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Proteorhodopsin quantification in marine bacteria by LCMS measurement of the retinal chromophore

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Proteorhodopsin quantification in marine bacteria by LCMS measurement of the retinal chromophore

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Content Page 1 of 58

Proteorhodopsin Quantification in Marine Bacteria by LCMS Measurement of the 1
Retinal Chromophore 2
3
Master’s Thesis 4
Brian J. Seegers 5

Ocean Sciences Program 6
Department of Earth Sciences 7
USC Dornsife College of Letters, Arts and Sciences 8
University of Southern California 9
Trousdale Parkway, Zumberge Hall of Science 117 10
Los Angeles, California 90089-0740 11
bseegers@usc.edu 12
Page 2 of 58

Contents 13
1 Abstract ................................................................................ 3 14
2 Acknowledgements ............................................................... 4 15
3 Introduction .......................................................................... 4 16
4 Materials and Procedures .................................................... 6 17
5 Assessment .......................................................................... 10 18
6 Discussion ........................................................................... 13 19
7 Comments and Recommendations ..................................... 15 20
8 Figures and Figure Legends ............................................... 17 21
9 Tables .................................................................................. 22 22
10 Appendices.......................................................................... 24 23
10.1 Standard Preparation and Evaluation ............................................ 24 24
10.2 Supplemental Tests ....................................................................... 25 25
10.2.1 Peak Corroboration ................................................................ 25 26
10.2.2 Cell disruption and Extraction Time ...................................... 26 27
10.2.3 Method Reproducibility ......................................................... 27 28
10.3 Light Extraction ............................................................................ 27 29
10.4 Hexane Extraction ........................................................................ 28 30
10.5 Appendix Figures and Figure Legends ......................................... 30 31
10.6 Appendix Tables........................................................................... 36 32
11 References ........................................................................... 41 33
12 Bibliography ....................................................................... 47 34
35
36
Page 3 of 58

1 Abstract 37
It is well known that proteorhodopsins (PRs) are present in microbial populations 38
throughout the global oceans. However, no method exists for the routine quantification 39
of proteorhodopsins in marine microbial populations. As a result, basic questions remain 40
as to the function and importance of PRs in marine biogeochemical cycles. To better 41
understand the roles of PRs marine systems, we developed a LCMS method for the 42
quantification of the PR photochrome retinal. Tests on cultures of the representative, PR 43
containing, marine Vibrio species (AND4-WT) and a strain engineered with the PR genes 44
removed (AND4- Δ pr d) indicates these organisms contain a free retinal pool and a PR- 45
bound pool of retinal. Analysis of the 0.2 µm fraction filtered from seawater samples 46
with this new LCMS methods indicates that these retinal pools are also present in marine 47
communities and that the retinal concentrations can vary over relatively short temporal 48
and spatial scales. This method is a precise measurement of free retinal and the PR- 49
bound retinal pools, and enables the calculation of the number of PRs present in a sample 50
population. Culture and field results from this study indicate AND4-WT cells have an 51
average 12,300 PR molecules cell
-1
, and seawater samples from San Pedro Basin to have 52
an average of 3,200 PR molecules cell
-1
. These values are consistent with previously 53
reported values of 3,400 to 24,000 molecules cell
-1
. Application of this method will 54
enable routine quantification of PRs in mixed microbial assemblages and will allow 55
investigations into PR distribution and function. 56
Page 4 of 58

2 Acknowledgements 57
This work was made possible by support from the Sañudo-Wilhelmy and Furhman 58
laboratories at USC. Funding was provided, in part, by the Wrigley Institute for 59
Environmental Studies at Catalina Island. I would like to thank the committee members 60
for their guidance and assistance with the project. Additionally I would like the thanks 61
the captain and crew of the SV Blissfully for their assistance in sample collection. 62
Finally I would like to thank my family for all their support. 63
3 Introduction 64
Rhodopsins are simple energy-harvesting photoproteins consisting of a membrane 65
embedded opsin-like protein covalently bound to a single chromophore retinal (Spundich 66
and Jung 2005). This protein pigment complex functions as a light-driven proton pump, 67
creating energy for the cell by generating a proton motive force (Oesterhelt and 68
Stoeckenius 1971). Before 2000, this type of retinal based phototrophy was thought to be 69
restricted to archaea inhabiting niche environments, such as hypersaline ponds. In a 70
keystone study, Béjà et al. (2000) revealed the presence of a rhodopsin-like gene 71
(homologs to the archaeal “bacteriorhodopsins”) harbored in an abundant marine 72
Gammaproteobacteria (SAR86), thus labeling the protein as proteorhodopsin (PR). 73
Shortly after this discovery, PRs were found to be widespread throughout surface oceans 74
(Béjà et al. 2001), and subsequent metagenomic studies further confirmed their wide 75
distribution (de la Torre et al.,, 2003, Man et al.,, 2003, Sabehi et al., 2003, Sabehi et al., 76
2004, Sabehi et al., 2007, Venter et al., 2004, Moran and Miller, 2007, Rusch et al., 2007, 77
Campbell et al., 2008, Sharma et al., 2006, Finkel et al., 2012). Although functional 78
analyses of PR expressed in E. coli provides a clear picture of its function as a proton 79
Page 5 of 58

pump and in ATP synthesis (Béjà et al., 2000, Walter et al., 2007, Martinez et al., 2007), 80
the ecological importance of PRs in native marine bacteria in the field has not been fully 81
evaluated. Current knowledge on the importance of PR relies on sequence data or 82
physiological studies of a limited number of whole-genome sequenced bacteria in pure 83
cultures (Giovannoni et al., 2005; Gomez-Consarnau et al., 2007, 2010; Stingl et al., 84
2007; Steinder et al., 2011), and some inferences about native bacterial growth in light- 85
dark experiments (Schwalbach et al., 2005). Metagenomic survey data indicates PR 86
phototrophy may be a fundamental process in virtually all ecosystems where solar energy 87
is available (Moran and Miller, 2007) and that PR phototrophy is prominently a 88
prokaryotic process (Finkel et al., 2012). However this data cannot confirm gene 89
expression or protein function within these environments, and thus significant questions 90
remain as to the role of PR phototrophy in bacterial survival and biogeochemical cycles 91
within the ocean’s photic zone. 92
To further investigate the role of PR phototrophy, recent studies used 93
metaproteomic approaches to determine in situ protein expression (Giovannoni et al., 94
2005; Sowell et al., 2009; Morris et al., 2010; Kirchman and Hanson, 2012). The first of 95
these studies revealed PR expression in the alpha-proteobacterial SAR11 clade from 96
enriched microbial membrane fractions from samples collected in coastal waters of the 97
Northeast Pacific (Giovannoni et al., 2005). A subsequent study employed similar 98
techniques to investigate protein expression on a larger oceanic scale (Morris et al., 99
2010). This study revealed PR expression in a number of microbial clades and 100
determined that clade specific expression varies across water types. A further study 101
utilized this data to determine energetic benefits of PR-based phototrophy to microbes 102
Page 6 of 58

