Generalized Schemes for High-Throughput Manipulation of the
Desulfovibrio vulgaris Genome
S.R. Chhabra1,7,!,‡, G. Butland2,!, ‡, D. Elias3,!, J-M. Chandonia1, O-Y Fok1,7, T. Juba3, A. 3
Gorur2, S. Allen5, C.M. Leung2, K. Keller3, S. Reveco1,7, G. Zane3, E. Semkiw3, R. 4
Prathapam2, B. Gold2, M. Singer2, M. Ouellet1,7, D. Sazakal5 , D. Jorgens2, M.N. Price1, 5
E. Witkowska5, H.R. Beller4,7, A.P. Arkin1,6,7, T.C. Hazen4,7, M.D. Biggin8, M. Auer2, 6
J.D. Wall3 and J. D. Keasling1,6,7. 7
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley,
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California2;
Biochemistry and Molecular Microbiology and Immunology Departments, University of
Missouri Columbia, Missouri3;
Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California4
Departments of Cell and Tissue Biology, University of California, San Francisco,
Departments of Chemical Engineering and Bioengineering, University of California,
Joint BioEnergy Institute, Emeryville, California7.
Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California8;
! These authors contributed equally to this work.
‡ Correspondence may be addressed to email@example.com or firstname.lastname@example.org
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Appl. Environ. Microbiol. doi:10.1128/AEM.05495-11
AEM Accepts, published online ahead of print on 9 September 2011
35 The ability to conduct advanced functional genomic studies of the thousands of
sequenced bacteria has been hampered by the lack of available tools for making high-36
throughput chromosomal manipulations in a systematic manner that can be applied across 37
diverse species. In this work, we highlight the use of synthetic biological tools to 38
assemble custom suicide vectors with reusable and interchangeable DNA “parts” to 39
facilitate chromosomal modification at designated loci. These constructs enable an array 40
of downstream applications including gene replacement and creation of gene fusions with 41
affinity purification or localization tags. We employed this approach to engineer 42
chromosomal modifications in a bacterium that has previously proven difficult to 43
manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of 44
over 700 strains. Furthermore, we demonstrate how these modifications can be used for 45
examining metabolic pathways, protein-protein interactions, and protein localization. The 46
ubiquity of suicide constructs in gene replacement throughout biology suggests that this 47
approach can be applied to engineer a broad range of species for a diverse array of 48
systems biological applications and is amenable to high-throughput implementation. 49
Keywords: Functional Genomics/ Microbial Chromosomal Engineering/ Obligate
The rate and depth of characterization of bacterial species has increased over the 63
last few years due to advances in genome sequencing technology and the application of 64
high-throughput functional genomics approaches that identify and quantify mRNA 65
transcripts, expressed proteins, and cellular metabolites. To answer important, far ranging 66
questions in functional genomics [e.g., assessments of gene essentiality or cell-wide 67
genetic interactions (epistasis) and protein-protein interactions (PPI)], experimental 68
validation will require rapid and efficient genetic engineering of the strain of interest. Of 69
the more than 1400 bacterial genomes sequenced so far (3), relatively few transposon-70
mediated knockout libraries have been reported (15, 20, 22, 24, 25, 30, 31, 34) and 71
systematic, large-scale, single-gene deletion collections exist for only E. coli K12 (5) and 72
Acinetobacter baylyi ADP1 (20). Furthermore, large-scale Tandem-Affinity-Purification 73
(TAP)-based PPI identified with proteins produced from chromosomally tagged genes 74
have been reported only for E. coli K12 (14). Genome-wide genetic interaction screening, 75
which requires an ordered gene knockout library, has also recently been reported, but was 76
only applied in E. coli (13, 43). In summary, there is currently no systematic approach 77
for making large-scale, targeted chromosomal manipulations that can be readily applied 78
across a diverse range of bacteria for functional genomics studies. 79
Here we present a scheme for high-throughput manipulation of bacterial genomes 80
that is both inexpensive and flexible due to the use of interchangeable “parts” for making 81
different kinds of chromosomal modifications, including gene deletions and tagged genes 82
for the study of PPI and protein localization and other applications (Fig. 1). Our goal was 83
to create a systematic approach for chromosomal modification that could be applied to a 84
wide range of bacteria with minimal need for methodological alteration. For this reason, 85
the direct use of phage-based recombination systems in candidate microbes of interest 86
was deemed unsuitable, as this could require species-specific additional host mutations 87
and extensive development work for individual species. Rather, we chose to leverage a 88
common theme for chromosomal modification: the use of non-replicating gene 89
modification (“suicide”) constructs. Suicide constructs have been used successfully for 90
chromosomal modification in a wide range of bacterial species and generally require only 91
host-based RecA-mediated homologous recombination (35). Our approach for high-92
throughput construction of suicide vectors was based on Sequence and Ligation 93
Independent Cloning (SLIC) (29), heretofore used for plasmid-based (rather than 94
chromosomally based) metabolic engineering and heterologous protein expression 95
We applied this approach to the sulfate-reducing bacterium (SRB) Desulfovibrio 97
vulgaris Hildenborough. D. vulgaris, which has been the subject of recent functional 98
genomics studies (7, 16-18, 32, 38). Proposed stress response models of this bacterium 99
are based on gene expression data alone and need to be complemented by other 100
experimental data types. The ability to create targeted gene deletions in a systematic 101
manner in this organism will help fill gaps in metabolic pathways and greatly assist in the 102
functional annotation of unknown genes. In addition, the ability to generate TAP- or 103
visualization-tagged genes will facilitate the development of the corresponding 104
interactome and allow the mapping of protein complex localization within the cell. Here 105
we compare the protocols for facile chromosomal engineering of D. vulgaris to achieve 106
these objectives and provide proof-of-principle data for protein complex isolation, gene 107
deletion and sub-cellular localization (Fig. 4). 108
Materials and Methods
Mutant Strain Generation. Design principles for the three schemes are shown in Fig. S1. 110
Laboratory Information Management System (LIMS) tools were developed for the 111
Gateway® and SLIC schemes as detailed in the Supplemental Material. The Gateway® 112
scheme involves generation of a library of entry vectors which serve as the source of 113
mobile DNA fragments that are directionally incorporated in destination vectors of 114
choice after the LR reaction. Entry vectors in this study were generated by directional 115
TOPO cloning of desired DNA fragments into pENTR/dTOPO (Invitrogen Inc., 116
Carlsbad, CA) and transformed in One Shot Top10 Chemically Competent E. coli 117
(Invitrogen) as per the manufacturer’s instructions. The DNA fragments for TOPO 118
cloning were generated by PCR amplification of the respective regions from genomic 119
