The Yersinia pestis gcvB gene encodes two small regulatory RNA molecules.

BACKGROUND: 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.
RESULTS: 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.
CONCLUSION: 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.

Background

Yersinia pestis is the causative agent of plague, an infectious disease that results in lymphatic and blood infections [1]. 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 [1]. 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 [4]. Natural LCRS-negative mutants of Y. pestis occur, resulting in avirulence of the bacteria [1]. 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 [1].

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 [5]. 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 [6]. 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 [10] and in Brucella abortus an hfq mutation is lethal [9]. These results suggest that sRNAs and Hfq likely play important roles in the virulence of certain Gram-negative pathogens.

The E. coli gcvB gene encodes sRNAs that are not translated in vivo [11]. A strain carrying a deletion of gcvB has constitutive synthesis of OppA and DppA, periplasmic binding proteins of the two major peptide transport systems normally repressed in cells grown in rich medium [12-14]. In addition to OppA and DppA, several other proteins were shown to increase or decrease in response to GcvB RNA levels, but the specific proteins were not identified [11]. Nevertheless, the results show that the GcvB RNAs are regulatory and possibly serve as global regulators. A computer search of the Y. pestis sequence showed that Y. pestis has a gcvB gene that shares considerable sequence homology with the E. coli gcvB sequence (Fig. 1). Thus, the GcvB RNAs from E. coli likely have functional counterparts in Y. pestis. The results of this study show that the Y. pestis gcvB gene encodes two sRNAs that, in turn, have regulatory activity. In addition, a deletion of the gcvB gene from the Y. pestis chromosome alters growth rate and colony morphology.

Figure 1

Comparison of the E. coli and Y. pestis gcvA/gcvB control regions and gcvB genes. Ec, E. coli; Yp, Y. pestis. Bases that are identical are boxed in gray. The E. coli promoter -10 and -35 sequences are underlined for gcvA [29] and overlined for gcvB [11]. Arrows indicate transcription start sites and directions of transcription of gcvA and gcvB. The GcvA binding region is indicated above the sequence [30]. The deduced Y. pestis -10 and -35 promoter sequences are underlined for gcvA and overlined for gcvB, and the deduced GcvA binding site is indicated above the sequence. Two Rho-independent transcription terminators for the E. coli and Y. pestis gcvB genes are indicated by inverted arrows. The fusion points for transcriptional fusions gcvB+53::lacZ (TF-1), gcvB+164::lacZ (TF-2) and gcvB+251::lacZ (TF-3) are indicated by vertical arrows.

Results and discussion

Identification of the Y. pestis gcvB gene

The E. coli gcvB gene is divergently transcribed from gcvA, which encodes the activator protein for gcvB expression (Fig. 1) [11]. Thus, we used computer searches of genome sequences with the gcvA gene product as a query to predict gcvB homologs in other organisms. We identified GcvB-like RNA sequences in the genera Yersinia, Salmonella, Haemophilus, Vibrio, Pasteurella, Shigella, Erwinia, Klebsiella, Photorhabdus and Actinobacillus. Despite considerable sequence variation in many of these homologs, they are predicted by the mfold algorithm [15] to assume a similar secondary structure. A comparison of three of these GcvB RNAs is shown in Fig. 2. The location of the putative Y. pestis gcvB gene adjacent to and divergent from gcvA, its 77% sequence similarity to the E. coli gcvB sequence and its predicted secondary structure make it a likely homolog of gcvB in Y. pestis. Furthermore, identical gcvB sequences can be found in all other Y. pestis strains presently in the data base, both virulent and avirulent strains.

Figure 2

Secondary structures of GcvB RNAs with 77% (Y. pestis) and 53% (V. cholerae) identity to the E. coli GcvB RNA as predicted by the mfold algorithm [15].

