Functional Analysis of Two 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (Hdr) Isozymes in Burkholderia glumae BGR1

Abstract

The terpenoids are biosynthesized by polymerization of isoprenoid units consisting of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The Hdr (4-Hydroxy-3-methylbut-2-enyl diphosphate reductase) is the ultimate enzyme in MEP pathway converting (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) to IPP and DMAPP. Despite most free-living bacteria have a trend of genome size reduction in evolution from generalist to specialist, Burkholderia glumae, a Gram negative rice-pathogenic bacterium, harbors 2 hdr isogenes while lacking isopentenyl diphosphate isomerase (idi). Molecular and genetics analysis of hdr gene region suggest that each hdr isogene was found in respective putative operon. The Bghdr1 gene and FKBP-type peptidyl-prolyl cis-trans isomerase (slp) were polycistronic, as were the Bghdr2 gene and hopanoids associated radical S-adenosyl methionine (SAM) domain containing protein (hpnH). Hdr2 isozyme was placed in a clade different from Hdr1, and only 56.1 % identity was found between them. Nevertheless, both hdr genes could complement E. coli hdr deletion mutant (DYTL1). BgHdr1 and BgHdr2 catalyzed reduction of HMBPP into IPP and DMAPP at a virtually same ratio of 2:1, in contrast to 5:1 ratio of other bacterial Hdrs so far characterized in vitro enzyme assay. The kcat and Km values of BgHdr1 and BgHdr2 were 187.0 min-1 and 6.0 μM and 66.6 min-1 and 21.2 μM, respectively. Therefore, Hdr1 was 10 times more efficient than Hdr2: kcat/KM of Hdr1 was 31.1 µM-1 min-1 and Hdr2 3.1 µM-1 min-1. The transcript message level of hdr2 was 1.6 ~ 1.8 folds higher than hdr1 at 6 and 10 h after inoculation. The different expression level was further confirmed by Northern blot throughout whole growth phase. To identify the physiological role of each isogene, hdr knock-out (KO) mutants were constructed. On LB medium, the growth rates of 2 mutants were not different from that of BGR1 (wild-type). The colony size of hdr1 knock-out mutant (HDR1KO) was smaller than that of BGR1 and hdr2 knock-out mutant (HDR2KO). The HDR1KO grew slower than HDR2KO and BGR1 under conditions such as minimal nutrient, high temperature, acidic environment, and in planta. Especially, in rice plant, the HDR1KO was less virulent than due to the reduced colonization ability. The difference in phenotype was evident in protein composition of the KO mutants. In SDS-PAGE, several cellular protein bands of HDR1KO moved faster than those of HDR2KO and BGR1. The proteins that showed electrophoretic mobility shift was identified through LC MS/MS. The major change was observed in GroEL, which exhibited acetylation at Lys390 upon disruption of hdr1. To answer if the change was cued by the differential function of hdr isogenes or by the regulation in the promoter level, HDR1KO complementation assay was performed as follows. The 6 plasmids were constructed by combination of putative hdr1 promoter, hdr1 operon promoter, and hdr2 promoter and two hdr open-reading frames (ORF).The GroEL acetylation of HDR1KO was rescued by the vectors harboring putative hdr1 operon promoter, regardless of hdr ORF, The same vectors also rescued hypersensitive reaction (HR) ability of HDR1KO. The hdr2 upstream region, the putative hdr2 operon promoter, was predicted to have a ToxR (LysR-type regulator) binding site by genome anlaysis. By electrophoretic mobility shift assay and yeast-1-hybride assay, ToxR was confirmed to bind and activate hdr2 operon. The overall regulation points in MEP pathway were shown in transcriptome analysis. 4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase (cmk) was up-regulated in both KO mutants, whereas (E)-4-hydroxy-3-methyl-but-2-enyl 4-diphosphate synthase (hds) was down regulated in HDR2KO. Upregulation of undecaprenyl diphosphate phosphatase (upp) and undecaprenyl diphosphate synthase (ups) and downregulation of squalene synthase1 (sqs1) and squalene synthase2 (sqs2) were found in HDR2KO. The most positively affected in the HDR1KO was tox operon genes that were responsible for toxoflavin biosynthesis. The toxin accumulation in the cell was higher in hdr1 KO mutant than hdr2 KO or BGR1. In the HDR2KO, the most up-regulated were stress-related ribosomal protein and SAM decarboxylase genes.