(Kirchman and Hanson, 2012). Investigations of gene expression though metaproteomics 103
further suggests the significant potential of PR phototrophy in global biogeochemical 104
cycles, however the complexity of these methods prevents the routine investigation into 105
PR distribution and function. Numerous methods exist for the routine quantification of 106
chlorophylls and bacteriochlorophylls (Jeffrey et al., 1997, Goericke, 2002), however, 107
there currently are no methods for the quantification of retinal in marine microbial 108
assemblages. Here we describe a new LCMS method for the extraction and 109
quantification of of the PR photochrome retinal. This method enables precise calculation 110
of the number of PR proteins present in a samples and enables routine measurement of 111
PRs in cultures and natural marine microbial assemblages. 112
4 Materials and Procedures 113
All rhodopsins have a single retinal chromophore associated with the polypeptide 114
opsins (Rave n 2009), creating a 1:1 relationship between the number of retinal molecules 115
and the number of PR proteins. A methanol treatment of filters with PR-containing 116
prokaryotic cells extracts only the non-covalently-bonded (free retinal) from the cells. An 117
extraction of the filters with methanol plus hydroxylamine extracts both free retinal and 118
as well as the covalently bound retinal in the PRs (Oesterhelt et al., 1974). As there is 119
one retinal molecule per PR protein, the number of PR proteins in a sample can be 120
determined by the difference between the number of free retinal molecules extracted 121
using methanol and the retinal extracted using methanol plus hydroxylamine (free retinal 122
and retinal extracted from the rhodopsin pigment protein complex). 123
Sampling 124
Page 7 of 58

Culture and field samples were used for the development of this retinal 125
quantification method. For method development a PR-containing member of the genus 126
Vibrio, (strain AND4) was utilized. It has been demonstrated that this PR-containing 127
representative of marine bacteria uses PR to prolong survival under starvation conditions 128
(Gomez-Consarnau et al., 2010). A mutant strain of AND4, with the PR genes removed, 129
was also utilized. In this mutant strain (AND4- Δ pr d), the genes for the PR protein were 130
removed but the retinal synthesis genes are still present. Cultures of these two AND4 131
strains were used to evaluate aspects the retinal quantification method, including 132
corroboration of pigment peaks, cell disruption methods, pigment extraction times, 133
pigment extraction efficiency, and instrument precision (see Appendix 10.2). 134
The retinal quantification method was further validated with field samples 135
collected in the San Pedro Basin between Los Angeles, California and Catalina Island. 136
Surface samples were collected using pole samplers with 4 liter brown polyethylene 137
(HDPE) sampling bottles. All retinal samples were complemented with samples for 138
bacterial cell counts and measurement of chlorophyll-a concentrations (Table 1). 139
Cultures Samples 140
Cultures were prepared from AND4-WT (wild type) and AND4 Δ pr d (mutant) 141
stocks stored in glycerol (20% final concentration) at -80˚C. Cells were grown on agar 142
and individual colonies picked and grown in Zobell Media under full light with 143
continuous, gentle agitation. Cell concentrations were determined by staining with 144
Acridine Orange, filtering onto black 0.2 µm polycarbonate filters (Nuclepore Track- 145
Etched Membrane Filters, 25 mm; Whatman), and counting by epifluorescence 146
microscopy. Cells were harvested, by filtration onto 0.22 µm membrane filters 147
Page 8 of 58

(Durapore, 47 mm; EMD Millipore), once cultures had reached stationary phase. Filters 148
were run fresh or stored at -80˚C prior to time of analysis. 149
Field Samples 150
Seawater samples for retinal analysis (4 – 8 liters) were filtered immediately after 151
collection. The bacterial fraction was isolated from larger particles using a series of in- 152
line filters (20.0 µm [Magma Nylon Membrane Filters, 47 mm; GE Osmonics] and 2.0 153
µm [Nuclepore Track-Etched Membrane Filters, 47 mm; Whatman]) and was collected 154
on 0.22 µm membrane filters (Durapore, 47 mm; EMD Millipore). These size fractions 155
were selected to allow for analysis of pigments in the microplankton, nanoplankton, and 156
picoplankton communities. Samples were filtered at a low flow rate (flow rate < 50 ml 157
minute
-1
), using a peristaltic pump. All samples were run fresh, stored in liquid nitrogen, 158
or flash frozen in liquid nitrogen and stored at -80˚C prior to time of analysis. Chl a 159
concentrations were determined using standard fluorometric methodologies (Holm- 160
Hansen and Riemann, 1978). 161
Sample Extraction 162
To facilitate extraction, bacterial cells were disrupted by sonication (Bronson 163
Digital Sonifier 450 with microtip). Filters were placed in 3 ml of methanol. The micro- 164
tip was submerged in methanol and the sample sonicated at 50% amplitude for 30 165
seconds on ice. For retinal oxime measurements, 100 µl of 1 M hydroxylamine was 166
added to the methanol filter mixture after sonication (El-Sayed et al., 2002, Kane et al., 167
2008). Samples were allowed to extract at -20˚C for 24 hours (see Appendix 11.1.2). 168
Prior to analysis, samples were centrifuged at 5,000g for 10 minutes at 4˚C, and the 169
supernatant was pipetted into sample vial for LCMS analysis. 170
Page 9 of 58

LCMS Setup and Calibration 171
The LCMS system consists of a ThermoTSQ Quantum Access electro-spray 172
ionization triple quadruple mass spectrometer, coupled to a Thermo Accela High Speed 173
Liquid Chromatography unit. For retinal quantification, the LCMS system was set up 174
with a C18 column (Supelco Discovery 15 cm, 3 µm, 4.6 mm) and ran a 12-minute 175
gradient [solvents, A-acetonitrile:Milli-Q water, 0.5% acetic acid (60:40 v/v), B-acetone; 176
gradient (time, %A), (0 min, 80%), (1 min, 50%), (10 min, 0%), (11min, 80%); flow rate 177
250 µl min
-1
] (Goericke, 2002). The flow rate was set at 250 µL min
-1
with a 100 µL 178
sample loop (El-Sayed, et al., 2002). The mass spectrometer was tuned to retinal and 179
retinal oxime using all-trans retinal (Santa Cruz Biotechnology, CAS 116-31-4). Retinal 180
oxime was prepared by adding hydroxylamine to a solution of all trans-trans retinal in 181
methanol, with a hexane extraction to separate retinal oxime from excess hydroxylamine 182
(Kane et al., 2008). This liquid to liquid extraction step with hexane was only for 183
instrument tuning purposes. The mass spectrometer was run in ‘Selected Reaction 184
Monitoring’ mode with a retinal parent of 285 mass/z and products of 91.1, 105.1, and 185
119.1 mass/z, and a retinal oxime parent of 300 mass/z and products of 94.2, 158.6, and 186
161.5 mass/z. 1 M hydroxylamine solutions and calibration curves for retinal and retinal 187
oxime were prepared daily. The concentration of retinal standard was calculated from the 188
extinction coefficient 42,800 M
-1
cm
-1
at 380 nm (Imasheva et al., 2008). Five point 189
calibration curves (ranging from approximately 5 nM to 150 nM) were run before and 190
after sample runs. The average slope and offsets were determined by fitting a linear 191
regression and used to quantify retinal and retinal oxime values (see Appendix 10.1). 192
Page 10 of 58

