- Research article
- Open Access
Analysis of a taurine-dependent promoter in Sinorhizobium meliloti that offers tight modulation of gene expression
© Mostafavi et al.; licensee BioMed Central Ltd. 2014
Received: 27 August 2014
Accepted: 13 November 2014
Published: 25 November 2014
Genetic models have been developed in divergent branches of the class Alphaproteobacteria to help answer a wide spectrum of questions regarding bacterial physiology. For example, Sinorhizobium meliloti serves as a useful representative for investigating rhizobia-plant symbiosis and nitrogen fixation, Caulobacter crescentus for studying cell cycle regulation and organelle biogenesis, and Zymomonas mobilis for assessing the potentials of metabolic engineering and biofuel production. A tightly regulated promoter that enables titratable expression of a cloned gene in these different models is highly desirable, as it can facilitate observation of phenotypes that would otherwise be obfuscated by leaky expression.
We compared the functionality of four promoter regions in S. meliloti (P araA , P tauA , P rhaR , and P melA ) by constructing strains carrying fusions to the uidA reporter in their genomes and measuring beta-glucuronidase activities when they were induced by arabinose, taurine, rhamnose, or melibiose. P tauA was chosen for further study because it, and, to a lesser extent, P melA , exhibited characteristics suitable for efficient modulation of gene expression. The levels of expression from P tauA depended on the concentrations of taurine, in both complex and defined media, in S. meliloti as well as C. crescentus and Z. mobilis. Moreover, our analysis indicated that TauR, TauC, and TauY are each necessary for taurine catabolism and substantiated their designated roles as a transcriptional activator, the permease component of an ABC transporter, and a major subunit of the taurine dehydrogenase, respectively. Finally, we demonstrated that P tauA can be used to deplete essential cellular factors in S. meliloti, such as the PleC histidine kinase and TatB, a component of the twin-arginine transport machinery.
The P tauA promoter of S. meliloti can control gene expression with a relatively inexpensive and permeable inducer, taurine, in diverse alpha-proteobacteria. Regulated expression of the same gene in different hosts can be achieved by placing both tauR and P tauA on appropriate vectors, thus facilitating inspection of conservation of gene function across species.
The class Alphaproteobacteria encompasses a multitude of noteworthy taxa with diverse physiologies. Members of the group range from human and livestock pathogens, such as Rickettsia and Brucella, to rhizobial species, such as Sinorhizobium meliloti and Agrobacterium tumefaciens (also known as Ensifer meliloti and Rhizobium radiobacter, respectively), that benefit or damage agricultural crops . The possible progenitor of present-day mitochondria, and several highly abundant marine groups, including Roseobacter, SAR116, and SAR11, also belong to this class . In-depth molecular studies of various representatives have led to significant insights regarding cell cycle progression , organelle biogenesis , metabolic pathways , microbe-host interactions ,, and potential for biofuel production .
The availability of genetic tools in readily culturable bacteria facilitates the investigation of their physiologies. In particular, an inducible promoter that allows tight titration of gene expression enables functional analysis of cellular factors that are toxic at low levels or essential for viability. For example, investigators working with Escherichia coli, a gamma-proteobacterium, possess an array of such tools at their disposal: some commonly used ones are the arabinose-inducible PBAD promoter ,, the rhamnose-inducible P rhaB promoter , and various derivatives of the original lac and tet promoters ,. Within the alpha-proteobacteria group, Caulobacter crescentus is perhaps one of the representative members most amenable to genetic manipulation, with two well characterized, regulatable promoters, inducible by xylose or vanillate -. Identification of these control elements has helped C. crescentus emerge as a prominent model for study of bacterial cell biology .
In contrast, the agriculturally important alpha-proteobacterium S. meliloti, which forms mutualistic symbiosis with legume plants, lacks a selection of such fully developed induction systems. Following reciprocal exchange of chemical signals between a suitable plant host and the endosymbiont, S. meliloti infects and colonizes nodules that develop along the plant root; the bacteria enter membrane-bound compartments within cortical cells and differentiate into bacteroids, capable of converting atmospheric dinitrogen to ammonia ,. The ability of S. meliloti to fix nitrogen for its hosts, especially alfalfa, improves crop yield, reduces expensive reliance on synthetic fertilizers, and enriches soil fertility in an ecologically sound manner ,. Better tools for manipulating the bacterium would facilitate investigation of this model rhizobial-plant symbiosis. Our goal is to identify expression systems that can be modulated and efficiently shut off, thus permitting characterization of phenotypes that would otherwise be obscured by leaky expression. We are particularly interested in a system that is functional and easily transferrable among different alpha-proteobacteria. Derivatives of the E. coli lac promoter, inducible by isopropyl-beta-D-thiogalactopyranoside (IPTG), appear to serve this purpose ,. Nevertheless, additional candidates that would enlarge the repertoire of genetic tools deserve further examination.
We used transcriptional fusions to the genomic loci of these four promoters to assess their induction profiles in both complex and defined media. Our results suggested that P tauA and P melA offered both tighter regulation and wider ranges of expression compared to the other promoters. Further characterization of the P tauA promoter indicated that the surrounding tau genes are required for taurine metabolism and affect expression from the promoter. We demonstrate the utility of the promoter by showing that P tauA allows modulated expression of reporter genes in two other model alpha-proteobacteria, C. crescentus and Zymomonas mobilis. Moreover, we verified that P tauA enables tight shut-off and depletion of essential genes in S. meliloti. Thus, this study establishes the feasibility of using P tauA as a regulatory switch in diverse alpha-proteobacteria.
