Development and application of schemes for high-throughput generation of suicide constructs.
Traditional mutagenesis approaches with suicide constructs have generally been regarded as cumbersome due to difficulties with vector construction (i.e., cloning of large sections of homologous DNA from either side of the locus to be modified) (34
). We therefore tested three recombination-based approaches, the Gateway system (Invitrogen, Carlsbad, CA), “recombineering” (18
), and sequence- and ligation-independent cloning (SLIC) (28
), for the generation of suicide constructs in a high-throughput manner (see Fig. S1 in the supplemental material). In all examples, suicide vectors designed for modifying the D. vulgaris
host chromosome were first generated in E. coli
and then transformed into D. vulgaris
, resulting in a modified host chromosome through single or double homologous recombination events integrating all or part of the nonreplicating delivery vector.
Suicide vector construction via the Gateway scheme was realized through two steps (see Fig. S1A in the supplemental material). In the first step, the sole homology region of the target locus was directionally cloned into a TOPO entry vector. The second step involved an LR recombination reaction with the directional placement of the homology region from the entry clone into a custom-designed destination vector. The destination vector included the sequences for the modification of the host cell and a suitable origin of replication. Destination vectors with different insertion sequences, such as TAP tags for elucidation of PPI, or visualization tags, such as SNAP (O6-alkylguanine-DNA alkyltransferase; New England BioLabs, Ipswich, MA), that allow protein localization may be used with a given library of entry clones. This powerful attribute of the Gateway scheme allows the facile exchange of the tags once a library of TOPO entry clones has been constructed.
Importantly, the introduction of a single region of homologous DNA in the construction of the entry clones allows only a single recombination event with the host chromosome that incorporates the entire plasmid. When creating tagging constructs for genes located at the beginning of their operons, we incorporated the native promoter sequence in addition to the target gene to be modified in the suicide vector to allow the expression of downstream genes. Necessary promoter sequences for each gene were assumed to be present within 300 bp upstream of the target gene. From a practical standpoint, this scheme is therefore limited to genes located at the terminal ends of their respective operons, where downstream polarity effects can be minimized. These caveats render the Gateway scheme useful for the rapid modification of a select class of target genes with a range of fusion tags. In order to be able to modify genes in a locus-independent manner, however, we leveraged two other schemes, “recombineering” and SLIC, to generate suicide vectors with two homology regions that permitted marker exchange.
In the recombineering approach, we utilized the bacteriophage lambda general recombination system (λred
) to modify genes carried on recombinant plasmids selected from an ordered genomic library of D. vulgaris
. The expression of λred
in E. coli
has been shown to mediate the efficient integration of linear DNA molecules into the host chromosome or plasmids through short regions (~40 bp) of sequence homology (46
). In this scheme (see Fig. S1B in the supplemental material), a linear DNA molecule that contained homology regions 1 and 2 (HR1/HR2) flanking the marker to be exchanged or the part to be inserted (insertion part [IP]) was generated by PCR. In the example shown, the IP is an affinity purification tag and a kanamycin resistance cassette expressed from its own promoter. Plasmid constructs and an HR1-IP-HR2-containing PCR product were transformed together into an E. coli
strain in which the λred
system was induced. The λred
recombinase facilitates recombination between the short 40-bp homology regions of the target loci flanking the IP and identical regions in the plasmid containing the fragment of chromosomal DNA with the genes to be modified. The length of the chromosomal regions of homology available for double-crossover events between modified plasmid constructs and the host chromosome varies with the location of the target gene within the genomic DNA insert in the suicide vector but is generally sufficient for detectable recombination with the genome being modified, with the exception of genes located at the termini of the insert DNA in the suicide vector. During the development of this modification procedure, it was noted that many isolated strains contained both a modified plasmid, into which the IP fragment had integrated, and an unmodified plasmid. Furthermore, when higher plasmid concentrations were used, multimeric plasmids were often isolated, a phenomenon previously reported when plasmids were used with the λred
). These factors led to increased processing requirements to generate a pure modified plasmid construct. Therefore, while suicide constructs made by this approach can be used for inserting or deleting sequences through marker exchange by double crossovers, the ready availability of a comprehensive ordered genomic library is an essential prerequisite. Due to the lack of such a comprehensive set of library constructs for D. vulgaris
and the inefficiencies of isolating recombineered plasmid constructs (compared to SLIC), this approach was not considered further in this study.
The third approach involves the de novo
assembly of suicide vectors by the SLIC procedure (28
). Vectors assembled by this technique are composed of four parts: two corresponding to the homology regions (HR1/HR2) from the host chromosome, an IP dictated by the application of choice, and a vegetative origin of replication and selection part (RSP). To obtain a suicide vector, the replication origin was functional only in E. coli
but not in the strain targeted for manipulation (43
) (see Fig. S1C in the supplemental material). The advantage of this approach lies in the reusability of parts for various applications. The IP and RSP regions remain constant for each specific application, whereas the HR1 and HR2 regions vary. Alternative IP regions, such as molecular barcodes, purification tags, antibiotic markers, and origins of replication, are incorporated into vector constructions depending on the downstream application. The RSP region is the most generic part used for suicide vector construction. However, a modification of the RSP to include an oriT
(origin of transfer) sequence is possible if the suicide vectors are to be transferred by conjugation from E. coli
to the target microbe.
