It is known that Escherichia coli K-12 is cryptic [Phn^–] for utilization of methyl phosphonate [MePn] and that Phn⁺ variantscan be selected for growth on MePn as the sole P source . Variantsarise 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 instabilityin 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 high-frequency reversible gene switch, producing phase variation which either allows ["on"form] or blocks ["off" form] MePn utilization . The switch canalso block usage of other metabolizable alkyl phosphonates,including the naturally occurring 2-aminoethylphosphonate . AllK-12 strains, obtained from collections, appear in the "off"form even when bearing mutations in mutS, mutD, or dnaQ whichare known to enhance slip strand events between repetitive sequences.The ability to inactivate the phnE gene appears to be uniqueto K-12 strains since the B strain is naturally Phn⁺ and lacksthe 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-Plyase, 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 inE . 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 potentiallyencode regulatory proteins [5] . Several phn genes appear toencode components of solute transporters, and it has been deducedthat, 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 encodesthe 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 insertionin the coding region of the phnE gene in the K-12 strain relativeto the B strain, causing truncation of the phnE product . Theyalso observed that the 8-bp sequence is one element in the directtriply repeated sequence in the K-12 strain comprising two typesof octamer variants in the arrangement 5'-ABB-3', where A correspondsto the sequence 5'-CGCTGGCG-3' and B corresponds to the sequence5'-TGCTGGCG-3' [Fig . 1] . Makino et al . isolated variants ofE . coli K-12 able to use MePn as the sole P source [Phn⁺], andthese were found to have deletions of octamer B, which, theypostulated, occurred via a strand slippage event during DNAreplication [14] . The nature of the variation in the phnE genein E . coli is investigated in more detail in this work.

 
Bacterial strains and growth conditions. All the strains of E . coli used in this study [Table 1] were routinely cultivated under aerobic conditions at 37°C in Luria-Bertani broth [LB] with agar added to 0.8%, wt/vol, foragar plates except for Phn⁺ variants of K-12 strains [see below] and temperature-sensitive mutants for which the permissive temperature for growth was 30°C . The minimal medium used was modified Neidhardt’s medium [MNM] and was based on that of Neidhardtet al . [15], but, in order to study the metabolism of differentphosphorous sources, the normal phosphate buffer component wasreplaced with MOPS [morpholinepropanesulfonic acid] . MNM containedMOPS [40 mM], glucose [11.1 mM], NH₄Cl [9.5 mM], Tricine [4mM], thiamine · HCl [29.6 µM], FeSO₄0 ·7H₂0 [10 µM], CaCl₂ [0.5 µM], MnCl₂ [0.8 nM], CoCl₂[0.3 nM], CuSO₄ [0.16 nM], ZnSO₄ [0.1 nM], [NH₄]₆Mn₇O₂₄ [30pM], and H₃BO₄ [4 pM] . The medium was adjusted to pH 7.4 . MNMwas solidified for plates [MNM-agarose] with electrophoresisgrade agarose [1%, wt/vol] because it is especially low in phosphatecompared to purified agars . MNM and MNM-agarose were supplementedwith different P sources at 0.5 mM . Phn⁺ variants of K-12 strainswere isolated, purified, and maintained on MNM-agarose containingMePn unless otherwise stated . Culture dilutions were made inMNM lacking added P sources . Phosphorous compounds were obtainedfrom the following sources . MePn was from Fluka Chemie AG; 2-AEPn,aminomethylphosphonate [AMPn], phosphonoacetate, phosphonoformate,phosphonomycin, and o-phospho-L-serine were from Sigma ChemicalCo.; 1-AEPn, 1-aminopropylphosphonate [1-APPn], 3-APPn, tert-butylphosphonate,ethylphosphonate [EPn], phosphonomethylglycine [glyphosate],phenylphosphonate [PhPn], and propylphosphonate [PPn] were fromAldrich Chemical Co.; and N-butanephosphonate was from LancasterSynthesis, Morecambe, United Kingdom

 
Estimation of the frequency of Phn⁺ variants. The frequency of Phn⁺ variants in populations of E . coli strains was estimated by comparing the number of CFU arising when populations were diluted appropriately and plated onto MNM-agarose containing MePn compared to the number on MNM-agarose containing eitherP_i or o-phospho-L-serine [positive controls] or no added P source[negative control] . The inocula for these experiments were firstgrown to stationary phase on MNM containing P_i and then washedin MNM containing no added P source . Dilutions were performedwith MNM containing no added P source . Colonies arising on phosphateor phosphonates were counted at 4 and 10 days, respectively,because growth of colonies on phosphonates was generally slowerthan growth on P_i.