Results and discussion
Comparison of promoter regions in complex and defined media
To identify a promoter region that allows controlled expression of desired genes, we compared the levels of inducible expression from four different potential promoters: P araA , P tauA , P rhaR , and P melA . These regions were chosen according to the criteria described above after reviewing previously published studies for candidates. Each promoter region was transcriptionally fused to the uidA gene [encoding the beta-glucuronidase (GUS) reporter enzyme] on a suicide plasmid, which was subsequently integrated into the S. meliloti genome via homologous recombination (Figure1). We designed the constructs to minimize disruption of the target genomic regions, such that the surrounding genes and their regulation remained intact, by integrating between divergently transcribed genes. Strains carrying the reporter constructs were grown in complex (PYE, LB) or defined (M9, M9 + CAA) media for three hours to mid-log phase, with or without appropriate inducers: arabinose for P araA , taurine for P tauA , rhamnose for P rhaR , and melibiose for P melA . For comparison in M9 minimal medium, strains were first grown in M9 supplemented with glucose (M9G), washed with M9, and then grown for three hours in M9G or M9 plus inducer as the sole carbon source. Samples were subsequently harvested for measurement of GUS activities.
The P rhaR promoter demonstrated obvious elevation in expression when induced with rhamnose, increasing 10-fold (from 1.1 to 11 Miller units) in PYE complex medium and 38-fold (from 0.4 to 15.2 Miller units) in M9 minimal medium. However, P rhaR also showed the lowest levels of expression, whether induced or uninduced, when compared to other promoters under similar conditions. We only measured expression from P rhaR in PYE and M9 and did not test it in LB or M9 + CAA because other promoter regions all exhibited lower expression in LB compared to PYE and in M9 + CAA compared to M9. The results in PYE and M9 media had already suggested that the rhaR promoter region would not permit regulating genes that require high levels of expression.
The remaining two promoter regions, P tauA and P melA , showed significant increases in GUS activities when induced. For P tauA , the increase ranged from 8-fold in M9 + CAA to 41-fold in M9, while for P melA the increase ranged from 8-fold in PYE to 19-fold in M9. Both demonstrated low levels of basal expression and robust expression when induced. The notable exception occurred for P melA in PYE, which exhibited higher levels of basal expression, suggesting leakiness in that particular medium (Figure2A). Nevertheless, both P tauA and P melA emerged as useful candidates for modulating expression of desired genes.
Effects of tauR, tauC, and tauY on P tauA expression
Next, we examined the effects of representative genes in the tau region (tauR, tauC, and tauY) on P tauA expression. tauR (SMb21525) encodes a transcriptional regulator of the GntR family, tauC (SMb21528) encodes the permease component of a probable taurine ABC transporter, and tauY (SMb21529) encodes the large subunit of the putative taurine dehydrogenase ,. Genetic analyses suggested that orthologs of the TauABC transporter in E. coli and Rhodobacter capsulatus participate in taurine utilization -. Also, previous studies indicated that the TauR ortholog in R. capsulatus (49% identity, 62% similarity, 0% gap ) activates expression of genes necessary for taurine dissimilation , and S. meliloti P tauA requires its cognate TauR for expression in Rhizobium leguminosarum. We expected similar behaviors in S. meliloti.
To assess the effects of the tau deletions, we used two similar plasmids carrying the P tauA -uidA reporter fusion, one with tauR also on the plasmid (pJC479), and the other without (pJC478). Wild-type and mutant strains transformed with the plasmids were grown in PYE in the presence or absence of taurine for three hours and then harvested to measure gene expression (Figure5B). Except for the ΔtauR mutant, all strains showed similar patterns of expression with both reporter plasmids: low basal activities in the absence of taurine and high levels of expression when induced. The ΔtauR mutant exhibited induction when tauR was present on the reporter plasmid but no induction when tauR was absent, confirming that TauR is the transcriptional activator for the P tauA promoter, as previously shown . Behavior of the ΔtauC mutant was indistinguishable from that of the wild type, suggesting that the ABC transporter is not required for taurine to enter the cell in complex medium. Finally, whether induced or uninduced, the ΔtauY mutant displayed the highest levels of reporter expression compared to other strains grown under similar conditions. These results are consistent with an increased accumulation of taurine, and thus enhanced expression, in the ΔtauY mutant due to its inability to degrade the sulfonate compound. Higher basal expression may be attributed to low levels of taurine in the PYE complex medium itself.
