Journal of Bacteriology, November 2003, p . 6728-6731, Vol . 185, No . 22

The yabJ gene is located downstream from purR and overlaps it by 4 bp, suggesting that the two genes are translationally coupled . YabJ belongs to an uncharacterized protein family (PROSITE accession no . PS01094) . Published data concerning the role of yabJ in PurR function appear conflicting . The results of Weng et al . (11) showed no effect of yabJ inactivation on regulation of the pur operon . However, work by Weng and Zalkin (12) and by our laboratory (5) suggested that yabJ is required for repression of two PurR targets, the pur operon and single-gene purine biosynthetic operon purA, respectively . The recent results of Saxild et al . (6) showed no difference in expression of either purA or another PurR target gene, glyA, between a yabJ mutant and wild-type B . subtilis, which is consistent with the observation of Weng et al . (11) .

In the present study, we provide data that conclusively establish PRPP as a regulator of PurR function in vivo . In addition, we show that the orientation of the marker gene downstream from purR in the yabJ mutant strain used in our earlier work (5) has a strong influence on regulation of purA .

Influence of Nm^r orientation on regulation of purA. The bacterial strains and plasmids used in this work are listed in Table 1 . purR6H is the purR gene with an additional six histidine codons in its 3' end . The same nucleotide sequence has been used in the overexpression and purification of PurR and its mutant forms in our previous work (5) . The integration vector pN6H2 was previously used to construct the yabJ mutant strain N6H (5) . In N6H, the neomycin resistance marker, Nm^r, is oppositely oriented with respect to purR6H (Fig . 1) . To test the effect of the Nm^r orientation on the function of purR6H, the marker was reversed by excision with XbaI and subsequently rejoined with the remaining XbaI vector fragment . The orientation of the Nm^r gene was verified by restriction digestion . The resulting vector, pUN6H, was integrated into the chromosome of PAL1 (Fig . 1) by a double-crossover type of homologous recombination in the same manner as pN6H2 in the previous work (5), generating new yabJ mutant strain UN6H (Fig . 1) . The integration was verified by PCR .

 
The strains PAL1 (wild-type purR and yabJ), N6H, and UN6H were assayed for purA promoter activity with the integrated purA-lucGR promoter-reporter system (5) based on luciferase encoded by lucGR . Overnight cultures were grown in minimal medium (2) supplemented with 1 mM adenine, 1 mM guanosine, or both adenine and guanosine (1 mM each) or with no added purine compounds . The medium contained either chloramphenicol (strain PAL1) or neomycin (strains N6H and UN6H) at 5 µg/ml . One milliliter of overnight culture was used to inoculate 50 ml of the medium described above . At an A₆₅₀ of 0.2, the culture was diluted 1:50 and grown again to an A₆₅₀ of 0.2 . A 1-ml sample was removed and assayed for luciferase activity as previously described (5), with the following exceptions: bacterial cells were lysed with 2x cell culture lysis reagent (Promega) containing 2 mg of lysozyme/ml and 5 mg of bovine serum albumin/ml, a 50-µl aliquot of lysate was mixed with 100 µl of luciferase assay reagent (Promega), and the maximum light output was recorded and converted to light units with the standard curve obtained by assaying known amounts of QuantiLum recombinant luciferase (Promega) . The results are expressed as light units per 10⁸ CFU .

The levels of luciferase activity in the constructed strains are given in Table 2 . The data show that, in the yabJ mutant strain N6H, the basal expression was about sixfold higher than in the PAL1 strain . There was no significant repression by addition of adenine, which is consistent with the earlier results (5) . Induction by addition of guanosine was not as clear as in our previous work, probably due to slightly different growth protocols (5) . Nevertheless, the results show that the phenotype of N6H is clearly distinct from that of PAL1 . In contrast, both the basal expression and regulation in UN6H (yabJ mutant with reversed Nm^r gene) were similar to those of PAL1, which indicates the importance of the Nm^r orientation to regulation of purA . The results also verify that the six His residues in the carboxyl terminal of PurR do not affect the PurR function in vivo .