Molecular biology techniques . [i] Extraction of genomic DNA. Template DNAs for PCRs were extracted from cultures as follows. One-tenth milliliter of culture was diluted with 0.9 ml of sterile distilled water in a 1.5-ml microcentrifuge tube . The tube wasboiled for 5 min and snap-chilled in ice . The boiled cell suspensionswere diluted 100-fold in sterile water, and 5 µl was sufficientto set up PCRs.

[ii] AFLP analysis. Deletions or insertions in phnE were monitored by amplified-fragmentlength polymorphism [AFLP] analysis as follows . Fragments ofphnE of 200 or 192 bp containing the triple or double octamericrepeat regions, respectively, were amplified from samples ofliquid cultures or colonies by PCR using oligonucleotide primerset 1 [Fig. 1], comprising EcphnEF2 [5'-Cy 5-TTACCAGCCCGTTCGCCGCC-3'] and EcphnER2 [5'-CCTTCCACCGGGCCAGGTTCAAT-3'] . Amplifications were carried out with Bio-X-Act DNA polymerase [Bioline UK Ltd.]with the following thermal cycle: 30 cycles of 95°C for30 s, 60°C for 1 min, and 68°C for 1 min . Products werepurified with a PCR fragment purification kit [QIAGEN] and checkedby gel electrophoresis in a 1% Tris-acetate-EDTA-agarose gel.Fragments were diluted and run together with standard-size fragments[50-bp ladder; Amersham] on a 6%, wt/vol, polyacrylamide sequencinggel in an ALF-Express automated sequencer [Amersham Pharmacia],and the gel image was visualized with ALF-Express software.Fragments were detected, sized, and quantified with AllelLinks,version 1.00 [Amersham Pharmacia] . The relative proportionsof the two alleles of phnE were estimated from a standard curveprepared from AFLP analysis of different mixtures of the K-12strain MC4100 and the B strain BL21[DE3], prepared by mixing individual cultures grown in LB prior to preparation of templates DNA.

DNA sequencing. To sequence the region of phnE containing the triply or doublyrepeated region, a 600-bp fragment was amplified by PCR witholigonucleotide primer set 2 [Fig. 1], consisting of EcphnES1[5'-GCGGATCCCGCAGCTG-3'] and EcphnER [5'-ACGGTCGCCGAGCGGACGTT-3'].The amplification protocol used was that described for the AFLPanalysis above . The DNA was purified with a QIAGEN PCR purificationkit and then sequenced with an ALF-Express automated sequencerand a cycle sequencing strategy using Cy 5-labeled EcphnEF andEcphnER primers.

 
Occurrence of Phn⁺ variants of E . coli K-12. The frequencies of occurrence of Phn⁺ variants in populations of common laboratory E . coli K-12 strains DH5 and MC4100, grownin LB, were estimated by determining the CFU appearing 10 daysafter plating washed populations at suitable dilutions on MNM-agarosecontaining MePn and comparing them to those appearing on MNM-agarosecontaining P_i or o-phospho-L-serine or no added P source, asa negative control . Phn⁺ variants were observed to be presentat the surprisingly high levels of 3.4 and 8.6% of the totalCFU of DH5 and MC4100, respectively, of those observed on theplates containing P_i or o-phospho-L-serine . Plates with no addedP source contained only pinpoint colonies, presumably growingon traces of utilizable P in the medium.

AFLP analysis of genotypic events in E . coli K-12 Phn variants. AFLP analysis was used to monitor the status of the phnE genein different populations of strains . DNA fragments spanning that segment of the phnE gene in K-12 which contains the triple octameric repeat were amplified with primer set 1 [Fig . 1] fromculture lysates of organisms grown in various media . When strainswere grown in LB, as expected, a 200-bp fragment was amplifiedfrom the K-12 strain [DH5] while a 192-bp fragment was amplifiedfrom the B strain [BL21[DE3]] [Fig . 2] . When the analysis wasperformed on various mixtures of cultures of these two strains,both 200- and 192-bp fragments were amplified and the peak areaswere approximately proportional to those for the prepared mixtures[Fig . 2] . This establishes that AFLP can be used to detect bothforms of phnE if present in populations . AFLP analysis was thenused to examine phnE in cultures growing in MNM containing alternative P sources . Studies were performed on both MC4100 and DH5 . Resultswere essentially identical . Fragments of 200 bp were amplifiedonly from cultures grown to stationary phase in MNM containingP_i and o-phospho-L-serine as the sole added P sources . However,populations of Phn⁺ variants which had grown to stationary phasein MNM with MePn as the sole added P source gave rise to twofragment types, the major species being 192 bp and the minorspecies being 200 bp . Therefore, stationary-phase populationsof purified Phn⁺ variants unexpectedly contained a mixture oftwo phnE alleles, of which the major form probably containedthe expected 8-bp deletion reported by Makino et al . [14] . Weextended these studies by examining the state of phnE in culturesin both logarithmic and stationary phases of growth with MePnas the sole P source . In the log-phase populations we coulddetect only fragments of 192 bp, but, in stationary-phase populations,both 192- and 200-bp fragments were detected [Fig . 3] . Thissuggested that the deletion in phnE may be reversible and ofhigh frequency and that it is potentially linked to later stagesof the growth phase.

