Figure 1.
Methylation reactions in the biosynthetic pathway of MK.
The length of the isoprenoid side chain (n) varies depending on the species. SAM, S-adenosyl-L-methionine; SAH, S-adenosyl-L-homocysteine; DMK, demethylmenaquinone; MK, menaquinone.
Figure 2.
Plant growth test of the symbiotic ability of the disruptant HK116 on A. sinicus.
(A) Control plant not inoculated Rhizobium. (B) Plant inoculated by HK116 formed pseudonodules and yellow leaves. (C) Control plant inoculated by the wild type 7653R. (D) Plant inoculated by complemented strain HK116C restored nitrogen-fixing ability.
Table 1.
Symbiotic phenotypes of M. huakuii wild type strain 7653R, mutant strain HK116 (Nod+ fix−), and complemented strain HK116C.
Figure 3.
Paraffin section and ultrastructure observations of 5-week-old nodules formed by wild type strain 7653R, disruptant HK116, and complemented strain HK116C.
(A) Paraffin section of normal nitrogen-fixation nodules formed by wild type strain 7653R. (B) Paraffin section of nodules induced by disruptant HK116 strains in which the dmtH gene was disrupted and nitrogen-fixation ability lost; bacteroids are mainly located in II zone. (C) Paraffin section of nodules induced by the complemented strain HK116C with restored nitrogen-fixation ability. (D) Ultrastructure of normal bacteroids in nodules induced by wild-type 7653R. (E) Ultrastructure of aberrant bacteroids in nodules induced by disruptant HK116, with smaller size, incrassated membrane, and visible PHB granules in bacteroid cells. (F) Ultrastructure of function-restored bacteroids in nodules induced by the complemented strain HK116C.
Figure 4.
Expression patterns of dmtH gene in free-living cells and symbiotic nodules.
Gene expression levels were examined by real-time RT-PCR. Nodules were collected on different days after inoculation with 7653R. Expression levels of dmtH were highest in nodules at 25 dai, and hardly expressed in free-living cells. Histograms represent quantification of the products normalized to the constitutive control rnb. The experiment was repeated three times.
Figure 5.
HPLC analyses of MK and UQ from wild type strain 7653R and disruptant HK116 under aerobic and microaerobic conditions.
(A) UQ-10 was accumulated in 7653R a under aerobic condition. (B) UQ-10 was accumulated in HK116 under aerobic condition. (C) MK-7 was accumulated in wild type strain 7653R when culturing conditions were shifted to microaerobic for 24 hours. (D) MK-7 was not produced in disruptant HK116 under microaerobic condition.
Figure 6.
Mass spectrum of quinine compounds from M. huakuii 7653R cultured under microaerobic condition.
(A) The mass spectrum of MK7 which showed that MK7 was accumulated in M. huakuii 7653R under microaerobic respiration. (B) The mass spectrum of UQ-10 which showed that UQ-10 was accumulated in M. huakuii 7653R.
Figure 7.
Overexpression of the dmtH gene in E. coli and target protein DmtH purification.
The DmtH protein was separated on an SDS-polyacrylamide gel. E. coli Rosetta 2(DE3) harboring pET28a was used as the control (lane 1). E. coli Rosetta 2(DE3) harboring pETdmt was incubated with 28 KDa DmtH protein (lane 2). DmtH protein was incubated after purification (lane 3). Protein markers are on the right.
Figure 8.
HPLC analyses of quinone compounds for testing DmtH peotein activity.
(A) DMK-8 was extracted from E.coli JC7623Δ4-1(ubiE: Kanr) but MK-8 was not formed; (B) MK-8 was synthesized in vitro in the reaction system by purified DmtH protein, SAM and E.coli JC7623Δ4-1 cell homogenate at 37°C for 1 h.