Random mutagenesis of RMC26 generated four mutants with the desired phenotype that had disruptions in srlE
, which encodes a protein in the sorbitol PTS. PCR using genomic DNA from the mutants as a template confirmed an insertion in the srlE
region. Insertion of a CAT cassette into srlE
produced a MVA-Ara-dependent phenotype in RMC26, confirming its involvement in ME utilization. During PTS-mediated phosphorylation, a phosphate group from PEP is first transferred to the N1 position of a histidine residue of EI and subsequently transferred to the N1 position of a histidine in HPr, with neither of these steps being sugar specific. The phosphate is then transferred from HPr to a histidine N3 of EIIA in the sugar-specific transport complex, followed by transfer to a cysteine residue of EIIB and finally to the sugar molecule (45
). Because of the coordinated mechanism of the EIIA, EIIB, and EIIC subunits in a PTS system, srlA
were also implicated along with srlE
in ME utilization. Insertions of CAT cassettes into srlA
gave a MVA-Ara-dependent phenotype similar to that seen for srlE
, confirming the role of all three genes in ME utilization.
Incorporation of ME and DX into isoprenoids was reported before many of the steps in the MEP pathway had been discovered (6
). It is now clear that both compounds must be phosphorylated before they are utilized as substrates in the MEP pathway. The sorbitol PTS provides a mechanism for import and phosphorylation of ME. In vitro experiments have demonstrated that d
-xylulokinase (encoded by xylB
), the enzyme responsible for phosphorylating d
-xylulose, also phosphorylates DX (46
), although the transport mechanism for uptake by cells has not been reported. The viability of CR5 when DX is present in the growth medium indicates that the mechanism for utilization of DX in the MEP pathway is different from that for ME. One could presumably identify the gene (or genes) responsible for transporting exogenous DX by performing a similar set of experiments described in this work except using DX instead of ME in the screening process.
Involvement of the srlE gene product in the utilization of exogenously supplied ME for isoprenoid biosynthesis was verified biochemically by analysis of Q8. S. enterica serovar Typhimurium strain RMC26 readily incorporated the deuterium label from ME into the isoprenoid side chain of Q8. Interestingly, RMC26, which synthesizes isoprenoids from either ME or MVA, had a substantial preference for utilization of ME when incubated with a mixture of MVA-Ara and deuterium-labeled ME. In contrast, incubation of CR5, where SrlE in the sorbitol PTS system is disabled, with the same mixture of substrates produced Q8 with no deuterium in the side chain. Thus, a functional copy of the SrlE protein is required for ME utilization.
The sorbitol PTS complex also appears to be responsible for phosphorylation of ME. In particular, the SrlE protein is the EIIB subunit of the sorbitol PTS and is analogous to protein that transfers phosphate to the sugar in the glucose PTS (18
). Although d
-sorbitol and ME are polyhydroxyl compounds, the absolute stereochemistries of their chiral centers do not precisely map onto one another, and it is not clear why the operon responsible for the utilization of sorbitol is also responsible for the transport and phosphorylation of ME.
In some cases overproduction of enzymes in the MEP pathway stimulates the synthesis of isoprenoids (25
). For example, higher levels of lycopene are obtained when isopentenyl diphosphate isomerase is overexpressed (25
). The level of lycopene can also be increased when DXP synthase and MEP synthase are overproduced (44
). It might be possible to further stimulate the synthesis of lycopene from ME by overproduction of the SrlA, SrlE, and SrlB proteins along with DXP synthase and MEP synthase.
It is notable that no insertions were seen in known biosynthetic genes in the MEP pathway even though directed knockouts of the genes in RMC26 gave strains with the same phenotype as CR5 (unpublished results). We were able to specifically disrupt each of the MEP pathway genes beyond MEP synthase in RMC26, with each exhibiting the phenotype of growth on MVA-Ara and no growth on ME. This would imply that we should be able to identify mutations that render each of the genes inactive, yet all of the isolated mutants appear to be involved in ME uptake and phosphorylation. In addition to the four srlE mutants, the five unknown mutants do not have an absolute requirement for the MVAoperon and readily lose it through recombination when crossed with a tightly linked marker, such as thiI::Tn10d-Tet. If the random insertions were in MEP pathway genes, the MVA operon would be essential to provide cellular isoprenoids, as demonstrated by the inability of CR4 to lose the MVA operon in the same cross. If it is assumed that there are 1,000 genes encoded by the S. enterica serovar Typhimurium chromosome, with five being potential MEP pathway targets in our screen (ispD through ispH) and five belonging to the proposed ME transport system (srlA, srlB, srlE, and the genes encoding HPr and EI), an estimated 100 mutants exhibiting the phenotype of growth on MVA-Ara and no growth on ME would be expected for completely random insertions. Our finding of only nine mutants out of 200,000 is low. We have no evidence to explain this discrepancy.