The Y. pestis gcvB gene encodes two sRNAs

The E. coli gcvB gene encodes two sRNA transcripts that are not translated in vivo [11]. 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 gcvB gene from Y. pestis possesses two possible Rho-independent transcription terminators, which if functional, would allow the production of two sRNAs of about 130 nts and 206 nts (Fig. 1). Three transcriptional gene fusions of the Y. pestis gcvB gene to lacZ were created to determine if these putative terminator sites function as transcription terminators in vivo. The three fusions, designated λgcvBYp+53::lacZ, λgcvBYp+164::lacZ and λgcvBYp+251::lacZ, were used to lysogenize E. coli strain GS162. The lysogens were then grown in Luria-Bertani broth (LB) [16] to mid-log phase of growth and the cultures assayed for β-galactosidase activity. About 44% of the β-galactosidase activity seen when the fusion precedes both terminators (λgcvBYp+53::lacZ) is lost when the fusion point follows terminator t1 (λgcvBYp+164::lacZ), implicating t1 as a site of transcription termination in vivo (Table 1). The remaining activity that escapes termination by t1 is not seen in GS162λgcvBYp+251::lacZ, indicating t2 also functions as a terminator in vivo (Table 1). When the 206 nts preceding terminator t2 for gcvB were analyzed, there were only short open reading frames (ORFs) that could encode polypeptides of 36 amino acids or less. These ORFs all lack good translational start sites and were not tested to determine if they encode small polypeptides. The E. coli GcvB RNAs are not translated into polypeptides [11]. Thus, we conclude that the products of the gcvB gene in Y. pestis are two sRNAs that are not translated, although the results do not completely rule out the possibility that the Y. pestis GcvB sRNAs encode small peptides. In E. coli, about 90% of the transcripts that initiate at the gcvB promoter terminate at terminator t1, and the remaining 10% terminate at terminator t2 [11]. A comparison of the E. coli and Y. pestis t1 sites shows that an additional 2 bps occur between the predicted GC-rich stem-loop structure and the run of T residues in the Y. pestis t1 site that are not present in the E. coli sequence, suggesting that the Y. pestis t1 site is likely less functional as a transcription terminator than the E. coli t1 site (Fig. 1). Nevertheless, the results are consistent with the Y. pestis gcvB gene encoding two sRNA molecules of about 130 and 206 nts and in roughly equal amounts.

Table 1

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 [16]. Activity is expressed in Miller units.

Transformant	Relevant genotype	β-Galactosidase activity
GS162	WT	1 ± 0.1
GS162λgcvBYp+53::lacZ	WT	369 ± 40
GS162λgcvBYp+164::lacZ	WT	182 ± 7
GS162λgcvBYp+251::lacZ	WT	1 ± 0.3

We used Northern blotting to confirm that the gcvB locus in Y. pestis encodes sRNA transcripts of about 130 and 206 nts. Two small RNA molecules were detected in RNA isolated from Y. pestis KIM6 grown in HIB medium using a probe specific for the gcvB locus (Fig. 3). These results are consistent with the in vivo results with the Y. pestis gcvB transcriptional fusions.

Figure 3

Northern blot analysis of GcvB from Y. pestis strain KIM6. Total cell RNA was isolated from strain KIM6 grown in HIB at 30°C to an O.D.600 of 0.7 and probed with a 32P-labeled GcvB specific DNA probe as described in Methods. Two gcvB transcripts of about 206 and 130 nucleotides identified are indicated with arrows. Their sizes were determined based on their mobilities relative to the mobility of the E. coli GcvB RNA and 5S rRNAs (not shown).

Regulation of the Y. pestis gcvB gene

The E. coli gcvB gene is activated by GcvA in the presence of glycine and repressed by GcvA + GcvR in its absence; this repression is enhanced by the addition of purines [11]. The regulation of the Y. pestis gcvB gene was tested with respect to the effects of glycine and purine supplementation to the growth medium and with respect to the GcvR and GcvA proteins and the GcvB RNAs. For these experiments we used the λgcvBYp+53::lacZ fusion to lysogenize appropriate E. coli host strains. The lysogens were grown in glucose minimal (GM) or GM supplemented with glycine or inosine to mid-log phase of growth and assayed for β-galactosidase levels. In the wild-type (WT) GS162λgcvBYp+53::lacZ lysogen, the addition of glycine to GM growth medium resulted in an 11.5-fold induction of β-galactosidase expression, whereas the addition of the purine inosine resulted in a 2.5-fold repression below the unsupplemented GM level (Table 2, line 1). In the gcvA mutant lysogen GS1118λgcvBYp+53::lacZ, the β-galactosidase levels were low and non-inducible by glycine (Table 2, line 2). The addition of inosine had no significant effect in the gcvA mutant strain. In the gcvR mutant lysogen GS1053λgcvBYp+53::lacZ, the β-galactosidase levels are constitutively high under all three growth conditions (Table 2, line 3). The results suggest that activation of the Y. pestis gcvB gene requires the GcvA protein and that repression requires the GcvR protein. Whether the negative regulation by GcvR requires a direct interaction of GcvR with GcvA as in E. coli [17,18] awaits further investigation. Furthermore, there appears to be no autoregulation of gcvB by its own sRNA products as the gcvB mutant lysogen GS1144λgcvBYp+53::lacZ shows normal regulation of the gcvBYp+53::lacZ fusion (Table 2, line 4).

