there is intense interest in using biotechnology for the production of fuels and chemicals from renewable resources ( 1 , 2 ) . current fuels and chemicals derive largely from petroleum hydrocarbon feedstocks that are becoming scarcer and more expensive .
thus , a major goal of commodity biotechnology is to produce hydrocarbons via bacterial metabolism ( 3 ) .
recent studies have identified the genes involved in cyanobacterial medium - chain alkane biosynthesis ( 4 ) .
other studies have described bacterial polyolefin biosynthesis via the head - to - head condensation of fatty acyl groups ( 57 ) .
while these studies lay the groundwork for future bacterial hydrocarbon production , obtaining bacterial strains suitable for mass production will require rapid screening tools for facilitating the iterative processes of directed microbial engineering and random mutagenesis .
bioinformatics has revealed approximately 70 bacterial genomes that contain ole genes , and a number of those organisms have been demonstrated to produce long - chain hydrocarbons ( 8) .
hydrocarbon biosynthesis has been studied with bacteria of the genus shewanella ( 7 , 9 ) .
shewanella  oneidensis strain mr-1 produces a single polyolefinic hydrocarbon , 3,6,9,12,15,19,22,25,28-hentriacontanonaene , and contains the oleabcd genes .
the ole gene cluster was shown to be essential for polyolefin biosynthesis ( 10 ) , and the isolated expression of olea , which encodes a fatty acyl - coenzyme a ( coa ) condensase , produces a long - chain ketone in s.  oneidensis .
additionally , experiments with a strain of s.  oneidensis mr-1 showing a deletion of the ole genes suggested that the polyolefin may be involved in adaption to lowered temperature ( 7 ) .
foreign olea genes expressed in s.  oneidensis result in the production of a variety of ketones , depending on the source .
for example , the expression of olea from stenotrophomonas maltophilia resulted in the production of more than 15 ketones derived from the condensation of different fatty acids ( 7 ) .
the long - chain ketones , ranging from 26 to 30 carbons in length , are of interest as fuel precursors or novel chemicals .
chen et al . ( 11 ) previously described a high - throughput method using nile red to detect neutral lipids in microalgae .
the present study describes a similar high - throughput method for identification of mutant strains of s.  oneidensis and escherichia  coli that produce higher levels of hydrocarbons or ketones . while nonfluorescent in water , nile red fluoresces in a hydrophobic environment ( 12 ) .
nile red has previously been used to screen bacteria , cyanobacteria , haloarchaea , and microalgae for those producing fatty acids and esters ( 11 , 1320 ) .
nile red has also been used to screen for isoprenoid - derived hydrocarbon production in microalgae ( 21 ) and for triacylglyceride production in saccharomyces 
 cerevisiae ( 22 ) .
previous studies typically used agar plates with nile red in the medium ( 1418 , 23 ) . in those examples , strains making more fatty acids or esters could be identified using fluorescence microscopic techniques . in initial agar plate studies , both wild - type s.  oneidensis and a derivative expressing s.  maltophilia olea
were compared to the non - hydrocarbon - producing oleabcd deletion ( oleabcd ) strain ( see table  s1 in the supplemental material ) . on plates
, the three strains showed a low level of nile red fluorescence that could not be differentiated ( data not shown ) . unlike for the fatty esters , which comprise up to 80% of the cell mass and
are typically deposited in cellular granules , it is possible that the level of hydrocarbon production in these shewanella strains may be too low to differentiate on plates .
because colony fluorescence could not be differentiated , subsequent studies were conducted with liquid culture to test nile red for use as a high - throughput screening vehicle .
stationary - phase liquid cultures ( luria broth [ lb ] ) of s.  oneidensis expressing s.  maltophilia olea on a pbbad vector , s.  oneidensis with an empty pbbad vector , and the oleabcd strain with an empty pbbad vector were grown in microtiter plates with lb - kanamycin ( 50  g / ml ) .
nile red in dimethyl sulfoxide ( dmso ) ( 1  mg / ml ) was added to give a final concentration of 1  g / ml . expression of olea under the control of the arabad promoter was induced with 0.2% arabinose at an optical density at 600 nm ( od600 ) of 0.5 to 0.8 , and strains harboring empty vectors were mock induced .
the plates were incubated overnight at 30c in a microtiter plate orbital shaker with 70% humidity ( optimization of dye contact time is described in fig . 
fluorescence intensity was measured with a fluorescence spectrophotometer ( molecular devices spectramax m2 ) using an excitation wavelength of 580  nm and an emission wavelength of 640  nm .