Somewhat unexpectedly, deletion of tauC, which encodes a component of the putative taurine transporter, did not affect P tauA expression in PYE medium; because tauC is required for taurine utilization (Figure5A), we decided to assess the effects of the tau mutations over a range of inducer concentrations in both complex and defined media. To facilitate measurement of promoter activity, we generated a plasmid bearing tauR as well as a P tauA -mCherry reporter fusion. Wild-type and deletion strains carrying the plasmid were grown for 20hours with different concentrations of taurine in PYE or M9G prior to measurement of mCherry fluorescence. For all strains in PYE rich medium (Figure5C), an increase in fluorescence directly correlated with an increase in taurine concentration. As expected, since the tauR mutation is complemented by a copy of tauR on the plasmid, the wild-type Rm1021 and ΔtauR strains behaved similarly. Moreover, in agreement with the GUS assay results (Figure5B), the ΔtauY mutant exhibited much higher levels of expression compared to the other strains, reaching saturation at relatively low levels of inducer (1020mM taurine). The ΔtauC mutant displayed slightly lower expression of P tauA -mCherry compared to the wild type, with the difference becoming more noticeable at higher concentrations of taurine. This subtle difference in expression suggests that while TauC and its associated ABC-type transporter are not absolutely required for taurine import in complex medium, they may enhance the process.
In contrast, TauC is required in M9G minimal medium for import of taurine to induce P tauA expression. Only basal levels of fluorescence were observed with the ΔtauC mutant, regardless of taurine concentration, compared to the wild type and other tau mutants, where fluorescence correlated with increasing taurine concentration (Figure5D). Similar to results obtained with the GUS reporter (Figure4B), wild-type Rm1021 required much lower levels of taurine to achieve full induction in M9G compared to PYE. The ΔtauY mutant again exhibited the highest levels of expression. Intriguingly, in M9G medium the ΔtauR mutant showed consistently higher expression relative to wild-type Rm1021 and reached levels similar to that of the ΔtauY mutant at higher taurine concentrations. We do not have a clear explanation for this observation. The Rm1021 strain actually carries two copies of tauR (one genomic, one plasmid-borne) and should activate expression more readily than the ΔtauR mutant, which only has one copy of the transcriptional regulator gene on the plasmid. One possibility is that genomic deletion of tauR influences expression of surrounding genes, such as reducing the levels of taurine dehydrogenase, and thus affects expression from the reporter plasmid.
Taken together, our results indicate that tauR encodes a transcriptional activator necessary for expression from the P tauA promoter. TauY is involved in the degradation of taurine, most likely by forming part of the taurine dehydrogenase. The ABC transporter that contains TauC is required to import taurine in minimal, but not complex, medium. We speculate that the ABC transporter encoded by SMc02829-2832 serves as an alternative taurine uptake pathway in complex medium, because SMc02832 expression is induced by taurine, valine, isoleucine, and leucine .
Taurine-dependent expression in C. crescentus and Z. mobilis
The same P tauA -uidA reporter strain (with tauR) was used to determine the possible range of expression in PYE medium (Figure6B). GUS activity increased with taurine concentration and reached maximum at approximately 10mM taurine. Thus, much lower levels of the inducer were needed to saturate the P tauA expression system in C. crescentus relative to S. meliloti. Expression varied approximately 19-fold, from 150 to 2800 Miller units. Similar results were obtained using a reporter plasmid carrying tauR-P tauA -mCherry: fluorescence levels increased with taurine concentration and approached maximum at 10mM taurine in PYE (Figure6C) and 0.15mM taurine in M2G minimal medium (Figure6D). Therefore, the P tauA promoter should be useful for modulating gene expression in C. crescentus.
Depletion of essential proteins in S. meliloti
The second example we chose was tatABC, three contiguous genes that encode essential components of the twin arginine transport system in S. meliloti. We took an approach similar to that for pleC. Because tatA is relatively small in size, and we wanted >500bp of homology for efficient recombination, both tatA and the 5 portion of tatB were fused to P tauA on the suicide plasmid. The resultant depletion strain has tatABC under control of P tauA , but also an intact copy of tatA under control of its native promoter (Figure8B). Presumably TatB and TatC are depleted in this strain when expression is blocked in the absence of taurine.
Together with previously published results ,, our analysis demonstrates that the P tauA promoter of S. meliloti Rm1021, when paired with its cognate transcriptional activator TauR, can provide robust tuning of gene expression in diverse model organisms of the class Alphaproteobacteria, including members of Rhizobiales (R. leguminosarum Rlv3841, R. etli CE3, and S. fredii NGR234), Rhodobacterales (R. sphaeroides WS8N), Sphingomonadales (Z. mobilis ZM4), and Caulobacterales (C. crescentus NA1000). Import of taurine does not appear to pose a challenge, despite the absence of known or obvious transporters for the inducer in many of the organisms tested. P tauA and tauR, as well as other promoter regions described above, can be readily amplified from plasmids reported here and elsewhere , and cloned into other vectors that are more suitable for the end user. Therefore, this study adds to the repertoire of tools available for analyzing genetic models of Alphaproteobacteria. Because taurine has been shown to activate expression of genes other than those in the tau region, such as rpoE1 and rpoE4, in S. meliloti, the addition of taurine as an inducer may cause ancillary and potentially undesirable effects, particularly in untested organisms. Thus, appropriate control strains, such as one harboring an empty vector, should be included for comparison when examining the physiological consequences of inducing a gene of interest under the control of P tauA .