Table 2

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 [16]. Activity is expressed in Miller units.

Lysogen	Relevant genotype	β-galactosidase activity for cells grown in:
		GM	GM + glycine	GM + inosine
GS162λgcvBYp+53::lacZ	WT	15 ± 2	173 ± 2	6 ± 3
GS1118λgcvBYp+53::lacZ	ΔgcvA	2 ± 1	3 ± 1	2 ± 1
GS1053λgcvBYp+53::lacZ	gcvR	620 ± 58	419 ± 13	440 ± 174
GS1144λgcvBYp+53::lacZ	ΔgcvB	10 ± 2	140 ± 24	6 ± 2

Y. pestis gcvA encodes an activator protein for gcvB expression

Since activation of the Y. pestis gcvBYp+53 fusion in E. coli was dependent on GcvA (Table 2), we determined if the Y. pestis gcvA gene also encodes an activator protein for gcvB expression. We assumed this would be the case, as the E. coli and Y. pestis GcvA proteins are 88% identical at the amino acid sequence level. The Y. pestis gcvA gene was cloned into plasmid pACYC177 and tested for its ability to complement an E. coli gcvA mutant. The E. coli strain GS1132 carries a deletion of the gcvA gene [11]. This strain was lysogenized with an E. coli λgcvB::lacZ transcriptional gene fusion and subsequently transformed with the control plasmid pACYC177, or pACYC177 carrying either the E. coli or the Y. pestis gcvA gene. The cells were grown in LB to mid-log phase of growth and assayed for β-galactosidase activity. As reported previously [11], expression of the E. coli gcvB::lacZ fusion was increased about 400-fold in the presence of the E. coli gcvA gene (Table 3, line 3). The Y. pestis gcvA gene also complemented the E. coli ΔgcvA strain, restoring gcvB::lacZ expression to nearly the same level as seen with the E. coli gcvA gene (Table 3, line 4). These results show that the Y. pestis gcvA gene codes for an activator protein capable of activating expression of an E. coli gcvB::lacZ fusion.

Table 3

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 [16]. Activity is expressed in Miller units.

Lysogen	Relevant genotype	β-Galactosidase activity
GS1132λgcvB::lacZ	Δ(gcvA gcvB)	<1
GS1132λgcvB::lacZ[pACYC177]	Δ(gcvA gcvB)	<1
GS1132λgcvB::lacZ[pGS335]	Δ(gcvA gcvB)/gcvAEc	399 ± 22
GS1132λgcvB::lacZ[pgcvAYp-p177]	Δ(gcvA gcvB)/gcvAYp	254 ± 21

The Y. pestis gcvR gene encodes a repressor protein for gcvB expression

Since deletion of the gcvR gene in E. coli results in constitutive expression of the Y. pestis gcvBYp+53 fusion (Table 2), we tested if the Y. pestis gcvR gene encodes a repressor for gcvB expression. We assumed this would be the case, as the E. coli and Y. pestis GcvR proteins are 75% identical at the amino acid sequence level. The E. coli strain GS1053 carries a Tn10 element inserted into the gcvR gene [19]. This strain was lysogenized with an E. coli λgcvB::lacZ+50 transcriptional gene fusion [11] and subsequently transformed with the control plasmid pACYC177, or pACYC177 carrying either the E. coli gcvR gene or the Y. pestis gcvR gene. The cells were grown in GM media to mid-log phase of growth and assayed for β-galactosidase activity. Expression of the E. coli gcvB::lacZ fusion is constitutive in the absence of a functional GcvR protein (Table 4, lines 1 and 2). The gcvB::lacZ fusion, however, was repressed in the presence of either pGS601, carrying E. coli gcvR, or pgcvRYp-p177, carrying Y. pestis gcvR (Table 4, lines 3 and 4).

Table 4

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 [16]. Activity is expressed in Miller units.

