MATERIALS AND METHODS

Mutant strains and plasmids: Two E. coli mutant strains which were used in this study namely a tryptophan mutant strain JW2326 (aroC) and a threonine mutant strain JW0001 (thrA). The genotypes of the mutant strains are JW2326 [Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ–, ΔaroC737::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514] and JW0001 (thrA). Both mutant strains are obtained from National Bio-Resource Project (NBRP) Japan. The EST clone carrying the expected gene encoding for chorismate synthase, having the GenBank accession number of AK099850 and clone number of J013110C10 was collected from Rice Genome Resource Center (RGRC), Japan. The source of E. coli mutant strains were obtained from National Bioresource Project (NBRP), Japan.

DNA sequence analysis: The sequence analysis of rice EST clone (J013110C10) which was derived from rice cDNA library¹⁸ from developing seeds prepared in pBluescript SK-that was done by using different bioinformatics tools. The DNA sequencing and sequence analysis were described previously¹⁹. The nucleotide and amino acid sequences were compared with various sequences present in GenBank and were analyzed by using Nucleotide-BLAST²⁰ and CLUSTALW a multiple sequence alignment program²¹ and GeneDoc. Motifs regions were searched by the Genome Net computation Service at Kyoto University (http://www.genome. ad.jp) and the phylogenic tree analysis was performed by MEGA 4 program²².

Polymerase chain reaction: Specific primers were designed from the sequence of OsCS information surrounding the translational start and stop codons of OsCS to amplify the full-length open reading frame (ORF) and to construct a recombinant DNA to express the gene product in E. coli. Polymerase chain reaction (PCR) was conducted in accordance with the method described previously¹⁹. The ORF region of chorismate synthase was amplified by own designed forward and reverses primers by using the PCR. After the purification of plasmid DNA from the bacterial culture harvested from LB ampicillin broth media the cDNA was amplified using the designed primers from the OsCS sequene; OsCS-F-(5’-GGAGCTCTCACCAACCAAACCAACAAC-3’) and OsCS-R-(5’-GGGTACCCTGTATAACGGATTCCTTGG-3’) both of the primers have the SacI and KpnI restriction sites. PCR was performed by using the MY Cycler TM PCR system (Bio Rad, United States) for 40 cycles carried out by denaturation at 95°C for 10 min, annealing at 58°C for 1 min and extension at 72°C for 1 min. The PCR products were analyzed by gel electrophoresis on 1% (w/v) agarose gel.

Growth curve determination in E. coli: To determine the growth of E. coli mutant with OsCS, the transformed mutant strain JW2326 harbouring pB:OsCS, control plasmid and wild type with control plasmid were inoculated in 1 mL of LB ampicillin (Amp) broth in different tubes and incubated for overnight at 37°C with shaking. The overnight culture was inoculated in the 100 mL of M9 minimal medium containing 1 mM IPTG, 20% glucose, Amp (50 μg mL^–1) and all 19 amino acids except tryptophan. The E. coli growth was monitored at 37°C at 1 h interval by using measurement the optical density with a UV-spectrophotometer (UV1101, Biochrom, England) at 595 nm (OD₅₉₅).

Bathophenanthroline: Bathophenanthroline was used to check the inhibitory activity on CS. This compound is also known as 4, 7 diphenyl-1, 10 phenanthroline and has molecular weight of 332.40 (Sigma-Aldrich company, USA). About 100 mL of M9 minimal medium contains 1 mM IPTG, 20% glucose, Amp (50 μg mL^–1) and all 19 amino acids except tryptophan including 0.005 mM bathophenanthroline. The media was turned into red color due to formation of iron complex. The activity of E. coli was also checked with this media simultaneously media without bathophenanthroline.