In its native host, P tauA permits depletion of essential proteins and tight regulation of potentially toxic genes, especially if combined with careful manipulation of translational efficiency; specifically, Harrison et al.  was able to reduce the level of expression from P tauA by modifying sequences adjacent to the ribosome binding site such that they form RNA secondary structures. Overexpression of the gene under P tauA control can be conducted in the ΔtauY background, in which inducer level remains high due to the inability to degrade taurine. Considering the ranges of expression observed, similar levels of control in other host organisms should be achievable with appropriate adjustments, including alterations of copy number via placement in the genome or on plasmids. The possibility of using the same construct to control expression in different alpha-proteobacteria can expedite examination of gene function across species: for example, we can use the same plasmid to determine whether an essential gene from one organism can complement null mutations in its orthologs. Although P tauA currently does not appear regulatable outside of the alpha-proteobacteria group, for instance in E. coli or Pseudomonas aeruginosa, further investigation and engineering may broaden the range of its functionality.
Bacterial strains, growth conditions, and genetic techniques
Strains used in this study
Relevant genetic markers, features, and/or description
Construction, source, or reference
Escherichia coli cloning strain: F − φ80lacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 hsd R17 (rk-, mk+) deoR thi-1 supE44 gyrA96 relA1
Escherichia coli cloning strain: F − mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 endA1 recA1 deoR Δ(ara, leu)7697 araD139 galU galK nupG rpsL
MT607/pRK600; Escherichia coli helper strain for mobilizing RK2/RP4-derived plasmids
Caulobacter crescentus syn-1000; previously called CB15N, a synchronizable derivative of wild-type CB15
Zymomonas mobilis wild-type strain, previously called CP4
Sinorhizobium meliloti SU47 derivative, SmR (progenitor of strains listed below)
SMb20895::pJC445 (P araA -uidA)
Mate pJC445 into Rm1021, select for NmR
SMb21526::pJC446 (P tauA -uidA)
Mate pJC446 into Rm1021, select for NmR
SMc02323::pJC447 (P rhaR -uidA)
Mate pJC447 into Rm1021, select for NmR
SMb21648::pJC455 (P melA -uidA)
Mate pJC455 into Rm1021, select for NmR
SMc02369::pJC468 (P tauA -pleC)
Mate pJC468 into Rm1021, select for NmR
SMc02066::pJC470 (P tauA -tatAB)
Mate pJC470 into Rm1021, select for NmR
Allelic replacement using pJC507
Allelic replacement using pJC508
Allelic replacement using pJC509
Plasmids used in this study
Relevant genetic markers, features, and/or description
Construction, source, or reference
Broad-host-range vector, KmR
RK2-derived broad-host-range vector with E. coli rrnB terminator preceding polylinker, TcR
Counter-selectable vector for allelic replacement, sacB, GmR
RK2-derived broad-host-range vector with C. crescentus P van promoter, TcR
Narrow-host-range vector with C. crescentus P van promoter and mCherry, TcR
pUC119-derived suicide vector with uidA cassette, NmR/KmR ApR
pVO155-P araA , for fusing uidA to sequence upstream of araA
pVO155-P tauA , for fusing uidA to sequence upstream of tauA
pVO155-P rhaR , for fusing uidA to sequence upstream of rhaR
pVO155-P melA , for fusing uidA to sequence upstream of melA
pVO155-P tauA -pleC, for integrating into chromosome and placing pleC under control of P tauA
pVO155-P tauA -tatAB, for integrating into chromosome and placing tatB and tatC under control of P tauA
pRVMCS-5-derived plasmid carrying P tauA
pRVMCS-5-derived plasmid carrying tauR-P tauA
pCM130-P tauA -uidA
pCM130-tauR-P tauA -uidA
pRVMCS-5-derived plasmid carrying tauR-P tauA -mCherry
pBBR1 MCS-2-tauR-P tauA -mCherry
pCM130-P tauA -tauC
pCM130-P tauA -tauY
Mobilization of plasmids from E. coli to S. meliloti , C. crescentus, or Z. mobilis was accomplished by triparental mating, with the help of strain MT616, carrying pRK600 ; nalidixic acid or streptomycin was used to select against E. coli donor and helper strains. Electroporation of plasmid DNA into C. crescentus was achieved using cuvettes with 1-mm gaps and a Bio-Rad Gene Pulser II with the following settings: 400 Ω, 25 μF, and 1.5 kV . Deletions in the S. meliloti genome were generated by homologous recombination and allelic replacement, using a two-step selection procedure with the vector pJQ200sk ,. Plasmid integrations and genomic deletions were screened and confirmed by PCR. Construction of depletion strains required the presence of 100mM taurine in the medium while selecting for transconjugants.
Standard techniques were used for cloning and analysis of DNA, PCR, and transformation into E. coli,. Plasmids and DNA fragments were isolated using commercial kits from Qiagen. KOD polymerase (Novagen) or MangoMix (Bioline) was used for high- or low-fidelity PCR amplification, respectively, while other enzymes used to manipulate DNA came from New England BioLabs. Elim Biopharmaceuticals provided oligonucleotide synthesis and DNA sequencing services. Details of plasmid construction are described in Additional file 1. Oligonucleotide sequences are provided in Additional file 2.
Beta-glucuronidase (GUS) reporter assays
We monitored expression of the uidA reporter, under the control of various promoters, by assessing beta-glucuronidase activities as previously described , except lysozyme was omitted and 400 μL 1 M sodium carbonate was added to stop each reaction. Enzymatic activity in each biological sample was measured in duplicates or triplicates, and measurements from at least three independent biological samples were averaged.
