Bacillus subtilis possesses two isogenes encoding glutamate racemases, the poly-γ-glutamate synthesis-linking Glr enzyme and the YrpC isozyme, and produces abundant amounts of the Glr enzyme. The YrpC isozyme, but not the Glr enzyme, was found to influence the activity of DNA gyrase, as did the MurI-type glutamate racemase of Escherichia coli, which is involved in peptidoglycan synthesis during cell division.
DNA gyrase (i.e. bacterial DNA topoisomerase II) participates in various types of DNA processing, such as DNA replication and gene expression . It is structurally and functionally conserved among almost all bacteria and the strict modulation of its intracellular activity is crucial for proper cell division. The investigation of DNA gyrase modulators, therefore, increases the understanding of the mechanism of cell division. In order to search for a DNA gyrase modulator, the phenotypic analyses of a clone where a gene of interest is forcibly expressed and of a mutant defective in the interesting gene are available . Escherichia coli is the most suitable host for the study of DNA gyrase modulators, since it offers ease of genetic manipulation  and there are various assays for its intercellular DNA topoisomerases [1,4–6]. A recent study showed that the E. coli glutamate racemase functions as an endogenous inhibitor for DNA gyrase .
Glutamate racemase is also ubiquitously inherited in bacteria. It is involved in the synthesis of peptidoglycans (alternatively, mureins)  and is thus named MurI. Its activity, however, usually cannot be detected in bacteria, except in some lactobacilli and bacilli . Biochemical studies suggest that MurI is strictly controlled so that it can operate (or be produced) only during peptidoglycan synthesis in cell division [2,10,11]. Many bacteria including E. coli possess only one glutamate racemase gene, murI, whereas Bacillus subtilis has two glutamate racemase genes, namely glr and yrpC. In cell growth of the MurI-overproducing E. coli clone, abnormal prolongation of the lag phase, in which most of machineries essential for DNA replication would be synthesized , has been observed [2,7]. Similarly, the enforced production of the yrpC gene product, YrpC, resulted in the suppression of the proliferation of the E. coli clone cells [11,12]. In contrast, the production of the glr gene product, Glr, did not affect the growth rate of the E. coli clone [9,11]. Glr is associated with the supply of d-glutamate, the important component of both poly-γ-glutamate  and peptidoglycans in B. subtilis, and YrpC has been characterized as the glutamate racemase isozyme . Unlike Glr, it is difficult to purify YrpC directly from B. subtilis cells due to its limited production. These findings implied that a certain important function other than the d-glutamate supply, which has been found in the MurI-type glutamate racemase , is probably conserved in YrpC but not in Glr.
In this study, we examined the in vivo effects of YrpC on DNA processing and then compared with those of Glr. This paper also describes inhibition of the activity of DNA gyrase by the YrpC isozyme.
2Materials and methods
Supercoiled pBR322, calf thymus DNA topoisomerase I, and isopropyl-β-d-thiogalactopyranoside (IPTG) were purchased from Takara Shuzo, Kyoto, Japan. A protein assay kit was obtained from Bio-Rad, CA, USA. Relaxed pBR322 was prepared from supercoiled pBR322 with the DNA topoisomerase I by the method of Ferro et al. . DNA gyrase was prepared as described previously . All other chemicals were of analytical grade.
2.2Bacteria and plasmids
E. coli JM109 was purchased from Takara Shuzo, Japan. A vector plasmid pTrc99A (an E. coli expression vector having the Trc promoter operating in the presence of IPTG) were obtained from Amersham Pharmacia Biotech, Buckingham, UK. Both plasmids pBSGR2 (a pTrc99A derivative carrying the B. subtilis glr gene) and pYRPC1 (a pTrc99A derivative carrying the B. subtilis yrpC gene) were constructed according to the strategies reported previously [9,11]. The E. coli JM109 clones harboring pTrc99A, pBSGR2, and pYRPC1 were used as a negative control, the Glr overproducer, and the YrpC overproducer, respectively.
The E. coli clones were inoculated in 10 ml of Luria–Bertani broth  containing only ampicillin (final concentration 50 μg ml−1) or both ampicillin and IPTG (1 mM) and cultured at 37°C until the turbidity at 600 nm of the culture broth reached 2.1.
2.4Measurement of plasmid copy number
Plasmid copy numbers were determined densitometrically with a Digital Science EDAS 120 LE system (Gibco BRL, Grand Island, NY, USA) as described previously . The whole DNA was isolated by the method of Saito and Miura  and the plasmid DNA was prepared by the alkaline-sodium dodecyl sulfate method . The plasmid copy number was defined to be one when 1 ng of plasmid DNA was contained in 1 μg of whole DNA on the basis of the difference in molecular sizes between the plasmid DNA used and the chromosomal DNA of E. coli.
2.5Determination of plasmid superhelicity
Plasmid DNAs (5 μg) were electrophoresed in 0.9% (w/v) agarose gel (14×14×0.5 cm) submerged in a TPE buffer [90 mM Tris–phosphate, 2 mM ethylenediamine tetraacetic acid (EDTA), pH 8.0] containing chloroquine (13 μg ml−1) at 4 V cm−1 at 25°C for 14 h (more negatively supercoiled DNAs migrated more rapidly under the conditions ). The resulting gel was stained with ethidium bromide (EtBr) (1 μg ml−1).