RESULTS

Sequence analysis of OsCS: The EST clone (clone ID: J013110C10) obtained from the RGRC was analyzed to determine the nucleotide sequence using the designed primers and bioinformatics tools. The results of data analysis with the cDNA sequence and complete rice genome indicated that an ORF was identical to that of the rice genomic region located on chromosome III (Os03g0254800). The cDNA sequence (OsCS) contained a full-length open reading frame consisting of 1,317 bp and encoded for 438 amino acid residues and approximate molecular weight of 46.9 kDa. The expected isoelectric point of the protein was 7.922. The similar sequences of different species such as rice, Zea maize, Hordeum vulgare, Arabidopsis and many bacteria were analyzed. The most of the regions of OsCS are highly conserved in plant species sharing similar domains, but OsCS share few conserved regions compared to similar proteins of bacteria. The comparison of the predicted amino acid sequence of OsCS with the deduced sequences from A. thaliana and E. coli showed high homology with identity values of 74 and 48%, respectively (Fig. 2). Motif analysis of the amino acid sequence of OsCS revealed that there are three motifs found in the amino sequence which are located at 64...79 (GESHGGGVGCVISGCP), 172...188 (GRSSARETIGRVAAGAL) and 379...395 (RHDPCVVPRAVPMVESM), respectively (Fig. 2). The patterns of the three motifs on the OsCS amino acid sequence are G-[DES]-S-H-[GC]-x(2)-[LIVM]-[GTIVLAMS]-x-[LIVTM]-[LIVM]-[DEST]-[GH]-x-[PV], [GE]-x(2)-S-[AG]-R-x-[ST]-x(3)-[VT]-x(2)-[GA]-[STAVY]-[LIVMF] and R-[SHF]-D-[PSV]-[CSAVT]-x(4)-[SGAIVM]-x-[IVGSTAPM]-[LIVM]-x-E-[STAHNCG]-[LIVMA], respectively. The phylogenetic tree derived from the related sequences showed that OsCS is grouped with Zea mays and Hordeum vulgare in monocotyledon plants and divergent and evolved from ancestor bacterial CS using the Mega 4.1 neighbor joining program²². The branching pattern and Numbers at the nodes indicate levels of bootstrap support based on neighbor joining analyses of 1000 resample data sets using the same program (Fig. 3).

Fig. 2:

Multiple sequence alignment of CS was analyzed by using Boxshade program The amino acid sequences of chorismate synthase (CS) from different organisms that were collected from NCBI database for preparing amino acid sequence alignment of CS from Oryza sativa (OsCS accession No. AK099850), Arabidopsis thaliana (AtCS; NP_564534), Escherichia coli (EcCS; WP_020239114) and Bacillus cereus (BcCS; WP_046392823)

Expression of OsCS can complement the aroC mutant of E. coli: The recombinant DNA, pB::OsCS was constructed using the ORF of a PCR-amplified OsCS fragment. After the transformation of E. coli with the recombinant DNA, OsCS activity was observed in vivo in a medium containing IPTG and 19 amino acids excluding Trp. Functional complementation was carried out using the CS mutant of E. coli strains JW2326 to prove the enzyme activity of the pB::OsCS product of OsCS. To evaluate the viability of E. coli cells by OsCS activity, the OsCS-expressing cells were cultured for overnight at 37°C with shaking and the diluted portion of culture was plated on agar medium containing the 19 amino acids except Trp with Amp (50 mg mL^–1). The viable colonies greatly differed on the agar medium among the plasmids . The aroC mutant of E. coli JW2326 with OsCS could grow colonies under the conditions without Trp in contrast with the mutant with control plasmid showed no growth in the media without Trp due to no CS activity. This showed that the OsCS was able to functionally complement to the aroC mutant of E. coli. To determine the OsCS activity in detail, a growth activity was performed to check whether the gene would increase the sensitivity of bacterial cells to Trp. The pB::OsCS construct was transformed into the aroC mutant strain JW2326 of E. coli. A control plasmid was also transformed into the mutant strain to differentiate the activity of aroC in both cases. A transformed E. coli strain of thrB mutant (JW0001) with control plasmid was used as wild-type and negative control. The bacterial cells were grown initially incubated overnight at 37°C with 1 mL LB media with Amp and then inoculated in MM media having 1 mM IPTG, Amp (50 μg mL^–1) and all 19 amino acids excluding Trp. The wild type thrB grew normally under MM media without Trp as it is evident by increased in the growth exponentially in the media with Trp.

Fig. 3:

Phylogenetic tree: Phylogenetic analysis of OsTS related proteins using Clustal W and Mega 4.1 program. GenBank accession numbers of above protein sequences are as follows: KHN17131 (GsCS from Glycine soja), NP_001148583 (ZmCS; Zea mays), AES62175 (MtCS; Medicago truncatula); NP_564534 (AtCS; Arabidopsis thaliana), ADR70879 (HbCS; Hevea brasiliensis); XP_002312018 (PtCS; Populus trichocarpa), AGW47887 (NtCS; Nicotiana tabacum), XP_002529571 (RcCS; Ricinus communis), ABA54870 (FsCS; Fagus sylvatica), AK099850 (OsCS; Oryza sativa), NP_001148583 (ZmCS; Zea mays), ACF36173(HvCS; Hordeum vulgare), WP_046392823 (BcCS; Bacillus cereus), BAM51143 (BsCS; Bacillus subtilis), WP_020239114 (EcCS; Escherichia coli), CDQ35237 (VhCS; Virgibacillus halodenitrificans), WP_023559308 (BpCS; Brevibacillus panacihumi) and WP_020429593 (PrCS; Paenibacillus riograndensis)

The E. coli mutant strains harboring control plasmid showed no growth in the media without Trp. The E. coli strain JW2326 expressing the pB:OsCS grew normally in the same media as it is evident by S-shaped growth curve. The E. coli mutant strains harboring control plasmid do not grow in the medium without Trp as they were not able to synthesize the Trp due to lacking of aroC gene. The wild type JW0001 harboring control plasmid grows normally in the media without Trp. The same E. coli strains JW2326 containing pB::OsCS grew well because the aroC mutants E. coli was able to synthesize Trp using rice CS expressed by the pB::OsCS plasmid (Fig. 4a). This is a consequence of pB::OsCS activity. From the above finding, it was concluded that OsCS expression could be functionally complement the aroC mutant of E. coli.