 
The possibility that UN6H would permit normal regulation of purA by producing a neomycin resistance protein-YabJ fusion protein substituting functionally for intact YabJ is very small since the construct has several stop codons in each of the three forward frames between the Nm^r gene and the yabJ gene with the first 38 codons deleted . However, it is possible that Nm^r insertion introduces an alternative initiation codon for the truncated yabJ, resulting in functional YabJ (provided that a corresponding mRNA is produced) . To exclude this possibility, two strains having a yabJ deletion different from that of UN6H were constructed . First, the Nm^r gene of pBEST 501 (3) was inserted into the SmaI site of pGEM-7Zf(+) . Next, a chromosomal fragment of PAL1 from 54123 to 55430 (numbered according to the B . subtilis complete genome sequence, section 1; EMBL accession no . Z99104) was amplified by PCR and inserted into SacI and BamHI sites . Next, a 117-bp in-frame deletion was introduced into yabJ by amplifying a chromosomal fragment of PAL1 from 55548 to 56200 and inserting this fragment into a BamHI site . In the construct, the BamHI site replaces the deleted 117-bp fragment of yabJ . The orientation of the insert was verified by restriction digestion . Finally, a chromosomal fragment from 55741 to 56435 was inserted into XhoI and SphI sites . The Nm^r gene in the resulting vector, pTMP1, was oppositely oriented with respect to purR . The orientation of the Nm^r gene was reversed as in the construction of pUN6H, to get the vector pTMP2 . pTMP1 and pTMP2 were digested by SphI and SacI and integrated into PAL1 by a double-crossover type of homologous recombination, producing strains TMP1 and TMP2, respectively (Fig . 1) . The purA promoter activity was determined as described above . The results show that TMP2 is regulated similarly to PAL1 (Table 2) . It is highly unlikely that both UN6H and TMP2 could produce a protein substituting for YabJ functionally . Since the putative binding site is located between the subunits of the homotrimer of YabJ (9), most probably neither of the constructs could produce a protein with YabJ activity . Moreover, TMP1, which is identical to TMP2 except for the orientation of Nm^r gene (Fig . 1), shows significantly weaker regulation than TMP2 (Table 2) . This further supports the fact that the orientation of the Nm^r, rather than disruption of yabJ, affects the regulation of purA . The results explain our previous data (5) and also the results of Weng and Zalkin (12), who used the same integration vector as we had, pNPR1 (Nm^r gene opposite to purR) .

Effect of the Nm^r orientation on expression of PurR. To study the effect of Nm^r orientation on regulation of purA in more detail, the level of the PurR protein was examined by immunoblotting . The His-tagged PurR protein used for immunization was overexpressed and purified by affinity chromatography as previously described (8) with the following exceptions: Escherichia coli BL21(DE3) (Novagen) was used for overexpression, the ÄKTA fast protein liquid chromatography system with a 5-ml HiTrap chelating column (Amersham Pharmacia) loaded with Ni²⁺ ions was used for affinity chromatography, and bound PurR was eluted with a 100-ml gradient of 0 to 0.3 M imidazole . The 50-ml cultures of PAL1, N6H, UN6H, and NMW (negative control) were grown as described above and harvested at an A₆₅₀ of 0.2 . The cells were resuspended in 2 ml of 50 mM Tris-Cl, pH 8.0, and sonicated for four 10-s bursts . The crude extract was prepared by centrifuging the lysate at 20,000 x g and 4°C for 30 min, and the protein concentration in the supernatant was determined by the method of Bradford (1) . Along with 1 ng of purified PurR, 0.23 µg of total protein in the crude extract from each culture was run on a sodium dodecyl sulfate-8-to-25% polyacrylamide gradient PhastGel (Amersham Pharmacia) and electroblotted onto a Hybond-P polyvinylidene difluoride membrane (Millipore) by a PhastTransfer apparatus (Amersham Pharmacia) according to the manufacturer's instructions . PurR was detected by using PurR-immunized rabbit serum produced at Eurogentec and the horseradish peroxidase-based SuperSignal West Pico rabbit immunoglobulin G detection kit (Pierce Biotechnology) . Western blocking reagent (Roche) was used at a concentration of 5% to block the membrane . The blot was exposed on Hyperfilm ECL (Amersham Pharmacia) . The results of immunoblotting are shown in Fig . 2 . Antiserum against PurR showed some cross-reactivity to proteins other than PurR . Nevertheless, no cross-reacting protein of the size of PurR could be detected in the crude extract of purR mutant strain NMW, indicating that the band present in all samples except NMW represents merely PurR . The PurR levels of strains PAL1 and UN6H were similar . In contrast, N6H had significantly lower PurR level, suggesting that the oppositely oriented Nm^r gene disturbs PurR expression . To determine the relative PurR level with respect to that for the PAL1 strain, total protein amounts of 0.1, 0.05, 0.025, and 0.0125 µg of PAL1 extract, 0.05 and 0.02 µg of UN6H extract, 0.1 µg of N6H extract, and 0.5 ng of purified PurR were assayed by immunoblotting as described above, and the densities of the bands representing PurR were measured by using an MCID M5 image analyzer (Imaging Research) . Calculated from the measured densities, the PurR levels of UN6H and N6H with respect to that of PAL1 were approximately 3:4 and 1:4, respectively .

