A Switch in the Mechanism of Communication between the Two DNA-Binding Sites in the SfiI Restriction Endonuclease

While many Type II restriction enzymes are dimers with a single DNA-binding cleft between the subunits, SfiI is a tetramer of identical subunits. Two of its subunits (a dimeric unit) create one DNA-binding cleft, and the other two create a second cleft on the opposite side of the protein. The two clefts bind specific DNA cooperatively to give a complex of SfiI with two recognition sites. This complex is responsible for essentially all of the DNA-cleavage reactions by SfiI: virtually none is due to the complex with one site. The communication between the DNA-binding clefts was examined by disrupting one of the very few polar interactions in the otherwise hydrophobic interface between the dimeric units: a tyrosine hydroxyl was removed by mutation to phenylalanine. The mutant protein remained tetrameric in solution and could bind two DNA sites. But instead of being activated by binding two sites, like wild-type SfiI, it showed maximal activity when bound to a single site and had a lower activity when bound to two sites. This interaction across the dimer interface thus enforces in wild-type SfiI a cooperative transition between inactive and active states in both dimers, but without this interaction as in the mutant protein, a single dimer can undergo the transition to give a stable intermediate with one inactive dimer and one active dimer.
Introduction
Type II restriction endonucleases recognise specific sequences in DNA, typically palindromic sites 4 to 8 bp long, and cut the DNA at specified positions within or close to the site.1 Their reactions usually (but not always2) require Mg2+ as a cofactor.3 Some Type II endonucleases are dimers of identical subunits that interact symmetrically with their palindromic sites.4,5 These have a single DNA-binding cleft at the subunit interface, and they act at individual copies of their target sites.6–8 The best-studied restriction enzymes, such as EcoRV and BamHI,8–14 and those most widely used in vitro as tools for molecular biology,15 all function in this manner. Consequently, these are often considered as “standard” restriction enzymes. However, many Type II endonucleases differ from the standard in that they are fully active only after interacting with two copies of their recognition site.16–20 The Type II enzymes that need two sites fall into two subtypes, IIE or IIF.21 The Type IIE restriction enzymes bind two (or more22) copies of their recognition sequence yet cleave only one.23 They contain two DNA-binding clefts, catalytic and allosteric, but the catalytic cleft is inactive unless cognate DNA is also bound at the allosteric site.17,22 In contrast, the Type IIF enzymes form complexes with two DNA sites, at equivalent loci in the protein,24,25 and then cut both sites in a concerted reaction.26–30
The first restriction enzyme found to act concertedly at two DNA sites was the SfiI endonuclease.26,27 SfiI recognises the sequence GGCCNNNN↓NGGCC (where N is any base and ↓ is the point of cleavage),31 but it cleaves substrates with two copies of this sequence more rapidly than DNA with a single copy. Moreover, in steady-state reactions at low enzyme concentrations, SfiI converts DNA with two sites directly to the final product cut at both sites, without liberating intermediates cut at one site:26,27 the intermediates remain bound to the enzyme until it has cut both strands at both sites.32 On DNA with two sites, SfiI loops out the DNA between the sites,33–35 but it can also bind simultaneously to two separate DNA molecules that each have one copy of the recognition sequence.36,37 The rates of their reactions on one-site substrates increase sigmoidally with DNA concentration, indicative of positive cooperativity.36 The interaction with two sites is obligatory, as virtually no DNA is cleaved by any complex of SfiI with a single recognition site.36,38 Many genetic events—such as DNA replication and recombination, and the regulation of gene expression—often depend on proteins interacting with two sites at separate locations in the DNA, and the SfiI restriction enzyme has become one of the principal test systems for analysing the mechanisms of long-range communications between distant DNA sites.39
Many restriction enzymes are now known to belong to the Type IIF family: examples include Cfr10I,28 NgoMIV,24 Bse643I,40–42 BspMI,29 SgrAI,43,44 Mly113I and BbeI,19 and almost all of the Type II enzymes that cut DNA bilaterally on either side of their recognition sites, such as BcgI and AloI.20 All of these enzymes need to interact with two recognition sites for full activity. Proteins that interact with two DNA sites generally prefer sites in cis, on the same molecule of DNA, to sites in trans, on separate DNA molecules, simply because two sites in cis will almost always be in closer proximity than sites in trans.39,45 Consequently, all of these enzymes are capable of cleaving DNA with two sites more rapidly than DNA with one site.
Most Type IIF enzymes, including SfiI, are known from analytical ultracentrifugation (AUC) studies to exist in solution as tetramers of identical subunits.26,28,29,34 [SgrAI is an exception, but while it is a dimer in solution, two dimers bound to separate sites associate to form a tetramer before cutting DNA.44] In the crystal structures of the tetrameric enzymes, two of the subunits (a primary dimer) constitute one DNA-binding cleft, and the other two subunits constitute a second identical cleft.24,25,28,40 The two primary dimers are arranged back-to-back, so that their DNA-binding clefts are on the opposite sides of the protein (Figure 1(a) and (b)). The primary dimers in SfiI are comparable to the dimeric restriction enzyme BglI,46 an enzyme whose recognition site, GCCNNNN↓NGGC, is a truncated SfiI site. The structures of the individual subunits of SfiI and the arrangement of the two subunits in its primary dimer are similar to those in BglI, likewise its motifs for DNA sequence recognition and catalysis.47 The subunit interface within the dimer has, however, a much smaller area in SfiI than in BglI. In addition, SfiI and BglI have very different surfaces opposite the DNA-binding cleft: polar and solvent-exposed in the case of dimeric BglI,46 but almost completely hydrophobic and buried in tetrameric SfiI.25
In previous studies, the mode of communication between the two DNA-binding clefts in another tetrameric Type IIF enzyme, Bse634I, was examined by mutating selected amino acids at the dimer–dimer interface.41,42 A mutation that converted the tetramer into a dimer yielded an enzyme with much the same properties as a standard dimeric restriction enzyme: while wild-type (wt) Bse634I cleaves DNA with two target sites at a rapid rate and DNA with one site at a slow rate, the dimeric mutant cleaved both substrates at equally rapid rates.41 Another mutant at the dimer interface of Bse634I cleaved both one-site and two-site substrates at diminished rates, while a third cleaved both substrates at elevated rates, but in both cases the two-site DNA was still cleaved more rapidly than the one-site DNA.42 In this study, we report on the effect of a single amino-acid substitution at the dimer–dimer interface of the SfiI endonuclease, a conservative change from a tyrosine to a phenylalanine that removes just one hydroxyl group per subunit. In contrast to the previous mutants of Bse634I, this mutation yields an enzyme that cleaves DNA with one cognate site more rapidly than DNA with two sites. It thus switches SfiI from an enzyme that is activated by binding two DNA sites to one that shows its maximal activity on binding a single site.
Results
Subunit communications in SfiI
In the crystal structure of SfiI bound to two copies of its recognition sequence, two subunits (a primary dimer) bind one duplex, with each monomer contacting one of the two GGCC elements of the sequence.25 The other two subunits bind the second duplex on the opposite side of the tetramer in a back-to-back arrangement (Figure 1(a) and (b)). The interface between the two monomers within each dimer has a surface area (1650 Å2) that is not only smaller than that for the related dimeric enzyme BglI (3500 Å2)46 but which is also smaller than those for other tetrameric restriction enzymes (viz. 3100 Å2 for Bse634I).40 Many of the amino acids at the interface within each dimer are polar in nature and interact with the opposite monomer.25
In contrast, the subunit interactions at the interface between the primary dimers have a larger contact area in SfiI than in the other tetramers (3450 Å2 compared to 1700 Å2 for Bse634I). In SfiI, the inter-dimer interface is composed almost entirely of nonpolar residues:25 the amino acids in one subunit that lie within 5 Å of the opposite subunit across the dimer interface (Figure 1(c)) coincide with a patch on this surface that is devoid of either positively or negatively charged side chains48 (Figure 1(d)). The subunit interactions across the inter-dimer interface are thus mainly van der Waals contacts between hydrophobic side chains, although they also include some contacts between peptidyl main-chain carbonyl and amino groups. However, there appear to be only two side-chain-to-side-chain hydrogen bonds across the inter-dimer interface: between Gln3 in one subunit and Gln26 in the opposite partner, likewise between Tyr68 and Gln30 (Figure 1(c)). Gln3 is located at the outside edge of the inter-dimer interface (Figure 1(c)) while Tyr68 is positioned near the centre of the contact area (Figure 1(c) and (d)). Moreover, the aromatic side chain of Tyr68 protrudes from the surface of each subunit into a pocket in the opposite subunit (Figure 1(b)). Given the paucity of directional interactions across this interface, it seemed plausible that Tyr68 might play a pivotal role in the communication between the two DNA-binding clefts. To test this possibility, the hydroxyl moiety from the Tyr68 side chain was removed by using site-directed mutagenesis to replace the tyrosine with phenylalanine to yield the mutant Y68F.
Enzyme stability and quaternary structure
DNA-cleavage reactions of the Y68F mutant were initially carried out at 50 °C (the standard temperature for SfiI assays) in the same way as for wt SfiI: the enzyme was diluted into dilution buffer (Materials and Methods), incubated at 50 °C and then added to a solution of DNA and MgCl2.26,32 However, Y68F lost activity rapidly between dilution and subsequent addition to the DNA: after 2 and 5 min in dilution buffer at 50 °C, its activity had fallen by factors of 10 and 1000, respectively, while the wt enzyme retained full activity (data not shown). However, when the Y68F enzyme was mixed with the DNA substrate before initiating the reaction with MgCl2, it retained full activity. For wt SfiI, no differences were observed between reactions initiated by adding diluted enzyme to the mix of DNA and MgCl2 and reactions initiated by adding MgCl2 to premixed enzyme and DNA. All subsequent analyses of Y68F were therefore carried out in the presence of cognate DNA, and all DNA-cleavage reactions were initiated by adding Mg2+ to mixtures of enzyme and DNA.
Since the Y68F protein is unstable in the absence of DNA, its oligomeric state was determined with DNA present. The DNA was a 21-bp duplex with the recognition sequence for SfiI, HEX-21 (Table 1). This duplex carries a chromophoric label, hexachlorofluorescein (HEX), attached to the 5′ end of the “top” strand. HEX-21 has an absorbance peak at 539 nm, a wavelength at which neither protein nor nucleotides have any intrinsic absorbance.