DNA of wild-type D. vulgaris Hildenborough using a proofreading DNA polymerase, 120
Pfu Turbo, (Stratagene Inc., La Jolla, CA). All plasmid extractions were performed using 121
QIAprep Spin Miniprep Kits (Qiagen Inc., Valencia, CA). Design rules for primers used 122
in amplification of desired DNA fragments are described in the Supplemental Material. 123
Amplified targets were visualized on E-gels® (Invitrogen) before proceeding with 124
dTOPO cloning. Up to three colonies were picked from ampicillin (100 µg/ml) 125
containing LB-agar plates bearing the transformed cells for sequence verification of entry 126
vectors. Sequence-verified constructs were used in the subsequent LR recombination step 127
(as per the manufacturer’s protocol) along with custom destination vectors. The design 128
strategy for custom destination vectors is described in the Supplemental Material. 129
Sequence-verified dTOPO constructs were coupled with custom destination vectors 130
through LR recombination to generate Gateway® constructs that carried the desired DNA 131
fragments with the corresponding tag sequence appended at their 3’ ends. Products of the 132
LR recombination reaction were transformed into One Shot TOP10 chemically 133
competent E. coli and plated on ampicillin (100 µg/ml) and kanamycin (50 µg/ml) 134
containing LB-agar plates. Two colonies were randomly picked from the resulting plates 135
for sequence verification of the Gateway® constructs. Sequence verified suicide 136
constructs were transformed in competent D. vulgaris cells for chromosomal integration 137
through homologous recombination as described below. 138
The λ Red recombination system has been utilized to perform both chromosomal 139
and plasmid modification in E. coli (19, 41, 48). Here we utilized E. coli strain SW105, 140
which expresses λ Red recombination functions when subjected to heat-shock, to enable 141
the recombination of a PCR product into plasmids carrying fragments of D. vulgaris 142
genomic DNA (45). For our desired application, this PCR product encoded a SPA tag and 143
a kanamycin-resistance gene, and was identical to that used to create chromosomal SPA-144
tagged genes in E. coli (51). Five initial D. vulgaris target genes present on an 8.7-kb 145
fragment spanning the entire apsBA-qmoABC-DVU0857 region were provided on 146
pMO9034 (J. Wall, U. Missouri) which is a derivative of pCR8/GW/TOPO (50). A 147
further 18 D. vulgaris genes were provided on three genomic DNA fragments each 148
cloned into pUC19 and designated pDVH2-36, pDVH2-37, pDVH2-39. Details on the 149
PCR products employed and subsequent transformation in E. coli are described in the 150
Supplemental Material. Transformed cells were plated onto selective LB medium 151
containing kanamycin and colonies isolated. Isolates were then cultured, plasmid DNA 152
prepared and subjected to restriction analysis to confirm the integration of the PCR 153
product at the correct locus in the plasmid target construct. 154
The SLIC technique was employed as described previously (29). Specifically 155
four DNA fragments were pieced together to form the final suicide constructs. The same 156
Inserted Part (IP, composed of the tag and one selection marker) and Selection Part (SP, 157
composed of a plasmid origin of replication and a second selection marker) were used for 158
a given library of insertion or deletion suicide constructs. The other two variable regions 159
were generated by PCR amplification of the respective regions from genomic DNA of 160
wild-type D. vulgaris Hildenborough using a proofreading DNA polymerase, Phusion 161
High Fidelity DNA Polymerase (Finnzymes Inc., Woburn, MA). Design rules for primer 162
design are detailed in the Supplemental Material. Sizes of amplified PCR products were 163
verified using agarose gels and confirmed products were purified using the QIAquick® 164
PCR Purification Kit (Qiagen) and subsequently quantified using a NanoDrop 2000 165
(NanoDrop products, Wilmington, DE). 1 μg of each part was treated with 1μl of 0.5 U 166
T4 DNA polymerase in 1 x Buffer 2 (NEB) and 1 X BSA Buffer (NEB) in a 20 μl 167
reaction at room temperature for 30 minutes. The reaction was stopped by adding 1/10 168
volume of 10 mM dCTP followed by an annealing step of all four parts at 37ºC for 30 169
minutes. The annealed mixture was next chemically transformed in DH10B competent 170
cells and plated on agar plates bearing spectinomycin and kanamycin (both at 50 μg/ml) 171
antibiotic resistance markers. Two colonies were picked after an overnight incubation 172
(37ºC) for sequence verification of suicide constructs. 173
Transformation of D. vulgaris strains. For transformations, cells were grown in 174
MOYLS4 medium in an anaerobic growth chamber (Coy Laboratory Products, Inc., 175
Grass Lake, MI) with an atmosphere composed of nitrogen, <5 % hydrogen, and <2 % 176
oxygen at 33°C. MOYLS4 medium contained 8 mM magnesium chloride, 20 mM 177
ammonium chloride, 0.6 mM calcium chloride, 1 mM potassium phosphate (dibasic), 1 178
mM sodium phosphate (monobasic), 0.06 mM ferrous chloride, 0.12 mM EDTA, 30 mM 179
TRIS (pH7.4), 60 mM sodium lactate, 30 mM sodium sulfate, 1 ml/L Thauer's vitamin 180
solution (12), 6 ml/L trace element solution and 1.0 g/L yeast extract. The trace element 181
solution was modified by addition of 2.5 mM manganese chloride, 1.26 mM cobaltous 182
chloride, 35 uM sodium selenite, and 24 uM sodium tungstate. MOYLS4 was adjusted to 183
pH7.2 with 12M HCl and 1.2 mM sodium thioglycolate was added as a reductant prior to 184
sterilization in the autoclave. For plates, 15 g/L agar was added to MOYLS4 before 185
sterilization. Immediately prior to pouring plates of molten MOYLS4 0.38 mM titanium 186
citrate, prepared and stored anaerobically, was added along with antibiotics as 187
appropriate. To prepare D. vulgaris cells for transformation, 1.0 ml of a freezer stock 188
(early stationary-phase cells in 10% vol glycerol /vol MOYLS4) was added to 10 ml 189
MOYLS4 and allowed to grow overnight. The 10-ml overnight culture was diluted to 190
100 ml in MOYLS4 and allowed to grow to an OD600 between 0.3 and 0.7 at 33 oC. The 191
culture was harvested by centrifugation for 12 min at 3,000 x g at 4 oC, supernatant was 192
discarded, and the cells resuspended in 50 ml of chilled, sterile wash buffer (30 mM Tris- 193
HCl buffer, pH 7.2, non-degassed). A second centrifugation, under the same conditions, 194
was used to wash the cells and the supernatant discarded. The pellet of electrocompetant 195
cells was resuspended in 1.0 ml wash buffer and a 100-µl aliquot was used for 196
electroporation. About 1 μg (10 μl) of the plasmid was added to the cells, mixed, and 197
100 μl of the mixture transferred to a 1-mm gapped electroporation cuvette (Molecular 198
BioProducts, San Diego, CA). The cuvette was transferred to the ECM 630 199
electroporator (BTX, Holliston, MA) and electroporated at 1500V, 250Ω, and 25µF. The 200
electroporated cells were diluted into 1 ml MOYLS4 medium containing 0.1% wt/vol 201
yeast extract. The putative transformants were transferred to the anaerobic chamber, 202
opened momentarily for headspace exchange, and allowed to recover overnight at 33 oC. 203
Aliquots (100 μl and 900 μl) of the recovered cells were then added to Petri plates, 204
followed immediately by ~25 ml of reduced, molten MOYLS4 with 15 g/L agar 205
containing the kanamycin analogue G418 (RPI corp., Mt. Prospect, IL; 400 µg/ml) in the 206
anaerobic chamber. Two reductants were used. Sodium thioglycolate (1.2 mM) was 207