Chromosome modifications by suicide vectors may be characterized as “marked” or “unmarked” depending on the presence or absence of suitable selection markers in the host chromosome after the modifications have been introduced. In either case, chromosomal modifications at the 3′ end of a target gene may alter the expression or translation of co-operonic downstream genes. In the case of the marked approach, the incorporation of a selection marker and its cognate promoter may introduce a second transcription initiation site. For genes located in close proximity to each other, one must also consider the possibility of a displaced ribosomal binding site (RBS). The SLIC approach, through appropriate design, may be used to correct these problems for both marked and unmarked approaches. Systems for unmarked modifications, such as the Cre-lox
recombination system (6
) or the levansucrase-dependent sucrose sensitivity system (11
), could easily be implemented by the incorporation of the respective parts (loxP
sites and sacB
) into IP or SP regions of SLIC-generated suicide vectors. In hosts where sucrose sensitivity is not a strong selection factor or a residual “scar” is not desired, alternative counterselection systems are available. For D. vulgaris
, sensitivity to the toxic pyrimidine analogue 5-fluorouracil allows selection against the expression of upp
, encoding the salvage enzyme uracil phosphoribosyl transferase. This marker has been successfully used in a number of microbes (25
We compared 550 distinct target genes for tagging with suicide vectors assembled by the Gateway and SLIC strategies and generated 297 (54%) and 468 (85%) sequence-verified plasmids, respectively. In general, we observed that the SLIC strategy yielded a higher percentage of confirmed D. vulgaris mutant strains (304 strains/468 plasmids constructed; ~65%) than the Gateway scheme (70 strains/297 plasmids constructed; ~24%). For the recombineering strategy, we generated 18 sample suicide vectors, which resulted in 9 confirmed mutant strains. Given the apparent superiority of the SLIC approach, it was the method of choice for the further manipulation of the D. vulgaris chromosome to study the effects of gene deletions, to identify physical interactions of proteins, and to localize selected tagged proteins. To date, we have generated a library of over 700 engineered strains of D. vulgaris using the methodologies described in this work.
Screening for protein-protein interactions by tandem affinity purification.
With plasmids constructed by the SLIC procedure, we introduced sequences into the chromosome encoding TAP tags in frame at the 3′ ends of several genes from D. vulgaris. Engineered strains expressing native levels of C-terminally TAP-tagged fusion proteins were used to examine the protein complexes isolated with the tagged baits inferred to represent functional PPI. We validated conserved interactions in several essential complexes, such as the F1-ATPase, the RNA polymerase, the chaperone DnaK, and others ( and see Table S1 in the supplemental material).
Fig. 2. 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 masses, and (more ...)
Next, we examined potential interacting partners of proteins associated with the D. vulgaris
nucleoid (). Well-known components of the E. coli
nucleoid include DNA-binding proteins such as Fis, HNS, Dps, IHF (IhfAB), and HU (HupAB). By the very nature of their inherent DNA-binding capabilities, these highly abundant proteins are involved in the modulation of cellular processes such as transcriptional regulation, maintenance of DNA architecture, replication, recombination, and stress protection (1
Given the common set of functions attributed to these proteins, it is not surprising that they exhibit a high level of interactions with each other. Indeed, proteins precipitated with TAP-tagged baits of HU and IHF from D. vulgaris
suggest a closely knit interaction subnetwork comprising many of these DNA-binding proteins. Intriguingly, the D. vulgaris
genome appears to lack the diversity of nucleoid protein domains reported for E. coli
, such as Fis (COG2901), HNS (COG2916), and Dps (COG783) and their corresponding interacting partners (8
). In contrast, D. vulgaris
encodes twice as many proteins with the “bacterial nucleoid DNA-binding protein” domain, COG776, as those found in E. coli
). In order to compare the E. coli
and D. vulgaris
subnetworks associated with COG776 family proteins, we identified interacting partners of D. vulgaris
-tagged baits, Hup-3 and IhfB. With the exception of DVUA0004 and DVU1134, all members of the COG776 family appeared to interact with the tagged baits and potentially with each other ( and see Table S2 in the supplemental material).
Unlike topoisomerases from E. coli
, members of the D. vulgaris
“topoisomerase” family (TopA and TopB) did not appear to copurify with the tagged HU proteins. This was also confirmed when TopB was used as the bait and none of the COG776 family proteins were observed as interacting partners. In E. coli
, HU (HupAB) was reported previously to introduce negative supercoiling in covalently closed circular DNA in the presence of topoisomerase I (TopA) (36
). From these results, it appears that mechanisms of DNA architecture maintenance and global regulatory controls in D. vulgaris
may differ from those in E. coli
Gene deletions: examining the methionine biosynthesis pathway of D. vulgaris.