5 Assessment 193
AND4-WT and AND4 Δ pr d 194
To demonstrate the ability of the new LCMS method to accurately quantity PR, 195
cultures of the Vibrio strain AND4-WT and AND4 Δ p r d were grown under similar 196
conditions. Cells were harvested (in triplicate) by filtration. Retinal was extracted using 197
two treatments: 1) methanol only treatment and 2) methanol plus hydroxylamine 198
treatment. 199
Results (Table 1) indicate the methanol plus hydroxylamine treatment extracts 200
significantly more retinal (t-Test: p-value =0.009) from the AND4-WT cells than the 201
methanol only treatment (Figure 1-A-B), and that there is no significant difference (t- 202
Test: p-value =2.1) between extraction treatments for the AND4- Δ pr d cells (Figure 1-C). 203
Retinal concentrations in AND4-WT were higher across both treatments (retinal 22.1 204
±2.2 nM [mean ±SD], retinal oxime 61.2 ±4.8 nM) than AND4- Δ pr d cultures (retinal 2.9 205
±0.2 nM, retinal oxime 2.3 ±0.7nM). The concentration of retinal in AND4-WT cells 206
extracted in methanol only treatment was 22.1 ±2.2nM. The retinal concentration for 207
cells extracted in methanol plus hydroxylamine treatment increased nearly 3 fold (61.2 208
±4.8nM). The difference between these treatments normalized to cell counts concludes 209
the concentrations of PR to be 10,074 ±1,754 PR proteins cell
-1
. A subsequent test of a 210
separate AND4-WT culture produced similar results, with a final PR concentration of 211
14,555 ±1,171 PR proteins cell
-1
(Figure 1-D). These results agree well with reported 212
values of PR concentrations in marine prokaryotes, values calculated using 213
methodologies which are not applicable for routine analysis (Table 2). 214
Retinal Spatial and Temporal Distributions 215
Page 11 of 58

Metagenomic studies have found PR genes to be present in nearly all surface 216
marine environments (de la Torre et al.,, 2003, Man et al.,, 2003, Sabehi et al., 2003, 217
Sabehi et al., 2004, Sabehi et al., 2007, Venter et al., 2004, Rusch et al., 2007, Campbell 218
et al., 2008, Sharma et al., 2006, Finkel et al., 2012), and metaproteomic data suggests PR 219
proteins to be present across a variety of ocean regimes (Morris et al., 2010). To evaluate 220
the utility of the new LCMS method for retinal measurement, samples were collected 221
along a natural gradient in nutrients extending from eutrophic waters within Santa 222
Monica Bay (Los Angeles, CA) across the San Pedro Channel to Santa Catalina Island 223
(Figure 2). During the summer season, this region experiences a strong, northward flow 224
of offshore waters (Di Lorenzo 2003) which results in stratified, relatively low 225
productivity waters mid San Pedro Channel. Field samples were collected during repeat 226
transects (Trans01 and Trans02) across a chlorophyll-a gradient (ranging from 3.84 to 227
0.23 µg L
-1
), with additional samples collected during two separate 72 hour sampling 228
efforts at an oligotrophic site (average chlorophyll-a concentration 0.32 µg L
-1
) located 229
near Santa Catalina Island (Figure 3-B). This sampling strategy permitted the evaluation 230
of the new method over a range of trophic conditions and enabled the investigation of 231
retinal distribution and variability over both spatial and temporal scales (Figure 3). These 232
samples were extracted using the methanol only extraction method and not the methanol 233
plus hydroxylamine. Thus the results represent the free retinal found in the cells and not 234
the retinal covalently bound in the PR protein. 235
Trace levels of retinal were detected in the bacterial fraction (0.2 – 2.0 µm) of all 236
samples. Retinal concentrations ranged from 4.4 ±0.2 pM to 12.2 ±0.2 pM, with an 237
average of 7.0 pM at the Catalina site (Figure 3-A). The retinal concentration increased 238
Page 12 of 58

two fold over the course of the first sampling period (Diel01) and remained relatively 239
constant during the second sampling period (Diel02). Transect retinal concentrations 240
ranged from 7.6 ±0.9pM to 16.1 ±0.9 pM, with an average of 14.0 pM (Figure 2-B). 241
Retinal distribution patterns were similar across both transects, with concentrations being 242
highest close to land (both the mainland and the island) and decreasing with distance 243
from land with the exception of Trans02, Station 5 (Figure 2-B). This station exhibited 244
both the highest and lowest concentration of retinal encountered during the transects. 245
However, these values are similar to the range of concentrations found at the nearby 246
Catalina site, indicating the potential for significant temporal variability. 247
Pigment ratios were used to investigate the relationship between free retinal and 248
chl-a (Figure 3-C). At the Catalina site, the molar ratio of free retinal to chl-a averaged 249
2.1%, ranging from 1.1 to 3.5%. A similar average free retinal chl-a ratio of 2.5% was 250
found for the transects; however the range was much greater, ranging from 0.3% to 4.9%. 251
The free retinal chl-a ratio distribution patterns are similar for both transects, with the 252
lowest values at the station closest to mainland (Station 1) and the highest being mid San 253
Pedro Channel (Station 4). Overall, the free retinal chl-a ratio decreases with increasing 254
chl-a (Figure 4). These findings may suggest the relative importance of PR phototrophy 255
for energy transduction into the system increases as chl-a decreases. 256
To estimate the PR bound retinal in field samples, an additional study was 257
conducted. In this final study, samples were collected in triplicate at a near shore station 258
close to the first transect station (Figure 2). These samples were extracted using both the 259
methanol only and the methanol plus hydroxylamine treatments. The retinal 260
concentration normalized to bacterial cell counts measured from the methanol-only 261
Page 13 of 58

extraction was similar to the concentrations measured at the near shore station in the 262
previous field study (Figure 5-A). Results from the methanol plus hydroxylamine 263
treatments indicate retinal concentration to be significantly (t-Test: p-value=0.002) higher 264
than the methanol only treatments, as was seen in the marine Vibrio culture studies 265
(Figure 1-A-B). The concentration of retinal in samples extracted using the methanol 266
only treatment was 37.0 ±1.0 pM, while the retinal concentration for cells extracted in 267
methanol plus hydroxylamine treatment was 49.9 ± 3.8pM, a 35% increase. The 268
difference between these treatments normalized to bacterial cell abundances indicates the 269
concentrations of PR to be 2,816 ±876 PR proteins cell
-1
(Figure 5-C). This number falls 270
within the range of reported PR cell
-1
values (Table 2); however, it is likely to be an 271
underestimation of the actual concentration of PR proteins cell
-1
as the number of 272
bacterial cells containing PR is not known. It is estimated that 15% to >70% of all 273
surface bacteria contain PR (Sabehi et al., 2003, Rusch et al., 2007, Campbell et al., 274
2008). A previous study reported PR concentrations as protein per liter of seawater (Béjà 275
et al., 2001). Assuming a 1:1 ratio of retinal molecules to PR proteins and a molecular 276
weight of PR as 27,000, the calculated concentration of PR proteins per liter of seawater 277
for these samples is 0.35 µg L
-1
. This is precisely the value reported in by Béjà et al., 278
(2001), suggesting the LCMS retinal method iseffective in determining the concentration 279
of PR in seawater samples. 280
6 Discussion 281
Several studies have estimated the number of PR molecules cell
-1
using different 282
methodologies which are not easily applicable to routine measurement of experimental or 283
field samples (Table 2). Béjà et al. (2001) estimated SAR86 to have approximately 284
Page 14 of 58