The sedimentation of HEX-21 to equilibrium was measured by recording in the AUC the absorbance of the samples at 539 nm as a function of centrifugal radius. The samples comprised HEX-21 alone or HEX-21 with either Y68F or wt SfiI (the DNA was present at half the concentration of DNA-binding sites in the enzyme). In all three cases, the increase in absorbance with centrifugal radius was fitted to the equation for a single homogenous species49 to yield apparent Mr (relative molecular mass) values. By itself, HEX-21 gave an Mr of about 16,000, close to that expected for a HEX-labelled DNA of 21 bp (data not shown). The best fits to the data in the presence of wt SfiI or Y68F gave Mr values of 154,203 and 156,274, respectively (Figure 2), which are both close to that predicted for the SfiI tetramer bound to two duplexes (151,367).
The removal of the hydroxyl group from the side chain of Tyr68, by replacing it with Phe, diminished the stability of the protein: upon dilution to low protein concentrations, the mutant lost activity, possibly due to subunit dissociation at low concentrations. Nevertheless, as with wt SfiI, the Mr of Y68F bound to a 21-bp DNA matched that expected for the tetramer bound to two duplexes.
DNA-binding studies
Gel retardation was used to compare the DNA–protein complexes formed by Y68F and by wt SfiI (Figure 3). As in previous studies,36 two duplexes of different lengths were employed: HEX-21, the 21-bp DNA used in the AUC, and an elongated version, HEX-35 (Table 1). The complexes of enzyme bound to either duplex, or to both, were separated from each other, and from the free duplexes, by electrophoresis through polyacrylamide. The DNA was detected by HEX fluorescence.
The two duplexes were added, at a constant total concentration, to fixed amounts of either Y68F or wt SfiI, to give mixtures that contained twice the molarity of duplex over enzyme tetramer. The binding buffer36,37 contained Ca2+: this ion can promote specific binding by Mg2+-dependent enzymes without supporting DNA cleavage.10–12 The addition of HEX-21 alone to either protein gave a single retarded complex, as did the addition of HEX-35 alone (Figure 3, left-hand and right-hand lanes, respectively). The complex with HEX-21 had a faster electrophoretic mobility than that with HEX-35. When both duplexes were added, three complexes were observed: one with the same mobility as that with HEX-21, one equal to the complex with HEX-35 and a third with an intermediate mobility (Figure 3, central lanes). The yields of the three complexes varied with the ratio of the two duplexes in a binomial manner, with the 1:2:1 distribution occurring when the ratio of the concentrations of HEX-21 to HEX-35 was close to 1:1. The same pattern was observed with Y68F.
The intermediate complex, with a mobility in between that with HEX-21 alone and that with HEX-35 alone, must contain one molecule of HEX-21 and one molecule of HEX-35. The complexes with the highest and the lowest mobilities therefore correspond, respectively, to the SfiI tetramer bound to two molecules of HEX-21 and to two molecules of HEX-35. Both Y68F and wt SfiI can thus form complexes that contain two DNA duplexes bound to a tetrameric protein. However, both proteins were saturated at the DNA concentrations used here, so it remains to be determined whether there are any differences in affinity and/or cooperativity between wt and mutant proteins.
Cleavage of two-site plasmid
The optimal substrates for SfiI are supercoiled (SC) plasmids that have two copies of its recognition sequence.27 A single tetramer of SfiI binds to two sites in cis and traps the intervening DNA in a loop.33–35 It then cleaves all four of its target phosphodiester bonds before dissociating from the DNA: the initial product liberated from the enzyme is the final product cut in both strands at both sites, to the virtual exclusion of intermediates cut at one, two or three bonds.32 To find out if the Y68F mutant acts like wt SfiI in this respect, the reactions of both enzymes were examined on a plasmid with two SfiI sites, pGB1.26 The reactions were initially conducted under steady-state conditions, with [E0] < [S] (Figure 4(a) and (b)), to be able to observe directly the nature of the DNA liberated from the enzyme, rather than the enzyme-bound intermediates generated during the reaction.
Under these conditions, wt SfiI converted almost all of the SC plasmid directly to the two linear products (L1 and L2) cut in both strands at both sites: none of the open circle (OC) form of the DNA, cut in one strand, and only a small amount of the full-length linear (LIN) form of the DNA, cut in both strands at one site, were released from the enzyme during the reaction (Figure 4(a)). To allow comparisons between substrates with one SfiI site and substrates with two SfiI sites, all steady-state rates are recorded here in terms of moles of SfiI sites cleaved per mole of enzyme tetramer per minute (mol/mol/min). The velocity of wt SfiI on this two-site plasmid was 2.2 mol/mol/min (Figure 4(a)), the Vmax for this reaction.26,27 The Y68F mutant also cleaved the two-site plasmid in a highly concerted manner: it liberated very little of either the nicked OC form or the LIN product cut at one site. Instead, it converted this DNA directly to the final products cut at both sites, L1 and L2 (Figure 4(b)). However, the rate at which Y68F cleaved pGB1 (0.11 mol/mol/min) was 20 times slower than wt SfiI.
It is unlikely that the 20-fold difference is due to a difference in Km as the gel-shift experiments (Figure 3) had shown that the DNA concentrations used here were sufficient to saturate either enzyme. However, the rate-limiting step for the complete reaction pathway for wt SfiI (the process that determines its steady-state velocity) is the final dissociation of the products cut at both sites: all of the preceding stages, including the four DNA-cleavage steps, are relatively rapid.32 The reduction in reaction velocity could thus be due to either product release from Y68F being 20-fold slower than from wt, or the rates of the DNA-cleavage steps in its reaction pathway being reduced to values below that for product release by wt SfiI. These possibilities can be distinguished by determining whether Y68F generates a pre-steady state burst of product formation. If the DNA cleavage steps are faster than product release, the enzyme will create a burst of enzyme-bound product, to a concentration equal to that of the enzyme, prior to a slow steady-state phase, the rate of which is limited by product release.50 On the other hand, if the DNA-cleavage steps are rate-limiting for Y68F, there will be no burst, and the reaction will proceed instead at the linear steady-state rate from time zero.
The reactions of both wt and mutant enzymes were studied under conditions where the presence of a burst phase is readily detected: with an enzyme concentration (2 nM) approaching that of the DNA (5 nM) so that the enzyme-bound product can constitute a significant fraction of the total product, and at a reaction temperature (30 °C) where SfiI has a very slow turnover rate32, thus allowing the reactions at elevated enzyme concentrations to still be monitored. Under these conditions, both wt and Y68F enzymes cleaved a fraction of the two-site substrate rapidly before entering a slower phase during which the concentration of the substrate declined linearly with time: the decline was more rapid with wt SfiI than with Y68F (Figure 4(c)). Hence, on a DNA with two sites, both enzymes are rate-limited by product release, but Y68F releases the doubly cut product more slowly than wt SfiI. [For both enzymes, the amount of substrate consumed in the initial burst phase was about 70% of the enzyme concentration rather than 100%: this was as expected,32 as the addition of SfiI to a two-site substrate leads not only to DNA with one SfiI tetramer bridging the two sites in cis but also to DNA carrying a tetramer at each site, which resists cleavage.33,35]
Cleavage of one-site plasmid
The turnover rate of wt SfiI on a plasmid with one site is typically about 10 times slower than on a plasmid with two sites.26,32,33 To see if the Y68F mutant behaved like wt, both enzymes were tested against pGB1/S1, a plasmid that carries one of the two SfiI sites from pGB1.26 During these reactions, the SC substrate was cleaved directly to the final product, LIN DNA, with a double-strand break at the SfiI site, without liberating the nicked OC form (data not shown): only the decline in the substrate concentration is shown here (Figure 5).
The first tests employed the same enzyme concentration (0.5 nM; Figure 5(b)) as that used for the steady-state reactions on the two-site plasmid (Figure 4(a) and (b)). For wt SfiI, most of the reaction proceeded with a linear decline in the concentration of the substrate with time and, as expected, its reaction velocity was 10-fold lower than that on the two-site plasmid (0.21 compared to 2.2 mol/mol/min). However, the same concentration of Y68F yielded markedly biphasic kinetics: a rapid phase that could be fitted to a single exponential to give a rate constant of 0.7 min− 1, followed by a slower linear phase with a velocity of 0.10 mol/mol/min (Figure 5(b)). The amount of substrate consumed during the fast phase (about 1.5 nM) was larger than the amount of enzyme in this reaction (0.5 nM), so the fast phase cannot be due to a pre-steady-state burst of product formation stoichiometric with the enzyme. Instead, the fast phase must reflect multiple turnovers of an active form of the enzyme that decays exponentially to a less active form, the latter being responsible for the slow linear phase of the reaction. The initial rate for the utilisation of the one-site plasmid by Y68F, in the fast phase, was about sevenfold faster than its reaction on the two-site plasmid. Moreover, even during the slow linear phase, Y68F gave virtually the same rates on the one-site and two-site plasmids (0.11 and 0.10 mol/mol/min, respectively).
To see whether the biphasicity varied with enzyme concentrations, both Y68F and wt SfiI were tested against the one-site plasmid at lower (0.05 nM; Figure 5(a)) and at higher (1.5 nM; Figure 5(c)) concentrations. At each level of wt SfiI, the majority of the SC substrate was utilised at a single zero-order rate: the rates increased in direct proportion to the enzyme concentration. Low concentrations of Y68F also gave, for most of the reaction, a linear decline in the concentration of the one-site substrate with time, at a velocity (0.15 mol/mol/min; Figure 5(a)) that was similar to the slow phase of the reaction at 0.5 nM Y68F (0.10 mol/mol/min; Figure 5(b)). In contrast, at high levels of Y68F (Figure 5(c)), virtually all of the one-site substrate was consumed rapidly, at a similar rate to the fast phase from the reaction with 0.5 nM protein.
The active form of Y68F that gives rise to the rapid phase of cutting the DNA with one site is possibly the tetrameric enzyme bound to a single copy of its recognition sequence. This then may decay to a less active form upon binding a second copy to give the synaptic complex, the tetramer bound to two recognition sites. The synaptic complex will form more readily on a two-site DNA than on a one-site DNA,39 so both wt and mutant enzymes are likely to cleave DNA with two sites by forming a synaptic complex bridging two sites in cis. However, despite the difficulty of synapsing sites in trans, wt SfiI cleaves DNA with one site mainly by forming synaptic complexes between sites in separate DNA molecules and not by acting at individual sites.36,38 The complex of wt SfiI bound to one site is therefore either inactive or never present at a significant level. The Y68F mutant may differ from wt in one or both of these aspects.