added prior to autoclaving, while titanium citrate (1.2 mM, prepared under nitrogen and 208
stored anaerobically) was added just prior to pouring the medium over the cells in the 209
Petri plates. Colonies were typically observed after 5 d incubation. Selection and storage 210
of mutants as well as protocols for Southern blot and IP western analysis to confirm 211
chromosomal manipulations are described in the Supplemental Material. 212
Protein-Protein Interactions using Tandem Affinity Purification. 213
Affinity-tagged D. vulgaris strains were cultured in 2 x 1L LS4D medium (7) in 214
stationary bottles present in an anaerobic chamber. Sequential peptide affinity 215
purification was performed using tagged D. vulgaris strains exactly as previously 216
reported for E. coli (51). The alternative Streptavidin-TEV-FLAG (STF) tag purifications 217
were performed using the SPA protocol (14) with the following modifications. The STF 218
purification is identical to the SPA procedure until immediately following TEV 219
incubation and removal of tagged proteins from the anti-FLAG beads. For STF 220
purification, after the TEV eluate was drained into a new column, 100 µl 50 % slurry 221
Streptactin Superflow beads (IBA GmbH) were added and incubated for 3 hours with 222
rotation at 4 °C (17). The beads were then washed with 1.4 ml wash buffer I (100 mM 223
Tris-Cl pH7.9, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 10 mM 2-224
mercaptoethanol, Roche Complete protease inhibitor), followed by 400 µl of the wash 225
buffer without the Triton X-100, and proteins eluted using 300 µl of elution buffer 226
containing 2.5 mM desthiobiotin (Sigma). Purified eluates were subsequently digested 227
with trypsin and analyzed mass spectrometry to obtain protein identifications as detailed 228
in the Supplemental Material. 229
Growth studies on knockout mutants for examining methionine biosynthesis. Growth 230
study manipulations were done in an anaerobic growth chamber (Coy Laboratory 231
Products, Inc.) which contained approximately 95% N2 and 5% H2. D. vulgaris wild-type 232
and mutant strains were grown at 37ºC from freezer stocks to early stationary phase in 5 233
ml MO medium supplemented with 60 mM lactate, 30 mM sulfate, and 0.1% (wt./vol.) 234
yeast extract (MOYLS4(60/30)). DVU0171 and DVU1585 gene deletion mutants were 235
grown in MO medium supplemented with 60 mM lactate, 30 mM sulfate, 40 mM sulfite, 236
and 0.2% (wt./vol.) yeast extract. To select for growth of the mutant strains, the media 237
were supplemented with G418 antibiotic at 400 μg/ml. To impose starvation conditions, 238
two consecutive triplicate 2% subcultures were then grown in 5mL defined medium 239
supplemented with 60 mM lactate and 30mM sulfate [MOLS4(60/30)] (50) plus G418 at 240
400 μg/ml for mutant selection. As the DVU0890 deletion mutant exhibited little or no 241
growth in subcultures, screening of the DVU0890 deletion mutant was performed without 242
first subculturing in defined medium. To screen for auxotrophic mutant strains, triplicate 243
15 mL culture tubes containing 5 mL MOLS4(60/30) medium amended with 0.3 mM 244
amino acids were inoculated with 5% (vol./vol.) of the strains and sealed with rubber 245
stoppers. Controls were prepared by inoculation of MOLS4(60/30) and MOYLS4(60/30) 246
media. For comparison of growth, ODs were monitored at 600 nm and final total protein 247
concentrations were measured using the Bradford Assay (9). LC-glucose plates were 248
streaked to check for aerobic contamination of the initial growth culture and periodically 249
of the subcultures. Gene deletion mutations were verified by PCR prior to and again 250
upon completion of the growth studies. 251
Labeling of AGT-Tagged Proteins with SNAP Fluorophore. 252
The AGT tag or the SNAP-tag (New England Biolabs, Ipswich, MA) is a highly 253
engineered modified version of AGT (alkylguanine DNA alkyltransferase), a human 254
DNA repair protein with a molecular weight of 20 kD. It is a visualization tag similar to 255
green fluorescent protein but unlike the latter has been shown to work effectively under 256
anaerobic conditions. It forms a highly stable, covalent thioether bond with fluorophores 257
or other substituted groups when appended to benzylguanine. This reaction is highly 258
specific, i.e., expressed SNAP-tag fusion proteins can be labeled even in the presence of 259
complex protein mixtures such as found in cells or in cleared bacterial lysates. 260
Anaerobically grown cells cultures were harvested at mid-logarithmic phase with an 261
optical density at 600 nm [OD600] of 0.3 to 0.4 and centrifuged under anaerobic 262
conditions at room temperature for 10 minutes at 5000 x g. The pellet was resuspended 263
under anaerobic conditions in sterile LS4D medium in the range of 400 μl to 600 μl at 264
OD600 of 0.5 (+/- 0.05) in order adjust each sample to the same relative cell density, as 265
determined by initial optical density readings. The SNAP fluorophore reagent (New 266
England Biolabs, Ipswich, MA) in DMSO was added to the cell suspensions to reach a 267
final reagent concentration of 5 μM. Cell solutions with the fluorophore reagent were 268
protected from light and incubated at 30° C for at least 60 minutes to ensure all cells were 269
exposed to the labeling reagent. Following the incubation period, the cells were 270
centrifuged at room temperature twice at 5000 x g for 10 minutes and resuspended in 271
sterile LS4D medium to remove any excess fluorophore reagent from the cells. 272
SDS-PAGE and In-Gel Fluorescence Detection. Labeled and washed cells were 273
subjected to Pefabloc protease (Sigma-Aldrich Co., St. Louis, MO) and DNase (Sigma-274
Aldrich Co., St. Louis, MO) treatment, at a concentration of 400 μM and 0.2 mg/mL, 275
respectively and stored on ice. Ice-cold cell suspensions were lyzed using a Branson 276
sonicator (Branson Ultrasonics, Danbury, CT) for 1-2 minutes at 40% duty cycle with 277
output of 3. After lysis, the samples were flash frozen in liquid nitrogen and stored at -278
20˚C. For SDS PAGE analysis, samples were mixed with 5X SDS loading buffer stock 279
and boiled for 1 minute prior to loading into the pockets of a precast 4-20% Tris-HCL 280
SDS-PAGE well (Thermo Fisher Scientific Inc., Pittsburgh, PA) and run at 140 V for 281
approximately 50 minutes. A Pageruler prestained protein marker (Fermentas Inc., Glen 282
Burnie, MD) was run on the gels, as this marker has a 72 kD protein which emits a 283
fluorescent signal at 488 nm excitation, and therefore allowed easy correlation of in-gel 284
fluorescence detection and Coomassie-stained gels. The gels were imaged for 285
fluorescence using a Bio-Rad Molecular phosphor-imager with an external laser source 286
using Alexa 488 filter settings (Bio-Rad Laboratories, Hercules, CA), followed by 287
Coomassie staining (0.5% Coomassie Brilliant Blue R-250, 30% ethanol, 10% glacial 288
acetic acid in ddH20), and destaining in 30% ethanol, 10% glacial acetic acid in ddH20 289
before imaging on a lightbox with a Canon A540 digital camera. For selected samples, 290
both intact cells as well as cell lysates were labeled and their in-gel fluorescence values 291
were compared to ensure that the labeling reagent had ready in-vivo access to all tags. 292
Details on cellular imaging using epifluorescence and deconvolution microscopy are 293
provided in the Supplemental Material. 294
Results and Discussion
Development and application of schemes for high-throughput generation of suicide