For comparison of different promoters, S. meliloti strains were grown in PYE, LB, or M9 liquid media overnight, diluted to the desired optical density, and induced for three hours, to mid-log phase, with 0.2% (13.3mM) L-arabinose, 0.063% (5mM) taurine, 0.2% (11.0mM) L-rhamnose monohydrate, 0.2% (5.6mM) D-melibiose monohydrate, or 0.2% (3.4mM) D-raffinose pentahydrate. For induction in M9, strains were grown in M9 supplemented with 0.2% glucose (M9G), washed in M9 buffer without carbon source, and diluted into M9G or M9 plus inducer as the sole carbon source. For induction in M9 supplemented with 0.2% casamino acids (M9 + CAA), strains were either first grown in M9 + CAA without glucose and then diluted into the same medium with or without inducer; or they were first grown in M9 + CAA supplemented with 0.2% glucose, washed with M9 + CAA, and diluted into M9 + CAA supplemented with either glucose or the inducer.
For comparison of melibiose and raffinose as inducers of P melA , strain JOE3334 was grown in PYE in the presence or absence of the inducer for three hours to mid-log phase, for 20hours to stationary phase, or for 12hours to mid-log or stationary phase, by diluting the starter PYE cultures appropriately.
For induction with different concentrations of taurine, S. meliloti strains were grown for three hours in PYE or M9G to mid-log phase. For induction with taurine concentrations above 10mM in PYE, cultures were adjusted to the desired optical density by mixing appropriate volumes of the starter culture, 0.5M taurine, water, and double-strength PYE (2X PYE), to prevent excessive dilution of nutrients in the medium. Similar procedures were used to measure GUS activity in C. crescentus, except the strains were induced for two hours.
Measurement of mCherry fluorescence
To monitor mCherry expression in S. meliloti, we cultivated strains harboring pBBR1 MCS-2 or pJC514 overnight in PYE or M9G supplemented with neomycin (5 μg ml−1) and then diluted the cultures in media containing different concentrations of taurine, to a final volume of 0.8mL in each well of a 48-well microtiter plate. The plates were shaken for 20hours, and 150 μL of each culture was transferred to a 96-well optic plate in duplicates for absorbance (535nm) and fluorescence (550nm excitation, 610nm emission) measurements with a Tecan Spectrafluor Plus reader. Expression in Z. mobilis was monitored in a similar fashion, except the strains were grown in RM or BM supplemented with kanamycin (100 μg ml−1). For C. crescentus, NA1000 carrying pJC503 or pRVMCS-5 was induced in PYE or M2G (M2 plus 0.2% glucose) containing oxytetracycline (0.5 μg ml−1) and different concentrations of taurine for four hours. Absorbance readings confirmed similar levels of growth for all samples of the same species. Fluorescence readings were adjusted by subtracting the background fluorescence of strains carrying only the vector (pBBR1 MCS-2 or pRVMCS-5). Average background fluorescence readings (in arbitrary units) were approximately 10000 and 21000 for S. meliloti in PYE and M9G, 23000 and 30000 for C. crescentus in PYE and M2G, and 14000 and 22000 for Z. mobilis in RM and BM. Measurements of at least four biological samples, grown on separate days, were averaged following appropriate subtraction of background readings.
MM, JCL, TS, JAB, ITG, TTT, SNK, ZH, and JCC carried out experiments and performed data analysis. MM, JCL, and JCC wrote the manuscript. All authors read and approved the final manuscript.
We thank past and present members of the Chen lab for their technical assistance, Jeffrey M. Skerker for providing Z. mobilis ZM4, and Robert F. Fisher and Melanie J. Barnett for critical appraisal of the manuscript. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under Award Number SC3GM096943. JCL, TS, and JAB were funded by the following programs: NIH MARC (T34-GM008574), NIH MBRS-RISE (R25-GM059298), NIH MS-PhD Bridge (R25-GM048972), and the Genentech Foundation MS Dissertation Scholarship. Additional financial support came from the College of Science and Engineering, San Francisco State University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or other funding agencies.
