The Yersinia pestis gcvB gene encodes two small regulatory RNA molecules
© McArthur et al; licensee BioMed Central Ltd. 2006
Received: 27 March 2006
Accepted: 12 June 2006
Published: 12 June 2006
In recent years it has become clear that small non-coding RNAs function as regulatory elements in bacterial virulence and bacterial stress responses. We tested for the presence of the small non-coding GcvB RNAs in Y. pestis as possible regulators of gene expression in this organism.
In this study, we report that the Yersinia pestis KIM6 gcvB gene encodes two small RNAs. Transcription of gcvB is activated by the GcvA protein and repressed by the GcvR protein. The gcvB-encoded RNAs are required for repression of the Y. pestis dppA gene, encoding the periplasmic-binding protein component of the dipeptide transport system, showing that the GcvB RNAs have regulatory activity. A deletion of the gcvB gene from the Y. pestis KIM6 chromosome results in a decrease in the generation time of the organism as well as a change in colony morphology.
The results of this study indicate that the Y. pestis gcvB gene encodes two small non-coding regulatory RNAs that repress dppA expression. A gcvB deletion is pleiotropic, suggesting that the sRNAs are likely involved in controlling genes in addition to dppA.
Yersinia pestis is the causative agent of plague, an infectious disease that results in lymphatic and blood infections . The Y. pestis genome has been sequenced [2, 3]. Y. pestis carries three plasmids of approximately 9.5, 70, and 100 kilobasepairs and each carries genes necessary for or that contribute to the pathogenicity of the bacterium . The 70 kilobasepair plasmid encodes the low-calcium response stimulon (LCRS). Components of the LCRS include Yops (secreted anti-host proteins) and a type III secretion apparatus, or Ysc. The type III secretion apparatus is responsible for the translocation of the Yops to host cells that in turn down-regulate the response of the host phagocytic cells to infection . Natural LCRS-negative mutants of Y. pestis occur, resulting in avirulence of the bacteria . Besides the three plasmids, another pathogenicity factor is pigmentation. Cells of Y. pestis adsorb hemin at 26°C but not at 37°C and are pigmented (Pgm+) and virulent. Spontaneous nonpigmented (Pgm-) mutants of Y. pestis have been isolated. The Yersiniabactin iron transport system is part of the pgm locus, and its loss results in a Pgm- mutant that is avirulent in mice unless hemin, ferrous sulfate, or ferric chloride is injected into mice along with the bacterial challenge .
Recently, a new class of molecules has been shown to regulate gene expression in bacteria, small non-coding regulatory RNAs (sRNAs). These sRNAs have gained much attention as recent genome-wide studies have identified sRNAs in a wide variety of organisms . Most of these bacterial sRNAs are between 50 and 400 nucleotides (nts) in length and play important roles in global regulation [6, 7]. Hfq is a small RNA binding protein and sRNAs in particular are targets for Hfq . Binding of these sRNAs by Hfq in some way facilitates base pairing between the sRNAs and their respective target RNAs [8, 9]. In Vibrio cholerae, sRNAs (Qrr RNAs) have been shown to regulate virulence genes  and in Brucella abortus an hfq mutation is lethal . These results suggest that sRNAs and Hfq likely play important roles in the virulence of certain Gram-negative pathogens.
Results and discussion
Identification of the Y. pestis gcvB gene
The Y. pestis gcvB gene encodes two sRNAs
The E. coli gcvB gene encodes two sRNA transcripts that are not translated in vivo . To determine if the gcvB gene in Y. pestis is functional and possibly encodes sRNAs, we initially constructed plasmid pgcvBYp+53::lacZ, carrying a transcriptional fusion of the gcvB gene at basepair (bp) +53 to lacZ. Plasmid pgcvBYp+53::lacZ and the vector alone were transformed into Y. pestis strain KIM6, the transformants grown in heart-infusion broth (HIB) + ampicillin (AP) to mid-log phase of growth and the cultures assayed for β-galactosidase activity. The KIM6 and KIM6 [pMC1403] control transformant gave 6 ± 0.3 and 6 ± 1 units of β-galactosidase activity, respectively, whereas the KIM6 [pgcvBYp+53::lacZ] transformant gave 5,985 ± 118 units of β-galactosidase activity. The results suggest that the gcvB gene is expressed in Y. pestis.
The Y. pestis gcvB gene encodes two sRNAs. Cells were grown in LB to an OD600 of ~0.5 and assayed for β-galactosidase activity . Activity is expressed in Miller units.