Reactions in trans
If a restriction enzyme cleaves a plasmid with a single target by acting at that target alone, then the addition of an oligoduplex carrying the recognition sequence will reduce the rate at which that enzyme cleaves the plasmid, as the duplex will act as a competitive inhibitor.20,44 On the other hand, a restriction enzyme that cleaves plasmids with a single site by spanning two sites in trans will be activated by adding an appropriate concentration of the specific duplex, as the synaptic complex can then be formed more readily with one molecule of plasmid and one molecule of duplex rather than with two molecules of plasmid.17,26,28,30 [Higher concentrations of duplex will, however, lead to synaptic complexes with two molecules of the duplex and thus inhibit plasmid cleavage.] Hence, if the fast phase of the reaction of Y68F is due to its reaction at an individual site, it should be inhibited by adding even low concentrations of cognate duplex. But if it is due to a reaction in trans spanning separate molecules of DNA, it should be activated by low levels of the duplex.
Previous experiments had shown that 25 nM duplex activated wt SfiI against 5 nM one-site plasmid.26 This amount of duplex was therefore added to the reaction of Y68F on a one-site plasmid and, for comparison, the wt reaction (Figure 6). HEX-21 (Table 1) was again used as the duplex. Relatively high enzyme concentrations were employed so that Y68F cleaved almost all of the DNA in the fast phase (Figure 5(c)). The plasmid with one site was cleaved by wt SfiI more rapidly in the presence of HEX-21 than in its absence (Figure 6(a)). In marked contrast, the fast phase of the reaction of Y68F on the one-site plasmid was much slower in the presence of HEX-21 than in its absence (Figure 6(b)). In further experiments at this high concentration of Y68F, activated cleavage of the one-site plasmid was not observed at any level of duplex tested (from 1 nM to 1 μM). However, at 0.05 nM Y68F, an enzyme concentration that results in most of the one-site plasmid being cleaved in the slow phase (Figure 5(a)), the addition of HEX-21 enhanced the reaction rate (data not shown).
It thus seems likely that the fast phase of the reaction of Y68F on the plasmid with one SfiI site is indeed due to the tetrameric protein bound to an individual site, while the slow phase of the Y68F reaction and the monophasic reaction of wt SfiI on the one-site DNA are both due to a tetramer bound to two sites in trans. If so, the Y68F mutant bound to one SfiI site gives a much higher reaction velocity than wt SfiI at a solitary site. However, if a reaction in free solution is carried out by adding a one-site DNA to a protein that can bind two sites, then, as there exists no physical means to prevent the protein from binding two separate molecules of DNA, one can never completely exclude the possibility that the observed reaction involves the protein bridging sites in trans.41,42
Reactions at individual sites
To determine unequivocally whether a protein capable of binding two DNA sites is active after binding just one, the protein must be barred from contacting two DNA molecules at the same time. This can be achieved by immobilising the DNA on a solid surface at a low density, so as to hold the DNA molecules separate from each other.41,42 This strategy was applied to SfiI by using a 30-bp duplex with a single cognate site, BIO-30 (Table 1). The site in this duplex is the same as that in the one-site plasmid pGB1/S1 with respect to spacer and flanking sequences. The duplex carried a biotin tag on the 5′ end of one strand, and this was used to attach it to a streptavidin-coated bead. The ratio of streptavidin on the bead to biotinylated DNA was set at 125:1, conditions that result in the mean distance between adjacent DNA molecules on the surface of the bead being considerably longer than that between the two DNA-binding clefts in the SfiI tetramer. This leaves the individual DNA chains too far apart to allow SfiI to form a synaptic complex with two duplexes. The BIO-30 duplex also contained a 32P label at the 5′ end of the other strand, so that cleavage of this strand could be monitored. These experiments employed enzyme concentrations in excess of immobilised DNA and thus are, effectively, single-turnover reactions. The observed rates of DNA cleavage thus reflect the phosphodiester hydrolysis step in the reaction pathway.
Even though wt SfiI generally acts on DNA substrates with a single site by bridging two DNA molecules in trans (Figures 3(a) and 6(a)), it was still able to cleave a one-site DNA even when the individual molecules of that DNA were isolated from each other by immobilisation on the bead (Figure 7). The extent of cleavage of the immobilised substrate by wt SfiI was fitted to a single exponential to give an apparent rate constant of 0.1 min− 1. This rate constant for phosphodiester hydrolysis by wt SfiI at a single site is very much slower than measured previously for the same process in its synaptic complex with two sites (0.1 s− 1).32 Even though the immobilised duplex used here differs from the plasmids used previously for reactions in bulk solution, this 60-fold difference is consistent with other studies that have shown that the activity of wt SfiI bound to two sites is ≥ 30 times higher than that at a single site.36
The single-turnover reaction of Y68F on the immobilised DNA with one SfiI site also gave an exponential decline in substrate concentration, with an apparent rate constant of 0.7 min− 1 (Figure 7), much faster than wt SfiI. Hence, the Y68F mutant bound to a single site cleaves that site more rapidly than wt SfiI at an individual site (Figure 7). Moreover, the rate constant for phosphodiester hydrolysis by Y68F bound to the immobilised DNA equals that from the fast phase of the reaction of Y68F on the one-site plasmid in bulk solution (Figure 5(b)). Although these experiments employed different types of DNA substrates—in one case an oligoduplex attached to a coated bead, and in the other case a plasmid in free solution—they still support the view that the fast phase from the reaction of Y68F on the one-site plasmid is due to the enzyme bound to a single site.
Discussion
The tetrameric SfiI restriction endonuclease26,34 is now one of the principal experimental systems used for the analysis of long-range interactions between distant DNA sites.39 Two of its subunits (a primary dimer)24,28 form one DNA-binding cleft, and the other two form a second cleft on the opposite side of the protein: the clefts are separated by about 80 Å.25 SfiI recognises a palindromic DNA sequence31 and the two subunits in each primary dimer interact symmetrically with the cognate site, in much the same way as a standard dimeric restriction enzyme with a single DNA-binding cleft. However, virtually no DNA is cleaved by the complex of wt SfiI with one recognition site: instead, almost all of its reactions are due to the complex with two sites.33 Moreover, it has to be the scissile site in both clefts: a complex carrying the recognition sequence in one cleft and, in the other cleft, either a noncognate sequence 1 bp different or a phosphorothioate derivative of the correct sequence, fails to cleave either DNA.36,38 Hence, information about the occupancy of each DNA-binding cleft, and the susceptibility of the sequence in each cleft, must somehow be transmitted through the protein to the other cleft 80 Å away.
The transfer of information between the two DNA-binding clefts in a tetrameric restriction enzyme must occur through the interface between the two primary dimers.41,42 Inspection of the crystal structure of SfiI bound to two cognate duplexes suggests that Tyr68 may play a pivotal role at this interface (Figure 1). The aromatic ring of this tyrosine sits in a hydrophobic pocket in the opposite subunit, and its hydroxyl group forms an inter-subunit hydrogen bond across the dimer interface.25 The removal of the hydroxyl function from Tyr68, by the conservative mutation to phenylalanine, resulted in a protein, Y68F, that retains the tetrameric structure of wt SfiI and its ability to bind two DNA sites at the same time, to cleave two sites in cis concertedly (Figures 2, 3 and 4). In these respects, Y68F is like wt SfiI, though its turnover rate on the two-site plasmid is about 20-fold lower than wt, on account of its slow dissociation from the doubly-cut product (Figure 4(c)). However, Y68F differs markedly from wt on DNA with one SfiI site (Figure 5). The wt enzyme acts more slowly on one-site than on two-site DNA because it acts on the one-site DNA in trans, bridging two separate molecules of the DNA, and on the two-site DNA in cis, looping out the intervening DNA. In contrast, the steady-state reaction of Y68F on the plasmid with one SfiI site gave biphasic kinetics—a fast phase that was considerably faster than its reaction on the two-site plasmid, but which then declined exponentially to a slower phase whose rate matched that on the two-site DNA. Both phases involve multiple turnovers of the enzyme. Further experiments showed that the fast phase is due to Y68F acting at individual sites and that the slower phase is due to its synaptic complex with two DNA sites (Figures 6 and 7). Y68F can thus cleave one-site DNA without the need to bridge two separate molecules of the DNA (Figure 6). The net effect of this Tyr→Phe mutation is thus to switch SfiI from an enzyme showing high activity at two sites and low activity at one site to the exact opposite—low activity at two sites and high activity at one site. It inverts the ratio of its turnover rates on two-site versus one-site substrates from 10:1 for wt SfiI to approximately 1:10 for Y68F (as measured from its fast phase on the one-site DNA).
Kinetic model for SfiI cooperativity
Cooperative action by an oligomeric protein is generally accounted for by either the Monod–Wyman–Changeux (MWC) or the Koshland–Nemethy–Filmer (KNF) scheme.51,52 In both schemes, each subunit of the oligomer exists in either an inactive low-affinity tense (T) state or an active high-affinity relaxed (R) state. In the following, the terms T and R apply to a primary dimer of SfiI, a single DNA-binding unit, so that the tetramer is noted as T/T, T/R or R/R (Figure 8(a)): the subscript “S” will denote bound substrate. In the MWC model, the T→R transition occurs in all or none of the subunits so that the protein never contains both T and R subunits at the same time. On the other hand, the KNF model proposes sequential T→R transitions upon ligand binding to each subunit, allowing hybrid oligomers containing both T and RS subunits. [To keep the Y68F protein active, the reactions reported here were carried out by adding Mg2+ to mixtures of enzyme and DNA and thus might appear to start from an enzyme–DNA complex rather than from free enzyme. However, these reactions had the same kinetics as those started by adding enzyme to the DNA–Mg2+ mix, so the dissociation of any DNA bound in the pre-equilibrium must be rapid compared to the subsequent steps. Hence, these reactions can be considered as starting from the free enzyme. In the absence of metal ions as in the pre-equilibria, SfiI binds weakly and nonspecifically to DNA.36]
The properties of wt SfiI, its cooperative binding to two DNA duplexes and its enhanced activity on two-site over one-site substrates, can be accounted for readily on either the MWC or the KNF model. The wt enzyme almost always needs to bind two copies of its recognition site before it can cleave DNA; thus, on DNA with one site, it acts primarily in trans, bridging two DNA molecules. The wt reaction on a one-site DNA thus proceeds largely via the RS/RS state, to the virtual exclusion of T/RS or R/RS forms. On a DNA with two sites, the wt enzyme binds both sites in cis to again yield the RS/RS state, but in this case the transition from the free T/T form to the doubly-liganded RS/RS structure will occur much more readily than on a one-site DNA due to the local concentration effect for sites in cis.39,45 In contrast, the Y68F mutant of SfiI cleaves DNA readily after binding just one site, so that a substrate-induced conformational change in one primary dimer, to give the hybrid T/RS state, is sufficient for Y68F activity. The presence of hybrid species is allowed by the KNF scheme but not by the MWC scheme; thus, if both Y68F and wt SfiI follow the same mechanism, it has to be a KNF scheme.