24,000 PR molecules cell
-1
, while Giovannoni et al. (2005) found the smaller SAR11 285
strain of Peligabacter to have 10,000 PR molecules cell
-1
. Kirchman and Hanson (2012) 286
report an average of 3,400 PR molecules cell
-1
in surface waters across South Atlantic. 287
PR cell
-1
results generated using the LCMS retinal quantification method agree with these 288
estimates (Table 2), with AND4-WT concentrations averaging 12,315 PR molecules cell
-1
289
(ranging from 10,074 to 14,555 PR cell
-1
) and seawater values (from the Near Shore 290
Station) of approximately 3,182 PR molecules cell
-1
. 291
As the hydroxylamine extraction could only be utilized on one set of samples 292
collected in the San Pedro Basin, PR concentrations cannot be calculated across the study 293
area. However, the values for non-covalently bound retinal can provide some insight into 294
the distribution of retinal and PR across the coastal ocean. Because the non-covalently 295
bound retinal (normalized to total bacterial cells) values are similar across all 296
measurements made at all near mainland stations (Figure 5-A), and because the sample 297
set (Near Shore Stations) for which the hydroxylamine extraction was also conducted 298
produced higher retinal oxime values than retinal values (retinal 37.0±1.0 pM, retinal 299
oxime 49.9±3.8 pM; similar to results seen in the Vibrio culture studies), it can be 300
assumed that PR can be quantified in field samples using this retinal extraction and 301
LCMS quantification method. The distribution of the non-covalently bound retinal 302
across the San Pedro Basin supports previous studies indicating PR genes are ubiquitous 303
across surface waters (Moran and Miller 2007). The full implementation of this new 304
method is a precise measurement of the photochrome of the PR protein pigment complex. 305
Therefore PR genes are expressed across a variety of ocean regimes and the 306
concentration of PR photosynthetic units can vary significantly over relatively short 307
Page 15 of 58

spatial and temporal scales. These results show that our new technique can be used to 308
effectively measure retinal concentrations in natural microbial populations, thus 309
providing data essential for understanding the ecological significance of PRs in the 310
marine environment. 311
Because our methanol extraction protocol is similar to the extractions being used 312
for chlorophyll and bacteriochlorophyll analyses, the new analytical protocol will allow 313
for the simultaneous measurement of the three pigments in natural samples. Furthermore, 314
because this method separates the samples into 20.0, 2.0, and 0.2 µm fractions it can be 315
utilized in investigations of PR in prokaryotic and eukaryotic communities. We believe 316
that the application of our technique in lab experiments will allow extrapolation to 317
broader levels of ecosystem function, which combined with field sampling in different 318
oceanographic regimes, will explain how environmental variables influence the synthesis 319
of retinal. 320
7 Comments and Recommendations 321
These are the first direct quantitative estimates of retinal in marine systems. This 322
new extraction and LCMS method is the only method with the potential for routine 323
assessment of PR activity across marine ecosystems. The two step extraction method 324
allows for the precise measure of non-covalently bound (free retinal) and retinal bound in 325
the PR pigment protein complex; enabling the study of factors influencing retinal 326
synthesis and PR phototrophy. The size fractionation enables the investigation of PR 327
activity in the prokaryotic and eukaryotic microbial fractions. Implementation of this 328
method with laboratory culture experiments, manipulative field experiments, and survey 329
type expeditions will advance the understanding of the function of PR in microbial 330
Page 16 of 58

assemblages. 331
The data presented establishes our method for the determination of the 332
concentration of PR proteins within mixed microbial assemblages via the quantification 333
of the photochrome retinal as valid. The generated results are predicted by previous 334
metagenomic and proteomic studies, indicating PR proteins are present in both coastal 335
eutrophic environments and offshore oligiotrophic environments. This study 336
demonstrates the ability of this LCMS retinal measurement method to quantify PR 337
proteins not only across diverse environments but also over short spatial and temporal 338
time scales. Incorporating this technique into routine oceanographic measurements is 339
necessary to more fully evaluate the impact of PR phototrophy on global biogeochemical 340
cycles. 341
342
Page 17 of 58

8 Figures and Figure Legends 343
344
Figure 1. Results comparing differences in extraction treatments from samples collected 345
from cultures of the Vibrio species AND4-WT and AND4- Δ pr d. MeOH indicates 346
extraction with methanol only. Hydroxylamine indicates extraction with methanol plus 347
hydroxylamine. A-B) Retinal concentration from two separate AND4-WT cultures. C) 348
Retinal concentration found in culture of AND4- Δ p r d. D) PR cell
-1
in AND4-WT 349
Cultures01 (A) and Culture02 (B). PR cell
-1
is determined by the difference between 350
total retinal (hydroxylamine) and free retinal (methanol). 351
Page 18 of 58

352
Figure 2. Map of the study area. Stations are laid over a SeaWIFS chlorophyll image 353
from the time of data collection (early July, 2012). Two transect studies sampled at 354
stations 1 to 5. Near Shore Station was used to collect samples for verification of the 355
LCMS method using field samples. All diel study samples were collected at Catalina 356
Station. 357
358
Page 19 of 58

359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
Figure 3. Results for parameters measured at the Catalina Stations (left) and Transects 380
(right). Grey bars denote darkness at the Catalina site. Station 1 is near the CA mainland 381
and station 5 is near Catalina Island. A) Distribution of non-covalently bound (free) 382
retinal. B) Distribution of chl-a. C) The molar ratio of free retinal and chl-a across the 383
study area. 384
385
Page 20 of 58

386
Figure 4. The free retinal chl-a molar ratio plotted against concentration of chl-a. All 387
Catalina Station and Transect data included (Linear regression results: p= 0.014 r2= 388
0.43). 389
390
Page 21 of 58

391
Figure 5. Comparison of results from both extraction treatments in field samples. A) 392
Comparison of non-covalently bound (free) retinal, normalized to cell counts, from the 393
three near shore samplings (Trans01 Stations 1, Trans02 Station2, and Near Shore 394
Station). B) Comparison of extraction treatments from samples collected at Near Shore 395
Station. MeOH indicates extraction with methanol only. Hydroxylamine indicates 396
extraction with methanol plus hydroxylamine. C) PR cell
-1
at near shore station. PR cell- 397
1 is determined by the difference between total retinal and free retinal. 398
399
Page 22 of 58

