Response of Desulfovibrio vulgaris to acid medium

1. RESULTS MICROARRAY RESULTS Figure 1. Growth at different initial pHs Table 1. Growth of D. vulgaris on acid medium Final protein yields and OD of cultures grown on acid media ------------------------------------------------------------------------------------------------------------------ Medium pH Protein (µg/ml; % control) ------------------------------------------------------------------------------------------------------------------ Exp. 1 Exp. 2 Exp. 3 Avg. % 7.0 97 (100%) 94 (100%) 93 (100%) 100% 6.5 84 (87%) 81 (86%) 83 (89%) 86% 6.0 55 (57%) 54 (57%) 48 (51%) 55% 5.5 50 (52%) 43 (46%) 47 (51%) 50% 5.25 39 (40%) 37 (39%) 32 (34%) 38% 5.0 29 (30%) 26 (28%) 28 (30%) 29% Figure 3. Acid pH shift at 0.4 OD 600 nm D. vulgaris growth and lactate consumption Figure 2. D. vulgaris growth and pH change on acid medium* * Medium initially buffered with 30 mM PIPES Table 2. Lactate consumption for pH adjustment? Figure 4. Effect of supplements on acid resistance: Thiamine, KCl, Arg, Lys, and Glu. RESULTS & CONCLUSIONS Figure 5. Growth of WT, ΔFur, and PspA- mutants inoculated at acid pHs Wall Lab Supplements: 2 µM Thiamine, 8 mM KCl, 6 mM each Arg, Lys, and Glu. Figure 7. Growth on acid media containing Formate Figure 8. Growth on acid media containing Sodium sulfide Figure 6. Growth on acid media containing acetate Pink line=HK and RR genes including other regulator genes Up-regulated gene cluster arrays Red (up-regulated; Z=1.2 or higher) Down-regulated gene cluster arrays Pink (up-regulated and logR=2.6 or higher; Z=1.2 or higher) Green (Down-regulated; Z=1.2 or higher) Blue (Down-regulated and logR=-1.6 or lower ; Z=1.2 or higher) Phage genes Maga-plasmid (pH 5.5; 240/0 min. array) DVUA0020-0028 DNA restriction/modification DVUA0098 DVUA0129-0135 CRISPR (Cas) DVUA0067-0073 Cell envolope (glycosyltransferase) DVUA0092 Response of Desulfovibrio vulgaris to acid medium H.-C. Bill Yen1, E. Alm2, K. Huang2, T.C. Hazen3, A. Arkin3, J. Zhou4, and J. D. Wall1* 1University of Missouri-Columbia, Columbia, MO; 2MIT, Cambridge, MA; 3Lawrence Berkeley National Laboratory, Berkeley, CA; and 4University of Oklahoma, Norman, OK DVU0043-0050 Flagellar DVU0102-0107 DVU0404-0425 Trk/potassium uptake Polyamine operon (spe) DVU2971-3020 Hydrogenase Phage shock (pspA/C/F) Glycosyl transferase (Cell envolope) DVU3031-3062 DVU3080-3084 Transcriptional regulators DVU3117 DVU3141 DVU3202 Hydrolase DVU3234 DVU3242 DVU3279-3285 DVU3300-3346 La-protease (Ion); metal transporter kdp operon (kdpD/C/B/A/F) DVU3363 DVU3381-3390 Zinc resistance DVU0026 DVU0082-0086 DVU0120-0136 Membrane protein DVU0239 DVU0252 DVU0281 DVU0307-0335 Flagellar DVU0355 DVU3186 DVU0159-0160 Thioesterase Isomerase DVU3108 DVU3124 DVU3314-3240 DVU0228 DVU241 DVU3358 DVU3274 DVU0298 DVU0127 DVU0138 DVU3395 DVU0001 D. vulgaris chromosome (pH 5.5; 240/0 min. arrays) DVU0407 DVU0189-0236 Phage#1 DVU0427 DVU0443 Exonucleas DVU2937-2960 Transcriptional regulators ABSTRACT DVU0454 DVU2919-2936 Translation/ribosome DVU0475 DVU0483 The anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough is currently being explored for environmental bioremediation applications. Among the environmental constraints likely to be encountered in contaminated sites will be acid conditions. Therefore, the effect of low pH on the metabolic capacities of D. vulgaris is being explored. Transcriptional profiling was performed for cells exposed to pH 5.5. Cells inoculated into acid pH media had a longer lag phase and lower final protein yields. Lactate appeared not to be completely oxidized at acid pH. When cultures were shifted to low pH at OD 0.4, no cell growth occurred below pH 