A simplified three-state version of the KNF model for cooperative action by a dimer, as opposed to the full nine-state version, is shown in Figure 8(a): in this scheme, the substrate-binding and the T→R steps always occur together so that the unliganded T/R and R/R states, the partially liganded T/TS and R/RS states and the fully liganded TS/TS and TS/RS forms are all excluded, leaving only T/T, T/RS and RS/RS. It also coalesces the DNA-cleavage and product-release steps.
To examine whether the scheme in Figure 8(a) can describe the reactions of both wt and Y68F enzymes on both one-site and two-site substrates, the differential equations for the change in concentration of each species with time were solved by numerical integration, using a range of different values for each of the six rate constants: k1 and k− 1 for the first DNA-binding event; k2 and k− 2 for the second DNA-binding event; and k3 and k4 for DNA cleavage/product release from the singly- and doubly-liganded protein. Numerous trials were undertaken, with different values, until a match between model and experiment had been obtained. The values used for Y68F and for wt SfiI differed from each other, but in both cases, the same sets of rate constants were used for their reactions on the one-site and two-site substrates. The distinction between one and two sites was modelled instead by multiplying k2[S], the association rate for the second DNA, by a factor Ω to account for the local concentration of one DNA site in the vicinity of another being higher for sites in cis than for sites in trans: by definition, Ω = 1 for sites in trans and, by selection, Ω = 100 for sites in cis. Even though the latter value was selected arbitrarily, it reflects experimental measurements45 and large (10-fold) variations in this figure made essentially no difference to these calculations (data not shown).
For both enzymes, a set of rate constants was found that yielded progress curves that closely matched the experimental data for that enzyme on both one-site and two-site substrates (Figure 8(c)) for wt; Figure 8(d) for Y68F: the corresponding experimental data are shown in Figures 4 and 5. To match the experimental data, it was necessary to stipulate that both rate constants leading to the formation of T/RS (k1 and k− 2) were smaller for wt SfiI than for Y68F and that, conversely, both rate constants leading away from T/RS (k− 1 and k2) had to be smaller for Y68F. In particular, for Y68F, k2 had to be held at a low value, comparable to that for the DNA-cleavage/product-release steps from the T/RS intermediate (k3), in order to simulate the biphasic reaction profile on the one-site DNA (Figure 5(b)). [If k2 was set either much smaller or much larger than k3, the substrate concentration declined linearly with time. In addition, in order to account for the slower turnover rate of Y68F on the two-site plasmid, k4 had to be fixed at a lower value than for wt SfiI, but this concurs with the experimental data: k4 encompasses both DNA cleavage by RS/RS and product release, and the release of the doubly-cut product is slower from Y68F than from wt SfiI (Figure 4(c)).] However, the models used the same values for k3, the rate constant for DNA cleavage by the complex with one DNA (the T/RS state). The fact that the T/RS state leads to some DNA cleavage by Y68F, but virtually none by wt SfiI, is due to the differing extents to which this state is populated during their reactions.
In this scheme (Figure 8(a)), the principal effect of replacing Tyr68 with Phe is to reduce the differences in free energy between the T/RS state and either the T/T or the RS/RS structures. With wt SfiI, the T/RS state is very strongly disfavoured relative to either T/T or RS/RS in thermodynamic terms, and it also has a short lifetime so that wt SfiI cleaves DNA with one site by forming the RS/RS complex with two sites in trans. Even when a high value is allotted to the wt enzyme for k3 (the rate constant for DNA cleavage by the T/RS state), virtually all of the DNA is still cleaved by wt via its RS/RS state. In contrast, the T/RS state for Y68F is more stable thermodynamically than that for the wt enzyme and it also has a longer lifetime, due to a low value for k2, the rate constant for the T/RS→RS/RS step. Hence, with Y68F, the one-site DNA is cleaved initially via the T/RS state, but because the T/RS→RS/RS transition still has a negative ΔG°, it will eventually form the RS/RS state with two DNA molecules in trans.
Structural model for SfiI cooperativity
How does this Tyr→Phe change stabilise the T/RS intermediate? At present, a crystal structure is available for wt SfiI bound to two cognate duplexes, but not for the free protein without DNA. Given the scheme in Figure 8(a), the solved structure is presumably akin to the RS/RS state, although it may denote a precursor to the catalytically active form.25 Nevertheless, the cartoon of RS/RS (Figure 8(a)) relates to the known crystal structure (Figure 1(a)). In this structure, the two subunits that make up the primary dimers are in close proximity at the DNA-binding surface but lie distant from each other at the dimer interface.
In almost every case to date where crystal structures are available for a restriction enzyme in both DNA-bound and free forms,1,4,5 the DNA-binding cleft has an open configuration in the absence of DNA, but it closes up around the DNA in the complex. Hence, it is possible that the two DNA-binding clefts in SfiI also have open configurations in the absence of DNA but become narrower upon binding cognate DNA. If so, then the movement of two subunits towards each other across the DNA-binding cleft might result, given rigid body motion, in these subunits moving away from each other at the interface with the other primary dimer (as illustrated by the T/T→ RS/RS transition in Figure 8(a)). The interface between the dimers is relatively flat and is almost entirely hydrophobic in nature,25 which might permit each subunit to slide or rotate past the opposite subunit in the other dimer. A similar scheme for intersubunit motion has been proposed for the subunit rotation step in site-specific recombination by γδ resolvase.53 However, in the crystal structure of wt SfiI bound to two duplexes, the hydroxyl groups of Tyr68 in all four subunits make direct hydrogen bonds to Gln30 in their partner subunits across the dimer interface. In order to satisfy the hydrogen-bonding potentials of both Tyr68 and Gln30, these interactions are likely to be present not only in the RS/RS state, as observed in the crystal structure, but also in the free protein, the T/T state, even though the free protein may have a different geometry at the dimer interface. On the other hand, when only one of the two primary dimers changes conformation from T to R upon DNA binding, the Tyr68 and Gln30 residues in the liganded RS dimer will no longer be in register with those in the unliganded T dimer (Figure 8(a)). This dislocation may destabilise the T/RS state, as neither Tyr68 nor Gln30 will have a suitably positioned hydrogen-bonding partner.
In the above scheme, one function of Tyr68 in wt SfiI is to ensure that whenever one of the two dimers binds DNA and undergoes the T-to-RS transition, the other dimer is forced to follow suit and to undergo immediately its own switch from T to R, so as not to accumulate the highly unstable T/RS state. However, without the Tyr68–Gln30 interaction, as in the Y68F mutant, the T-to-RS transition in one dimer would no longer compel the other dimer to undergo straightaway its own T-to-R switch. The hybrid T/RS form is more stable and has a longer lifetime in the reaction of Y68F compared to wt, so it accumulates sufficiently and exists long enough to allow for some DNA cleavage events while in this state.
Materials and Methods
Mutagenesis
Plasmids containing the genes coding for the SfiI restriction endonuclease, pRRS-SfiIR+, and the SfiI modification methyltransferase, pSYX33-SfiIM+, were provided by Ira Schildkraut (New England Biolabs) and were used to transform Escherichia coli ER2353, first with pSYX33-SfiIM+ and then with pRRS-SfiIR+. The mixture of plasmids isolated from this strain was employed for site-directed mutagenesis of the SfiI restriction gene by the QuikChange method (Stratagene). The resultant PCR products were used to transform E. coli ER2353 (pSYX33-SfiIM+). The plasmids were isolated from the transformants, and the derivatives of pRRS-SfiIR+ were sequenced across the entire gene for the mutant SfiI endonuclease (University of Dundee Sequencing Service): only the Y68F mutation had been introduced.
Proteins and DNA
Wt SfiI and Y68F were purified from E. coli ER2238 cells that had been transformed successively with pSYX33-SfiIM+ and pRRS-SfiIR+ (or a derivative) as described previously.26 The purified enzymes were stored at − 20 °C. Concentrations of wt SfiI and the Y68F mutant were assessed by absorbance at 280 nm using an extinction coefficient of 123,200 M− 1 cm− 1, where M refers to the tetramer.34 All SfiI molarities are thus cited for the tetramer with Mr = 124,176. Protein structures were analysed with INSIGHT II v. 2005 (Accelrys, San Diego), and surface charge was evaluated with GRASP v. 1.2.48
The plasmids pGB1/S1 and pGB1,26 which contain one or two SfiI sites, respectively, were used to transform E. coli ER2267, and the resulting transformants were grown in M9 minimal media containing 37 MBq/l [methyl-3H]thymidine (GE Healthcare). The covalently closed form of the plasmid was purified by density gradient centrifugations.32–35 The preparations contained mostly the SC form of the monomeric plasmid, with generally < 10% as either dimer or OC. DNA concentrations were assessed by absorbance at 260 nm.