Lysogen	Relevant genotype	β-Galactosidase activity
GS1053λgcvB::lacZ	GcvR	308 ± 19
GS1053λgcvB::lacZ[pACYC177]	GcvR	384 ± 175
GS1053λgcvB::lacZ[pGS601]	gcvR/gcvREc	11 ± 1.5
GS1053λgcvB::lacZ[pgcvRYp-p177]	gcvR/gcvRYp	14 ± 1.2
GS1131λgcvB::lacZ	ΔgcvA ΔgcvR	2.2 ± 0.2
GS1131λgcvB::lacZ[pgcvRYp-p322]	ΔgcvA ΔgcvR/gcvRYp	2.8 ± 0.1
GS1131λgcvB::lacZ[pgcvAYp-p177]	ΔgcvA ΔgcvR/gcvAYp	393 ± 8
GS1131λgcvB::lacZ[pgcvAYp-177 pgcvRYp-p322]	ΔgcvA ΔgcvR/gcvAYp gcvRYp	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

The E. coli gcvB gene negatively regulates the dppA and oppA genes [11]. In addition, many other genes were shown to be either negatively or positively regulated by the GcvB RNAs [11]. Thus, the E. coli GcvB RNAs are likely global regulators of gene expression. Y. pestis has homologs of dppA and oppA. To determine if the Y. pestis GcvB RNAs are regulatory, we transformed an E. coli Δ gcvB λdppA::lacZ lysogen with pgcvBYp-p322, the transformant and the parent lysogen were grown in LB to mid-log phase of growth and assayed for β-galactosidase levels. As expected, deletion of gcvB caused an increase in dppA::lacZ expression (Table 5, line 2). However, pgcvBYp-p322 complemented the E. coli Δ gcvB mutation, repressing the E. coli dppA::lacZ fusion (Table 5, line 3). Thus, the Y. pestis GcvB RNAs regulate the E. coli dppA::lacZ fusion. We then tested the regulatory activity of the GcvB RNAs in Y. pestis directly. A single-copy plasmid carrying a Y. pestis dppA::lacZ fusion was used to transform Y. pestis strain KIM6 and KIM6Δ gcvB. The transformants were grown in HIB + AP to mid-log phase of growth and assayed for β-galactosidase levels. Deletion of the gcvB gene resulted in a 7.3-fold increase in dppA::lacZ expression (Table 5, compare lines 4 and 5). The results suggest that the Y. pestis GcvB RNAs are regulatory molecules. However, the mechanism of GcvB RNA repression of dppA has not been determined. Although there is a region of 13–14 nucleotides in the Y. pestis GcvB RNA that can potentially base-pair with both the E. coli and Y. pestis dppA mRNAs near their ribosome binding sites, further studies are necessary to determine if base-pairing of GcvB RNA and dppA mRNA is part of the regulatory mechanism. Furthermore, in E. coli, the 206 nucleotide GcvB RNA is required for repression of oppA and dppA [11]. We are constructing a plasmid that will only produce the 130 nucleotide Y. pestis GcvB RNA to determine whether the 130 or 206 nucleotide RNA species is required for activity in Y. pestis.

Table 5

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 [16]. 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.

Lysogen	Relevant genotype	β-Galactosidase activity
GS162λdppAEc::lacZ	WT	103 ± 24
GS1144λdppAEc::lacZ	ΔgcvB	554 ± 81
GS1144λdppAEc::lacZ[pgcvBYp-p322]	ΔgcvB/gcvBYp	154 ± 32
KIM6[pdppAYp::lacZ]	WT	62 ± 13
KIM6ΔgcvB[pdppAYp::lacZ]	ΔgcvB	455 ± 7

Deletion of the Y. pestis gcvB gene slows growth rate and alters colony morphology

The KIM6Δ gcvB strain routinely gave smaller colonies on HIB plates than the parent KIM6 strain. Thus, we investigated the growth of KIM6Δ gcvB to determine the effect of the ΔgcvB mutation on growth rate. The parent strain KIM6, KIM6ΔgcvB and KIM6ΔgcvB [pgcvBYp-sc] were grown in HIB broth at 37°C. The generation times were then calculated. The KIM6 generation time at 37°C was 135 ± 15 minutes whereas KIM6ΔgcvB had a generation time of 194 ± 20 minutes (Fig. 4). The presence of pgcvBYp-sc in KIM6ΔgcvB complemented the gcvB deletion, as the generation time was reduced to 155 ± 8 minutes, close to the generation time of strain KIM6. Thus, deletion of the gcvB gene impairs the ability of Y. pestis to grow as well as the parent strain on either solid media or in liquid media. This is in contrast to E. coli gcvB deletion mutants that have no observable phenotype. The KIM6Δ gcvB strain also showed a different colony morphology from WT KIM6. WT KIM6 colonies appear smooth and sticky, whereas the KIM6Δ gcvB colonies appear dry and compact. The presence of pgcvBYp-sc in KIM6ΔgcvB again complemented the gcvB deletion, as the phenotype was restored back to the WT colony morphology.