- Lee KB, Liu CT, Anzai Y, Kim H, Aono T, Oyaizu H: The hierarchical system of the `Alphaproteobacteria: description of Hyphomonadaceae fam. nov., Xanthobacteraceae fam. nov. and Erythrobacteraceae fam. nov. Int J Syst Evol Microbiol. 2005, 55 (Pt 5): 1907-1919. 10.1099/ijs.0.63663-0.View ArticlePubMedGoogle Scholar
- Ferla MP, Thrash JC, Giovannoni SJ, Patrick WM: New rRNA gene-based phylogenies of the Alphaproteobacteria provide perspective on major groups, mitochondrial ancestry and phylogenetic instability. PLoS One. 2013, 8 (12): e83383-10.1371/journal.pone.0083383.PubMed CentralView ArticlePubMedGoogle Scholar
- Ardissone S, Viollier PH: Developmental and environmental regulatory pathways in alpha-proteobacteria. Front Biosci (Landmark Ed). 2012, 17: 1695-1714. 10.2741/4013.View ArticleGoogle Scholar
- Komeili A: Molecular mechanisms of compartmentalization and biomineralization in magnetotactic bacteria. FEMS Microbiol Rev. 2012, 36 (1): 232-255. 10.1111/j.1574-6976.2011.00315.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Vuilleumier S, Chistoserdova L, Lee MC, Bringel F, Lajus A, Zhou Y, Gourion B, Barbe V, Chang J, Cruveiller S, Dossat C, Gillett W, Gruffaz C, Haugen E, Hourcade E, Levy R, Mangenot S, Muller E, Nadalig T, Pagni M, Penny C, Peyraud R, Robinson DG, Roche D, Rouy Z, Saenampechek C, Salvignol G, Vallenet D, Wu Z, Marx CJ:Methylobacterium genome sequences: a reference blueprint to investigate microbial metabolism of C1 compounds from natural and industrial sources. PLoS One. 2009, 4 (5): e5584-10.1371/journal.pone.0005584.PubMed CentralView ArticlePubMedGoogle Scholar
- Jones KM, Kobayashi H, Davies BW, Taga ME, Walker GC: How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model. Nat Rev Microbiol. 2007, 5 (8): 619-633. 10.1038/nrmicro1705.PubMed CentralView ArticlePubMedGoogle Scholar
- Kondorosi E, Mergaert P, Kereszt A: A paradigm for endosymbiotic life: cell differentiation of Rhizobium bacteria provoked by host plant factors. Annu Rev Microbiol. 2013, 67: 611-628. 10.1146/annurev-micro-092412-155630.View ArticlePubMedGoogle Scholar
- Todhanakasem T, Sangsutthiseree A, Areerat K, Young GM, Thanonkeo P: Biofilm production by Zymomonas mobilis enhances ethanol production and tolerance to toxic inhibitors from rice bran hydrolysate. N Biotechnol. 2014, 31 (5): 451-459. 10.1016/j.nbt.2014.06.002.View ArticlePubMedGoogle Scholar
- Guzman LM, Belin D, Carson MJ, Beckwith J: Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995, 177 (14): 4121-4130.PubMed CentralPubMedGoogle Scholar
- Morgan-Kiss RM, Wadler C, Cronan JE: Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity. Proc Natl Acad Sci USA. 2002, 99 (11): 7373-7377. 10.1073/pnas.122227599.PubMed CentralView ArticlePubMedGoogle Scholar
- Sibley MH, Raleigh EA: A versatile element for gene addition in bacterial chromosomes. Nucleic Acids Res. 2012, 40 (3): e19-10.1093/nar/gkr1085.PubMed CentralView ArticlePubMedGoogle Scholar
- Lutz R, Bujard H: Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2regulatory elements.Nucleic Acids Res 1997, 25(6):12031210.,Google Scholar
- Brosius J, Erfle M, Storella J: Spacing of the −10 and −35 regions in the tac promoter. Effect on its in vivo activity. J Biol Chem. 1985, 260 (6): 3539-3541.PubMedGoogle Scholar
- Thanbichler M, Iniesta AA, Shapiro L: A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus. Nucleic Acids Res. 2007, 35 (20): e137-10.1093/nar/gkm818.PubMed CentralView ArticlePubMedGoogle Scholar
- Meisenzahl AC, Shapiro L, Jenal U: Isolation and characterization of a xylose-dependent promoter from Caulobacter crescentus. J Bacteriol. 1997, 179 (3): 592-600.PubMed CentralPubMedGoogle Scholar
- Stephens C, Christen B, Watanabe K, Fuchs T, Jenal U: Regulation of D-xylose metabolism in Caulobacter crescentus by a LacI-type repressor. J Bacteriol. 2007, 189 (24): 8828-8834. 10.1128/JB.01342-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Curtis PD, Brun YV: Getting in the loop: regulation of development in Caulobacter crescentus. Microbiol Mol Biol Rev. 2010, 74 (1): 13-41. 10.1128/MMBR.00040-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Barnett MJ, Fisher RF: Global gene expression in the rhizobial-legume symbiosis. Symbiosis. 2006, 42 (1): 1-24.Google Scholar
- Peterson TA, Russelle MP: Alfalfa and the nitrogen cycle in the Corn Belt. J Soil Water Conserv. 1991, 46 (3): 229-235.Google Scholar
- Graham PH, Vance CP: Legumes: importance and constraints to greater use. Plant Physiol. 2003, 131 (3): 872-877. 10.1104/pp.017004.PubMed CentralView ArticlePubMedGoogle Scholar
- Khan SR, Gaines J, Roop RM, Farrand SK: Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing. Appl Environ Microbiol. 2008, 74 (16): 5053-5062. 10.1128/AEM.01098-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Pini F, Frage B, Ferri L, De Nisco NJ, Mohapatra SS, Taddei L, Fioravanti A, Dewitte F, Galardini M, Brilli M, Villeret V, Bazzicalupo M, Mengoni A, Walker GC, Becker A, Biondi EG: The DivJ, CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti. Mol Microbiol. 2013, 90 (1): 54-71.PubMed CentralPubMedGoogle Scholar