All oligonucleotides were obtained from Sigma Genosys as HPLC-purified samples. Oligonucleotides were annealed to give the duplexes shown in Table 1 by heating a mixture of two oligonucleotides with complementary sequences to 95 °C and then slowly cooling overnight to room temperature. The mixtures generally contained more of the “bottom” strand than the “top” strand to ensure that all of the top strand was incorporated into the duplex. The bottom strand of the BIO-30 duplex (Table 1) was phosphorylated at its 5′ end by using T4 polynucleotide kinase (Roche) and [γ-32P]ATP (GE Healthcare), as described before.30,36
Analytical ultracentrifugation
Sedimentation to equilibrium was done at 20 °C in a Beckman XL-A analytical ultracentrifuge using an An60-Ti rotor with six channel centrepieces.44 The three sample channels contained 100 μl of HEX-21 or HEX-21 with either Y68F or wt SfiI in AUC buffer [10 mM Tris–HCl, 50 mM NaCl, 10 mM CaCl2 and 1 mM dithiothreitol (DTT); pH 7.9]. The proteins and the DNA, both at 2.5 μM, had been dialysed previously against AUC buffer, and the reference channels contained 110 μl of the dialysate. Centrifugation was carried out sequentially at 10,000, 15,000 and 25,000 rpm. After 16 and 20 h at each speed, the differences in absorbance at 539 nm between samples and references were recorded as a function of centrifugal radius (r). At all speeds, the profiles at 16 and 20 h were identical, showing that equilibrium had been reached. Centrifugation was then continued for 8 h at 40,000 rpm to obtain the baseline offset. For each sample, plots of A539 against centrifugal radius at varied speeds were fitted globally to the equation for a single homogenous species,49to give values for the molecular mass (M, given here as Mr values): Ar and A0 are the absorbances at r and at the reference r0, respectively; ν¯ is the partial specific volume; ρ is the buffer density; ω is the angular velocity; and B is the baseline offset. Values for ν¯ and ρ were obtained as before.44
DNA-binding studies
Binding reactions were performed by adding aliquots of wt SfiI or Y68F in dilution buffer to either HEX-21 or HEX-35, or to mixtures of the two duplexes, to give solutions that had, in 20 μl of binding buffer, 5 nM SfiI tetramer and a total duplex concentration of 10 nM. Dilution buffer is composed of 20 mM Tris–HCl, 10 mM β-mercaptoethanol, 0.1 mM EDTA, 10% vol/vol glycerol, 1 mM spermine and 0.2% vol/vol Triton X-100 (pH 7.5). Binding buffer is composed of 20 mM Tris–HCl, 25 mM NaCl, 2 mM CaCl2, 5 mM β-mercaptoethanol and 100 μg/ml bovine serum albumin (pH 7.5). After 30 min at room temperature, the samples were mixed with 10 μl of binding buffer containing 4% (wt/vol) Ficoll 400 and applied to 8% polyacrylamide gels in 45 mM Tris–borate (pH 8.3) and 2 mM CaCl2, as described previously.36 After electrophoresis, the gels were scanned in a Molecular Dynamics PhosphorImager with illumination at 550 nm.
Enzyme assays
For kinetic experiments, the final reaction mixtures contained 5 nM 3H-labelled DNA, either pGB1/S1 or pGB1, and various concentrations of either wt SfiI or the Y68F mutant in 200 μl of reaction buffer at 50 °C. Reaction buffer is composed of 10 mM Tris–HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT and 100 mg/ml bovine serum albumin (pH 7.9). The reactions were carried out by adding 10 μl of MgCl2 (200 mM) to 190 μl of enzyme and DNA in reaction buffer lacking MgCl2 (apart from some trial reactions initiated by adding enzyme to DNA in reaction buffer). Aliquots (15 μl) were removed from the reactions at various times after adding MgCl2 (one was removed before adding MgCl2 to serve as a zero time point) and vortexed immediately with 10 μl of an EDTA stop mix.18–20 The samples were analysed by electrophoresis through agarose under conditions that separate the various products from each other and from the SC substrate. The concentrations of each form at each time point were determined by scintillation counting.32–36 All values given here are presented as the means from three independent experiments, with error bars to denote standard errors. Zero-order rates were evaluated using GRAFIT (Erithacus Software) to fit the initial phases of substrate utilisation and/or product formation to linear plots. For some reactions (viz. Figure 5(b)), the concentration of DNA substrate was fitted to an initial exponential decline followed by a zero-order phase. For the various reaction mechanisms considered here (Figure 8), extents of substrate utilisation were modelled by solving the differential equations for the time-dependent changes in the concentrations of each species during the course of the reaction by numerical integration in BERKELEY MADONNA.
Some reactions on pGB1/S1 also contained the oligoduplex HEX-21. These typically contained, in 200 μl of reaction buffer at 50 °C, 1.5 nM enzyme (either Y68F or wt SfiI), 5 nM pGB1/S1 (3H-labelled) and 25 nM HEX-21: samples were taken at varied times after initiating the reaction with MgCl2 and analysed as described above to determine the amount of pGB1/S1 that had been converted to its linear form.
Immobilised oligonucleotides
Streptavidin-coated magnetic beads from Promega (10 pmol of streptavidin) were mixed with 32P-labelled BIO-30 (0.08 pmol) in 80 μl of SSC,41 washed in SSC and resuspended in 200 μl of reaction buffer without MgCl2. The requisite concentrations of either wt SfiI or the Y68F mutant were then added before initiating the reactions with MgCl2 (final concentration, 10 mM). Aliquots (15 μl) were taken from the reactions at varied times and quenched with loading dye (10 mM NaOH, 100 mM EDTA, 95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanol). After incubating first at 95 °C for 10 min and then on ice for 15 min, the samples were analysed by denaturing gel electrophoresis through a 12% polyacrylamide gel in TBE buffer [45 mM Tris–borate and 1 mM EDTA (pH 8.3)] at ∼ 40 V/cm and 55 °C. The gels were fixed in 20% (vol/vol) acetic acid and 20% (vol/vol) methanol, dried, exposed overnight and scanned in a PhosphorImager. The amounts of intact and cleaved 32P strands of the duplex were quantified using ImageQuant (Molecular Dynamics), and the decline in substrate concentration with time fitted to a single exponential in GRAFIT.

restriction enzyme
cooperativity
auc, analytical ultracentrifugation
dtt, dithiothreitol
hex, hexachlorofluorescein
knf, koshland–nemethy–filmer
lin, linear
mr, relative molecular mass
mwc, monod–wyman–changeux
oc, open circle
sc, supercoiled
wt, wild type
allostery
dna–protein interaction
protein–protein interaction


Tetraether membrane lipids of Candidatus “Aciduliprofundum boonei”, a cultivated obligate thermoacidophilic euryarchaeote from deep-sea hydrothermal vents

The lipid composition of Candidatus “Aciduliprofundum boonei”, the only cultivated representative of archaea falling in the DHVE2 phylogenetic cluster, a group of microorganisms ubiquitously occurring at hydrothermal vents, was studied. The predominant core membrane lipids in this thermophilic euryarchaeote were found to be composed of glycerol dibiphytanyl glycerol tetraethers (GDGTs) containing 0–4 cyclopentyl moieties. In addition, GDGTs with an additional covalent bond between the isoprenoid hydrocarbon chains, so-called H-shaped GDGTs, were present. The latter core lipids have been rarely reported previously. Intact polar lipid analysis revealed that they predominantly consist of GDGTs with a phospho-glycerol headgroup.
Introduction
Deep-sea hydrothermal vents are unique environments, which are thought to represent models for both the origin of life on Earth and exploration of life on other planets. These vents contain ecosystems, which are predominantly fueled by geochemical energy and are host to many newly described free-living microbes, which are often associated with actively venting porous deep-sea vent deposits or “chimneys”. The steep chemical and thermal gradients within the walls of these deposits provide a wide range of microhabitats for microorganisms with suitable conditions for aerobic and anaerobic thermophiles and mesophiles (e.g. McCollom and Schock 1997). Indeed, both culture-dependent and -independent approaches have exposed a vast diversity of Bacteria and Archaea associated with deep-sea vent deposits (e.g. Reysenbach and Shock 2002; Schrenk et al. 2003). Numerous Archaea have been isolated from these deposits but few of these are found in environmental 16S rRNA gene clone libraries and most detected environmental clones in these environments have no representatives available in pure culture.
In particular, one archaeal lineage is widespread at deep-sea vents, namely the “deep-sea hydrothermal vent euryarchaeotic” lineage DHVE2, and is frequently associated with actively venting sulphide deposits (e.g. Nercessian et al. 2003; Hoek et al. 2003; Reysenbach and Shock 2002; Takai et al. 1999, 2001). In some cases it has been reported as the most dominant clone type in archaeal clone libraries (Hoek et al. 2003), yet the physiology of these organisms was unclear. Recently, Reysenbach et al. (2006) isolated and cultivated a member of the DHVE2 phylogenetic DHVE2 cluster, Candidatus “Aciduliprofundum boonei”, and showed it to be an obligate thermoacidophilic sulphur and iron reducing heterotroph capable of growing from pH 3.3 to 5.8 and between 60 and 75°C. This provided the first evidence that thermoacidophiles may be key players in sulphur and iron cycling at deep-sea vents.
Archaea are not only unique in their 16S rRNA phylogenetic position in the tree of life, but also synthesize specific membrane lipids. Analysis of cultivated hyperthermophilic Archaea showed that their membrane is predominantly composed of isoprenoid glycerol dibiphytanyl glycerol tetraethers (GDGTs) with additional cyclopentyl moieties (e.g. Structures I–VI in Fig. 1). The structural differences from diacyl membrane lipids of non-thermophilic Eukarya and Bacteria, i.e. ether bonds and the formation of a monolayer rather than a bilayer, have been suggested to contribute to the stability of membranes of hyperthermophiles at high temperatures and low pH (e.g. De Rosa and Gambacorta 1988; van den Vossenberg et al. 1998; Macalady et al. 2004). This suggests that members of the DHVE2 cluster may be also synthesizing GDGT membrane lipids. Therefore, in this study we analyzed the lipid composition of the only cultivated representative of DHVE2, Aciduliprofundum boonei, and examined both the core GDGT lipid composition as well as its intact polar lipid composition.
Fig. 1HPLC/APCI/MS base peak chromatogram of GDGT core lipids in extract released after base hydrolysis (using KOH/methanol mixture) of the cell material of Candidatus “Aciduliprofundum boonei”. Inset shows the atmospheric pressure chemical ionization mass spectrum of GDGT VII. Note that the position of the covalent bond between the isoprenoid hydrocarbon chains in GDGTs VII–XI is tentative (Morii et al. 1998)
Materials and methods
Culture conditions
Aciduliprofundum boonei was grown as previously described (Reysenbach et al. 2006). Cells were harvested at late log phase of growth. Cell pellets were freeze-dried and stored frozen until analysis.