Figure 4

Effects of the ΔgcvB mutation on Y. pestis growth rates. Y. pestis strains KIM6 (◆), KIM6ΔgcvB (■), and KIM6ΔgcvB[pgcvBYp-sc] (▲) were grown in HIB (+ AP for the pgcvBYp-sc transformant) at 37°C. The experiment was repeated three times. The curves show the results of a representative experiment.

In E. coli, many genes respond to the GcvB RNAs [11]. 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.

Conclusion

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.

Methods

Bacterial strains, plasmids and phage

Bacterial strains, plasmids and phage used in this study are listed in Table 6 or are described in the text.

Table 6

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.

Strains/plasmids/phage	Relevant genotype	Source/reference
Strains*
GS162	WT	This laboratory
GS1053	gcvR::Tn10	[19]
GS1118	ΔgcvA:ΣaadA	This laboratory
GS1131	ΔgcvA:ΣaadA ΔgcvR:ΣKNR	[11]
GS1132	Δ(gcvA gcvB):ΣaadA	[11]
GS1144	ΔgcvB:ΣCMR	This laboratory
KIM6	lcr-	[31]
KIM6ΔgcvB	ΔgcvB:ΣCMR	This study
Plasmid
pGS366	Single-copy translational lacZ fusion vector	This laboratory
pgcvBYp-p322	Carries Y. pestis gcvB in pBR322	This study
pgcvBYp-sc	Carries Y. pestis gcvB in a single-copy vector	This study
pgcvAYp-p177	Carries Y. pestis gcvA in pACYC177	This study
pgcvRYp-p177	Carries Y. pestis gcvR in pACYC177	This study
pgcvRYp-p322	Carries Y. pestis gcvR in pBR322	This study
pdppAYp::lacZ	Y. pestis dppA::lacZ fusion in pGS366	This study
pGS335	Carries E. coli gcvA in pACYC177	This lab
pGS601	Carries E. coli gcvR in pACYC177	This lab
Phage
λdppA::lacZ	λgt2 with E. coli dppA::lacZ translational fusion	[11]
λgcvB::lacZ	λgt2 with E. coli gcvB+50::lacZ transcriptional fusion	[11]
λgcvBYp+53::lacZ	λgt2 with Y. pestis gcvB+53::lacZ transcriptional fusion	This study
λgcvBYp+164::lacZ	λgt2 with Y. pestis gcvB+164::lacZ transcriptional fusion	This study
λgcvBYp+251::lacZ	λgt2 with Y. pestis gcvB+251::lacZ transcriptional fusion	This study

Media

For E. coli strains, the complex medium used was LB [16] and the defined medium used was the minimal salts of Vogel and Bonner [20] 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 [21]. 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), 20μg ml-1; tetracycline (TC), 10 μg ml-1.

β-galactosidase assays

β-galactosidase assays were performed on mid-log phase cells (OD600~0.5) as described by Miller [16]. Each experiment was repeated at least twice, with each sample assayed in triplicate.

DNA manipulation

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

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 [22].

Construction of gcvA, gcvB and gcvR plasmids

The Y. pestis gcvB gene was cloned as follows. PCR primer YP-GCVB1F has an artificial EcoRI 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 HindIII 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 EcoRI and HindIII, the 400 bp fragment carrying gcvB isolated from a 1% agarose gel and ligated into the EcoRI and HindIII sites of plasmid pBR322 [23], 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 HindIII 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 HindIII 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 HindIII site of plasmid pACYC177 [24], generating plasmids pgcvAYp-p177 and pgcvRYp-p177. In a second construct of gcvR, both the upstream and downstream primers contained artificial EcoRI sites and the PCR amplified fragment was cloned into the EcoRI 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 BamHI 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 HindIII site was included at the end of the primer to allow the cloning of the 202 bpBamHI-HindIII fragment into the BamHI-HindIII sites of the lacZ transcriptional reporter plasmid pQF50 [25]. 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 BamHI-HindIII 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 [26], generating plasmids pgcvBYp+53::lacZ, pgcvBYp+164::lacZ and pgcvBYp+251::lacZ, and subsequently transferred to phage λgt2 [27] as described [11], 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 EcoRI 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 SmaI 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 EcoRI and SmaI 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 [28]. Y. pestis strain KIM6 was transformed with plasmid pKD46, which encodes the Red recombinase of phage λ[28]. 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 [28]. 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 [28]. The strain was designated KIM6Δ gcvB.

Authors' contributions

SM carried out most of the genetic experiments and wrote the first draft of the manuscript. SP carried out the genetic experiments with gcvR and also performed the Northern analysis. GS carried out the computer search to identify putative gcvB genes in other organisms and was the principal investigator and supervised the project.