9 Tables 400
Table 1. All culture and field sample data used in validation of the LCMS retinal 401
quantification method. 402
Test Sample Retinal Retinal oxime Free Retinal molecules cell
-1
Total Retinal cell
-1
Bacterial cell ml
-1
PR cell
-1*
Chl-a µg L
-1
Culture01 AND4-WT 22.1 ± 2.2 nM 61.2 ± 4.8 nM 5,400 ± 550 15,000 ± 1,200 9,600 ± 1,300 2.46E+09 -
Culture01 AND4-Δprd 2.9 ± 0.2 nM 2.3 ± 0.7 nM 400 ± 50 300 ± 100 - 4.21E+09 -
Culture02 AND4-WT 27.3 ± 2.6 nM 80.8 ± 3.9 nM 7,400 ± 700 22,000 ± 1,000 14,500 ± 1,300 2.22E+09 -
Transect Shore Station 37.0 ± 1.0 pM 49.9 ± 3.8 pM 8,100 ± 300 11,000 ± 800 2,800 ± 900 2.74E+06 -
Transect Tran01 Sta01 16.1 ± 0.9 pM - 4,400 ± 250 - - 2.22E+06 1.11
Transect Tran01 Sta02 14.1 ± 1.3 pM - 3,900 ± 350 - - 2.16E+06 0.47
Transect Tran01 Sta03 10.2 ± 1.1 pM - 6,000 ± 600 - - 1.02E+06 0.31
Transect Tran01 Sta04 14.9 ± 0.9 pM - 9,700 ± 600 - - 9.25E+05 0.27
Transect Tran01 Sta05 15.8 ± 0.3 pM - 8,400 ± 150 - - 1.14E+06 0.56
Transect Tran02 Sta01 14.0 ± 1.3 pM - 2,700 ± 250 - - 2.15E+06 3.84
Transect Tran02 Sta01 10.8 ± 1.5 pM - 3,800 ± 500 - - 1.36E+06 2.02
Transect Tran02 Sta01 8.5 ± 1.2 pM - 3,200 ± 450 - - 1.14E+06 0.38
Transect Tran02 Sta01 12.0 ± 1.0 pM - 6,700 ± 530 - - 1.05E+06 0.23
Transect Tran02 Sta01 7.6 ± 0.9 pM - 3,500 ± 400 - - 7.76E+05 0.31
Diel Study Diel01 Event01 5.2 ± 0.6 pM - 1,500 ± 50 - - 2.15E+06 0.24
Diel Study Diel01 Event02 6.2 ± 1.3 pM - 4,900 ± 1,700 - - 7.76E+05 0.13
Diel Study Diel01 Event03 6.6 ± 1.0 pM - 2,000 ± 300 - - 1.98E+06 0.55
Diel Study Diel01 Event04 9.1 ± 0.1 pM - 3,200 ± 1,100 - - 1.43E+06 0.31
Diel Study Diel01 Event05 11.9 ± 0.1 pM - 5,100 ± 1,700 - - 1.17E+06 0.56
Diel Study Diel01 Event06 12.1 ± 0.2 pM - 3,700 ± 1,400 - - 1.60E+06 0.35
Diel Study Diel02 Event01 5.4 ± 0.4 pM - 3,100 ± 250 - - 1.04E+06 0.28
Diel Study Diel02 Event02 4.4 ± 0.2 pM - 2,500 ± 100 - - 1.03E+06 0.21
Diel Study Diel02 Event03 5.8 ± 0.6 pM - 2,800 ± 300 - - 1.25E+06 0.48
Diel Study Diel02 Event04 5.0 ± 0.2 pM - 2,523 ± 50 - - 1.20E+06 0.20
Diel Study Diel02 Event05 6.4 ± 0.9 pM - 3,400 ± 450 - - 1.14E+06 0.30
Diel Study Diel02 Event06 6.4 ± 0.7 pM - 3,800 ± 400 - - 1.03E+06 0.25
*PR cell-1 is determined by the difference between total retinal and free retinal. One retinal molecule per PR complex.
403
404
Page 23 of 58

Table 2. Number of proteorhodopsin (PR) molecules per bacterial cell. Summary of 405
results from several studies using a different methodologies. 406
Bacterial Taxa Region Method
PR cell
-
1
References
SAR86
(Gammaproteobacteria)
Pacific
coast Spectroscopic 24,000 Béjà et al. 2001
Pelagibacter Pure culture Spectroscopic 10,000
Giovannoni et al.
2005a
Bacteria S. Atlantic Metaproteomics 2,189
Kirchman and
Hanson 2012
Alphaproteobacteria S. Atlantic Metaproteomics 2,728
Kirchman and
Hanson 2012
HTCC2225
(Alphaproteobacteria) S. Atlantic Metaproteomics 3,393
Kirchman and
Hanson 2012
Pelagibacter S. Atlantic Metaproteomics 337
Kirchman and
Hanson 2012
Polaribacter S. Atlantic Metaproteomics 11,434
Kirchman and
Hanson 2012
AND4-WT Culture LCMS 12,300 This Study
AND4-Δprd Culture LCMS - This Study
Marine Sample
San Pedro
Basin LCMS 3,200 This Study
407
Page 24 of 58

10 Appendices 408
10.1 Standard Preparation and Evaluation 409
An all-trans retinal standard (Santa Cruz Biotech, CAS 116-31-4) is used for 410
retinal and retinal oxime quantification. The primary retinal standard is stored under 411
nitrogen at -20˚C and only opened under reduced light and nitrogen gas. The retinal 412
standards for calibration are made by preparing a primary, high concentration (µM) 413
dilution by dissolving a small amount of the all-trans retinal in methanol. The retinal 414
concentration of the primary dilution is determined spectrophotometrically using the 415
extinction coefficient 42,800 M
-1
cm
-1
at 380 nm (Imasheva et al., 2008). This all-trans 416
retinal primary dilution is used to make a five point retinal calibration curve, ranging 417
from approximately 5 nM to 150 nM, for retinal quantification on the LCMS. A five 418
point calibration curve, ranging from approximately 5 nM to 150 nM, is also used to 419
quantify retinal oxime. The retinal oxime calibration curve is prepared using the same 420
all-trans retinal primary dilution as the retinal calibration curve, however, 100 µl of 1 M 421
hydroxylamine (Hydroxylamine hydrochloride, Sigma-Aldrich ) is added to the initial 422
curve dilution (final hydroxylamine concentration ~30 nM). After hydroxylamine 423
addition, the solution is allowed to sit for 15 minutes, in the dark, at room temperature. 424
This ensures a complete conversion of all retinal to retinal oxime (Figure S1). The 425
hydroxylamine solution and all retinal and retinal oxime standards are prepared daily. 426
A test was conducted to ensure complete conversion of retinal to retinal oxime 427
and to determine the stability of the standards over time. The absorbance spectrum of a 428
primary dilution was measured immediately after preparation. The absorbance peak at 429
Page 25 of 58