Traditional mutagenesis approaches with suicide constructs have generally been regarded 299
as cumbersome due to difficulties with vector construction (i.e., cloning of large sections 300
of homologous DNA from either side of the locus to be modified) (35). We therefore 301
tested three recombination based approaches – Gateway® (Invitrogen, Carlsbad, CA), 302
‘Recombineering’ (19, 47) and Sequence and Ligation Independent Cloning (SLIC) (29), 303
for the generation of suicide constructs in a high-throughput manner (Fig. S1). In all 304
examples, suicide vectors designed for modifying the D. vulgaris host chromosome were 305
first generated in E. coli and then transformed into D. vulgaris resulting in a modified 306
host chromosome through single or double homologous recombination events integrating 307
all or part of the non-replicating delivery vector. 308
Suicide vector construction via the Gateway® scheme was realized through two 309
steps (Fig. S1-A). In the first step, the sole homology region of the target locus was 310
directionally cloned into a TOPO® entry vector. The second step involved a LR 311
recombination reaction with directional placement of the homology region from the entry 312
clone to a custom-designed destination vector. The destination vector included the 313
sequences for modification of the host cell and a suitable origin of replication. 314
Destination vectors with different insertion sequences such as TAP tags for elucidation of 315
PPI or visualization tags such as SNAP (O6-alkylguanine-DNA alkyltransferase, New 316
England Biolabs, Ipswich, MA) that allow protein localization may be used with a given 317
library of entry clones. This powerful attribute of the Gateway® scheme allows facile 318
exchange of the tags once a library of TOPO® entry clones has been constructed. 319
Importantly, the introduction of a single region of homologous DNA in the 320
construction of the entry clones allows only a single recombination event with the host 321
chromosome that incorporates the entire plasmid. When creating tagging constructs for 322
genes located at the beginning of their operons, we incorporated the native promoter 323
sequence in addition to the target gene to be modified in the suicide vector to allow the 324
expression of downstream genes. Necessary promoter sequences for each gene were 325
assumed to be present within 300 bp upstream of the target gene. From a practical 326
standpoint, this scheme is therefore limited to genes located at terminal ends of their 327
respective operons where downstream polarity effects can be minimized. These caveats 328
render the Gateway scheme useful for rapid modification of a select class of target genes 329
with a range of fusion tags. In order to be able to modify genes in a locus-independent 330
manner, however, we leveraged two other schemes, ‘Recombineering’ and SLIC, to 331
generate suicide vectors with two homology regions that permitted marker exchange. 332
In the ‘Recombineering’ approach, we utilized the bacteriophage lambda general 333
recombination system (λred) (19) to modify genes carried on recombinant plasmids 334
selected from an ordered genomic library of D. vulgaris. Expression of λred in E. coli has 335
been shown to mediate efficient integration of linear DNA molecules into the host 336
chromosome or plasmids through short regions (~40 bp) of sequence homology (47). In 337
this scheme (Fig. S1-B), a linear DNA molecule was generated by PCR that contained 338
homology regions 1 and 2 (HR1/HR2) flanking the marker to be exchanged or the part to 339
be inserted (insertion part, IP). In the example shown, the IP is an affinity purification tag 340
and a kanamycin-resistance cassette expressed from its own promoter. Plasmid constructs 341
and an HR1-IP-HR2-containing PCR product were transformed together into an E. coli 342
strain in which the λred system was induced. The λred recombinase, facilitates 343
recombination between the short 40bp homology regions of the target loci flanking the IP 344
and identical regions in the plasmid containing the fragment of chromosomal DNA with 345
the genes to be modified. The length of the chromosomal regions of homology available 346
for double crossover events between modified plasmid constructs and the host 347
chromosome varies with the location of the target gene within the genomic DNA insert in 348
the suicide vector but is generally sufficient for detectable recombination with the 349
genome being modified, with the exception of genes located at the termini of the insert 350
DNA in the suicide vector. During the development of this modification procedure it was 351
noted that many isolated strains contained both modified plasmid, into which the IP 352
fragment had integrated, and unmodified plasmid. Furthermore, when higher plasmid 353
concentrations were used, multimeric plasmids were often isolated, a phenomenon 354
previously reported when using plasmids with the λred system (40). These factors led to 355
increased processing requirements to generate a pure modified plasmid construct. 356
Therefore, while suicide constructs made by this approach can be used for inserting or 357
deleting sequences through marker exchange by double crossovers, ready availability of a 358
comprehensive ordered genomic library is an essential prerequisite. Due to the lack of 359
such a comprehensive set of library constructs for D. vulgaris, and the inefficiencies of 360
isolating recombineered plasmid constructs (compared to SLIC) this approach was not 361
considered further in this study. 362
The third approach involves de novo assembly of suicide vectors by the SLIC 363
procedure (Li and Elledge, 2007). Vectors assembled by this technique are composed of 364
four parts – two corresponding to the homology regions (HR1/HR2) from the host 365
chromosome, an insertion part (IP) dictated by the application of choice, and a vegetative 366
origin of replication and selection part (RSP). To obtain a suicide vector, the replication 367
origin was functional only in E. coli but not in the strain targeted for manipulation ((44); 368
Fig. S1-C). The advantage of this approach lies in the reusability of parts for various 369
applications. The IP and RSP regions remain constant for each specific application; 370
whereas, HR1 and HR2 regions vary. Alternative IP regions such as molecular barcodes, 371
purification tags, antibiotic markers, origins of replication are incorporated into vector 372
construction depending on the downstream application. The RSP region is the most 373
generic part used for suicide vector construction. However modification of the RSP to 374
include an oriT (origin of transfer) sequence is possible if the suicide vectors are to be 375
transferred by conjugation from E. coli to the target microbe. 376
Chromosome modifications by suicide vectors may be characterized as ‘marked’ 377
or ‘unmarked’ depending on the presence or absence of suitable selection markers in the 378
host chromosome after the modifications have been introduced. In either case, 379
chromosomal modifications at the 3’ end of a target gene may alter expression or 380
translation of co-operonic downstream genes. In case of the ‘marked’ approach, 381
incorporation of a selection marker and its cognate promoter may introduce a second 382
transcription initiation site. For genes located in close proximity to each other, one must 383
also consider the possibility of a displaced ribosomal binding site (RBS). The SLIC 384