Core lipid analysis
Cell material (74 mg dry weight) of A. boonei was hydrolyzed by refluxing in ca. 4 ml 1 M KOH in methanol for 1 h. The pH of the hydrolyzed extract was adjusted to pH 3 using 2 M HCl/methanol (MeOH) 1/1 (v/v) and transferred to a separatory funnel. The residual cell material was washed subsequently with 2 ml MeOH/water 1/1 (v/v), MeOH and dichloromethane (DCM, three times). The washings and 6 ml bidistilled water were added to the separatory funnel, which was shaken vigorously. The DCM layer was removed and the residual water/MeOH layer was re-extracted two times using DCM. The combined DCM layers were dried using a sodium sulfate column to yield the hydrolyzed extract. The extract released upon hydrolysis was condensed by rotary evaporation. Elemental sulphur was removed with activated Cu. The extract was methylated by diazomethane and filtered over a silicagel column using ethyl acetate. One aliquot of the eluate was silylated using BSTFA (with 1% TMCS) in pyridine (1/1, v/v) for 20 min at 60°C and subsequently analyzed by gas chromatography (GC) and GC/mass spectrometry (GC/MS). Another aliquot of the eluate was dissolved in hexane/isopropanol (99:1, v/v), ultrasonicated and filtered using a PTFE 0.45-μm filter prior to analysis for GDGTs on the high performance liquid chromatography (HPLC)/MS.
Part of the GDGT fraction was hydrogenated by dissolving it in ethyl acetate, adding a small amount of PtO2 and a drop of acetic acid and bubbling hydrogen for 1 h. Subsequently, the solution was stirred overnight and filtered over a small pipette column containing Na2CO3 (top) and MgSO4 (bottom). Ethyl acetate was evaporated and the hydrogenated GDGT fraction was dissolved in hexane/isopropanol (99:1, v/v), ultrasonicated and analyzed using HPLC/MS.
A second aliquot of the GDGT fraction was separated using HPLC with a preparative device (Foxy jr) and the fractions collected were analyzed by flow injection analysis mass spectrometry (Smittenberg et al. 2002). The fractions containing the unusual GDGT (see text below) were combined and treated with HI and LiAlH4 to release the ether bound hydrocarbon skeletons (Schouten et al. 1998). This fraction was analyzed by GC and GC/MS.
The total extract and the GDGT fractions treated with HI/LiAlH4 were analyzed by GC/MS using a Finnigan Trace GC ultra coupled to a Finnigan Trace DSQ mass spectrometer. A fused silica capillary column (25 m × 0.32 mm) coated with CP-Sil 5 (film thickness 0.12 μm) was used with helium as carrier gas. The oven was programmed at a starting (injection) temperature of 70°C, which rose to 130°C at 20°C/min and then to 320°C at 4°C/min, at which it was maintained for 20 min. The column was directly inserted into the electron impact ion source of the DSQ quadrupole mass spectrometer, scanning a mass range of m/z 50–800 at three scans per second and with an ionization energy of 70 eV.
HPLC/MS analyses were performed according to Hopmans et al. (2000). Analyses were performed using an Agilent (Palo-Alto, CA, USA) 1100 series LC-MSD SL equipped with an auto-injector and Chemstation chromatography manager software. Separation was achieved on a Prevail Cyano column (2.1 × 150 mm, 3 μm; Alltech, Deerfield, IL, USA), maintained at 30°C. Injection volumes were 10 μl. GDGTs were eluted isocratically with 99% A and 1% B for 5 min, followed by a linear gradient to 1.8% B in 45 min, where A = hexane and B = isopropanol. Flow rate was 0.2 ml/min. After each analysis the column was cleaned by back-flushing hexane/isopropanol (90:10, v/v) at 0.2 ml/min for 10 min. Detection was achieved using atmospheric pressure positive ion chemical ionization mass spectrometry (APCI-MS) of the eluent. Conditions for the HP 1100 APCI-MSD SL were as follows: nebulizer pressure 60 psi, vaporizer temperature 400°C, drying gas (N2) flow 6 l/min and temperature 200°C, capillary voltage −3 kV, corona 5 μA (∼3.2 kV). GDGTs were detected by mass scanning from m/z 950–1,450.
Intact polar lipid analysis
For analysis of the intact polar lipids, cell material of A. boonei were extracted using a modified Bligh–Dyer procedure (Bligh and Dyer 1959). To an aliquot of the cell material, a solvent mixture of phosphate-buffer (0.05 M, pH 7.4)/MeOH/DCM 0.8/2/1 (v/v) was added. The mixture was sonicated for 10 min after which DCM and phosphate buffer were added to a volume ratio of 0.9/l/1. After centrifuging (2,500 rpm, 5 min) the DCM layer was collected. The residue was re-extracted twice following the same procedure. The combined DCM layers were concentrated by evaporating solvent under a N2 stream, dried with Na2SO4 and the solvent was removed under a N2 stream. The residue was dissolved in a mixture of hexane/isopropanol (79:21, v/v), ultrasonicated and filtered using a RC 0.45-μm filter prior to analysis on the HPLC/electrospray ionization (ESI)–MS.
Intact core lipids were analyzed according to Sturt et al. (2004) with some modifications (Boumann et al. 2006). An Agilent 1100 series LC (Agilent, San Jose, CA, USA) was used, equipped with thermostatted autoinjector and column oven, coupled to a Thermo TSQ Quantum Ultra EM triple quadrupole mass spectrometer equipped with an Ion Max source with ESI probe (Thermo Electron Corporation, Waltham, MA, USA). Separation was achieved on an Inertsil diol column (250 mm × 2.1 mm, 5 μm particles; Alltech Associates Inc., Deerfield, IL, USA) maintained at 30°C. The following linear gradient was used with a flow rate of 0.2 mL min−1: 100–35% A:65% B over 45 min, maintained for 20 min, then back to 100% A for 20 min to re-equilibrate the column, where A = hexane/2-propanol/formic acid/14.8 M NH3aq ratios 79:20:0.12:0.04 (v/v/v/v) and B = 2-propanol/water/formic acid/14.8 M NH3aq ratios 88:10:0.12:0.04 (v/v/v/v). For MS detection, source parameters were optimized using loop injections of standard phospholipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine, 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-glycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphate, and soya l-α-phosphatidylinositol, Avanti Polar Lipids, Alabaster, AL, USA) into a stream of 0.2 ml/min of eluent A. ESI settings were as follows: capillary temperature 250°C, sheath gas (N2) pressure 49 (arbitrary units, auxiliary gas) (N2) pressure 21 (arbitrary units), spray voltage 4.2 kV, and source CID-14 V. Mass detection was achieved on the first MS scanning from m/z 1,000 to 2,000.
Results and discussion
Core lipids
GC/MS analysis of the lipid extract of hydrolyzed cell material of A. boonei did not reveal substantial amounts of dialkyl glycerol ethers, such as archaeol, suggesting that it does not accumulate these compounds in substantial amounts. By contrast, HPLC/APCI-MS analysis of the fraction revealed the abundant presence of a range of GDGTs (Fig. 1) suggesting that archaeol is effectively transformed into GDGTs. The main compound was GDGT II, containing no cyclopentyl moieties, with lower amounts of GDGTs III–VI, containing 1–4 cyclopentyl moieties, and small amounts of GDGT I. These GDGTs are well known compounds of thermoacidophilic Archaea in both the euryarchaeotal and crenarchaeotal groups (cf. De Rosa and Gambacorta 1988; Koga and Morii 2005). Cultivated species of the Order Thermoplasmatales, the closest phylogenetic relatives to A. boonei (Reysenbach et al. 2006), also contain GDGTs II–VI as their dominant membrane lipids (DeRosa and Gambacorta 1988; Uda et al. 2004; Macalady et al. 2004).
Besides GDGTs I–VI, another group of later-eluting compounds were present in the HPLC/MS chromatogram with mass spectra (insert Fig. 1) characteristic of GDGTs, i.e. a base peak likely representing the [M + H]+ ions and fragment ions with losses of 18 Da (hydroxyl group as water) and 74 Da (part of the glycerol moiety) (Hopmans et al. 2000). The main compound had a base peak ion of 1,300 with more minor compounds having base peak ions of 1,298, 1,296, 1,294 and 1,292, respectively. Remarkably, the distribution of these compounds is similar to that of GDGTs II–VI and their base peak ions are all 2 Da lower than their GDGT counterparts. This suggests that these compounds are GDGTs with an additional double bond or an additional cyclic moiety. To investigate the first hypothesis, we hydrogenated the polar fraction to remove any double bonds. However, subsequent analysis by HPLC/MS revealed no changes in GDGT composition, suggesting that these compounds did not contain any double bonds. The unknown GDGT was subsequently isolated by preparative HPLC and treated with HI/LiAlH4 to release the hydrocarbon skeletons (cf. Schouten et al. 1998). GC/MS analysis revealed, however, no distinct peaks suggesting that the hydrocarbon skeletons of the unknown GDGT do not consist of regular C40 isoprenoids. An explanation for these results can be that the hydrocarbon skeletons are covalently linked to each other, thereby forming a C80 hydrocarbon after ether-cleavage. The molecular weight of such a component is too high to elute over a regular GC column. Examples of such GDGTs have been previously reported, i.e. the so-called H-shaped GDGT (Morii et al. 1998) that contains a covalent bond between the two isoprenoid chains. We, therefore, analyzed a pure GDGT VII standard obtained from Methanothermus fervidus (a kind gift of Dr. T. Eguchi) by HPLC/MS. The mass spectrum of this standard was identical to that of the major unknown GDGT in A. boonei and co-injection revealed that they have identical retention times, thus unambiguously identifying this compound as GDGT VII. This suggests that the other later eluting GDGTs are also H-shaped isoprenoid GDGTs but with additional cyclopentyl moieties, i.e., GDGTs VII–XI. It must be noted that the exact position of the covalent bond in GDGT VII is tentative (Morii et al. 1998) and, thus, also the position of the covalent bonds in GDGTs VII-XI. Lutneas et al. (2007) recently determined the structure of structurally similar isoprenoid tetracids in which the covalent bond was between two methyl groups rather than between a methyl group and the aliphatic chain as suggested by Morii et al. (1998). Hence, future structural analyses are needed to unambiguously establish the position of the covalent bond.