1 ± 0.1
369 ± 40
182 ± 7
1 ± 0.3
Regulation of the Y. pestis gcvB gene
Regulation of the Y. pestis gcvB+53::lacZ transcriptional fusion in E. coli. Cells were grown in GM media with the indicated supplements to an OD600 of ~0.5 and assayed for β-galactosidase activity . Activity is expressed in Miller units.
β-galactosidase activity for cells grown in:
GM + glycine
GM + inosine
15 ± 2
173 ± 2
6 ± 3
2 ± 1
3 ± 1
2 ± 1
620 ± 58
419 ± 13
440 ± 174
10 ± 2
140 ± 24
6 ± 2
Y. pestis gcvA encodes an activator protein for gcvB expression
The Y. pestis gcvA gene encodes an activator protein. Cells were grown in LB to an OD600 of ~0.5 and assayed for β-galactosidase activity . Activity is expressed in Miller units.
399 ± 22
254 ± 21
The Y. pestis gcvR gene encodes a repressor protein for gcvB expression
The Y. pestis gcvR gene complements an E. coli gcvR mutation. Cells were grown in GM media to an OD600 of ~0.5 and assayed for β-galactosidase activity . Activity is expressed in Miller units.
308 ± 19
384 ± 175
11 ± 1.5
14 ± 1.2
2.2 ± 0.2
2.8 ± 0.1
393 ± 8
ΔgcvA ΔgcvR/gcvA Yp gcvR Yp
6.8 ± 0.4
In E. coli, the GcvA and GcvR proteins interact to form a repressor complex [17, 18]. The above results suggest that the Y. pestis GcvR protein interacts with the E. coli GcvA protein to form a repression complex. We tested if the Y. pestis gcvA and gcvR gene products also likely form a repressor complex to control expression of an E. coli gcvB::lacZ fusion. Strain GS1131λgcvB::lacZ carries Δ gcvR ΔgcvA mutations. Strain GS1131λgcvB::lacZ was transformed with plasmid pgcvAYp-p177, pgcvRYp-p322, or both plasmids. The vectors for pgcvAYp-p177 and pgcvRYp-p322 are pACYC177 and pBR322, respectively, to insure an excess of GcvRYp versus GcvAYp. The cells were grown in GM media + appropriate antibiotics, harvested in mid-log phase of growth and assayed for β-galactosidase activity. The Y. pestis gcvA gene complemented the ΔgcvA mutation, resulting in activation of the gcvB::lacZ fusion (Table 4, line 7). The Y. pestis gcvR gene complemented the gcvR mutation, as repression of the gcvB::lacZ fusion occurred in the pgcvAYp-p177 pgcvRYp-p322 double transformant (Table 4, line 8). These results suggest that the GcvA and GcvR proteins likely interact to form a repression complex in Y. pestis. In E. coli, GcvA also activates the gcvTHP operon and GcvA + GcvR repress the operon [17, 18]. Whether the Y. pestis GcvA and GcvR proteins also regulate the Y. pestis gcvTHP operon, or have additional regulatory roles, awaits further investigation.
The Y. pestis GcvB RNAs regulate the E. coli and Y. pestis dppA genes
Regulation of E. coli and Y. pestis dppA::lacZ translational gene fusions by the Y. pestis gcvB gene. Cells were grown in LB (E. coli) or in HIB (Y. pestis) at 37°C to an OD600 ~0.5 and assayed for β-galactosidase activity . Activity is expressed in Miller units. The parent strains KIM6 and KIM6ΔgcvB grown in HIB at 37°C showed <5 units of β-galactosidase activity.
103 ± 24
554 ± 81
154 ± 32
62 ± 13
455 ± 7
Deletion of the Y. pestis gcvB gene slows growth rate and alters colony morphology
In E. coli, many genes respond to the GcvB RNAs . The pleiotropic nature of the Y. pestis gcvB deletion suggests that the Y. pestis GcvB RNAs are likely global regulators as well. Identification of the specific genes regulated by the GcvB RNAs that are responsible for the altered phenotype will allow us to test directly their involvement in virulence of the organism. In addition, the GcvB sequences and regulatory regions from bp -90 to +1, which include the putative GcvA binding sites for activation of gcvB, are 100% identical in all Yersina pestis strains, and greater than 92% identical in other Yersinia species. Thus, expression of gcvB and the regulatory mechanisms of the GcvB RNAs are likely similar in all Yersinia species.
In summary, the Y. pestis gcvB gene is activated by the GcvA protein and repressed by the GcvR protein. The gcvB gene encodes two sRNAs that have regulatory activity, repressing dppA expression. A gcvB deletion is pleiotropic, suggesting that the GcvB RNAs possibly serve as global regulators in Y. pestis.
