Consideraciones éticas

Capsule expression by 1w4 is an all-or-none event; switcher cells either do not express a capsule, or express a full sized capsule. This uncompromising dichotomy implies that intracellular UTP levels do not hover uncertainly about the capsule threshold. Rather, UTP levels are maintained either stably below or above the capsule threshold, indicating the existence of self-perpetuating mechanisms for both phenotypic states (i.e. bistability; see section 1.1.2). The work in this chapter has shown that the molecular basis of bistability is restricted to within the pyrH-ndk-pyrG segment of the pyrimidine biosynthetic pathway. Thus, the transcription, translation or activity of one (or more) of these three genes holds the key to bistability.

In section 6.4.3.2, genetic regulation of the pyrimidine pathway was discussed. A main conclusion of this section was that the mechanisms controlling transcription of the genes in this pathway differ significantly between species. Since little experimental work has been previously undertaken in this area with Pseudomonas (and virtually none with P. fluorescens), insight into potential feedback loops involving pyrH, ndk and/or

pyrG in this pathway is limited. It is possible that a positive feedback loop (see section 1.1.2.1) exists in this region; for instance, the expression of any one of pyrH, ndk or

pyrG may be positively autoregulated. It is also possible that one component of a double-negative feedback loop (see section 1.1.2.1) exists in this region. Consider a situation where two genes, capsule-promoting gene a and capsule-repressing gene b, were subject to mutual transcriptional repression. In the presence of A, the product of gene a, transcription of b would be repressed and the cell would express a capsule. On the other hand, the presence of B, the product of gene b, transcription of a would be repressed and a capsule would not be expressed. It is conceivable that capsule- promoting gene a is present in the UTP biosynthetic pathway (i.e. pyrH or ndk). However, in the absence of likely candidates for gene b from the transposon mutagenesis screen of Chapter 4, this possibility has not been considered further.

Despite the lack of broadly applicable regulatory mechanisms, the pyrimidine biosynthetic pathway itself is almost impeccably conserved among all bacteria

investigated to date (Turnbough Jr. & Switzer, 2008). Thus, it is likely that reported enzyme functions are conserved in P. fluorescens, and the possibility that bistability results from post-transcriptional regulation involving PyrH, Ndk and/or PyrG could be investigated to a greater depth. The potential for bistability to originate from the promiscuous activity and enzymic memory of Ndk led to the development of the molecular model described in detail below. In switcher genotypes, the conditions governing net Ndk activity are almost certainly altered, given that the amount of one Ndk substrate (UDP) is severely reduced by the carB and pyrH mutations.

6.4.4.2.1 A molecular model based on Ndk activity and enzyme expression

A molecular model for capsule switching is illustrated in Figure 6.14. Consider a cell in which UDP levels are lower than those of other diphosphates. In this cell, Ndk will preferentially convert the higher-level diphosphates to triphosphates (an activity perpetuated by the enzymic memory of Ndk, see section 6.4.4.1). Thus, the cell with low UDP levels has correspondingly low UTP levels. Any UTP produced is utilized exclusively for DNA/RNA synthesis, ensuring no UTP-dependent capsules are expressed. Simultaneously, the cell senses that UDP/UTP levels are low, and feedback mechanisms are activated to increase the transcription and translation of the pyrimidine biosynthetic genes (Figure 6.1). As a result, high levels of the (mutant) pyrimidine pathway produce small amounts of UDP, which accumulate in the cell. When a wild- type level of UDP is reached, UDP is able to compete for binding sites on Ndk, and corresponding UTP levels increase. Even though UDP/UTP levels are now satisfactory, the increased quantities of pyrimidine biosynthetic enzymes still exist in the cell, allowing intracellular levels of UDP/UTP to increase to the capsule threshold (see Figure 6.13). If such over-compensation occurs, excess UTP is channelled through GalU into polysaccharide biosynthesis, generating the capsulated form.

The capsulated form is maintained as long as UDP/UTP levels are maintained above the capsule threshold. Importantly, although capsule biosynthesis requires the presence of large amounts of UTP, the process does not alter net levels of uridine nucleotides; UTP channelled into capsule biosynthesis does not form part of the final polymer, but instead

is recycled back into the system as UMP (see Figure 6.1). Thus, uridine nucleotide levels are lowered only by the synthesis of DNA and RNA at cell division. As a result, although a cell may switch during its lifetime, switching is more likely to occur upon replication. Thus, the self-perpetuation of each phenotypic state may be accounted for by a combination of competitive Ndk activity and temporal regulation of pyrimidine enzyme expression.

Aside from the flux-reducing effects of the switch-causing mutations, the above mechanism is consistent with experimental observations. Firstly, over-expression of ndk

was shown to reduce capsulation in both SBW25 and 1w4 (see Figure 6.12). In the above model, increasing the availability of Ndk would decrease competition for the enzyme, allowing lower levels of UDP to be immediately converted to UTP and preventing the accumulation of UDP/UTP beyond the capsule threshold. Also consistent with the model is the increase in capsulation observed in JG176, the transposon mutant genotype with an inactivational insertion in the 5' end of ndk (see section 4.3.1.5.2). In this genotype, ndk levels are likely to be very severely reduced (if not absent), with adk

acting as a substitute for ndk. Thus, competition for ndk/adk is likely to be even stronger in this strain, and the level of UDP required for successful competition closer to the capsule threshold. It is conceivable that a greater proportion of individuals would subsequently cross the threshold once UDP pools were restored. The capsule reducing effect of pyrH over-expression (see Figure 6.12) is less readily explained; it is possible that increase in PyrH increases the availability of the product, UDP, thus increasing UDP/UTP pools to a more usual level (although how this would be achieved in light of the low UMP substrate levels is unclear). It is also possible that over-expression alters the repressive effect of PyrH on the carAB operon (see section 6.4.3.2.4), although the details of such an effect remain unknown.

Finally, the proposed model is able to account for the presence of low-level capsulation in P. fluorescens SBW25 and 1s4. All features of the proposed model are present in ancestral strains; bistability in switcher types is merely activated by reduction of the uridine nucleotide pool. Given that the switch machinery is present in ancestral genotypes, it could potentially be activated at a lower level when the pyrimidine

nucleotide pool stochastically surpasses the ‘normal’ range. Additionally, activation of the same feedback machinery would increase in ancestral types if pyrimidine nucleotide pools were reduced. Such a reduction could occur as a result of genetic mutation, or as a result of environmental inhibition of the pyrimidine biosynthetic pathway. Interestingly, the activity of S. typhimurium CPSase has been shown to be sensitive to temperature: low temperatures result in lower CPSase activity (Han et al., 1990). Thus, activation of the feedback loop at 16˚C in SBW25 (see section 3.3.2.2.1) is consistent with the proposed model.

Figure 6.14: Model for the molecular basis of capsule switching. Switch-causing mutations lower

intracellular levels of UDP, resulting in poor competition for Ndk, low UTP levels and no capsule expression (left). Upregulation of the biosynthetic enzymes leads to accumulation of UDP, and eventual overcompensation leads to excess UTP being channelled into capsule biosynthesis (right). Downregulation and cell division reduces UTP, and back to the cap- state, where accumulation begins again. See text for further details.

Cap-

UTP accumulation (above threshold)

UTP depletion (below threshold)

High UDP levels:  Ndk utilizes UDP

 High UTP levels

 UTP-dependent capsule

Low UDP levels:

 Ndk does not utilize UDP

 Low UTP levels

 No capsule expression