380 nm confirms the standard is all-trans retinal (El-Sayed et al., 2002). Hydroxylamine 430
was added to an aliquot of the primary retinal dilution. The absorbance spectrum was 431
measured after 15 minutes. The shift in the absorbance peak to 360 nm confirms the 432
standard is retinal oxime (El-Sayed et al., 2002) and no shoulder at 380 indicates 433
complete conversion of retinal to retinal oxime (Figure S1). The standards were run 434
hourly over an 8 hour period. During this time, absorbance at 360 nm decreased < 3% 435
while absorbance at 380 nm decreased ~10% over the test period. These results indicate 436
retinal oxime to be more stable than all-trans retinal in methanol. 437
10.2 Supplemental Tests 438
A series of tests were performed to optimize retinal extraction and validate LCMS 439
quantification. These tests utilized the Vibrio strain AND4-WT as a representative, PR 440
possessing, marine bacteria. Tests investigating the extraction of free retinal and PR 441
bound retinal extraction methods described above. Extracted retinal was measured using 442
the new LCMS method and quantified using five point all-trans retinal and retinal oxime 443
calibration curves. 444
10.2.1 Peak Corroboration 445
Retinal and retinal oxime concentrations in samples were quantified using five 446
point calibration curves. All-trans retinal standards elutes off of the column at 5.5 447
minutes. The retention time of the AND4-WT sample extracted in methanol is consistent 448
with the all-trans retinal standard and is only detected on the channel tuned to all-trans 449
retinal (Figure S2-A). Analysis of the AND4-WT methanol extract with a retinal spike 450
produces one peak, consistent with the retinal standard. Analysis of the AND4-WT 451
Page 26 of 58

sample extracted in methanol plus hydroxylamine produced similar results, however, the 452
retention time of the retinal oxime standard is slightly shorter than retinal and the peak 453
was only detected on the channel tuned to retinal oxime.(Figure S2-B). 454
10.2.2 Cell disruption and Extraction Time 455
A test was performed to determine if sonication improved extraction efficiency 456
over filters simply left to soak in methanol. Cell disruption was achieved by sonication 457
(Bronson Digital Sonifier equipped with a micro-tip). Micro-tip was placed directly in 458
methanol with sample filter and run for 30 seconds (at 50% amplitude) on ice. Test was 459
run in triplicate using AND4-WT cells harvested from the same culture. All data is 460
reported in Table S1. 461
Test results indicate sonication alone resulted in higher retinal extraction at time 462
zero (methanol soak 19.2±2.2 nM, sonication 25.2±0.9 nM). After 24 hours the 463
concentration of retinal in both treatments increased (methanol soak 27.8±1.8 nM, 464
sonication 30.2±2.1 nM). However, there was no significant difference (t-Test: p- 465
value=0.2) between the filters sonicated and soaked and those simply soaked (Figure S3 466
A). Despite this finding, the sonication step was included in the extraction protocol as 467
the step yielded an increase in retinal (in some samples), and field samples will include a 468
variety of bacterial strains, some of which may require sonication for full extraction. 469
Further, sonication has been utilized for extraction of retinal form rhodopsins in previous 470
methods (El-Sayed, et al., 2002). 471
Optimal extraction time was determined using of AND4-WT cells. Cells for all 472
treatments were harvested from the same culture in triplicate. Cells were sonicated on ice 473
Page 27 of 58

and allowed to extract from 0 to 48 hours. Results indicate retinal extraction was 474
complete after 24 hours (Figure S3 B). 475
10.2.3 Method Reproducibility 476
To test for extraction consistency over a range of cell concentrations and to 477
validate the precision of the LCMS method, a number of filters of AND4-WT were 478
prepared from the same culture. Filters were collected in triplicate, sonicated on ice, and 479
extracted for 24 hours. Results indicate a near linear increase in retinal with an increase 480
in volume of culture filtered (Figure S4), with little variability between replicates 481
(average standard deviation of 2.83). This suggests consistent extraction of retinal and 482
precise measurement by the LCMS method over numerous samples (n=15) and injections 483
(Table S2). 484
10.3 Light Extraction 485
In rhodopsins, retinal is retained in the retinal binding pocket by a covalent bond 486
at the Schiff base (Grigorieff et al., 1996). Organic solvents alone will not free the bound 487
retinal molecule and thus hydroxylamine is utilized in the extraction of retinal from 488
rhodopsins (Oesterhelt et al., 1974). Several studies (Oesterhelt et al., 1974, Ismasheva et 489
al., 2008, Tamogami et al. 2012, Yoshizawa et al., 2012) include an illumination step in 490
the extraction process, however several other studies do not include this step (El-Sayed et 491
al., 2002, McCaffery et al., 2003, Kane et al., 2008). The LCMS retinal quantification 492
method was used to investigate the necessity of an illumination step in the extraction 493
process. Results are reported in Table S3. 494
Page 28 of 58

Samples for the illumination test were prepared using a culture of AND4-WT 495
cells. Cells were harvested, in duplicate for each treatment and time point, from the same 496
AND4-WT culture. All filters were extracted by sonication in methanol followed by an 497
addition of hydroxylamine (see Sample Extraction). Cells were extracted for 0, 1, 2, and 498
24 hours. Light treatments were illuminated with >530 nm light (300 watt halogen 499
projector bulb with Edmund Optics >530nm long pass filter, average irradiance 180 μmol 500
quanta m
-2
s
-1
). Samples were placed in a water bath and maintained at 20˚C during 501
illumination. Dark treatments were covered in aluminum foil and treated the same as 502
Light treatments. After determined extraction time passes, samples were centrifuged and 503
the supernatant pipetted into clean 15 ml centrifuge tubes and stored at -20˚C till LCMS 504
analysis. After 2 hours of illumination, the light and dark 24 hour samples were placed in 505
a freezer and filters were allowed to continue to extract. All samples were run after 24 506
hours and retinal oxime quantified using a 5 point calibration curve. Results (Figure S5) 507
do not indicate a significant difference between light and dark treatments after 24 hours 508
of extraction (t-Test: p-value=0.9) or between Time0 and Time24 (t-Test: p-value=0.1). 509
Thus it was concluded not to include the illumination step in the extraction protocol. 510
10.4 Hexane Extraction 511
The retinal oxime standard is prepared by adding hydroxylamine (final 512
concentration ~30 mM) to a methanol solution of all-trans retinal (see Standard 513
Preparation and Evaluation). Hydroxylamine is added in excess to ensure complete 514
conversion of all-trans retinal to retinal oxime (Figure S1). For LCMS tuning to retinal 515
oxime, the excess hydroxylamine must be removed from the methanol retinal oxime 516
solution. This is achieved using a hexane extraction step (Kane et al., 2008). A test was 517
Page 29 of 58

performed to explore if this liquid to liquid hexane extraction could be utilized in the 518
LCMS retinal quantification method to 1) prevent injection of excess hydroxylamine 519
(mM concentration) onto the column and 2) as a potential concentration step, which 520
would reduce the seawater filtration volume requirements. 521
Two separate tests were performed in triplicate. A retinal oxime standard was 522
prepared by adding hydroxylamine to a solution of all-trans retinal dissolved in methanol. 523
From this retinal oxime standard, a retinal oxime calibration curve was prepared (curve 524
ranged from approximately 23 nM to 150 nM). An aliquot (3 ml) of the highest retinal 525
oxime standard was extracted multiple times with hexane. 6 ml of hexane was added to 526
the 3 ml solution of retinal oxime standard. The mixture was shaken for 2 minutes then 527
centrifuged to facilitate phase separation. The hexane was pipetted off the retinal oxime 528
standard solution and collected in a 15 ml centrifuge tube. The hexane extraction and 529
collection was repeated three more times with 4 ml, 2 ml, and 2 ml of hexane. The 530
hexane was then driven off under at stream of nitrogen gas. Once dried, 3 ml of 531
methanol was added to the centrifuge tube and the tube was Vortexed for 30 seconds. 532
The methanol was pipetted into a sample vial and analyzed using the LCMS retinal 533
quantification method. Results are reported in Table S4. 534
For both tests, the hexane extraction treatment values were approximately half of 535
what the retinal oxime standards were (Table S6). This indicates the repeated hexane 536
extraction only removed approximately half of the retinal oxime from the methanol 537
solution. As a result, the hexane extraction step was not included in the LCMS retinal 538
quantification method. 539
Page 30 of 58

