Reversible Phase Variation in the phnE Gene, Which Is Required for Phosphonate Metabolism in Escherichia coli K-12

Abstract

It is known that Escherichia coli K-12 is cryptic (Phn ؊ ) for utilization of methyl phosphonate (MePn) and that Phn ؉ variants can be selected for growth on MePn as the sole P source. Variants arise from deletion via a possible slip strand mechanism of one of three direct 8-bp repeat sequences in phnE, which restores function to a component of a putative ABC type transporter. Here we show that Phn ؉ variants are present at the surprisingly high frequency of >10 ؊2 in K-12 strains. Amplified-fragment length polymorphism analysis was used to monitor instability in phnE in various strains growing under different conditions. This revealed that, once selection for growth on MePn is removed, Phn ؉ revertants reappear and accumulate at high levels through reinsertion of the 8-bp repeat element sequence. It appears that, in K-12, phnE contains a highfrequency reversible gene switch, producing phase variation which either allows ("on" form) or blocks ("off" form) MePn utilization. The switch can also block usage of other metabolizable alkyl phosphonates, including the naturally occurring 2-aminoethylphosphonate. All K-12 strains, obtained from collections, appear in the "off" form even when bearing mutations in mutS, mutD, or dnaQ which are known to enhance slip strand events between repetitive sequences. The ability to inactivate the phnE gene appears to be unique to K-12 strains since the B strain is naturally Phn ؉ and lacks the inactivating 8-bp insertion in phnE, as do important pathogenic strains for which genome sequences are known and also strains isolated recently from environmental sources.Escherichia coli can use P III compounds such as phosphite and organophosphonates, e.g., methylphosphonate (MePn) and aminoethylphosphonate (AEPn) as P sources. Their metabolism involves the enzyme C-P lyase, which appears to have a relatively broad substrate specificity (21). Whereas the mechanism of C-P lyase is not well understood, much is known about a cluster of 17 contiguous phn genes in E. coli, required for utilization of P III compounds (5, 21). The phnGHIJK genes within this cluster are thought to encode the core components of C-P lyase, while phnF and phnO potentially encode regulatory proteins (5). Several phn genes appear to encode components of solute transporters, and it has been deduced that, among these, the phnCDE genes encode an ABC type transporter. In this transporter, phnC encodes the ABC permease component, phnD encodes the periplasmic binding protein, and phnE encodes the integral membrane component.An interesting feature of the genetics of phosphonate metabolism in E. coli is that the B strain can use phosphonates whereas the K-12 strain is cryptic despite containing the entire phn gene cluster (21). The genetic basis for this crypticity was investigated by Makino et al. (14) and traced to an 8-bp insertion in the coding region of the phnE gene in the K-12 strain relative to the B strain, causing truncation of the phnE product. They also observed that the 8-bp sequence is one element in the direct t...

Alternative pathways for phosphonate metabolism in thermophilic cyanobacteria from microbial mats

“…Consequently, for all growth experiments described here, we used Syn OS-B 0 cells that had been acclimated to MePhn for at least 3 weeks (Adams et al, 2008). This acclimation requirement has been described for several other microorganisms, but it is not understood at the mechanistic level except in Escherichia coli (Iqbal et al, 2004). Syn OS-A cells were not initially subjected to an acclimation regime and it appears that they do not require an acclimation period to be able to use Phns.…”

Phosphorus Deprivation Responses and Phosphonate Utilization in a Thermophilic Synechococcus sp. from Microbial Mats

“…The E. coli K-12 strain is cryptic for MePhn utilization, and variants that can grow with MePhn as the sole P source are selected for after exposure to the Phn substrate followed by a relatively long lag phase, similar to the situation for OS-BЈ. The basis for this extended lag period of OS-BЈ is not known, although in E. coli, the lag phase represents the time that it takes to generate a slip strand event in the phnE gene; this lesion deletes one of three direct repeat sequences, thereby restoring the function of a membrane component of the Phn transporter (16). The OS-BЈ MePhn acclimation phenomenon could also represent a genetically based mechanism, as recent growth results indicate that acclimation requires the MePhn substrate presence in addition to P i starvation and that acclimation to the substrate is not lost over a prolonged period following growth of the cells on P i in the absence of MePhn (data not shown).…”

Bacterial Genome Instability

“…They control the formation of the capsule (428), the pili (429), flagella (430), adhesins (431), antifungal metabolites (432), iron acquisition factors (433), and other surface-exposed molecules (432,434), sometimes affecting motility (432) or colony morphology (434) and opacity (374). Nevertheless, these proteins can also be involved in general cellular pathways, such as DNA restrictionmodification (435), gene regulation (436), or metabolism (437). Phase and antigenic variation is thought to be essential for commensal, symbiotic, and pathogenic bacteria, as these processes can play an active role during the invasion (438) and infection (439) of an organism and help these bacteria to face the challenges raised by their hosts (440).…”