Anabolic Pathways

The genome sequence of L. monocytogenes contains all genes for the amino acid-, purine-, pyrimidine-, and several vitamin biosynthetic-pathways (those for biotin, riboflavin, thiamine, and lipoate are absent). The gene for the last enzyme in serine biosynthesis (serine-phosphate phosphatase) has not been annotated in the genome sequence, but all growth studies clearly show that L. monocyto-genes is not auxotrophic for serine. Surprisingly, previous studies (Phan-Thanh and Gormon, 1997; Premaratne et al., 1991) indicated the requirement of the BCAA (Ile, Val, and Leu) as well as cysteine, methionine, and arginine when L. monocytogenes was grown in defined minimal media with glucose as carbon and energy source.

Listeria monocytogenes lacks sulphate and nitrate reductases and hence is dependent on reduced N and S sources, which readily explains the growth requirement for cysteine and methionine. In the presence of cysteine, methionine can be biosynthesized de novo albeit at low rate (J. Slaghuis, personal communication). The ability of L. monocytogenes to biosynthesize Arg has been also demonstrated (Tsai and Hodgson, 2003; our own unpublished data), but again, addition of Arg to the minimal medium clearly enhances the growth rate. In the absence of Ile, the growth rate is very low in this minimal medium, and addition of Ile together with one of the other two BCAA (Leu or Val) is required to obtain efficient growth of L. monocytogenes in this culture medium. Our recently performed 13[C]-isotopolog perturbation studies (Eisenreich et al., 2006) using uniformly labeled 13[C]glucose show low-level biosynthesis of all three BCAA, even in the presence of externally added BCAA. This synthesis (especially that of Ile) is significantly enhanced in the presence of high PrfA concentration, which as discussed above reduces PTS-mediated glucose uptake and hence may inhibit PMF-dependent BCAA transport. These data clearly show that the biosynthesis of Ile, Leu, and Val is functional, but its efficacy is low in glucose-containing minimal medium.

The BCAA are indicators of the general nutritional status of the bacterial cell because their synthesis depends on several basic catabolic precursors (oxaloac-etate, pyruvate, and acetyl-CoA), and hence the rate of BCAA synthesis is an important factor in the overall bacterial physiology. In B. subtilis, the central BCAA biosynthesis operon (ilvB) is under complex control of the global regulators CcpA, CodY, and TnrA (Shivers and Sonenshein, 2005; Tojo et al., 2005) which also regulate many genes that respond to nutrient availability and growth rate (Molle et al., 2003). All three regulators have binding sites in the ilvB regulatory region. CcpA binding to a CRE site within this region activates the transcription starting at the ilvB promoter. CodY interacts directly with Ile and GTP, and these two components (indicators of efficient growth and high energy level in the bacterial cell) act as independent corepressors for CodY (Shivers and Sonenshein, 2005).

Listeria monocytogenes contains orthologous genes for CcpA, CodY, and GlnR (highly similar to TnrA), and it is therefore likely that similar control mechanisms may act in L. monocytogenes as in B. subtilis. The low rate of BCAA biosynthesis in glucose-containing minimal medium may therefore reflect the shortage of necessary catabolic intermediates (especially oxaloacetate) due to the interrupted citrate cycle.

Previous studies indicated that L. monocytogenes mutants auxotrophic for some amino acids, like Phe, Gly, and Pro, replicated within host cells like the parental L. monocytogenes strain, while a mutant deficient in all three aromatic amino acids was impaired in intracellular replication and virulence (Marquis et al., 1993). Similar results were obtained in our recent investigation with aro mutants which are defective in the basic pathway of all aromatic components. These mutants—in addition to exhibiting impaired cytosolic replication, cell-to-cell-spreading, and virulence—showed a predominantly anaerobic metabolism (Stritzker et al., 2004). The reason for this more unexpected result is apparently the lack of synthesis of menaquinone which is the only quinone produced by L. monocytogenes and hence its absence strongly impairs aerobic respiration.

Menaquinone biosynthesis involves the condensation of 1,4-dihydroxy-2-naphthoate with polyprenyl-PP which is produced by the isoprenoid biosynthesis pathway. The precursor of all isoprenoids, isopentenyl-PP, is biosynthesized either by the classical mevalonate or by the alternative 2-C-methyl-D-erythritol-4-phosphate (via gyceraldehyde-3-P and pyruvate) pathway. Interestingly, the L. monocytogenes genome carries the information for both pathways and both seem to be functional (Begley et al., 2004). It remains to be seen whether there is a preferential activation of one of the pathways when L. monocytogenes replicates inside host cells.

Comparative transcript profiling using RNA from extra- and intracellularly grown L. monocytogenes shows highly significant up-regulation of the genes for the biosynthesis of all essential amino acids; in particular, the aromatic amino acids and the three BCAA but not the nonessential ones (Joseph et al., 2006), suggesting that the latter ones are provided by the host cell. Strong up-regulation is also observed for the genes involved in purine and pyrimidine biosynthesis, in accordance with previous data (Klarsfeld et al., 1994; Marquis et al., 1993) indicating that nucleotides are not efficiently provided by the host cell to intra-cellularly growing L. monocytogenes.

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