10.5 Appendix Figures and Figure Legends 540
541
Figure S1. Absorbance spectrum of retinal and retinal oxime collected over an 8 hour 542
period. The retinal oxime absorbance peak is 360 nm. The retinal absorbance peak is 543
380 nm. 544
545
Page 31 of 58

546
Figure S2. Comparison of chromatographs of all-trans retinal, retinal oxime, and AND4- 547
WT sample extracts. A) All-trans retinal standard and AND4-WT methanol extract. B) 548
Retinal oxime standard and AND4-WT methanol extract. 549
550
Page 32 of 58

551
Figure S3 Results used to determine optimal cell extraction method. A) Results from 552
cells extracted in methanol and cells sonicated and extracted in methanol. B) 553
Concentration of retinal measured from cells sonicated and extracted for various lengths 554
of time. 555
556
Page 33 of 58

557
Figure S4. Concentration of retinal in AND4-WT samples. Linear increase in 558
concentration with increase in the volume of culture sampled. 559
560
Page 34 of 58

561
Figure S5. Extraction of free and covalently bound retinal with hydroxylamine. Test 562
evaluated light and dark extraction treatments. 563
564
Page 35 of 58

565
Figure S6. Liquid to liquid hexane extraction tests. The Standard sample is a retinal 566
oxime standard. The Hex.Ext. sample is the retinal oxime standard extracted in hexane, 567
dried, and resuspended in methanol. A) Test01 conducted in triplicate. B) Test02 568
conducted in triplicate. 569
570
Page 36 of 58

10.6 Appendix Tables 571
Table S1. Results of sonication versus methanol soak extraction treatment test and 572
extraction time optimization test. 573
Test Sample Retinal Free Retinal molecules cell
-1
Bacterial cell ml
-1
Soak vs Sonicate Soak-T0 19.3 ± 2.2 nM 6,500 ± 750 4.52E+09
Soak vs Sonicate Soak-T24 27.8 ± 1.8 nM 9,300 ± 600 4.52E+09
Soak vs Sonicate Sonicate-T0 25.2 ± 0.9 nM 8,400 ± 300 4.52E+09
Soak vs Sonicate Sonicate-T24 30.2 ± 2.1 nM 10,100 ± 700 4.52E+09
Extraction Time Hour00 41.1 ± 7.0 5,000 ± 250 4.70E+09
Extraction Time Hour12 50.5 ± 6.9 6,100 ± 200 4.70E+09
Extraction Time Hour24 63.2 ± 8.7 8,200 ± 460 4.70E+09
Extraction Time Hour48 67.2 ± 3.7 8,400 ± 500 4.70E+09
574
575
Page 37 of 58

Table S2. Results of retinal quantification by LCMS reproducibility test. 576
Sample Retinal Free Retinal molecules cell
-1
Bacterial cell ml
-1
2ml 23.8 ± 2.5 nM 6,500 ± 700 3.32E+09
4ml 42.7 ± 2.0 nM 5,800 ± 250 3.32E+09
6ml 62.0 ± 4.0 nM 5,600 ± 350 3.32E+09
8ml 85.9 ± 2.5 nM 5,800 ± 150 3.32E+09
10ml 93.7 ± 3.1 nM 5,100 ± 150 3.32E+09
577
578
Page 38 of 58

Table S3. Results of test comparing hydroxylamine extraction in the light and in the 579
dark. 580
Sample Retinal Free Retinal molecules cell
-1
Bacterial cell ml
-1
Hour00 22.7 ± 2.9 nM 5,600 ± 700 2.46E+09
Hour01-Light 25.2 ± 3.0 nM 6,299 ± 750 2.46E+09
Hour01-Dark 20.5 ± 2.7 nM 5,000 ± 650 2.46E+09
Hour02-Light 29.9 ± 1.3 nM 7,300 ± 300 2.46E+09
Hour02-Dark 24.4 ± 1.7 nM 6,000 ± 400 2.46E+09
Hour24-Light 19.5 ± 5.8 nM 7,200 ± 1,400 2.46E+09
Hour24-Dark 29.7 ± 5.8 nM 7,300 ± 1,400 2.46E+09
581
582
Page 39 of 58

Table S4. Results of the liquid to liquid hexane extraction test. Each test was performed 583
in triplicate. 584
Sample Retinal oxime
Test01-Standard 143.9 nM
Test01-Extraction 68.0 ± 5.3 nM
Test02-Standard 167.0 nM
Test02-Extraction 75.2 ± 7.1 nM
585
586
Page 40 of 58

