Gralnick et al. 10.1073/pnas.0505959103. |
Supporting Figure 6
Supporting Figure 7
Supporting Table 2
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10
Supporting Table 3
Fig. 6.
Type II secretion is required for maximal DMSO reduction. Anaerobic growth of Shewanella oneidensis strains with fumarate as the sole electron acceptor. Strains are MR-1 (n), JG91 (gspE, s), JG92 (gspJ, l), and JG94 (pilD, t). Error bars represent data range for duplicate cultures.
Fig. 7.
RT-PCR of gene pairs in the SO1427–SO1432 gene cluster. Primers used to amplify from cDNA can be found in Table 2. The order of samples analyzed was 1427–1428, 1428–1429, 1429–1430, 1430–1431, and 1431–1432. Lanes 1, 4, 7, 10, and 13 represent genomic DNA control. Lanes 2, 5, 8, 11, and 14 verify that no DNA contamination of the RNA used to generate cDNA (no reverse transcriptase added). Lanes 3, 6, 9, 12, and 15 are with cDNA as template. Outermost lanes are DNA size markers with the lowest shown being 500 base pairs.
Fig. 8.
Anaerobic growth of strains using fumarate as the anaerobic electron acceptor. Anaerobic growth of dmsB (SO1430, ¨) and dmsE (SO1427, s) deletion strains were compared with a mutant containing a polar insertion in SO1427 (t) and to wild-type MR-1 (n) by using either fumarate as the sole anaerobic electron acceptor. Error bars represent data range of duplicate independent cultures in all experiments.
Fig. 9.
Mutants defective in dmsB are poorly complemented by pDMSB-HA. Strains tested were MR-1 with pBBR1MCS-2 (n), dmsB with pBBR1MCS-2 (s), and dmsB with pDMSB-HA (t). Strains were tested anaerobically, with either DMSO (A) or fumarate (B) as the sole electron acceptor. Error bars represent the standard deviation of three independent cultures.
Fig. 10.
Shewanella oneidensis MR-1 can grow at 4°C anaerobically with DMSO. Growth was monitored either with DMSO (l) or with no electron acceptor added (s). Error bars represent the data range of two independent cultures.Table 2. DMSO reductase activity and expression of dmsFAB in MR-1, DdmsE, dmsE::Km
| Fold Expression Difference‡ | ||||
| Reductase Activity* | Fold Difference† | dmsF | dmsA | dmsB |
MR-1 | 1.1 | – | – | – | – |
D dmsE | 4.0 | 3.6 | 4.7 | 2.6 | 3.3 |
dmsE ::Km | 0.27 | 0.25 | 0.59 | 0.25 | 0.29 |
* Reductase activity reported as DA600 per min per OD600. Values represent the average of at least two independent cultures. Standard deviations were <10%. See Methods for experimental parameters.
† Fold difference between mutant strains and MR-1.
‡ Fold expression difference between mutant strains and MR-1 grown anaerobically with fumarate as the sole electron acceptor. Values derived from the average of duplicate cultures. Quantitative RT-PCR performed as described in Methods and standardized to envZ expression.
Table 3. Primers used in this study.
A. Primers for deletion construction | ||||
Gene deletion target | Primer orientation | Primer sequence | ||
D dmsE (SO1427)
| Out-upper Out-lower In-w/upper In-w/lower | GGACTAGTgtcaacacgccctaaccattcttc GGACTAGTgtttgcgctcggctgtcgtatc CCCATCCACTAAATTTAAATAAGTTTTAATTTTACGCCATCTCAT TATTTAAATTTAGTGGATGGGAGCGGCAGCAATTTTGCCCGTTGA | ||
D dmsB (SO1430) | Out-upper Out-lower In-w/upper In-w/lower | GGACTAGTgtggcgatcctaaaaagactc GGACTAGTcaataaaaataaccgcagacataa CCCATCCACTAAATTTAAATAATATTGTGTTTGTTGAGTCAT TATTTAAATTTAGTGGATGGGATAAACCCTGCAGAAGTGTAA | ||
B. Quantitative RT-PCR Primers | ||||
Gene target | Primer orientation | Primer sequence | ||
SO1427 | Left Right | CGAGCCAAAATACCGTTTGT GCACAGGCAATTTCTTCCAT | ||
SO1428 | Left Right | TGCTGCTACGCCTGATAATG ATCGCCATCTTGTAGCATCC | ||
SO1429 | Left Right | GGGGCGTACACACTCAAGTT AACCACGACCTAAAGCATCG | ||
SO1430 | Left Right | CCTGTAAGGACCGCATGACT AACACGGCGCCAAATAATAC | ||
SO4357 | Left Right | GTGGTGGAAATTGGACTGCT ACACACACCGGATTGCTACA | ||
SO4358 | Left Right | CGGGAATGATGGTGTATTCC CCATTCGTGCCGCTAATAAT | ||
SO4360 | Left Right | GCCATAATGCTCACGGAAGT GCATGGGGATTACCGTTATG | ||
C. RT-PCR Primers | ||||
Genes overlapped | Primer orientation | Primer sequence | ||
SO1427-1428 | Left Right | ACAATGTCATGCCGATGATG CTAATCCCAAGCCCACCTTT | ||
SO1428-1429 | Left Right | TGGATGACAGGCTGACTCTG CGCTTCATAGGTGTGCGTAA | ||
SO1429-1430 | Left Right | AGCCGCAGAAGATGCAGTAT GGCAAATTAGCGACACCATT | ||
SO1430-1431 | Left Right | TGGTTGCAGCGATAGACTTG TACGGGTTCATTTCGTGTCA | ||
SO1431-1432 | Left Right | TGACACGAAATGAACCCGTA ATCAGGCAGTTTCTGCTCGT | ||