Competition for nutritional resources masks the true frequency of bacterial mutants.

BACKGROUND: It is widely assumed that all mutant microorganisms present in a culture are able to grow and form colonies, provided that they express the features required for selection. Unlike wild-type Escherichia coli, PHO-constitutive mutants overexpress alkaline phosphatase and hence can hydrolyze glycerol-2-phosphate (G2P) to glycerol and form colonies on plates having G2P as the sole carbon source. These mutations mostly occur in the pst operon. However, the frequency of PHO-constitutive colonies on the G2P selective plate is exceptionally low. RESULTS: We show that the rate in which spontaneous PHO-constitutive mutations emerge is about 8.0 × 10-6/generation, a relatively high rate, but the growth of most existing mutants is inhibited by their neighboring wild-type cells. This inhibition is elicited only by non-mutant viable bacteria that can take up and metabolize glycerol formed by the mutants. Evidence indicates that the few mutants that do form colonies derive from microclusters of mutants on the selective plate. A mathematical model that describes the fate of the wild-type and mutant populations under these circumstances supports these results. CONCLUSION: This scenario in which neither the wild-type nor the majority of the mutants are able to grow resembles an unavoidable "tragedy of the commons" case which results in the collapse of the majority of the population. Cooperation between rare adjacent mutants enables them to overcome the competition and eventually form mutant colonies. The inhibition of PHO-constitutive mutants provides an example of mutant frequency masked by orders of magnitude due to a competition between mutants and their ancestral wild-type cells. Similar "tragedy of the commons-like" cases may occur in other settings and should be taken into consideration while estimating true mutant frequencies and mutation rates.

Ugp and PitA participate in the selection of PHO-constitutive mutants.

UNLABELLED: Mutations that cause the constitutive expression of the PHO regulon of Escherichia coli occur either in the pst operon or in the phoR gene, which encode, respectively, a high-affinity Pi transport system and a histidine kinase sensor protein. These mutations are normally selected on glycerol-2-phosphate (G2P) as the carbon source in the presence of excess Pi. The emergence of early PHO-constitutive mutants, which appear after growth for up to 48 h on selective medium, depends on the presence of phoA, which codes for a periplasmic alkaline phosphatase, while late mutants, which appear after 48 h, depend both on phoA and on the ugp operon, which encodes a glycerophosphodiester transport system. The emergence of the late mutants hints at an adaptive mutation process. PHO-constitutive phoR mutants appear only in a host that is mutated in pitA, which encodes an alternative Pi transport system that does not belong to the PHO regulon. The conserved Thr(217) residue in the PhoR protein is essential for PHO repression. IMPORTANCE: One of the principal ways in which bacteria adapt to new nutrient sources is by acquiring mutations in key regulatory genes. The inability of E. coli to grow on G2P as a carbon source is used to select mutations that derepress the PHO regulon, a system of genes involved in the uptake of phosphorus-containing molecules. Mutations in the pst operon or in phoR result in the constitutive expression of the entire PHO regulon, including alkaline phosphatase, which hydrolyzes G2P. Here we demonstrate that the ugp operon, another member of the PHO regulon, is important for the selection of PHO-constitutive mutants under prolonged nutritional stress and that phoR mutations can be selected only in bacteria lacking pitA, which encodes a secondary Pi transport system.

Alternative promoters in the pst operon of Escherichia coli.

The pst operon of Escherichia coli is composed of five genes pstS, pstC, pstA, pstB and phoU, that encode a high-affinity phosphate transport system and a negative regulator of the PHO regulon. Transcription of pst is induced under phosphate shortage and is initiated at the promoter located upstream of the first gene of the operon, pstS. Here, we show by four different technical approaches the existence of additional internal promoters upstream of pstC, pstB and phoU. These promoters are not induced by Pi-limitation and do not possess PHO-box sequences. Plasmids carrying the pst internal genes partially complement chromosomal mutations in their corresponding genes, indicating that they are translated into functional proteins.

The effect of IHF on sigmaS selectivity of the phoA and pst promoters of Escherichia coli.

The pst operon, a member of the PHO regulon of Escherichia coli, encodes a high-affinity phosphate transport system whose expression is induced when the cells enter a phase of phosphate starvation. The expression of pst is stimulated by the integration host factor (IHF). Transcription of the PHO regulon genes is initiated by the RNA polymerase complexed with sigma (D) (Esigma (D)). Owing to a cytosine residue at position -13 of the pst promoter its transcription can also be initiated by Esigma (S). Here, we show that inactivation of IHF in vivo abolishes the sigma (S)-dependent transcription initiation of the pst operon, indicating that both -13C residue and IHF are required to confer on pst the ability to be transcribed by Esigma (S). Introduction of a -13C residue in the promoter region of phoA, another PHO regulon gene that is not directly affected by IHF, did not affect its exclusive transcription initiation by Esigma (D).

A differential effect of sigmaS on the expression of the PHO regulon genes of Escherichia coli.

The RNA polymerase core associated with sigma(S) transcribes many genes related to stress or to the stationary phase. When cells enter a phase of phosphate starvation, the transcription of several genes and operons, collectively known as the PHO regulon, is strongly induced. The promoters of the PHO genes hitherto analysed are recognized by sigma(D)-associated RNA polymerase. A mutation in the gene that encodes sigma(S), rpoS, significantly increases the level of alkaline phosphatase activity and the overproduction of sigma(S) inhibits it. Other PHO genes such as phoE and ugpB are likewise affected by sigma(S). In contrast, pstS, which encodes a periplasmic phosphate-binding protein and is a negative regulator of PHO, is stimulated by sigma(S). The effect of sigma(S) on the PHO genes is at the transcriptional level. It is shown that a cytosine residue at position -13 is important for the positive effect of sigma(S) on pst. The interpretation of these observations is based on the competition between sigma(S) and sigma(D) for the binding to the core RNA polymerase.