 
In vivo effect of mutations at the PurR PRPP binding motif. The B . subtilis PurR is a member of the homologous PRT family and contains a PRPP binding sequence motif that is conserved in all members of this family (10) . Two acidic residues (Asp-203 and Asp-204 in PurR) are conserved in the PRPP binding motifs of the homologous PRT family, except uracil phosphoribosyltransferase . These acidic side chains form hydrogen bonds with the ribose hydroxyl groups (10) . The PRPP inhibition of PurR binding to the control region of purA has been previously determined for wild-type PurR and for mutant proteins PurR D203A and PurR D204A . In these experiments, these two mutant proteins each bound to the purA operator DNA with affinity similar to that for the wild type . Unlike that of the wild type, the binding of both PurR mutant proteins was relatively insensitive to inhibition by PRPP . The binding of the PurR D204A repressor to DNA was more resistant to inhibition by PRPP than was the binding of PurR D203A (5) . To examine the effect of mutations in the PRPP binding site of PurR in vivo, the orientation of the Nm^r gene in the integration vectors pN3A2 and pN4A2 (which are identical to pN6H2 except for the PurR mutations D203A and D204A, respectively) was reversed by the same method as with pN6H2 and the resulting vectors, pUN3A and pUN4A, were integrated into the PAL1 strain in the same manner as pUN6H, generating strains UN3A and UN4A, respectively . The integration and presence of the mutation were verified by PCR and sequencing . UN3A and UN4A were assayed for promoter activity as described above .

The data in Table 2 show that, in the absence of purine compounds, the purA promoter activity in UN3A and UN4A, the strains that express PurR mutant proteins resistant to PRPP inhibition, was about 1/10 that in the wild-type purR strain, PAL1 . In these two mutants, addition of adenine repressed purA by two- to threefold, whereas in PAL1 there was about fivefold repression of purA by adenine . On the other hand, addition of guanosine increased promoter activity by approximately the same amount as in PAL1 . The data are consistent with in vitro experiments showing that, although severely impaired, the inhibitory effect of PRPP was not completely lost in the mutant proteins PurR D203A and PurR D204A (5) .

The generation time of UN3A and UN4A cultures containing no adenine was about twice as long as the generation time of wild-type cultures or cultures containing adenine (data not shown) . This is most likely due to constant repression of purA, which is essential for both de novo synthesis of AMP and interconversion of GMP to AMP (4) .

Given both the inhibition of PRPP to the binding of PurR to its control region (11) and the effect of purine additions to the growth medium on intracellular PRPP concentration (7), it is reasonable to hypothesize that PRPP is a regulator of PurR in vivo . However, data supporting this hypothesis have been inconclusive to date (5, 12) . The data in Table 2 show clearly that a mutation in the purR gene resulting in defective inhibition by PRPP of PurR binding causes strong repression by PurR in vivo, proving that PRPP regulates PurR function in vivo .

Role of yabJ. The yabJ gene is located downstream from purR and overlaps it by 4 bp, suggesting that the two genes are translationally coupled . Therefore, it is tempting to speculate that yabJ has a role in purine metabolism . The previous results indeed suggest that yabJ is required for repression by PurR (5, 12) . However, the results of Weng et al . (11) and Saxild et al . (6) did not support the regulatory role of yabJ . This raised the possibility that integration itself has an effect on regulation of purA, regardless of yabJ . To examine the effect of Nm^r gene orientation, the gene was reversed to the same orientation as purR in the yabJ mutants . As can be seen from the data presented in Table 2, yabJ mutant strains had phenotypes similar to that of the wild-type strain when the Nm^r gene was at the same orientation as purR . Furthermore, the oppositely oriented Nm^r gene disturbed PurR expression, as can be seen in Fig . 2 . Thus, the previous results regarding the role of yabJ in regulation of purA (5) and the pur operon (12) were most likely due to the orientation of the marker, resulting in defective expression of PurR and hence impaired regulation of purA .

The present work conclusively shows for the first time that PRPP is a regulator of PurR function in vivo . Mutations in the purR gene resulting in defective PRPP inhibition of binding to the purA control region caused strong repression in all growth conditions . We also confirmed that there is currently no evidence of involvement of YabJ in regulation by PurR . However, given the close connection between purR and yabJ, one should not exclude the possibility that YabJ has a yet-unidentified role in purine metabolism .

This work was supported by the Finnish Ministry of Education and the Academy of Finland .

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