Bacterial strains, plasmids and phage
Bacterial strains, plasmids and phage. All E. coli strains listed also carry Δ(argF-lac)U169, pheA905, thi, araD129, rpsL150, relA1, deoC1, flb5301, ptsF25 and rpsR mutations.
Single-copy translational lacZ fusion vector
Carries Y. pestis gcvB in pBR322
Carries Y. pestis gcvB in a single-copy vector
Carries Y. pestis gcvA in pACYC177
Carries Y. pestis gcvR in pACYC177
Carries Y. pestis gcvR in pBR322
Y. pestis dppA::lacZ fusion in pGS366
Carries E. coli gcvA in pACYC177
Carries E. coli gcvR in pACYC177
λgt2 with E. coli dppA::lacZ translational fusion
λgt2 with E. coli gcvB+50::lacZ transcriptional fusion
λgt2 with Y. pestis gcvB+53::lacZ transcriptional fusion
λgt2 with Y. pestis gcvB+164::lacZ transcriptional fusion
λgt2 with Y. pestis gcvB+251::lacZ transcriptional fusion
For E. coli strains, the complex medium used was LB  and the defined medium used was the minimal salts of Vogel and Bonner  supplemented with 0.4% glucose. GM media was always supplemented with 50 μg ml-1 of phenylalanine and 1 μg ml-1 of vitamin B1, since all E. coli strains carry pheA, thi mutations. Where indicated, glycine and inosine were added at 300 μg ml-1 and 50 μg ml-1, respectively. For Y. pestis strains, HIB was used . Agar was added at 1.5% to make solid media. Antibiotics were added at the following concentrations: AP, 150 μg ml-1 for multi-copy plasmids and 50 μg ml-1 for single-copy plasmids; chloramphenicol (CM), 20μ g ml-1; tetracycline (TC), 10 μg ml-1.
β-galactosidase assays were performed on mid-log phase cells (OD600~0.5) as described by Miller . Each experiment was repeated at least twice, with each sample assayed in triplicate.
Plasmid DNA was isolated using Qiagen Miniprep kits as described by the manufacturer (Qiagen). Restriction enzyme digestions and DNA ligations were carried out according to the manufacturer (New England Biolabs). DNA sequencing was performed by the University of Iowa DNA Core Facility.
PCR reactions were performed in 100 μl volumes. Each reaction mixture contained 10 μl 10 × polymerase buffer, 10 μl 10 × dNTPs (0.2 mM each), 5 μl Y. pestis DNA (~15 ng), 100 pmoles of forward and reverse primers designed specifically for each reaction, 1 μl of vent polymerase, and sterile water to bring the volume to 100 μl. PCR reactions were carried out under the following conditions: 5 min pre-incubation at 95°C, and then 30 cycles of 95°C for 30 sec, 45°C for 30 sec, and 72°C for 2 min.
RNA extraction and Northern blot analysis
Y. pestis KIM6 was grown in HIB at 30°C to an O.D.600 of 0.7, the cells collected for 1 minute in a microcentrifuge and immediately frozen at -70°C. Total cellular RNA was isolated using the MasterPure™ RNA purification kit (Epicenter). The final RNA pellet was re-suspended in water treated with diethyl pyrocarbonate and kept at -70°C. The RNA concentration was measured with a spectrophotometer at 260 nm. RNA (10 μg) was separated through a 1.5% formaldehyde gel and blotted on to a Biodyne Plus Membrane (ISC BioExpress). The blot was hybridized with a PCR generated DNA fragment from bp +1 to +198 of the Y. pestis gcvB gene and 32P-labeled using the RediprimeTM II Random Prime Labeling System (Amersham Biosciences). Hybridization of the blot was at 58°C as described .