6.0, although medium pH increased ~ 0.6 pH units. This may be due to the accumulation of acid end products, such as acetate or formate. Addition of sodium sulfide on the other hand increased medium pH and allowed cell growth. When cells used for inoculation were pre-grown at pH 6 or 5, or were at stationary phase, they did not show acid adaptation as described for Eschesichia coli. Acid-grown D. vulgaris showed greater mobility during microscopic observations but did not increase mobility using a soft-agar plate assay. Addition of thiamine, potassium, or individual amino acids showed only a slight increase in acid resistance. A mutant with a null mutation in pspA (a phage shock gene) and a ΔFur mutant (an iron uptake regulatory gene) showed an increased sensitivity to acid, having a much longer lag phase before growth in pH 5.5 medium. DVU0512-0525 Flagellar DVU2866 DVU2828-2881 Phage#2 DVU0539-0564 ISDvu (orf) Transposase DVU2827-2840 Phage#2 DVU0572-0600 Met-sulfoxide reductase (msrB) DVU0602-0606 DVU2774-2788 DVU0617 DVU0628-0630 TetR DVU0651-0668 Cysteine biosynthesis DVU2752 DVU2717 DVU2687-2733 Phage#3 DVU2709 DVU0669-0722 DVU2686 Peptidase DVU2655 DVU2617-2642 Ala-tRNA synthase DVU2589-2654 Phage#8 DVU2605-2616 DVU0758-0803 • 1. Cells inoculated into acid media had longer lag phases and lower final protein yields. Lactate appeared not to be completely oxidized at acid pH. • 2. When cultures were pH shifted at OD 0.4, no cell growth occurred below pH 6.0, although medium pH increased ~ 0.6 pH unit. • 3. Addition of potassium, thiamine, and amino acids in pH 5.5 medium increased the acid resistance slightly. • Both ΔFur and PspA- mutants were more acid sensitive than the wild type. • Although many flagellar genes were increased in transcription at pH 5.5, an increase in motility could not be demonstrated (Data not shown). • Adding acetate or formate in acid media increased acid sensitivity. • Adding sodium sulfide in acid media increased medium pH greatly and allowed cell growth. • Additional observations: • I. Acid adaptation was not apparent, when cells were pre-grown at pH 6 or 5 before subjecting to a larger pH shift. • II. Stationary phase cell acid adaptation could not be demonstrated. • III. No obvious amino acid decarboxylase systems for acid resistance (e.g. glutamate and lysine) could be recognized in D. vulgaris DNA. • IV. F0F1 ATPase is not regulated by acid pH as observed in some Gram-positive bacteria. DVU0813 DVU2541-2576 Hybride cluster; Fe-S cluster; Alcohol dehydrogenase Fur regulon (feoA/B) DVU0817 DVU2520 DVU2500-2513 Cell wall synthesis DVU0857-0865 Flagellar/Isoprenoid DVU0872 DVU2456-2498 Flucculin; oxidoreductase Phosphate transport TrpF; Fe-S cluster; Thioesterase; lipoprotein DVU0916-0938 Endoribonuclase DVU2451 DVU2437-2446 Heat shock (hsp20) Flagellar DVU2400 DVU0985 DVU2371-2375 Omp85; lipoprotein DVU1010 DVU2358-2363 ThiE-2; ThiM DVU2351 DVU2324-2327 merP (mercuric-r) DVU1047-1051 DVU2320 DVU2300 DVU1080-1081 Fe-S cluster DVU2282 DVU1110 DVU2257-2272 DVU1098-1144 Phage#6 DVU1122-1144 DVU2212-2236 DVU1154 DVU01166 DVU2186-2208 Endoribonuclase DVU2174-2181 Phage#5-ISDvu DVU2151-2200 Phage#5 DVU2168 DVU1212 FxsA protein DVU2145-2146 Cm-acetyltransferase DVU1241 DVU1694-1757 Phage#7 DVU1476-1527 Phage#4 DVU2101-2107 Omp85; Fe-S cluster DVU1580 DVU1336-1344 Heat shock; Zur Isoprenoid DVU1980-1990 Met-sulfoxide reductase uvrA DVU2079-2091 Flagellin; GentR; ThiE-1 DVU2000-2011 ISDvu (transposase) DVU1869-1876 ClpB; dnaJ DVU2039-2047 Fic (cell division) DVU1599-1603 ClpS/ClpA DVU1564-1592 carD DVU1417-1447 Flagellin DVU1400-1402 LysR DVU1509 DVU1480 DVU1457 DVU1285 DVU1938-1946 DVU1902-1908 DVU1849 DVU1834 DVU1723-1739 DVU1646-1692