percentages of the nile red fluorescence signal relative to that of s.  oneidensis with the empty pbbad vector , and corrected for the optical density of the cultures ( relative fluorescence units [ rfu]/od600 unit ) , are reported for each strain , and standard deviations are shown for triplicate samples ( fig . 
the nile red signal for s.  oneidensis that is expressing s.  maltophilia olea is significantly increased over the background signal for both s.  oneidensis and the oleabcd strain ( analysis of variance [ anova ] ; p = 0.0164 ) .
these results are in agreement with results for gas chromatography with flame ionization detection ( gc - fid ) analysis of hydrocarbon and ketone production performed using a modified bligh and dyer method as described in the supplemental material ( fig .  1b ; see also fig . 
these results suggested that nile red fluorescence is a good indicator of increased long - chain hydrocarbon or ketone production , detectable above the background stain of membrane components .
( a ) percentages of mr-1 nile red fluorescence normalized to optical density ( od600 ) for s.  oneidensis mr-1 with empty pbbad ( blue ) , the ole strain with empty pbbad ( red ) , and s.  oneidensis with s.  maltophilia olea in pbbad ( gray ) .
( b ) gc - fid amounts for strains listed in panel a relative to the level for a 12-tricosanone standard normalized to optical density ( od600 ) and to the amount produced by s.  oneidensis with empty pbbad .
( c ) percentages of nile red fluorescence normalized to optical density ( od600 ) for e.  coli with empty pbbad ( blue ) and e.  coli with s.  maltophilia olea in pbbad ( gray ) .
( d ) gc - fid amounts for strains listed in panel c relative to the level for a 12-tricosanone standard normalized to optical density ( od600 ) and to the amount produced by s.  oneidensis with empty pbbad .
( e ) relative nile red fluorescence normalized to the optical densities ( rfu / od600 unit ) of 96 colonies of a 1:1:1 mixture of the s.  oneidensis strains listed in panel a. the five highest and the five lowest nile red signals are denoted by green and red bars , respectively .
the gc - fid chromatograms from the colonies producing the highest ( green ) and lowest ( red ) nile red signals are inset .
analogous experiments were carried out using bodipy 493/503 ( invitrogen ) , as similar dyes have also been used for hydrophobic - molecule screening ( 25 ) .
bodipy was found not to be as sensitive to changes in hydrocarbon and ketone production as nile red .
this may be a result of the smaller separation between the excitation and emission maximum wavelengths than for nile red .
furthermore , bodipy 493/503 is considerably more expensive and more sensitive to photobleaching than nile red ( 26 ) . for these reasons ,
nile red was chosen for further study . to examine whether nile red fluorescence could also be used as a method for detecting increased hydrocarbon production in e.  coli , we analyzed nile red fluorescence of e.  coli wm3064 harboring either s.  maltophilia olea on pbbad or an empty pbbad vector .
the e.  coli cultures were induced and assayed as described above for shewanella , with the exception that the cells were grown at 37c in the presence of diaminopimelic acid ( dap ) since wm3064 is a dap auxotroph .
the nile red signal was significantly ( anova ; p = 0.0004 ) higher in e.  coli expressing olea ( fig . 
these nile red data are in agreement with gc - fid analysis in which ketone production was observed in the strain expressing olea but not in the strain carrying an empty vector ( fig . 
, these data demonstrated that the fluorescent dye nile red could be used to identify bacterial strains producing long - chain hydrocarbons or ketones in a microtiter plate format . to test the feasibility of using nile red in a high - throughput screen , 96 colonies of a 1:1:1 mixed population of wild - type s.  oneidensis , s.  oneidensis expressing s.  maltophilia olea , and the oleabcd strain were screened with nile red as described above .
a distribution of nile red fluorescence signals ranging from 800 to 150 rfu / od600 unit was observed ( fig . 
extracts from the five colonies with the highest and lowest nile red fluorescence levels were analyzed by gc - fid to identify each strain by its previously determined hydrocarbon or ketone profile .
of the colonies giving the five highest nile red signals , all five produced increased amounts of ketones , indicating that all five were the strains expressing the s.  maltophilia olea gene .
the five colonies with the lowest nile red fluorescence levels either produced the single hydrocarbon signature of mr-1 or did not produce the hydrocarbon , as previously observed in the oleabcd strain ( fig .  1e , insets ; see also fig . 