approach, through appropriate design, may be used to correct these problems for both 385
marked and unmarked approaches. Systems for unmarked modifications such as the Cre-386
lox recombination system (6, 27) or the levansucrase-dependent sucrose sensitivity (11), 387
could easily be implemented by incorporation of the respective parts (loxP sites and 388
sacB) into IP or SP regions of SLIC generated suicide vectors. In hosts where sucrose 389
sensitivity is not a strong selection or a residual “scar” is not desired, alternative counter-390
selection systems are available. For D. vulgaris, sensitivity to the toxic pyrimidine 391
analog, 5-fluorouracil, allows selection against the expression of upp encoding the 392
salvage enzyme uracil phosphoribosyl transferase. This marker has been successfully 393
used in a number of microbes (26, 28). 394
We compared 550 distinct target genes for tagging with suicide vectors assembled 395
by the Gateway® and SLIC strategies and generated 297 (54%) and 468 (85%) sequence-396
verified plasmids, respectively. In general we observed that the SLIC strategy yielded a 397
higher percentage of confirmed D. vulgaris mutant strains (304 strains/468 plasmids 398
constructed; ~65%) as compared to the Gateway® scheme (70 strains/ 297 plasmids 399
constructed; ~24%). For the ‘Recombineering’ strategy we generated 18 sample suicide 400
vectors, which resulted in 9 confirmed mutant strains. Given the apparent superiority of 401
the SLIC approach, it was the method of choice for further manipulation of the D. 402
vulgaris chromosome to study effects of gene deletions, to identify physical interactions 403
of proteins, and to localize selected tagged proteins. To date we have generated a library 404
of over 700-engineered strains of D. vulgaris using the methodologies described in this 405
Screening for protein-protein interactions by tandem affinity purification. 407
With plasmids constructed by the SLIC procedure, we introduced sequences into the 408
chromosome-encoding TAP tags in-frame at the 3’ end of several genes from D. vulgaris. 409
Engineered strains expressing native levels of C-terminally TAP-tagged fusion proteins 410
were used to examine the protein complexes isolated with the tagged baits inferred to 411
represent functional PPI. We validated conserved interactions in several essential 412
complexes, such as the F1-ATPase the RNA polymerase, the chaperone DnaK and others 413
(Fig. 2A, Table S1). 414
Next, we examined potential interacting partners of proteins associated with the 415
D. vulgaris nucleoid (Fig. 2B). Well-known components of the E. coli nucleoid include 416
DNA-binding proteins such as Fis, HNS, Dps, IHF (IhfAB) and HU (HupAB). By the 417
very nature of their inherent DNA-binding capability, these highly abundant proteins are 418
involved in modulation of cellular processes such as transcriptional regulation, 419
maintenance of DNA architecture, replication, recombination and stress protection (1, 2, 420
10, 21). 421
Given the common set of functions attributed to these proteins, it is not surprising 422
that they exhibit a high level of interaction with each other. Indeed proteins precipitated 423
with TAP-tagged baits of HU and IHF from D. vulgaris suggest a closely knit interaction 424
sub-network comprising many of these DNA-binding proteins. Intriguingly the D. 425
vulgaris genome appears to lack the diversity of nucleoid protein domains reported in E. 426
coli such as Fis (COG2901), HNS (COG2916), and Dps (COG783) and their 427
corresponding interacting partners (8). In contrast, D. vulgaris encodes twice as many 428
proteins with the ‘Bacterial nucleoid DNA-binding protein’ domain, COG776, as are 429
found in E. coli (3). In order to compare the E. coli and D. vulgaris sub-networks 430
associated with COG776 family proteins, we identified interacting partners of D. vulgaris 431
tagged baits, Hup-3 and IhfB. With the exception of DVUA0004 and DVU1134, all 432
members of the COG776 family appeared to interact with the tagged baits and potentially 433
with each other (Fig. 2B, Table S2). 434
Unlike topoisomerases from E. coli, members of the D. vulgaris ‘Topoisomerase’ 435
family (TopA, TopB) did not appear to co-purify with the tagged HU proteins. This was 436
also confirmed when TopB was used as the bait and none of the COG776 family proteins 437
were observed as interacting partners. In E. coli, HU (HupAB) has been reported to 438
introduce negative supercoiling in covalently closed circular DNA in the presence of 439
topoisomerase I (TopA) (37). From these results, it appears that mechanisms of DNA 440
architecture maintenance and global regulatory controls in D. vulgaris may differ from 441
those in E. coli. 442
Gene Deletions: Examining the Methionine Biosynthesis Pathway of D. vulgaris. 443
While the genome sequence of D. vulgaris was published in 2004, several amino acid 444
biosynthesis pathways in this SRB remain to be elucidated. In this study we examined 445
putative alternative steps in methionine biosynthesis. At least 18 variant methionine 446
pathways have been proposed to originate from the common precursor – homoserine 447
(23). In examining the D. vulgaris genome for all known variant genes related to the 448
three major steps of methionine synthesis: (i) homoserine activation; (ii) sulfur 449
incorporation, and (iii) methylation, homologs corresponding only to step (iii) were 450
apparent – B12-dependent methionine synthase (DVU1585, metH) and methionine 451
synthase II (cobalamin-independent) (DVU3371, metE). We tested these and other genes 452
putatively involved in the production of the methionine precursor homoserine from L-453
aspartate. These included a putative aspartate kinase (DVU1913, lysC), homoserine 454
dehydrogenase (DVU0890, hom) (probable counterparts to bifunctional aspartate kinase 455
II/homoserine dehydrogenase from E. coli), a putative beta-cystathionase (DVU0171, 456
similar to patB (4)) and a protein with predicted methyltransferase activity (DVU3369, 457
similar to metW (20)). 458
We verified all gene deletion mutations by PCR as well as Southern blot analysis. 459
These gene deletion studies revealed that a majority of the putative methionine 460
biosynthesis pathway knockouts (DVU1585, DVU3371, DVU0890, DVU0171 and 461
DVU3369) did not result in methionine auxotrophy. A surprising result of this study was 462
that the mutant deleted for DVU0890, Δhom, was found to be auxotrophic for threonine 463
but not methionine (Fig. 3). This unexpected phenotype and the difficulty encountered in 464
isolation of a deletion of DVU1913 were interpreted to indicate that an unusual pathway 465
for methionine biosynthesis might be operational in this SRB. Further studies in this 466
direction are currently underway. 467
Protein Localization with Visualization Tags. 468
We engineered D. vulgaris strains to express proteins bearing a SNAP tag, which is 469
designed for subcellular visualization in anaerobic bacteria. Conventional Green-470
Fluorescent Protein derivatives require molecular oxygen for proper chromophore 471
formation and hence cannot be utilized under anaerobic culturing conditions. We 472
therefore explored the use of a modified SNAP tag that has a dead–end reaction with a 473
modified O6-benzylguanine (BG) derivative (33, 36). To validate the use of the AGT tag 474
based method for subcellular localization in anaerobic bacteria, we first compared SNAP 475
labeling of three AGT-tagged proteins from D. vulgaris: DsrC (DVU2776); MreB 476