 
Reversible switching in the phnE gene. Further evidence for reversibility of the 8-bp deletion in phnEwas obtained in the following experiment . Phn⁺ variants of MC4100 originally selected and purified on MNM containing MePn werefirst grown in MNM plus MePn and then serially subcultured inLB with a 0.03% inoculum level at each subculture . AFLP analysiswas performed on samples removed at stationary phase after eachround [Fig. 4] . In each set of populations derived from a Phn⁺ variant, the initial proportion of the 192-bp fragment was highbut subculture in LB resulted in a dramatic decrease in theproportion of the 192-bp forms and a reciprocal increase inthe proportion of the 200-bp forms [Fig . 4].

 
DNA sequencing of amplified fragments. To confirm that the 8-bp deletion in phnE observed in theseexperiments was identical to that described by Makino et al.[14], the sequences were determined for 600-bp fragments ofphnE amplified directly from boiled lysates of logarithmic-phasecultures of BL21[DE3] and MC4100 grown on P_i or on MePn andalso a Phn⁺ variant of MC4100 which had been repeatedly subcultured on LB and that therefore had apparently undergone reversion. These data [Fig . 5] confirmed that one octamer B sequence had been lost in the Phn⁺ variants selected on MePn and that the B strain contains only the AB repeat . The data also showed for the first time that the apparent reversion event detected byAFLP when Phn⁺ variants were cultivated in LB and not selected for MePn utilization involves precise restoration of an octamerB lost during the original selection process.

 
Behavior of the phnE gene in different K-12 strains. The status and behavior of the phnE gene in K-12 derivatives carrying mutations in recA [STL1671, STL2172, and STL2314], mutS [STL2172], dnaQ [STL2314 and NR9807], mutD [NR9458], andsbcB [STL1671] were examined . When cultures were cultivatedin MNM with P_i as the sole added P source, a 200-bp fragmentwas amplified from all strains with primer set 1 . Phn⁺ variantswere then isolated for each strain . All Phn⁺ variants yielded192-bp fragments after AFLP analysis using oligonucleotide set1 [Fig . 5].

Phn⁺ variants obtained with different phosphonate sources. A requirement for the 8-bp deletion in phnE for utilizationof other organophosphonates was also examined . In this experiment,Phn⁺ variants of MC4100 were isolated and purified on MNM-agarosewith alternative phosphonates provided as sole P sources . AFLPanalysis was performed on logarithmically growing populationswith primer set 1 . As with Phn⁺ variants selected and grownon MePn, fragments of 192 bp were amplified from all populationswhich grew well using EPn, AMPn, 2-AEPn, and 3-APPn as P sources[Fig. 3] . Also, as with Phn⁺ variants selected on MePn, allthese variants gave rise to mixtures of 192- and 200-bp fragments in stationary phase [data not shown] . Very slow growth was observed with PhPn and PPn . In the PhPn cultures, a 200-bp fragment was the sole fragment amplified, but in the PPn-grown organismstraces of the 192-bp forms were detected . No significant growthwas observed with the following phosphonates: N-butanephosphonate,tert-buty phosphonate, 1-AEPn, 1-EPn, 1-APPn, phosphonoacetate, phosphonoformate, and phosphonomycin.

Properties and behavior of the phnE gene in non K-12 E . coli strains. Eleven strains of E . coli originally isolated from raw wateror sewerage sludge on nutrient broth or LB were screened forpotential crypticity in phosphonate metabolism and for the occurrenceof the triple-repeat sequences in phnE . Their ability to useMePn as a sole P source on MNM was tested as for the K-12 strainspreviously . Of 11 independent isolates, four strains, 1, 8,A3, and D7, gave plating efficiencies significantly below 100% [at 5.7, 39, 35, and 1.3%, respectively] on MePn compared to the controls supplied with P_i . This suggested that these strains may be cryptic and may contain an inactivated phnE gene similar to that observed in K-12 strains . Therefore we determined the nucleotide sequences of the 600-bp fragments of the phnE genes surrounding the octameric repeats amplified from cultures notexposed to added phosphonates [Fig . 5] . However, although a few base substitutions affecting only the third base codingpositions were observed in the different phnE sequences, allstrains contained the double octameric repeat sequence in phnE,as found in the B strain [data not shown].