these results are consistent with the previous observation that both the wild - type and the oleabcd strains gave background levels of nile red fluorescence . unlike polyhydroxyalkanoates ( phas ) , which accumulate in granules inside the cells , the hydrocarbon and ketone products of the ole genes are thought to partition into the membrane ; however , this has not been investigated . with evidence that nile red is capable of detecting increased ketone production in vivo ,
nile red staining of ketone - producing strains was used to gain information regarding the cellular localization of the ketones using fluorescence microscopy .
a ketone producer strain of s. oneidensis expressing s.  maltophilia olea was incubated with 2  g / ml nile red and 1  g / ml 4,6-diamidino-2-phenylindole ( dapi ) , a dna dye , for at least 15  minutes .
these cells were adhered to microscope coverslips , mounted on glass slides , and visualized with a nikon c1si laser scanning confocal microscope ( fig .  2 ) .
cells that have been imaged as a cross section appeared as a ring of nile red ( fig . 
the nile red membrane stain surrounded the dapi nuclear stain ( fig .  2c ) .
these results are consistent with a localization of the ketones and hydrocarbons in cellular membranes and perhaps in some extracellular hydrophobic material .
confocal fluorescence micrographs of a ketone producer strain of s. oneidensis expressing s.  maltophilia olea .
( a ) nile red fluorescence ; ( b ) dapi fluorescence ; and ( c ) merged image of nile red and dapi .
these results showed that nile red fluorescence could be used to identify bacterial strains with increased levels of olefinic hydrocarbons or ketones that derive from the head - to - head hydrocarbon biosynthetic pathways .
it is likely that the methods described here will prove generally useful for high - throughput screening of bacteria to identify strains that are useful for mass production of hydrocarbons or ketones .
these results showed that nile red fluorescence could be used to identify bacterial strains with increased levels of olefinic hydrocarbons or ketones that derive from the head - to - head hydrocarbon biosynthetic pathways .
it is likely that the methods described here will prove generally useful for high - throughput screening of bacteria to identify strains that are useful for mass production of hydrocarbons or ketones .
text  s1 , doc file , 0.032 mb strains and plasmids used in this study .  
( a ) percentages of mr-1 nile red fluorescence normalized to optical density ( od600 ) for s.  oneidensis with empty pbbad and s.  oneidensis with s.  maltophilia olea in pbbad with the addition of nile red before ( red ) or after ( blue ) growth .
figure  s1 , doc file , 0.032 mb ( a ) percentages of mr-1 bodipy 493/503 fluorescence normalized to optical density ( od600 ) for s.  oneidensis with empty pbbad ( blue ) , the ole strain with empty pbbad ( red ) , and s.  oneidensis with s.  maltophilia olea in pbbad ( gray ) .
figure  s2 , doc file , 0.033 mb representative gc - fid chromatogram for s.  oneidensis wild - type mr-1 with empty pbbad ( a ) , the ole strain with empty pbbad ( b ) , and s.  oneidensis with s.  maltophilia olea on pbbad ( c ) .
figure  s3 , doc file , 0.125 mb representative gc - fid chromatogram for e.  coli with empty pbbad ( a ) and e.  coli with s.  maltophilia olea on pbbad ( b ) .
the peak at 18.5  minutes is the internal standard , and the peak at 20  minutes does not represent a ketone , as determined by mass spectrometry ( ms ) .
figure  s4 , doc file , 0.621 mb gas chromatograms from shewanella strains producing the five highest ( a ) and the five lowest ( b ) nile red fluorescent signals reported in fig .  1e .
 abstracta method for use in high - throughput screening of bacteria for the production of long - chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of nile red is described . 
 nile red has previously been used to screen for polyhydroxybutyrate ( phb ) and fatty acid esters , but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones . 
 the microtiter plate assay was evaluated using wild - type and recombinant strains of shewanella oneidensis and escherichia coli expressing the enzyme olea , previously shown to initiate hydrocarbon biosynthesis . 
 the strains expressing exogenous stenotrophomonas maltophilia olea , with increased levels of ketone production as determined by gas chromatography - mass spectrometry , were distinguished with nile red fluorescence . 
 confocal microscopy images of s.  oneidensis olea - expressing strains stained with nile red were consistent with a membrane localization of the ketones . 
 this differed from nile red staining of bacterial phb or algal lipid droplets that showed intracellular inclusion bodies . 
 these results demonstrated the applicability of nile red in a high - throughput technique for the detection of bacterial hydrocarbons and ketones . 
Observation
Implications.
SUPPLEMENTAL MATERIAL