H-shaped isoprenoid GDGTs have been only rarely reported in cultivated archaea. GDGT VII was first identified in the hyperthermophilic methanogen Methanothermus fervidus (Morii et al. 1998). They were further identified by fast atom bombardment-MS in 4 out of 17 species of the Order Thermococcales (Sugai et al. 2004). Until now, they have only been identified in hyperthermophilic euryarchaeota growing at neutral pH with optimal growth temperatures >80°C. The identification of H-shaped GDGTs in A. boonei now shows that these compounds are also synthesized at lower growth temperatures (70°C) and more acidic pH (4.5). The functional role of these GDGTs is not clear. Possibly, the introduction of a covalent cross-link between the alkyl chains will reinforce the strength of the monolayer membrane to protect the cell against membrane lysis at these high temperatures. The similar relative distribution of GDGTs II–VI compared to GDGTs VII–XI in A. boonei tentatively suggests a possible biosynthetic link, i.e., the latter GDGTs may be synthesized from GDGTs II–VI.
Intact polar lipids
HPLC/ESI-MS analysis of the Bligh–Dyer extract of cell material of A. boonei revealed one major peak and one minor peak (Fig. 2). The mass spectrum of the major peak is dominated by an ion at m/z 1,456 with minor ions 15 and 22 Da higher (insert Fig. 2). These latter ions are typical product of ammonia and sodium adductions, respectively, to protonated molecules in the ESI interface and suggests that the ion at m/z 1,456 represents the [M + H]+ ion (cf. Sturt et al. 2004). As the major core lipid is GDGT II with a [M + H]+ ion of 1,302, it suggests that the head group has a mass of 154 Da in agreement with a phospho-glycerol (PG) moiety (Sturt et al. 2004). The mass spectrum of the minor peak eluting slightly later is similar to that of the major peak except that all ions are shifted 2 Da lower. In view of the results obtained with the core lipids it suggests that this peak represents H-shaped GDGTs with a PG head group. PG moieties are not uncommon as head groups of GDGTs in hyperthermophilic Archaea, though they are usually accompanied by a hexose head group attached to the other glycerol moiety (Koga and Morii 2005 and references cited therein). However, these compounds were not detected in A. boonei although we cannot exclude that they might be present in minor amounts.
Fig. 2HPLC/ESI/MS base peak chromatogram of intact polar lipids in Bligh Dyer extract of cell material of Candidatus “Aciduliprofundum boonei”. Inset shows the electrospray ionization mass spectrum of the major peak in the chromatogram with tentative structural identification
Conclusions
Our analysis of lipid composition of Candidatus “Aciduliprofundum boonei”, the only cultivated representative of archaea falling in the DHVE2 phylogenetic cluster, showed it to be composed of GDGTs containing 0–4 cyclopentyl moieties. Surprisingly, GDGTs with an additional covalent bond between the isoprenoid hydrocarbon chains, the so-called H-shaped GDGTs were also present in substantial amounts. These GDGTs mainly occurred with a phospho-glycerol headgroup in the membrane of A. boonei.

aciduliprofundum boonei
thermoacidophile
dhve2 cluster
glycerol dialkyl glycerol tetraethers


Predicting Stricture in Morbidly Obese Patients Undergoing Laparoscopic Roux-en-Y Gastric Bypass: A Logistic Regression Analysis

Gastrojejunostomy stricture after Roux-en-Y gastric bypass occurs in 3 to 27% of morbidly obese patients in the USA. We questioned whether preoperative patient characteristics, including demographic attributes and comorbid disease, might be significant factors in the etiology of stricture. In this study from November 2001 to February 2006 (51 months), at a high-volume bariatric center, of the 1,351 patients who underwent laparoscopic gastric bypass, 92 developed stricture (6.8%). All but two were treated successfully by endoscopic dilation. All patients stopped nonsteroidal anti-inflammatory medications 2 weeks prior to surgery and did not restart them. The operative procedure included the use of a 21-mm transoral circular stapler to create the gastrojejunostomy; the Roux limb was brought retrogastric, retrocolic. In an effort to reduce our center’s stricture rate, late in the study, U-clips used at the gastrojejunostomy were replaced by absorbable sutures, and postoperative H2 antagonists were added to the treatment protocol. The change to absorbable polyglactin suture proved to be significant, resulting in a lower stricture rate. The addition of H2 antagonists showed no significant effect. Following the retrospective review of the prospective database, univariate and multivariate logistic regression analyses identified factors associated with the development of stricture. Gastroesophageal reflux disease and age were each shown to be statistically significant independent predictors of stricture following laparoscopic gastric bypass.
Introduction
Bariatric surgery, particularly Roux-en-Y gastric bypass (RYGB), has been proven safe1,2 and effective in achieving long-term weight loss,3,4 comorbidity reduction,3,5 and enhancement of quality of life.6 Since its introduction by Wittgrove et al. in 1994,7 laparoscopic Roux-en-Y gastric bypass (LRYGB) has been shown to be as reliable as open RYGB8–10 and less traumatic, requiring a briefer recovery time and shorter duration of stay.11 It is effective in achieving comparable weight loss at 1 year3,12–15 and is successful in reducing the comorbidities of morbid obesity.3,9,13 Laparoscopic RYGB has surpassed other weight-loss methods for the morbidly obese in frequency of use in the USA.14
Laparoscopic RYGB is a technically demanding procedure with a lengthy learning curve of approximately 100 cases.16,17 Gastrojejunostomy stricture following RYGB has been reported variously in 3 to 27% of patients.12,16–19 Actual incidence of this complication is a function of the accuracy of its diagnosis and reporting19 and has been shown to be relative to operative technique and surgeon experience.20 Other known etiologies of stricture following LRYGB are tension on the anastomosis, foreign body reaction, technical error in creation of the anastomosis, marginal ulcer, and leak with associated scarring.19
In 2003, Perugini and colleagues published an analysis of preoperative predictors of complications.20 They reported a 14.4% rate of gastrojejunostomy stricture (N = 27/188), greater than half of all complications. With multivariate analysis by stepwise logistic regression, they found that surgeon experience was the most significant predictor of complications in general (not of stricture specifically) and that sleep apnea and hypertension were also significant independent predictors of complications.20 Perugini et al. cautioned that the factors they identified (experience, sleep apnea, hypertension) as predictive of complications in general might, in fact, “ be specific for the complication of gastrojejunal stenosis”.20 Our study took up this question and, by univariate and multivariate logistic regression, analyzed preoperative predictors specifically for stricture.
Patients and Methods
Patients
A consecutive series of morbidly obese patients underwent LRYGB between November 12, 2001, and February 28, 2006 (51 months), in a dedicated, community-based, bariatric program awarded Center-of-Excellence status by the Surgical Review Corporation in 2005.
Institutional review board approval and informed consent were obtained prior to prospective data collection. In a retrospective analysis, preoperative patient characteristics, including whether the patient was taking any nonsteroidal anti-inflammatory medications on a chronic basis prior to surgery, were compared for two groups: those who developed gastrojejunostomy stricture (N = 17/92, 6.8%) and those who did not (N = 311/1,259, 93.2%). No patients were excluded from the study.
Patients where tested for Helicobacter pylori and treated if positive. Anti-inflammatory medications were withheld 2 weeks preoperatively and not resumed postoperatively. Procedures were performed by four bariatric surgeons. Stenosis, or stricture, indicated by progressive dysphagia, nausea, and vomiting, was diagnosed by endoscopy.
Surgical Technique
The procedure was performed using the transoral end-to-end anastomotic (EEA) technique similar to the Wittgrove technique.10 Using a Veress needle to an intra-abdominal pressure of 15 mm Hg, abdominal insufflation was performed. The patient was placed supine, in the reverse Trendelenburg position, and six abdominal trocars were introduced. The angle of His was dissected, and a 15-ml balloon was passed transorally and placed at the esophagogastric junction. The lesser sac was entered just below the balloon on the lesser curve of the stomach. A 15- to 20-ml gastric pouch was created with the Endo GIA 45-mm stapler (Ethicon Endo-Surgery, Cincinnati, OH, USA), using an average of three firings. A guidewire was introduced through the anterior abdominal wall and an endoscope was passed transorally. Cautery was used to pass the snare through the pouch and grasp the guidewire. The anvil of the 21-mm EEA Stealth stapler (Ethicon Endo-Surgery) was attached to the guidewire and brought down through the mouth. The ligament of Treitz was identified, and, approximately 20 cm distally, the jejunum was divided. A 100-cm Roux limb was created for patients with BMI < 55 kg/m2, and a 150-cm Roux limb was created for patients with BMI ≥ 55 kg/m2. The jejunojejunostomy was stapled side to side, and closed with the Endo GIA stapler. The Roux limb was brought retrocolic, retrogastric through the lesser curve of the stomach. The EEA circular stapler was introduced through the abdominal wall and the gastrojejunostomy was stapled end to end. The posterior wall of the gastrojejuostomy lays on soft tissue that holds the left gastric and vagus nerve and does not need reinforcement. All techniques for reinforcing the gastrojejunostomy were instituted only in the anterior wall of the anastamosis as a second layer. The initial technique was to sew a continuous second layer of the gastrojejunostomy anteriorly with 3–0 polyglactin absorbable suture. The angle is often technically demanding. In October 2005, the procedure was modified to one in which the gastrojejunostomy was oversewn with two Nitinol “U” clips that revert to preformed circular memory when placed, without additional suture. In October of 2005, these U-clips were replaced with two interrupted 3–0 polyglactin absorbable sutures. Finally, in January 2006, the addition of H2 antagonists for 90 days to the postoperative regimen was made. These technical changes were instituted in an attempt to lessen the stricture rate, which is followed monthly in our database.
Patients who experienced persistent postoperative nausea, vomiting, and intolerance to solid food were referred for upper gastrointestinal studies, followed by referral to the gastroenterologist for upper endoscopy. Patients with stricture were treated endoscopically with balloon dilation to a maximum of 15 mm.
Statistical Methods
Patient data were collected prospectively and included preoperative patient demographics and comorbidities, as well as postoperative complications extracted to identify potential influences on stricture rates. Variables included in the univariate logistic regression analysis are listed in Table 1.