(DVU0789; data not shown); FtsZ (DVU2499) from the respective engineered strains to 477
the unmodified wild-type strain. We confirmed specific labeling of tagged proteins using 478
two complementary methods: in-gel fluorescence detection SDS PAGE and fluorescence 479
microscopy. SDS PAGE analysis typically yielded single bands at the expected molecular 480
weight, indicating specific labeling of the tag, with little or no non-specific binding. 481
Interestingly, in our fluorescence micrographs we found a robust cell-to-cell variability in 482
labeling signal. To eliminate the possibility that the labeling reagent did not reach all 483
tagged proteins, we compared in-vivo labeled intact cells to in-vitro labeled whole-cell 484
extracts and observed no difference in the fluorescence signals between the two, as 485
judged by SDS PAGE gel analysis. This suggested efficient reagent access and specific 486
labeling of intracellular AGT-tagged proteins. In case of MreB and FtsZ unlike DsrC, the 487
chromosomal tagging appeared to alter the cellular morphology normally associated with 488
the wild-type strain. Morphological changes included either loss of vibrio-typic cell shape 489
(MreB-AGT; data not shown) or extensive elongation (FtsZ-AGT; Fig. S2), suggesting 490
diminished or altered protein function due to presence of the visualization tag. Our results 491
are comparable to GFP-based protein localization of FtsZ as demonstrated in E. coli (35). 492
To our knowledge this is the first account of specific tag-based fluorescence labeling for 493
the purpose of protein localization in an anaerobic bacterium. 494
Subsequently we expanded the method to fifteen additional proteins (Fig. 4). We 495
were able to decipher localization patterns for each of the fifteen SNAP-tagged proteins 496
presumably reflecting their respective biological roles in this SRB. ParA, MotA-1 and 497
MotA-3 localized exclusively to the poles, a subcellular area that has been referred to as a 498
“localization hotspot”; whereas, LytR, FtsH, FlgE and UvrB localized at the poles as well 499
as to additional regions in or towards the center of the cells. Hup-3 and PyrB showed a 500
patchy or spotty distribution along the length of the cells. The remaining proteins 501
displayed cytoplasmically uniform distribution. Orthologous counterparts of ParA and 502
FtsH from Caulobacter crescentus and E. coli have been experimentally visualized 503
previously (42, 46). For the remaining proteins only theoretical in-silico localization 504
predictions have been made to date (49). In these localization studies, we consistently 505
noted cell-to-cell variations in fluorescent signals in any given population, which may be 506
attributed to corresponding differences in expression levels (39). 507
In this work, we successfully established the use of a “parts” approach to generate 509
a library of over 700 engineered strains of the model sulfate reducer Desulfovibrio 510
vulgaris Hildenborough for advanced systems biology applications. We highlighted three 511
functional genomics tools including (a) gene deletions to study methionine biosynthesis, 512
(b) protein-protein interactions associated with chaperones and nucleoid proteins, and (c) 513
sub-cellular localization of select proteins to demonstrate the utility of our approach in 514
this SRB generally regarded as genetically intractable. One may extend the approach to 515
realize applications such as synthetic genetic arrays (13). in vivo expression profiling (39) 516
and others. The ubiquity of suicide constructs in gene replacement throughout biology 517
suggests that our approach may be applied to engineer a broad range of species for a 518
diverse array of systems biological applications and is amenable to high-throughput 519
This work received support from ENIGMA under Contract No. DE-AC02-05CH11231. 523
This work conducted at the Joint BioEnergy Institute was supported by the Office of 524
Science, Office of Biological and Environmental Research, of the U. S. Department of 525
Energy under Contract No. DE-AC02-05CH11231. We would like to thank Steven Ruzin 526
and Denise Schichnes of the Biological Imaging Facility at University of California, 527
Afflerbach, H., O. Schroder, and R. Wagner. 1998. Effects of the Escherichia
coli DNA-binding protein H-NS on rRNA synthesis in vivo. Mol Microbiol
Aki, T., and S. Adhya. 1997. Repressor induced site-specific binding of HU for
transcriptional regulation. Embo J 16:3666-3674.
Alm, E. J., K. H. Huang, M. N. Price, R. P. Koche, K. Keller, I. L. Dubchak,
and A. P. Arkin. 2005. The MicrobesOnline Web site for comparative genomics.
Genome research 15:1015-1022.
Auger, S., M. P. Gomez, A. Danchin, and I. Martin-Verstraete. 2005. The
PatB protein of Bacillus subtilis is a C-S-lyase. Biochimie 87:231-238.
Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A.
Datsenko, M. Tomita, B. L. Wanner, and H. Mori. 2006. Construction of
Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio
collection. Mol Syst Biol 2:2006 0008.
Banerjee, A., and I. Biswas. 2008. Markerless multiple-gene-deletion system for
Streptococcus mutans. Appl Environ Microbiol 74:2037-2042.
Bender, K. S., H. C. Yen, C. L. Hemme, Z. Yang, Z. He, Q. He, J. Zhou, K.
H. Huang, E. J. Alm, T. C. Hazen, A. P. Arkin, and J. D. Wall. 2007. Analysis
of a ferric uptake regulator (Fur) mutant of Desulfovibrio vulgaris Hildenborough.
Appl Environ Microbiol 73:5389-5400.
Blattner, F. R., G. Plunkett, 3rd, C. A. Bloch, N. T. Perna, V. Burland, M.
Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor,
N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y.
Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem 72:248-254.
10. Bradley, M. D., M. B. Beach, A. P. de Koning, T. S. Pratt, and R. Osuna.
2007. Effects of Fis on Escherichia coli gene expression during different growth
stages. Microbiology 153:2922-2940.
Bramucci, M. G., and V. Nagarajan. 1996. Direct selection of cloned DNA in
Bacillus subtilis based on sucrose-induced lethality. Appl Environ Microbiol
Brandis, A., and R. K. Thauer. 1981. Growth of Desulfovibrio Species on
Hydrogen and Sulfate as Sole Energy-Source. Journal of General Microbiology
Butland, G., M. Babu, J. J. Diaz-Mejia, F. Bohdana, S. Phanse, B. Gold, W.
Yang, J. Li, A. G. Gagarinova, O. Pogoutse, H. Mori, B. L. Wanner, H. Lo, J.
Wasniewski, C. Christopolous, M. Ali, P. Venn, A. Safavi-Naini, N. Sourour,
S. Caron, J. Y. Choi, L. Laigle, A. Nazarians-Armavil, A. Deshpande, S. Joe,
K. A. Datsenko, N. Yamamoto, B. J. Andrews, C. Boone, H. Ding, B. Sheikh,
G. Moreno-Hagelseib, J. F. Greenblatt, and A. Emili. 2008. eSGA: E. coli
synthetic genetic array analysis. Nat Methods 5:789-795.
Butland, G., J. M. Peregrin-Alvarez, J. Li, W. Yang, X. Yang, V. Canadien,
A. Starostine, D. Richards, B. Beattie, N. Krogan, M. Davey, J. Parkinson, J.
Greenblatt, and A. Emili. 2005. Interaction network containing conserved and
essential protein complexes in Escherichia coli. Nature 433:531-537.
Cameron, D. E., J. M. Urbach, and J. J. Mekalanos. 2008. A defined
transposon mutant library and its use in identifying motility genes in Vibrio
cholerae. Proc Natl Acad Sci U S A 105:8736-8741.
Chhabra, S. R., Q. He, K. H. Huang, S. P. Gaucher, E. J. Alm, Z. He, M. Z.
Hadi, T. C. Hazen, J. D. Wall, J. Zhou, A. P. Arkin, and A. K. Singh. 2006.
Global analysis of heat shock response in Desulfovibrio vulgaris Hildenborough.
Journal of bacteriology 188:1817-1828.
Chhabra, S. R., M. P. Joachimiak, C. J. Petzold, G. Zane, M. N. Price, B.-G.