- Poysti NJ, Loewen ED, Wang Z, Oresnik IJ:Sinorhizobium meliloti pSymB carries genes necessary for arabinose transport and catabolism. Microbiology. 2007, 153 (Pt 3): 727-736. 10.1099/mic.0.29148-0.View ArticlePubMedGoogle Scholar
- Geddes BA, Oresnik IJ: Inability to catabolize galactose leads to increased ability to compete for nodule occupancy in Sinorhizobium meliloti. J Bacteriol. 2012, 194 (18): 5044-5053. 10.1128/JB.00982-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Mauchline TH, Fowler JE, East AK, Sartor AL, Zaheer R, Hosie AH, Poole PS, Finan TM: Mapping the Sinorhizobium meliloti 1021 solute-binding protein-dependent transportome. Proc Natl Acad Sci USA. 2006, 103 (47): 17933-17938. 10.1073/pnas.0606673103.PubMed CentralView ArticlePubMedGoogle Scholar
- Brggemann C, Denger K, Cook AM, Ruff J: Enzymes and genes of taurine and isethionate dissimilation in Paracoccus denitrificans. Microbiology. 2004, 150 (Pt 4): 805-816. 10.1099/mic.0.26795-0.View ArticleGoogle Scholar
- Wilson JJ, Kappler U: Sulfite oxidation in Sinorhizobium meliloti. Biochim Biophys Acta. 2009, 1787 (12): 1516-1525. 10.1016/j.bbabio.2009.07.005.View ArticlePubMedGoogle Scholar
- Tett AJ, Rudder SJ, Bourdes A, Karunakaran R, Poole PS: Regulatable vectors for environmental gene expression in Alphaproteobacteria. Appl Environ Microbiol. 2012, 78 (19): 7137-7140. 10.1128/AEM.01188-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Harrison CL, Crook MB, Peco G, Long SR, Griffitts JS: Employing site-specific recombination for conditional genetic analysis in Sinorhizobium meliloti. Appl Environ Microbiol. 2011, 77 (12): 3916-3922. 10.1128/AEM.00544-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Arango Pinedo C, Gage DJ: Plasmids that insert into the rhamnose utilization locus, rha: a versatile tool for genetic studies in Sinorhizobium meliloti. J Mol Microbiol Biotechnol. 2009, 17 (4): 201-210. 10.1159/000242446.View ArticlePubMedGoogle Scholar
- Bringhurst RM, Gage DJ: An AraC-like transcriptional activator is required for induction of genes needed for alpha-galactoside utilization in Sinorhizobium meliloti. FEMS Microbiol Lett. 2000, 188 (1): 23-27.PubMedGoogle Scholar
- Gage DJ, Long SR: α-Galactoside uptake in Rhizobium meliloti: isolation and characterization of agpA, a gene encoding a periplasmic binding protein required for melibiose and raffinose utilization. J Bacteriol. 1998, 180 (21): 5739-5748.PubMed CentralPubMedGoogle Scholar
- Schlter JP, Reinkensmeier J, Barnett MJ, Lang C, Krol E, Giegerich R, Long SR, Becker A: Global mapping of transcription start sites and promoter motifs in the symbiotic alpha-proteobacterium Sinorhizobium meliloti 1021. BMC Genomics. 2013, 14: 156-10.1186/1471-2164-14-156.View ArticleGoogle Scholar
- Bringhurst RM, Cardon ZG, Gage DJ: Galactosides in the rhizosphere: utilization by Sinorhizobium meliloti and development of a biosensor. Proc Natl Acad Sci USA. 2001, 98 (8): 4540-4545. 10.1073/pnas.071375898.PubMed CentralView ArticlePubMedGoogle Scholar
- Bringhurst RM, Gage DJ: Control of inducer accumulation plays a key role in succinate-mediated catabolite repression in Sinorhizobium meliloti. J Bacteriol. 2002, 184 (19): 5385-5392. 10.1128/JB.184.19.5385-5392.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Arango Pinedo C, Bringhurst RM, Gage DJ:Sinorhizobium meliloti mutants lacking phosphotransferase system enzyme HPr or EIIA are altered in diverse processes, including carbon metabolism, cobalt requirements, and succinoglycan production. J Bacteriol. 2008, 190 (8): 2947-2956. 10.1128/JB.01917-07.View ArticleGoogle Scholar
- Garcia PP, Bringhurst RM, Arango Pinedo C, Gage DJ: Characterization of a two-component regulatory system that regulates succinate-mediated catabolite repression in Sinorhizobium meliloti. J Bacteriol. 2010, 192 (21): 5725-5735. 10.1128/JB.00629-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Becker A, Barnett MJ, Capela D, Dondrup M, Kamp PB, Krol E, Linke B, Rberg S, Runte K, Schroeder BK, Weidner S, Yurgel SN, Batut J, Long SR, Phler A, Goesmann A: A portal for rhizobial genomes: RhizoGATE integrates a Sinorhizobium meliloti genome annotation update with postgenome data. J Biotechnol. 2009, 140 (12): 45-50. 10.1016/j.jbiotec.2008.11.006.PubMed CentralView ArticlePubMedGoogle Scholar
- Eichhorn E, van der Ploeg JR, Leisinger T: Deletion analysis of the Escherichia coli taurine and alkanesulfonate transport systems. J Bacteriol. 2000, 182 (10): 2687-2695. 10.1128/JB.182.10.2687-2695.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- van der Ploeg JR, Weiss MA, Saller E, Nashimoto H, Saito N, Kertesz MA, Leisinger T: Identification of sulfate starvation-regulated genes in Escherichia coli: a gene cluster involved in the utilization of taurine as a sulfur source. J Bacteriol. 1996, 178 (18): 5438-5446.PubMed CentralPubMedGoogle Scholar
