Supplemental Material
Materials and Methods
Deletion of shc in R. palustris TIE-1. The Failsafe PCR System (Epicentre,
Madison, WI) and primers PW13 and PW14 were used to amplify the upstream region
of the shc gene (1 kilobase) from R. palustris TIE-1 genomic DNA. The PCR reaction
was purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and both
the PCR fragment and the cloning vector, pJQ200SK (1), were digested with NotI and
SpeI for 2 hours at 37oC. The restriction enzymes and buffer were removed from both
digestions with the QIAquick PCR purification kit and eluted in 30 l of water. A ligation
reaction was set up by mixing 15 l of NotI/SpeI cut PCR product, 2 l of NotI/SpeI cut
pJQ200SK, 2 l of 10X T4 DNA Ligase Buffer (New England Biolabs, Ipswich, MA), and
1 l T4 Ligase (New England Biolabs). The ligation reaction was incubated for 1 hour at
room temperature and then 1 l of the ligation reaction was electroporated into
electrocompetent E. coli S17-1 cells and plated on LB plus gentamicin at 20 g/ml.
Gentamicin resistant colonies were screened by PCR and verified by sequencing for
insertion of the shc upstream region into pJQ200SK. One positive clone, pPVW3, was
selected for further cloning.
The shc downstream region (1 kilobase) was PCR
amplified using primers PW15 and PW16 and cloned into pJQ200SK at SpeI and XmaI
as described above. The resulting plasmid, pPVW4, was verified to be correct by PCR
and sequencing. To generate the deletion plasmid, pPVW6, the shc upstream region
was subcloned from pPVW3 into pPVW4 at the NotI and SpeI sites.
To delete the shc gene in R. palustris TIE-1, pPVW6 was transferred from E. coli
S17-1 to TIE-1 via conjugation. TIE-1 was grown in 20 ml of YP (0.3% yeast extract,
0.3% peptone) to an OD600 of 0.3-0.4 and centrifuged for 10 minutes at 5000 x g at 4oC.
1.5 ml of the donor strain, S17-1 carrying pPVW6, was pelleted for 1 minute at 14000 x
g at room temperature. Each pellet was resuspended in 500 l of fresh YP and then
combined in one microfuge tube. The cell mix was centrifuged 1 minute at 14000 x g at
room temperature. The cell pellet was resuspended in 200 l of fresh YP and 50 l of
cell mixture was spotted onto YP plates (4 spots per plate). The plates were incubated
overnight at 30oC. After mating, the cells were scraped with a sterile pipet tip and
combined in 1 ml of YP. Serial dilutions of the cell mixture were plated on YP + 800
g/ml gentamicin and incubated for 5 days at 30 oC.
Because pPVW6 is a non-
replicating plasmid in TIE-1, only cells that have inserted the plasmid via homologous
recombination adjacent to the shc gene should be resistant to gentamicin.
Several
gentamicin colonies were screened by PCR for insertion of pPVW6 into the
chromosome. One colony was grown for 2 days in YP for 30 oC without gentamicin to
allow removal of pPVW6, and possibly deletion of shc, from the chromosome by
homologous recombination. Because insertion of pPVW6 conferred sucrose sensitivity
to the cell, removal of the plasmid should result in sucrose resistance. After 2 days of
non-selective growth, serial dilutions were plated on YP + 10% sucrose and incubated
for 5 days at 30oC. Sucrose resistant colonies were screened by PCR for loss of the
shc gene. Several colonies were found to have deleted shc and one was picked for
further characterization.
Figure S1. Chemoheterotrophic growth curves of TIE-1 and shc strains in YP medium
buffered at pH 4.5 and pH 5.0 as described in materials and methods. Each time point
represents the average of three replicate cultures (error bars represent standard
deviation and may not be visible beneath the data point marker). Each growth curve
was repeated two times and a representative growth curve is shown. The wild type and
shc strains are unable to grow chemoheterotrophically at pH 4.5 or lower and growth
of the shc mutant is impaired at pH 5.0.
Figure S2. Photoheterotrophic growth curves of TIE-1 and shc strains in FW medium
buffered at pH 4.5 and pH 5.0 as described in materials and methods. Each time point
represents the average of three replicate cultures (error bars represent standard error
and may not be visible beneath the data point marker).
Each growth curve was
repeated two times and a representative growth curve is shown. The wild type and shc
strains are unable to grow photoheterotrophically at pH 5.0 or lower.
Figure S1. Chemoheterotrophic growth of R. palustris TIE-1 and shc at acidic
pH
Figure S2. Photoheterotrophic growth of R. palustris TIE-1 and shc at acidic and
alkaline pH
Table S1. Key data for hopanoid analysis of R. palustris strains by hightemperature GC-MS.
Rta
M+ (RI)
Base
Peak
2-methylhopene
19.76
424(18)
189
205(73), 355(23), 381(25)
hopene
19.72
410(18)
189
191(93), 341(27), 367(23), 410(19)
21.61
484
205
189(81), 249(11), 409(5), 424(5)
21.61
484
205
189(32), 263(5), 355(4), 424(4)
tetrahymanol acetate
21.58
470(6)
191
189(46), 249(6), 395(5), 410(4)
unidentified BHP
25.37
550(5)
191
287(45), 392(15), 367(7), 369(6), 493(3)
2-methylBHtetrol
acetate
27.25
728(1)
205/95
253(10), 383(19), 433(5), 493(38), 713(1)
BHtetrol acetate
27.25
714(.5)
191
253(9), 369(18), 433(4), 493(34), 699(1)
BHaminotriol
28.41
713(4)
191
369(20), 492(65), 653(10), 698(1)
Analyte
20-methyltetrahymanol
acetate
2-methyltetrahymanol
acetate
Characteristic fragments (RI)
a. Retention time (min) on 15m DB-1HT column and using an Agilent 6890GC/5975MSD
References
1.
Quandt, J., and M. F. Hynes. 1993. Versatile suicide vectors which allow direct
selection for gene replacement in gram-negative bacteria. Gene 127:15-21.