Han, O.-Y. Fok, P. Hwu, D. Elias, S. M., J.-M. Chandonia, D. C. Joyner, T.
C. Hazen, A. Arkin, J. Wall, A. K. Singh, and J. D. Keasling. 2011. A
Network of Protein-Protein Interactions of the Model Sulfate Reducer
Desulfovibrio vulgaris Hildenborough. Submitted.
Clark, M. E., Q. He, Z. He, K. H. Huang, E. J. Alm, X. F. Wan, T. C. Hazen,
A. P. Arkin, J. D. Wall, J. Z. Zhou, and M. W. Fields. 2006. Temporal
transcriptomic analysis as Desulfovibrio vulgaris Hildenborough transitions into
stationary phase during electron donor depletion. Applied and environmental
Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of
chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad
Sci U S A 97:6640-6645.
de Berardinis, V., D. Vallenet, V. Castelli, M. Besnard, A. Pinet, C. Cruaud,
S. Samair, C. Lechaplais, G. Gyapay, C. Richez, M. Durot, A. Kreimeyer, F.
Le Fevre, V. Schachter, V. Pezo, V. Doring, C. Scarpelli, C. Medigue, G. N.
Cohen, P. Marliere, M. Salanoubat, and J. Weissenbach. 2008. A complete
collection of single-gene deletion mutants of Acinetobacter baylyi ADP1. Mol
Syst Biol 4:174.
21. Friedman, D. I. 1988. Integration host factor: a protein for all reasons. Cell
Gallagher, L. A., E. Ramage, M. A. Jacobs, R. Kaul, M. Brittnacher, and C.
Manoil. 2007. A comprehensive transposon mutant library of Francisella
novicida, a bioweapon surrogate. Proc Natl Acad Sci U S A 104:1009-1014.
Gophna, U., E. Bapteste, W. F. Doolittle, D. Biran, and E. Z. Ron. 2005.
Evolutionary plasticity of methionine biosynthesis. Gene 355:48-57.
Groh, J. L., Q. Luo, J. D. Ballard, and L. R. Krumholz. 2005. A method
adapting microarray technology for
Desulfovibrio desulfuricans G20 and Shewanella oneidensis MR-1 in anaerobic
sediment survival experiments. Applied and environmental microbiology
Jacobs, M. A., A. Alwood, I. Thaipisuttikul, D. Spencer, E. Haugen, S. Ernst,
O. Will, R. Kaul, C. Raymond, R. Levy, L. Chun-Rong, D. Guenthner, D.
Bovee, M. V. Olson, and C. Manoil. 2003. Comprehensive transposon mutant
library of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 100:14339-14344.
Keller, K. L., K. S. Bender, and J. D. Wall. 2009. Development of a Markerless
Genetic Exchange System in Desulfovibrio vulgaris Hildenborough and Its Use in
Generating a Strain with Increased Transformation Efficiency. Appl Environ
Kim, J. M., K. H. Lee, and S. Y. Lee. 2008. Development of a markerless gene
knock-out system for Mannheimia succiniciproducens using a temperature-
sensitive plasmid. FEMS Microbiol Lett 278:78-85.
Kristich, C. J., D. A. Manias, and G. M. Dunny. 2005. Development of a
method for markerless genetic exchange in Enterococcus faecalis and its use in
construction of a srtA mutant. Appl Environ Microbiol 71:5837-5849.
Li, M. Z., and S. J. Elledge. 2007. Harnessing homologous recombination in
vitro to generate recombinant DNA via SLIC. Nat Methods 4:251-256.
Liberati, N. T., J. M. Urbach, S. Miyata, D. G. Lee, E. Drenkard, G. Wu, J.
Villanueva, T. Wei, and F. M. Ausubel. 2006. An ordered, nonredundant library
of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc Natl
Acad Sci U S A 103:2833-2838.
Molina-Henares, M. A., J. de la Torre, A. Garcia-Salamanca, A. J. Molina-
Henares, M. C. Herrera, J. L. Ramos, and E. Duque. 2010. Identification of
conditionally essential genes for growth of Pseudomonas putida KT2440 on
minimal medium through the screening of a genome-wide mutant library. Environ
Mukhopadhyay, A., Z. He, E. J. Alm, A. P. Arkin, E. E. Baidoo, S. C.
Borglin, W. Chen, T. C. Hazen, Q. He, H. Y. Holman, K. Huang, R. Huang,
D. C. Joyner, N. Katz, M. Keller, P. Oeller, A. Redding, J. Sun, J. Wall, J.
Wei, Z. Yang, H. C. Yen, J. Zhou, and J. D. Keasling. 2006. Salt stress in
Desulfovibrio vulgaris Hildenborough: an integrated genomics approach. Journal
of bacteriology 188:4068-4078.
Nicolle, O., A. Rouillon, H. Guyodo, Z. Tamanai-Shacoori, F. Chandad, V.
Meuric, and M. Bonnaure-Mallet. 2010. Development of SNAP-tag-mediated
signature-tagged mutagenesis of
live cell labeling as an alternative to GFP in Porphyromonas gingivalis. FEMS
Immunol Med Microbiol 59:357-363.
Noble, S. M., S. French, L. A. Kohn, V. Chen, and A. D. Johnson. 2010.
Systematic screens of a Candida albicans homozygous deletion library decouple
morphogenetic switching and pathogenicity. Nat Genet 42:590-598.
Ortiz-Martin, I., A. P. Macho, L. Lambersten, C. Ramos, and C. R. Beuzon.
2006. Suicide vectors for antibiotic marker exchange and rapid generation of
multiple knockout mutants by allelic exchange in Gram-negative bacteria. J
Microbiol Methods 67:395-407.
Regoes, A., and A. B. Hehl. 2005. SNAP-tag mediated live cell labeling as an
alternative to GFP in anaerobic organisms. Biotechniques 39:809-810, 812.
Rouviere-Yaniv, J., M. Yaniv, and J. E. Germond. 1979. E. coli DNA binding
protein HU forms nucleosomelike structure with circular double-stranded DNA.
Stolyar, S., Q. He, M. P. Joachimiak, Z. He, Z. K. Yang, S. E. Borglin, D. C.
Joyner, K. Huang, E. Alm, T. C. Hazen, J. Zhou, J. D. Wall, A. P. Arkin, and
D. A. Stahl. 2007. Response of Desulfovibrio vulgaris to alkaline stress. Journal
of bacteriology 189:8944-8952.
Taniguchi, Y., P. J. Choi, G. W. Li, H. Chen, M. Babu, J. Hearn, A. Emili,
and X. S. Xie. 2010. Quantifying E. coli proteome and transcriptome with single-
molecule sensitivity in single cells. Science 329:533-538.
Thomason, L. C., N. Costantino, D. V. Shaw, and D. L. Court. 2007.
Multicopy plasmid modification with phage lambda Red recombineering. Plasmid
Thomason, L. C., A. B. Oppenheim, and D. L. Court. 2009. Modifying
bacteriophage lambda with recombineering. Methods Mol Biol 501:239-251.
Tomoyasu, T., K. Yamanaka, K. Murata, T. Suzaki, P. Bouloc, A. Kato, H.
Niki, S. Hiraga, and T. Ogura. 1993. Topology and subcellular localization of
FtsH protein in Escherichia coli. Journal of bacteriology 175:1352-1357.
Typas, A., R. J. Nichols, D. A. Siegele, M. Shales, S. R. Collins, B. Lim, H.
Braberg, N. Yamamoto, R. Takeuchi, B. L. Wanner, H. Mori, J. S.
Weissman, N. J. Krogan, and C. A. Gross. 2008. High-throughput, quantitative
analyses of genetic interactions in E. coli. Nat Methods 5:781-787.
Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7-derived system
for insertion mutagenesis and sequencing with synthetic universal primers. Gene
Warming, S., N. Costantino, D. L. Court, N. A. Jenkins, and N. G. Copeland.
2005. Simple and highly efficient BAC recombineering using galK selection.
Nucleic Acids Res 33:e36.
Werner, J. N., E. Y. Chen, J. M. Guberman, A. R. Zippilli, J. J. Irgon, and Z.
Gitai. 2009. Quantitative genome-scale analysis of protein localization in an
asymmetric bacterium. Proc Natl Acad Sci U S A 106:7858-7863.
Yu, D., H. M. Ellis, E. C. Lee, N. A. Jenkins, N. G. Copeland, and D. L.
Court. 2000. An efficient recombination system for chromosome engineering in
Escherichia coli. Proc Natl Acad Sci U S A 97:5978-5983.
48. Yu, D., J. A. Sawitzke, H. Ellis, and D. L. Court. 2003. Recombineering with
overlapping single-stranded DNA oligonucleotides: testing a recombination
intermediate. Proc Natl Acad Sci U S A 100:7207-7212.
Yu, N. Y., J. R. Wagner, M. R. Laird, G. Melli, S. Rey, R. Lo, P. Dao, S. C.
Sahinalp, M. Ester, L. J. Foster, and F. S. Brinkman. 2010. PSORTb 3.0:
improved protein subcellular localization prediction with refined localization
subcategories and predictive capabilities for all prokaryotes. Bioinformatics
Zane, G. M., H. C. Yen, and J. D. Wall. 2010. Effect of the deletion of
qmoABC and the promoter-distal gene encoding a hypothetical protein on sulfate
reduction in Desulfovibrio vulgaris Hildenborough. Appl Environ Microbiol
Zeghouf, M., J. Li, G. Butland, A. Borkowska, V. Canadien, D. Richards, B.
Beattie, A. Emili, and J. F. Greenblatt. 2004. Sequential Peptide Affinity (SPA)
system for the identification of mammalian and bacterial protein complexes. J
Proteome Res 3:463-468.
A. The SLIC approach (for double recombinations): Suicide vectors are assembled
directly from four ‘parts’ (■,■,■,■) using SLIC. Parts 1 and 2 (■,■) correspond to
homology regions (HR1/HR2) of the target loci on the chromosome. Parts 3 and 4
(■,■) correspond to an insertion part (IP) and a vector replication origin plus a
selection part (RSP) respectively. Different parts may be mixed and matched
depending on the choice of the application.
B. Example of a chromosomal modification in D. vulgaris Hildenborough using
the SLIC approach: Insertion of the SNAP tag at the 3’-end of DVU0172. The
suicide construct is assembled in E. coli using the SLIC technique from the
following parts: Part 1: 700bp upstream from the 3’ end of DVU0172 not
including its stop codon; Part 2: the AGT (visualization) tag followed by a
Kanamycin resistance gene; Part 3: 700bp downstream from the 5’ end of
DVU0172; Part 4: The replication origin of the vector (pUC) recognized only in E.
coli followed by a Spectinomycin resistance gene. The chromosomal modification
in D. vulgaris Hildenborough after double homologous recombination of the
transformed suicide vector is shown.
C. Utilizing the ‘parts’ based approach for enabling chromosomal modifications in
D. vulgaris Hildenborough using DVU1585 as the target gene. A set of reusable
‘parts’ (color coded) were employed for generating suicide constructs in E. coli
which were then transformed in D. vulgaris to examine the role of DVU1585 in
this sulfate reducer. Results of gene essentiality, protein-protein interactions and
protein localization are discussed in the text.
Conserved protein-protein interactions observed in this study. Chromosomally
tagged (STF) baits are shown in orange and prey proteins are shown in brown.
The relative sizes of interacting pairs are roughly proportional to their molecular
weights and arrows point from tagged baits to the respective prey. Conserved
interactions from the following complexes are shown: (1) The chaperonin
complex composed of the heat shock proteins DnaK (DVU0811), DnaJ
(DVU1876 and DVU3243), DafA (DVU1875), GrpE (DVU0812) and a
hypothetical protein (DVU2556); (2) The ATP synthase complex composed of
α(AtpA, DVU0777), β(AtpD, DVU0775), γ(AtpG, DVU0776), δ(AtpH,
DVU0778) and ε(AtpC, DVU0774) subunits; (3) The RNA polymerase complex
composed of α(DVU1329), β(DVU2928), β‘(DVU2929) subunits and σ54 factor
(DVU1628); (4) The glycyl-tRNA synthetase complex composed of α(GlyQ,
DVU1898) and β(GlyS, DVU1897) subunits; and (5) The binary interaction
between DNA Topoisomerase III (TopB, DVU2316) and single-strand binding
protein (SSB, DVU0222).
Comparison of interacting partners of DNA binding proteins (COG766 and
COG550) from E. coli and D. vulgaris. Arrows point from chromosomally tagged
baits in each organism to the respective prey. Green colored boxes indicate
presence of orthologs in both organisms. Orange colored boxes indicate proteins
unique to E. coli. Border colors represent proteins from the same COG category.
Optical density (600 nm) growth curve data for D. vulgaris HildenboroughΔDVU0890.
Growth of the mutant strain was restored in LS4 minimal medium by supplementation
with threonine but not methionine.
Predicted and observed localization of AGT tagged proteins in D. vulgaris. Each column
(L-R) depicts a representative image of an observed localization pattern in ten proteins
from D. vulgaris Hildenborough bearing chromosomally-inserted visualization tags
(AGT) at their respective C-termini. Fluorescently labeled cells were imaged by
deconvolution microscopy and images in the table represent an optical section through
the middle of the 3D deconvolved image stack (20-30 sections along the z axis).
Predicted localizations were obtained from PSORTb (www.psort.org/psortb/). PhsB
(DVU0172), a predicted cytoplasmic protein is uniformly distributed intracellularly.
Proteins localizing exclusively at both cell poles include MotA (DVU2608) and ParA
(DVU 3358). FlgE (DVU1443) and UvrB (DVU1605) proteins localize at four distinct
locations along the length of the cell. Hup-3 (DVU1795) and PyrB (DVU2901) proteins
show a patchy or spotty distribution. FtsH (DVU1278) localizes to the polar ends in
addition to a dispersed cytoplasmic distribution. LytR (DVU0596) displays a bipolar and
midband localization. MotA-1 (DVU 0050) has its localization signal restricted to one
polar end of the cell. A schematic representation of the observed localization pattern is
shown in the inset. Scale bars represent 400 nm for images of PhsB and MotA and 500
nm for the rest.
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