Bathophenanthroline inhibits the OsCS activity: Bathophenanthroline, a compound which has crystalline nature, was used as an inhibitor of OsCS activity. The bathophenanthroline (0.005 mM) was added to M9 minimal medium containing 1 mM IPTG, 20% glucose, Amp (50 μg mL^–1) and all 19 amino acids except Trp. The E. coli aroC mutant strain (JW2326) and wild type (JW0001) cultures were inoculated to check the inhibitory activity into the medium with bathophenanthroline. The inhibitory activity was determined the growth curve by measuring the optical density of bacterial culture at 595 nm at 1 h intervals for 16 h. The growth of mutant strain JW2326 harboring aroC gene was decreased due to the inhibitory effect of bathophenanthroline to CS enzyme. The growth with bathophenanthroline was rapidly decreased relative to the growth in the absence bathophenanthroline in MM media without Trp. The JW2326 strain harboring control plasmid showed no growth in the media containing bathophenanthroline. The wild type JW0001 strain harboring control plasmid have sustained growth rate in the media as relative to growth in the MM media without Trp (Fig. 4b).

Fig. 4(a-b):

(a) Growth curves of the aroC mutant strain JW2326 harboring pB:OsCS. Bacterial cells were grown at 37°C in MM containing 19 amino acids excluding Trp. Growth was monitored via optical density measurements at 595 nm (O.D 595) and (b) Growth curve of E. coli mutant strains JW2326 harboring pB:OsCS, control plasmid and wild type JW0001 harboring control plasmid. The bacterial strains were grown in M9 minimal media without tryptophan containing 0.005 mM bathophenanthroline. Growth was monitored via optical density measurements at 595 nm (O.D 595)

From the consequence, it was concluded that OsCS expression was able be functionally complement the aroC mutants E. coli and OsCS enzyme activity could be inhibited by bathophenanthroline.

DISCUSSION

Seven enzymes of shikimate pathway are effective targets for the development of antimicrobial and herbicidal compounds as a crucial pathway for synthesis of aromatic amino acid in bacteria and plants but not in mammals^1,23,24. An inhibitor of the pathway has a damaging effect on plant metabolism and possibly is a useful commercial herbicide with low mammalian toxicity¹⁵. The shikimate pathway is targeted for the development of herbicides. For example, glyphosate, a potent inhibitor, blocks the synthesis of aromatic amino acids²³ and inhibits the growth of both Gram-negative and Gram-positive bacteria in vitro^25,26. Glyphosate is being widely used as a nonselective, broad-spectrum, post-emergence herbicide that is readily translocated in plants and is biodegradable by soil microorganisms²⁷. Recent study showed that bacterial CS is an attractive target for new antibacterial drugs²⁸. Through the effort in the search of new targets for herbicide development in plants, these results showed that OsCS has enzyme activity in the biosynthesis of amino acids and is possible to an attractive target for new herbicides. In the complementation experiment on the E. coli with an inhibitor to CS, the growth of wild type JW0001 (control) is lower than JW2326 (mutant strain complemented with OsCS) when treated with bathophenanthroline. This phenomenon might be occurred by the difference in the sensitivity of the enzyme inhibitor against E. coli and rice chorismate synthases. It might be possible to apply a new herbicide development targeting OsCS which functions crucial role in aromatic amino acid synthesis in plants. Furthermore, it is necessary to study the shikimate pathway in rice so as to better uncover the formation and regulation mechanism of diverse flavonoids in rice crop. To elucidate the impact of the shikimate pathway on the production of specialized metabolites in rice, the manipulation of the rice gene encoding CS enzyme that controlling the conversion of primary to specialized metabolism could be an attractive tool for improving rice aroma and flavor qualities.

CONCLUSION

The OsCS from rice encodes chorismate synthase and its activity is inhibited by its inhibitor, bathophenanthroline.

ACKNOWLEDGMENTS

We wish to thank Rice Genome Resource Center (RGRC) and National Bio-Resource Project (NBRP), Japan for providing an EST clone J013110C10 and an aroC mutant (JW2326) of E. coli, respectively. This study was supported by the research grant of Jeju National University in 2012.

SIGNIFICANCE STATEMENT