Although the genome sequence of D. vulgaris
was published in 2004, several amino acid biosynthesis pathways in this SRB remain to be elucidated. In this study, we examined putative alternative steps in methionine biosynthesis. At least 18 variant methionine pathways have been proposed to originate from the common precursor, homoserine (22
). In examining the D. vulgaris
genome for all known variant genes related to the three major steps of methionine synthesis, (i) homoserine activation, (ii) sulfur incorporation, and (iii) methylation, homologs corresponding only to step 3 were apparent: vitamin B12
-dependent methionine synthase (DVU1585 [metH
]) and methionine synthase II (cobalamin independent) (DVU3371 [metE
]). We tested these and other genes putatively involved in the production of the methionine precursor homoserine from l
-aspartate. These included a putative aspartate kinase (DVU1913 [lysC
]), homoserine dehydrogenase (DVU0890 [hom
]) (probable counterparts to bifunctional aspartate kinase II/homoserine dehydrogenase from E. coli
), a putative beta-cystathionase (DVU0171, similar to patB
]), and a protein with predicted methyltransferase activity (DVU3369, similar to metW
We verified all gene deletion mutations by PCR as well as Southern blot analysis. These gene deletion studies revealed that a majority of the putative methionine biosynthesis pathway knockouts (DVU1585, DVU3371, DVU0890, DVU0171, and DVU3369) did not result in methionine auxotrophy. A surprising result of this study was that the mutant deleted for DVU0890, Δhom, was found to be auxotrophic for threonine but not methionine (). This unexpected phenotype and the difficulty encountered in the isolation of a deletion of DVU1913 were interpreted to indicate that an unusual pathway for methionine biosynthesis might be operational in this SRB. Further studies in this direction are under way.
Optical density (600 nm) growth curve data for the D. vulgaris Hildenborough (DvH) ΔDVU0890 strain. The growth of the mutant strain was restored in LS4 minimal medium by supplementation with threonine but not methionine.
Protein localization with visualization tags.
We engineered D. vulgaris
strains to express proteins bearing a SNAP tag, which is designed for subcellular visualization in anaerobic bacteria. Conventional green fluorescent protein derivatives require molecular oxygen for proper chromophore formation and hence cannot be utilized under anaerobic culturing conditions. We therefore explored the use of a modified SNAP tag that has a dead-end reaction with a modified O6
-benzylguanine (BG) derivative (32
). To validate the use of the AGT-tag-based method for subcellular localization in anaerobic bacteria, we first compared the SNAP labelings of three AGT-tagged proteins from D. vulgaris
, DsrC (DVU2776), MreB (DVU0789) (data not shown), and FtsZ (DVU2499), from the respective engineered strains to the unmodified wild-type strain. We confirmed the specific labeling of tagged proteins using two complementary methods: in-gel fluorescence detection by SDS-PAGE and fluorescence microscopy. SDS-PAGE analysis typically yielded single bands at the expected molecular masses, indicating the specific labeling of the tag, with little or no nonspecific binding. Interestingly, in our fluorescence micrographs, we found a robust cell-to-cell variability in the labeling signal. To eliminate the possibility that the labeling reagent did not reach all tagged proteins, we compared in vivo
-labeled intact cells to in vitro
-labeled whole-cell extracts and observed no difference in the fluorescence signals between the two, as judged by SDS-PAGE analysis. This suggested efficient reagent access and the specific labeling of intracellular AGT-tagged proteins. In the case of MreB and FtsZ, unlike DsrC, the chromosomal tagging appeared to alter the cellular morphology normally associated with the wild-type strain. Morphological changes included either the loss of the vibrio-typic cell shape (MreB-AGT) (data not shown) or extensive elongation (FtsZ-AGT) (see Fig. S2 in the supplemental material), suggesting diminished or altered protein function due to the presence of the visualization tag. Our results are comparable to those for the GFP-based protein localization of FtsZ, as demonstrated previously for E. coli
). To our knowledge, this is the first account of specific tag-based fluorescence labeling for the purpose of protein localization in an anaerobic bacterium.
Subsequently, we expanded the method to 15 additional proteins (). We were able to decipher localization patterns for each of the 15 SNAP-tagged proteins, presumably reflecting their respective biological roles in this SRB. ParA, MotA-1, and MotA-3 localized exclusively to the poles, a subcellular area that has been referred to as a “localization hot spot,” whereas LytR, FtsH, FlgE, and UvrB localized at the poles as well as at additional regions in or toward the center of the cells. Hup-3 and PyrB showed a patchy or spotty distribution along the length of the cells. The remaining proteins displayed a cytoplasmically uniform distribution. Orthologous counterparts of ParA and FtsH from Caulobacter crescentus
and E. coli
were experimentally visualized previously (41
). For the remaining proteins, only theoretical in silico
localization predictions have been made to date (48
). In these localization studies, we consistently noted cell-to-cell variations in fluorescent signals in any given population, which may be attributed to corresponding differences in expression levels (38
Fig. 4. Predicted and observed localizations of AGT-tagged proteins in D. vulgaris. Each column (left to right) depicts a representative image of an observed localization pattern in 10 proteins from D. vulgaris Hildenborough bearing chromosomally inserted visualization (more ...)