Construction of gcvA, gcvB and gcvR plasmids
The Y. pestis gcvB gene was cloned as follows. PCR primer YP-GCVB1F has an artificial Eco RI site and is complementary to the Y. pestis KIM6 DNA sequence beginning 114 bases upstream of the gcvB transcription start site. PCR primer YP-GCVB2R has an artificial Hin dIII site and is complementary to the Y. pestis DNA sequence beginning 45 bases downstream of the gcvB transcriptional termination site t2 (Fig. 1). Following PCR amplification, using Y. pestis chromosomal DNA as template, the amplified DNA was digested with Eco RI and Hin dIII, the 400 bp fragment carrying gcvB isolated from a 1% agarose gel and ligated into the Eco RI and Hin dIII sites of plasmid pBR322 , generating plasmid pgcvBYp-p322. The Y. pestis gcvA and gcvR genes were cloned using a similar strategy. For gcvA, both the forward and reverse primers contained artificial Hin dIII sites complementary to the Y. pestis sequence beginning 111 bases upstream of the gcvA transcription start site and 349 bases downstream of the gcvA translation stop codon. For gcvR, both the forward and reverse primers contained artificial Hin dIII sites complementary to the Y. pestis sequence beginning 313 bases upstream of the gcvR transcription start site and 198 bases downstream of the gcvR translation stop codon. The PCR amplified fragments were cloned into the Hin dIII site of plasmid pACYC177 , generating plasmids pgcvAYp-p177 and pgcvRYp-p177. In a second construct of gcvR, both the upstream and downstream primers contained artificial Eco RI sites and the PCR amplified fragment was cloned into the Eco RI site of plasmid pBR322, generating plasmid pgcvRYp-p322. Each gene was sequenced at the University of Iowa DNA Core Facility to verify that no bp changes were introduced during the PCR amplification procedure.
Construction of lacZ gene fusions
Three different transcriptional gene fusions of gcvB to the lacZ gene were constructed by PCR synthesis of fragments with common Bam HI termini 128 bp upstream of the gcvB transcription start site and 3 different fusion points within gcvB. In plasmid pBYp+53::lacZ, the downstream PCR primer hybridized to the gcvB sequence beginning at bp +53 relative to the predicted transcription start site (+1) of gcvB (Fig. 1). A synthetic Hin dIII site was included at the end of the primer to allow the cloning of the 202 bpBam HI-Hin dIII fragment into the Bam HI-Hin dIII sites of the lacZ transcriptional reporter plasmid pQF50 . Plasmids pBYp+164::lacZ and pBYp+251::lacZ were constructed similarly except that the downstream primers used for PCR synthesis hybridized to the gcvB sequence beginning at bp +164 and +251 (Fig. 1), and the 313 and 400 bp fragments produced were cloned into the Bam HI-Hin dIII sites of pQF50. Each fusion was sequenced at the University of Iowa DNA Core Facility to verify that the fusions were at the correct sites and that no bp changes were introduced during the PCR amplification procedure. Each gcvB transcriptional fusion was then subcloned into plasmid pMC1403 , generating plasmids pgcvBYp+53::lacZ, pgcvBYp+164::lacZ and pgcvBYp+251::lacZ, and subsequently transferred to phage λgt2  as described , generating phage λgcvBYp+53::lacZ, λgcvBYp+164::lacZ and λgcvBYp+251::lacZ, respectively.
A single-copy Y. pestis dppAYp::lacZ translational fusion was constructed in two steps. First, a dppAYp::lacZ translational fusion was constructed using an upstream PCR primer with an Eco RI site complementary to the Y. pestis DNA sequence beginning 300 bps upstream of the dppA transcription initiation site and a downstream primer that contains an artificial Sma I site and that hybridizes to the dppA sequence after the 15th codon relative to the translation initiation site. The 611 bp dppA fragment was cloned into the Eco RI and Sma I sites of the lacZ translational reporter plasmid pMC1403. The fusion was sequenced at the University of Iowa DNA Core Facility to verify that the fusion was at the correct site and that no bp changes were introduced during the PCR amplification procedure. The dppAYp::lacZ fusion, along with the lacY and lacA genes, was then cloned into the single-copy plasmid pGS366, designated pdppAYp::lacZ.
Chromosomal deletion of gcvB
A gcvB deletion was constructed on the Y. pestis chromosome essentially as described . Y. pestis strain KIM6 was transformed with plasmid pKD46, which encodes the Red recombinase of phage λ. PCR products were then generated using two primers with 50 nt extensions that are complementary to sequences that flank the gcvB gene and 20 nt priming sequences that are complementary to the template plasmid pKD32 and that flank the CMR gene and the FLP recognition sequence . The PCR fragment was gel purified and used to transform Y. pestis KIM6 [pKD46]. The cells were plated on HIB plates with CM and CMR recombinants were selected. One CMR recombinant was single colony purified, chromosomal DNA was prepared, and PCR analysis was used to verify that the gcvB gene was deleted and replaced with the CMR marker. The pKD46 plasmid is a temperature sensitive replicon and was cured by growth at 37°C . The strain was designated KIM6Δ gcvB.
We are indebted to S. Straley for providing Y. pestis strain KIM6. This work was supported by grant GM069506 from the National Institutes of Health.
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