2.6Enzyme and protein assays
DNA gyrase was assayed as follows. The assay mixture (100 μl) comprised 10 μmol of Tris–HCl (pH 8.0), 7 μmol of KCl, 1 μmol of MgCl2, 0.5 μmol of ATP, 0.5 μmol of spermidine hydrochloride, 0.5 μmol of dithiothreitol (DTT), 10 nmol of EDTA, 10 μg of bovine serum albumin (BSA), 0.5 μg of relaxed pBR322, and enzyme. The enzyme was replaced with water in a blank. The reaction was essentially performed at 37°C for 1 h and terminated by the phenol–chloroform extraction . The reaction mixture was concentrated to 20 μl and was then subjected to agarose gel electrophoresis without EtBr  in order to separate supercoiled species (as the reaction product) from relaxed species (as the substrate). DNAs in the gels were visualized by EtBr staining. The activity was assessed from both an increase in the density of bands corresponding to the supercoiled pBR322 thus formed and a decrease in that of the relaxed substrate. On the other hand, DNA topoisomerase I was assayed by the method of Bhaduri et al. . The assay mixture (100 μl) contained 10 μmol of Tris–HCl (pH 8.0), 7 μmol of KCl, 1 μmol of MgCl2, 0.5 μmol of DTT, 10 nmol of EDTA, 10 μg of BSA, 0.5 μg of supercoiled pBR322, and enzyme. The reaction, termination, and electrophoresis conditions were the same as those for the DNA gyrase assay. In contrast to the gyrase assay, the activity of DNA topoisomerase I was assessed from both an increase in the density of bands corresponding to the relaxed pBR322 thus formed and a decrease in that of the supercoiled substrate.
Protein concentrations were determined by means of the protein assay kit with BSA as a standard.
3Results and discussion
First, an examination was conducted to learn whether the production of Glr or YrpC alters the copy numbers and superhelicity of plasmid DNAs harbored in clone cells. Figs. 1 and 2 reveal that the enforced expression of the yrpC gene (eventually, the enforced production of YrpC) decreased dramatically in both the copy number and superhelicity of the plasmid pYRPC1. The enforced expression of the glr gene, however, did not apparently influence those of the plasmid pBSGR2. It is well known that multiplication of the plasmid DNA used as the vector plasmid, pTrc99A, depends entirely on the replication machineries of host cells . In some phenotypic changes that result from an aberrant production of the enzyme, the B. subtilis YrpC isozyme resembles the E. coli MurI glutamate racemase more than the B. subtilis Glr enzyme [2,7,11]. The results imply that YrpC, but not Glr, participates in DNA replication, as does the E. coli MurI enzyme.
It is assumed that the superhelicity of DNAs in cells is generally influenced by the intracellular activity for DNA supercoiling  and net supercoiling activity is practically determined in the balance of DNA topoisomerase I and gyrase activities [1,5,17]. Both activities were then assayed in cells of E. coli JM109 harboring pTrc99A (JM109/pTrc99A), JM109/pBSGR2, and JM109/pYRPC1 clones. As shown in Fig. 3A, there was apparently little difference in the activities of DNA topoisomerase I among the clones tested. In contrast, the DNA gyrase activity of the E. coli JM109/pYRPC1 clone (Fig. 3B, lane 3) was obviously low compared with those of other clones (Fig. 3B, lanes 1 and 2). These observations suggest that YrpC, but not Glr, modulates intercellular DNA gyrase activity.
Our recent data indicated that YrpC inhibited DNA gyrase in a dose-dependent fashion (Fig. 4). Furthermore, the data showed that during the DNA gyrase reaction in the presence of YrpC the reaction intermediates, e.g. the nicked forms of the substrate DNA were accumulated, as did the exogenous DNA gyrase inhibitors such as CcdB  and ParE . We have found that, similarly to the E. coli MurI glutamate racemase, the B. subtilis YrpC isozyme also contains the region showing high sequence homology with the ParE toxin (data not shown).
The fact that the gene encoding YrpC, which is obviously inferior to Glr in its catalytic efficiency in glutamate racemization (responsible for the d-glutamate supply) , has been retained in the B. subtilis genome implies that the function of glutamate racemase as an endogenous modulator (eventually inhibitor) for DNA gyrase is physiologically significant. Based on the multifunctionality of the MurI-type glutamate racemase, the coordination of cell wall synthesis and DNA replication during cell division has been proposed . YrpC may be considered as the MurI-type glutamate racemase from B. subtilis. An interesting study recently reported that a MurI-like protein was overexpressed in a human carcinoma (as a peptidoglycan-less organism) where abnormal DNA replication frequently took place during cell division . On the contrary, the Glr enzyme has been deprived of the function as a DNA gyrase modulator, so that certain strains of B. subtilis, e.g. B. subtilis (natto), possibly managed to produce abundant Glr to supply d-glutamate required in large quantities as the main substrate for poly-γ-glutamate synthesis . Recently, on the basis of the double crossover recombination strategy , we succeeded in constructing the glr-gene disruptant of B. subtilis, which possesses apparently little activity of glutamate racemase (leading to a decline in d-glutamate productivity), and found little difference in intercellular DNA supercoiling activity between the disruptant and the wild-type strain (data not shown).
The B. subtilis YrpC isozyme (in other words, the MurI enzyme) and the Glr enzyme share high sequence similarity (88%), but they are quite different from each other in the action to DNA gyrase. In addition to the construction of the yrpC-gene disruptant and its phenotypic analysis, the tertiary structures of both glutamate racemases from B. subtilis are now being studied. Such detailed analyses would provide deep insights into the regulatory mechanism of intercellular DNA gyrase activity by the MurI-type glutamate racemase as well as into the design of novel pharmaceuticals.