- Masepohl B, Fuhrer F, Klipp W: Genetic analysis of a Rhodobacter capsulatus gene region involved in utilization of taurine as a sulfur source. FEMS Microbiol Lett. 2001, 205 (1): 105-111. 10.1111/j.1574-6968.2001.tb10932.x.View ArticlePubMedGoogle Scholar
- Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL: NCBI BLAST: a better web interface. Nucleic Acids Res. 2008, 36 (Web Server issue): W5-W9. 10.1093/nar/gkn201.PubMed CentralView ArticlePubMedGoogle Scholar
- Wiethaus J, Schubert B, Pfander Y, Narberhaus F, Masepohl B: The GntR-like regulator TauR activates expression of taurine utilization genes in Rhodobacter capsulatus. J Bacteriol. 2008, 190 (2): 487-493. 10.1128/JB.01510-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Seo JS, Chong H, Park HS, Yoon KO, Jung C, Kim JJ, Hong JH, Kim H, Kim JH, Kil JI, Park CJ, Oh HM, Lee JS, Jin SJ, Um HW, Lee HJ, Oh SJ, Kim JY, Kang HL, Lee SY, Lee KJ, Kang HS: The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4. Nat Biotechnol. 2005, 23 (1): 63-68. 10.1038/nbt1045.View ArticlePubMedGoogle Scholar
- Fields AT, Navarrete CS, Zare AZ, Huang Z, Mostafavi M, Lewis JC, Rezaeihaghighi Y, Brezler BJ, Ray S, Rizzacasa AL, Barnett MJ, Long SR, Chen EJ, Chen JC: The conserved polarity factor podJ1 impacts multiple cell envelope-associated functions in Sinorhizobium meliloti. Mol Microbiol. 2012, 84 (5): 892-920. 10.1111/j.1365-2958.2012.08064.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Pickering BS, Oresnik IJ: The twin arginine transport system appears to be essential for viability in Sinorhizobium meliloti. J Bacteriol. 2010, 192 (19): 5173-5180. 10.1128/JB.00206-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Bastiat B, Sauviac L, Picheraux C, Rossignol M, Bruand C:Sinorhizobium meliloti sigma factors RpoE1 and RpoE4 are activated in stationary phase in response to sulfite. PLoS One. 2012, 7 (11): e50768-10.1371/journal.pone.0050768.PubMed CentralView ArticlePubMedGoogle Scholar
- Miller JH: Experiments in Molecular Genetics. 1972, Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
- Poindexter JS: Biological properties and classification of the Caulobacter group. Bacteriol Rev. 1964, 28: 231-295.PubMed CentralPubMedGoogle Scholar
- Ely B: Genetics of Caulobacter crescentus. Methods Enzymol. 1991, 204: 372-384. 10.1016/0076-6879(91)04019-K.View ArticlePubMedGoogle Scholar
- Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. 1998, John Wiley & Sons, New York, NYGoogle Scholar
- Goodman AE, Rogers PL, Skotnicki ML: Minimal medium for isolation of auxotrophic Zymomonas mutants. Appl Environ Microbiol. 1982, 44 (2): 496-498.PubMed CentralPubMedGoogle Scholar
- Finan TM, Kunkel B, De Vos GF, Signer ER: Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol. 1986, 167 (1): 66-72.PubMed CentralPubMedGoogle Scholar
- Evinger M, Agabian N: Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells. J Bacteriol. 1977, 132 (1): 294-301.PubMed CentralPubMedGoogle Scholar
- Swings J, De Ley J: The biology of Zymomonas. Bacteriol Rev. 1977, 41 (1): 1-46.PubMed CentralPubMedGoogle Scholar
- Meade HM, Long SR, Ruvkun GB, Brown SE, Ausubel FM: Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol. 1982, 149 (1): 114-122.PubMed CentralPubMedGoogle Scholar
- Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM: Four new derivatives of the broad-host-range cloning vector pBBR1 MCS, carrying different antibiotic-resistance cassettes. Gene. 1995, 166 (1): 175-176. 10.1016/0378-1119(95)00584-1.View ArticlePubMedGoogle Scholar
- Marx CJ, Lidstrom ME: Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria. Microbiology. 2001, 147 (Pt 8): 2065-2075.View ArticlePubMedGoogle Scholar
- Quandt J, Hynes MF: Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. Gene. 1993, 127 (1): 15-21. 10.1016/0378-1119(93)90611-6.View ArticlePubMedGoogle Scholar
- Oke V, Long SR: Bacterial genes induced within the nodule during the Rhizobium-legume symbiosis. Mol Microbiol. 1999, 32 (4): 837-849. 10.1046/j.1365-2958.1999.01402.x.View ArticlePubMedGoogle Scholar
- Griffitts JS, Long SR: A symbiotic mutant of Sinorhizobium meliloti reveals a novel genetic pathway involving succinoglycan biosynthetic functions. Mol Microbiol. 2008, 67 (6): 1292-1306. 10.1111/j.1365-2958.2008.06123.x.View ArticlePubMedGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2Google Scholar
- Beck S, Marlow VL, Woodall K, Doerrler WT, James EK, Ferguson GP: The Sinorhizobium meliloti MsbA2 protein is essential for the legume symbiosis. Microbiology. 2008, 154 (Pt 4): 1258-1270. 10.1099/mic.0.2007/014894-0.View ArticlePubMedGoogle Scholar
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