Table S5. All test data used for LCMS retinal quantification method development. 587
Test Sample Retinal Retinal oxime Free Retinal molecules cell
-1
Total Retinal cell
-1
Bacterial cell ml
-1
PR cell
-1
*
Culture01 AND4-WT 22.1 ± 2.2 nM 61.2 ± 4.8 nM 5,400 ± 550 15,000 ± 1,200 2.46E+09 9,600 ± 1,300
Culture01 AND4-Δprd 2.9 ± 0.2 nM 2.3 ± 0.7 nM 400 ± 50 300 ± 100 4.21E+09 -
Culture02 AND4-WT 27.3 ± 2.6 nM 80.8 ± 3.9 nM 7,400 ± 700 22,000 ± 1,000 2.22E+09 14,500 ± 1,300
Transect Shore Station 37.0 ± 1.0 pM 49.9 ± 3.8 pM 8,100 ± 300 11,000 ± 800 2.74E+06 2,800 ± 900
Transect Tran01 Sta01 16.1 ± 0.9 pM - 4,400 ± 250 - 2.22E+06 -
Transect Tran01 Sta02 14.1 ± 1.3 pM - 3,900 ± 350 - 2.16E+06 -
Transect Tran01 Sta03 10.2 ± 1.1 pM - 6,000 ± 600 - 1.02E+06 -
Transect Tran01 Sta04 14.9 ± 0.9 pM - 9,700 ± 600 - 9.25E+05 -
Transect Tran01 Sta05 15.8 ± 0.3 pM - 8,400 ± 150 - 1.14E+06 -
Transect Tran02 Sta01 14.0 ± 1.3 pM - 2,700 ± 250 - 2.15E+06 -
Transect Tran02 Sta01 10.8 ± 1.5 pM - 3,800 ± 500 - 1.36E+06 -
Transect Tran02 Sta01 8.5 ± 1.2 pM - 3,200 ± 450 - 1.14E+06 -
Transect Tran02 Sta01 12.0 ± 1.0 pM - 6,700 ± 530 - 1.05E+06 -
Transect Tran02 Sta01 7.6 ± 0.9 pM - 3,500 ± 400 - 7.76E+05 -
Diel Study Diel01 Event01 5.2 ± 0.6 pM - 1,500 ± 50 - 2.15E+06 -
Diel Study Diel01 Event02 6.2 ± 1.3 pM - 4,900 ± 1,700 - 7.76E+05 -
Diel Study Diel01 Event03 6.6 ± 1.0 pM - 2,000 ± 300 - 1.98E+06 -
Diel Study Diel01 Event04 9.1 ± 0.1 pM - 3,200 ± 1,100 - 1.43E+06 -
Diel Study Diel01 Event05 11.9 ± 0.1 pM - 5,100 ± 1,700 - 1.17E+06 -
Diel Study Diel01 Event06 12.1 ± 0.2 pM - 3,700 ± 1,400 - 1.60E+06 -
Diel Study Diel02 Event01 5.4 ± 0.4 pM - 3,100 ± 250 - 1.04E+06 -
Diel Study Diel02 Event02 4.4 ± 0.2 pM - 2,500 ± 100 - 1.03E+06 -
Diel Study Diel02 Event03 5.8 ± 0.6 pM - 2,800 ± 300 - 1.25E+06 -
Diel Study Diel02 Event04 5.0 ± 0.2 pM - 2,523 ± 50 - 1.20E+06 -
Diel Study Diel02 Event05 6.4 ± 0.9 pM - 3,400 ± 450 - 1.14E+06 -
Diel Study Diel02 Event06 6.4 ± 0.7 pM - 3,800 ± 400 - 1.03E+06 -
Light Extraction Hour00 22.7 ± 2.9 nM - 5,600 ± 700 - 2.46E+09 -
Light Extraction Hour01-Light 25.2 ± 3.0 nM - 6,299 ± 750 - 2.46E+09 -
Light Extraction Hour01-Dark 20.5 ± 2.7 nM - 5,000 ± 650 - 2.46E+09 -
Light Extraction Hour02-Light 29.9 ± 1.3 nM - 7,300 ± 300 - 2.46E+09 -
Light Extraction Hour02-Dark 24.4 ± 1.7 nM - 6,000 ± 400 - 2.46E+09 -
Light Extraction Hour24-Light 19.5 ± 5.8 nM - 7,200 ± 1,400 - 2.46E+09 -
Light Extraction Hour24-Dark 29.7 ± 5.8 nM - 7,300 ± 1,400 - 2.46E+09 -
Soak vs Sonicate Soak-T0 19.3 ± 2.2 nM - 6,500 ± 750 - 4.52E+09 -
Soak vs Sonicate Soak-T24 27.8 ± 1.8 nM - 9,300 ± 600 - 4.52E+09 -
Soak vs Sonicate Sonicate-T0 25.2 ± 0.9 nM - 8,400 ± 300 - 4.52E+09 -
Soak vs Sonicate Sonicate-T24 30.2 ± 2.1 nM - 10,100 ± 700 - 4.52E+09 -
Extraction Time Hour00 41.1 ± 7.0 - 5,000 ± 250 - 4.70E+09 -
Extraction Time Hour12 50.5 ± 6.9 - 6,100 ± 200 - 4.70E+09 -
Extraction Time Hour24 63.2 ± 8.7 - 8,200 ± 460 - 4.70E+09 -
Extraction Time Hour48 67.2 ± 3.7 - 8,400 ± 500 - 4.70E+09 -
Reproducibility 2ml 23.8 ± 2.5 nM - 6,500 ± 700 - 3.32E+09 -
Reproducibility 4ml 42.7 ± 2.0 nM - 5,800 ± 250 - 3.32E+09 -
Reproducibility 6ml 62.0 ± 4.0 nM - 5,600 ± 350 - 3.32E+09 -
Reproducibility 8ml 85.9 ± 2.5 nM - 5,800 ± 150 - 3.32E+09 -
Reproducibility 10ml 93.7 ± 3.1 nM - 5,100 ± 150 - 3.32E+09 -
Hexane Extraction Test01-Standard - 143.9 nM - - - -
Hexane Extraction Test01-Extraction - 68.0 ± 5.3 nM - - - -
Hexane Extraction Test02-Standard - 167.0 nM - - - -
Hexane Extraction Test02-Extraction - 75.2 ± 7.1 nM - - - -
*PR cell-1 is determined by the difference between total retinal and free retinal. One retinal molecule per PR complex.
588
589
Page 41 of 58

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Abstract (if available)

Abstract It is well known that proteorhodopsins (PRs) are present in microbial populations throughout the global oceans. However, no method exists for the routine quantification of proteorhodopsins in marine microbial populations. As a result, basic questions remain as to the function and importance of PRs in marine biogeochemical cycles. To better understand the roles of PRs marine systems, we developed a LCMS method for the quantification of the PR photochrome retinal. Tests on cultures of the representative, PR containing, marine Vibrio species (AND4-WT) and a strain engineered with the PR genes removed (AND4-Δprd) indicates these organisms contain a free retinal pool and a PR-bound pool of retinal. Analysis of the 0.2 µm fraction filtered from seawater samples with this new LCMS methods indicates that these retinal pools are also present in marine communities and that the retinal concentrations can vary over relatively short temporal and spatial scales. This method is a precise measurement of free retinal and the PR-bound retinal pools, and enables the calculation of the number of PRs present in a sample population. Culture and field results from this study indicate AND4-WT cells have an average 12,300 PR molecules cell⁻¹, and seawater samples from San Pedro Basin to have an average of 3,200 PR molecules cell⁻¹. These values are consistent with previously reported values of 3,400 to 24,000 molecules cell⁻¹. Application of this method will enable routine quantification of PRs in mixed microbial assemblages and will allow investigations into PR distribution and function.

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An investigation into low current density and microbially mediated arsenic electrocoagulation kinetics

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Creator Seegers, Brian Joseph (author)

Core Title Proteorhodopsin quantification in marine bacteria by LCMS measurement of the retinal chromophore

School College of Letters, Arts and Sciences

Degree Master of Science

Degree Program Ocean Sciences

Publication Date 07/18/2013

Defense Date 06/20/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag bacteria,LCMS,marine,OAI-PMH Harvest,pigments,proteorhodopsin,retinal

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Sañudo-Wilhelmy, Sergio A. (committee chair), Hammond, Douglas E. (committee member), Kiefer, Dale A. (committee member)

Creator Email brianseegers@gmail.com,bseegers@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-292876

Unique identifier UC11287989

Identifier etd-SeegersBri-1796.pdf (filename),usctheses-c3-292876 (legacy record id)

Legacy Identifier etd-SeegersBri-1796.pdf

Dmrecord 292876

Document Type Thesis

Rights Seegers, Brian Joseph

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA