -0.19 -0.32 -0.69 indole-3-glycerol phosphate synthaseDVU0469 trpF-1 0.37 -0.69 -0.62 N-(5-phosphoribosyl)anthranilate isomeraseDVU0470 trpB-2 -0.15 -0.94 -0.74 tryptophan synthase, beta subunitDVU0471 trpA 0.07 -0.49 -0.20 tryptophan synthase, alpha subunitFur regulationDVU2383 -0.50 -0.86 -2.26 tonB dependent receptor domain proteinDVU2384 -0.61 -0.70 -1.15 ABC transporter, periplasmic substrate-binding proteinDVU2385 ABC transporter, permease proteinDVU2386 oppC -0.09 -0.14 0.37 ABC transporter, permease proteinDVU2387 -0.46 0.02 ABC transporter, ATP-binding proteinDVU2388 tolQ-1 -0.57 -0.32 -0.94 tolQ protein, biopolymer transport proteinsDVU2571 -0.15 -0.40 -1.07 ferrous iron transport protein BDVU2572 -0.25 -0.62 -2.08 ferrous iron transport protein ADVU2573 -0.24 -0.93 -1.84 hypothetical proteinDVU2574 0.27 -0.50 -1.27 ferrous ion transport protein

Fig. 2 Operon structure

Salt adaptation:Salt adaptation: D. vulgaris cells were grown in the LS medium containing 500 mM NaCl (added before inoculation). Samples were taken at OD ~= 0.40 for microarray hybridization.

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.

Differences and similarities in gene expression were seen between salt adaptation and salt shock (Table 3).

Fig. 1

Fig. 3

Fig. 4 Fig. 5

Summary1. 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.

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.

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.

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.

trpADVU0460 DVU0461 DVU0462 trpG trpD trpC trpF-1 trpB-2aroA DVU0464 trpE

More confidence may be expected to incorporate operons or pathways for gene expression analysis.