 
We have established that Phn⁺ variants appear to be present at a surprisingly high frequency [>10^–2] in populations of all the E . coli K-12 strains tested here . AFLP analysis proved useful in monitoring the genotypic behavior of phnE in populations of various strains grown under different conditions, and significantly it revealed the reappearance of the phnE allele in populations of Phn⁺ variants, especially in stationary-phase cultures . Possibleexplanations for persistence of the phnE allele include cross-feedingof the Phn^– strains by the Phn⁺ variants and/or geneticheterogeneity in phnE in individual cells carrying more thanone chromosome copy . However, because our studies were conductedwith purified Phn⁺ isolates, we conclude that the phnE genein E . coli K-12 behaves like a reversible gene switch, causingphase variation in phosphonate metabolism . This appears to bethe first example of phase variation in a component of an ABCtransporter in a gram-negative bacterium [10] although it isinteresting that a high-frequency frameshift phase variationevent affects the proposed substrate-binding lipoprotein encodedwithin an ABC transporter operon in Mycoplasma fermentans [20].It is not clear why "off" forms start to accumulate rapidlylate in the growth cycle on phosphonates or once the selectionfor growth on phosphonates is removed . From a large number ofsuch studies with different strains, we estimate that the switchmay operate at frequencies as least as high as 10^–2 pergeneration in either direction but that the equilibrium of the switch strongly favors the "off" form unless selection for phosphonate utilization is applied.

The octameric sequence involved in this postulated slip strand event is, at 8 bp, relatively long, and interestingly it isthe most commonly occurring octamer in the genome of E . coliK-12 [2] . It also contains the core trimer 5'-CTG-3', thoughtto be the DnaG primase binding site [11, 18, 23, 24] . The apparenthigh instability in phnE in K-12 may be linked to the potential involvement of this octamer in the initiation of DNA replication.

Mutations in DNA replication, repair, and recombination influence instability in tandem repeat sequences [3, 12, 17] . All theK-12 mutants examined prior to selection for MePn utilizationexhibited the typical K-12 5'-ABB-3' or "off" form of phnE eventhough some carry mutations in functions known to increase deletionsbetween repetitive sequences, including mutations in recA andthe sbcB-encoded 3' exonuclease I and the dnaQ49^ts mutation,which affects DNA polymerase -subunit exonuclease activity andthe physical interaction of the -subunit with the polymerizing -subunit.

The physiological significance of the switch in E . coli K-12 remains unclear . In the "on" direction, the switch allows E. coli K-12 to use not only MePn but also EPn, AMPn, 2-AEPn, and 3-APPn; hence phnE is implicated in transport of all these organophosphonates.Elashvili et al . [6] have shown that the phnE gene is necessaryfor uptake of some organophosphates since the 8-bp deletionevent in phnE enabled the E . coli K-12 strain JA221 to utilizediisopropyl fluorophosphate and its hydrolysis product, diisopropylphosphate.

Surprisingly, the phnE switch appears to be confined to K-12 strains . E . coli strains for which genomes have been determined appear to contain the "on" form of phnE, including the uropathogenicstrain CFT073 [22], where the 5'-AB-3' repeat is perfectly conserved,and the enterohemorrhagic strain O157:H7 [9, 16], although here T substitutes for C at the seventh base in octamer A in the 5'-AB-3' sequence [Fig . 5] . We also found no evidence for the presence of the "off" form of phnE in several E . coli strainsisolated recently from environmental samples.

Although the phnE switch may be an artifact possibly arising from the repeated mutagenesis to which the K-12 strain has been subjected, it is possible that it might protect against theuptake of naturally occurring inhibitory phosphonates presentin the natural environment or in some way affect surface receptorsrequired for coliphage or lymphocyte recognition.

 
Samina Iqbal was supported by a grant from The Islamic Development Bank of Saudi Arabia, George Parker was supported by a researchgrant from Zeneca Agrochemicals, and Helen Davidson and Elham Moslehi-Rahmani were supported by summer vacation studentshipsfrom The Society for General Microbiology.

We thank Elizabeth Pontin and Mike Taylor for running the sequencer and help with the analysis software packages . We also most grateful for the provision of strains to Malgorzata Bzymek, Susan Lovett, Roel Schaaper, and Robert Wells and the E . coli Genetic Stock Center at Yale University.

 
* Corresponding author . Mailing address: Microbiology Division, School of Animal and Microbial Sciences, University of Reading, Reading RG6 6AJ, United Kingdom . Phone: 44-118-9316639 . Fax: 44-118-9316562 . E-mail: r.l.robson@rdg.ac.uk .

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