Table 1Univariate Analysis to Identify Preoperative Patient Characteristics Associated with StrictureVariableStricture Group [N (%)]Nonstricture Group [N (%)]P ValueNo. patients (N = 1,351)921,259Age, mean (SD)41.4 (11.6)44.4 (10.4)0.039aCardiac disease6 (6.5)107 (8.5)0.885Chronic depression21 (22.8)278 (22.1)0.634Chronic respiratory disease22 (23.9)318 (25.3)0.766Chronic venous insufficiency53 (57.6)692 (55.0)0.347Degenerative joint disease89 (96.7)1,209 (96.0)0.893Diabetes, type 226 (28.3)307 (24.4)0.184Ethnicity, N (%)––0.145 Caucasian76 (82.6)1,122 (89.1)– Other16 (17.4)137 (10.9)–GERD66 (71.7)715 (56.8)0.035aSex, N (%)––0.208 Female81 (88.0)1,042 (82.8)– Male11 (12.0)217 (17.2)–Hypercholesterolemia/hyperlipidemia40 (43.5)624 (49.6)0.958Hypertension41 (44.6)652 (51.8)0.838Infertility11 (12.0)96 (7.6)0.732Nonsteroidal anti-inflammatory medications17 (18.5)294 (23.4)0.284No. preoperative comorbidities5.2 (2.0)5.1 (1.9)0.259No. preoperative medications4.6 (3.6)4.2 (3.5)0.088Obstructive sleep apnea43 (46.7)564 (44.8)0.415BMI48.5 (8.5)49.2 (8.3)0.202Fasting blood sugar111.5 (45.4)108.9 (39.6)0.779HbA1C6.2 (1.8)6.1 (1.2)0.150Previous abdominal surgeries1.2 (1.1)1.2 (1.3)0.754Urinary stress incontinence52 (56.5)698 (55.4)0.322aSignificance at the 0.05 level
The SPSS™ software package (version 14.0, SPSS, Chicago, IL, USA) was used to perform all statistical analyses. Statistical significance was set at P < 0.05. Binary logistic regression analysis was used in both univariate and multivariate modeling to identify independent preoperative variables associated with the development of gastrojejunostomy stricture following LRYGB surgery. Univariate analysis using logistic regression was applied to identify significant associations with the dichotomous outcome variable (stricture). For comparison, Pearson chi-square test for categorical variables and Student’s t test for continuous variables were used. A priori preoperative factors of research interest (P < 0.21), as well as those found to be significantly associated with stricture (P < 0.05), were entered into multivariate analysis using forward stepwise logistic regression. Likelihood ratio tests were used for variable selection.
In the initial analysis of preoperative characteristics by univariate logistic regression, age, number of comorbidities, number of medications, BMI, fasting blood sugar, HbA1C, and previous abdominal surgeries were classified as continuous variables. With the exception of sex and ethnicity (dichotomized to male/female and Caucasian/non-Caucasian, respectively), all remaining variables were dichotomized (yes/no). In the final multivariate forward stepwise logistic regression model, both age and BMI were redefined as categorical variables: age was coded to reflect four levels (≤35, 36 to 45, 46 to 55, and ≥55 years) and BMI was coded to reflect two levels (<50 and ≥50).
Results
From November 12, 2001, to February 28, 2006 (51 months), 1,351 patients underwent LRYGB at a single institution. Mean preoperative patient age was 44 years, mean BMI was 49 kg/m2, 83% of patients were female, and mean preoperative weight was 305 lb. Patients were predominantly Caucasian (89%); 7% were Hispanic; 3% were African American; and 1% were of Native American, Asian/Pacific, and other ethnicities.
Operative technique was the same in all procedures, namely, a retrocolic, retrogastric, transoral, 21-mm, circular stapled gastrojejunostomy. Mean intraoperative time was 96 min and length of stay averaged 2.8 days; 97.6% of operations were accomplished laparoscopically and 2.4% were conversions to open procedures. There were two reoperations with revision of the gastrojejunostomy in the “stricture group.” The first was in a patient who developed a leak immediately postoperatively and returned to the OR for definitive management. The patient developed a stricture that was not amenable to dilation and, at 7 months, underwent a revision of her gastrojejunostomy. The second patient had a stricture that was dilated to 12–15 mm. Subsequent endoscopy showed no recurrent stricture. She continued to eat very large portions in multiple meals, developing recurrent bezoars that had to be cleared endoscopically. The pouch dilated over time, and even though the gastrojejunostomy was shown to be only large enough to pass an endoscope, we were obliged to revise the pouch to a smaller size. Since that time she has had no further problem with bezoars.
There were no deaths in the stricture group. In the “nonstricture group,” early mortality was 0.2% (two deaths <31 days postoperatively); intermediate mortality, 0.3% (four deaths between 31 and 90 days postoperatively); and late mortality, 0.4% (five deaths >90 days postoperatively). Ninety-two patients (6.8%) were diagnosed with stricture and underwent subsequent endoscopic dilation. Seventeen of 92 stricture patients (18.5%) were on nonsteroidal anti-inflammatory medications prior to surgery, whereas 294 of the 1,259 (23.4%) nonstricture patients were on nonsteroidal anti-inflammatory medications prior to surgery (Table 1). The difference in stricture rates between these two groups was not significant.
Our rate of stricture (92/1,351, 6.8%) was in the range of rates previously reported by Higa et al. (5.3%), Schauer et al. (4.7%), and DeMaria et al. (6.6%), as compiled by Perugini20, at the lower end of the national range of stricture prevalence. Other complications with the highest incidence were cholecystectomy (6.5% stricture group vs 3.0% nonstricture group), small bowel obstruction secondary to internal hernia (4.4 vs 1.9%), and intra-abdominal abscess (3.3 vs 2.0%). In the stricture and nonstricture groups, respectively, leakage occurred in 1.1 and 1.0% of patients, and port site infection occurred in 2.2 and 3.6% of patients. Peripheral neuropathy was 5.4% in the stricture group and 0.5% in the nonstricture group (Table 2).
Table 2Forward Stepwise Multivariate Logistic Regression Model for Complications of StrictureVariableRate, N (%)P ValueOdds Ratio (95% Confidence Interval)Stricture GroupNonstricture Group30-day readmission27 (29.35%)109 (8.66%)0.000a4.381 (2.666–7.197)Cholecystectomy6 (6.52%)38 (3.02%)0.181NSInternal hernia1 (1.09%)35 (2.78%)0.337NSSmall bowel obstruction4 (4.35%)24 (1.91%)0.500NSIntra-abdominal abscess3 (3.26%)25 (1.99%)0.691NSPeripheral neuropathy5 (5.43%)6 (0.48%)0.000a11.979 (3.423–41.929)Infection2 (2.17%)2 (3.57%)0.151NSaSignificance at the 0.05 level
Mean percent excess weight loss at 1 year following surgery was the same for both groups (−103.1 lb, 82.6% stricture group vs −115.8 lb, 82.0% nonstricture group). Mean numbers of medications were reduced from 4.6 to 1.6 and from 4.2 to 1.5 in the stricture and nonstricture groups, respectively, at the most recent postoperative examination.
By univariate logistic regression analysis, two preoperative characteristics were identified as factors associated significantly (P < 0.05 level) with stricture: gastroesophageal reflux disease (GERD) (P = 0.035) and age (P = 0.039) (Table 1). In addition to GERD and age, BMI (P = 0.202) and gender (P = 0.208) were incorporated into the final multivariate analysis. In the first multivariate model (Table 3), with age defined as a continuous variable, forward stepwise multivariate logistic regression confirmed GERD (P = 0.006, OR = 1.917, CI = 1.200–3.062) and age (P = 0.010, OR = 0.973, CI = 0.953–0.993) to be independent factors associated with stricture. Seventy-two percent of all stricture patients were diagnosed with GERD prior to surgery, and the mean age was 41 years, whereas, in the preoperative nonstricture group, 57% were diagnosed with GERD and the mean age was 44 years.
Table 3Significance and Odds Ratios for Age, GERD, Gender, and BMI in Multivariate Models to Predict StrictureVariableP ValueOdds Ratio (95% Confidence Interval)Multivariate model #1a Age0.010b0.973 (0.953–0.993)  GERD0.006b1.917 (1.200–3.062) Gender0.324NS Preoperative BMI0.086NSMultivariate model #2c Age overall0.015  Group 1: ≤35 years (reference category)1  Group 2: 36–45 years0.0150.508 (0.294–0.877)  Group 3: 46–55 years0.0040.431 (0.244–0.760)  Group 4: >55 years0.1260.596 (0.308–1.156) GERD0.0051.963 (1.227–3.141) Gender0.353NS Preoperative BMI0.100NSaAge classified as a continuous variablebSignificant at the 0.05 levelcAge classified as a categorical variable
In the second multivariate model (Table 3), age was classified as a categorical variable to refine our understanding of the impact of age on stricture. Using age group 1 (≤35 years) as the reference category, age groups 2 (36–45 years) and 3 (46–55 years) were significant at the 0.05 level [group 2, P = 0.015 (OR = 0.508, CI = 0.294–0.877); group 3, P = 0.004 (OR = 0.431, CI = 0.244–0.760)]. GERD remained significant (P = 0.005; OR = 1.963, CI = 1.227–3.141).
Three postoperative complications were found to be associated significantly with the presence of stricture by univariate logistic regression, including readmissions within 30 days of surgery (P = 0.000), peripheral neuropathy (P = 0.000), and pneumonia (P = 0.031). The postoperative multivariate model determined readmissions within 30 days of surgery (P = 0.000, OR = 4.381, CI = 2.666–7.197) and peripheral neuropathy (P = 0.000, OR = 11.979, CI = 3.423–41.929) to be statistically significant covariates of stricture.
Decreasing our rate of stricture by a protocol change to oversewing the gastrojejunostomy with polyglactin was shown, by chi square analysis, to be a significant decrease, with the rate of 6.8% diminishing to 2.8% (P = 0.028, OR = 0.358, CI = 0.143–0.895). The second protocol adjustment, administration of an H2 antagonist (prevacid) postoperatively, had no effect.
Discussion
Strictures contribute to almost half of all readmissions in the early perioperative period and require instrumentation and rehydration. They can also be associated with the development of a more serious complication, peripheral neuropathy, as is demonstrated by these data.
Specific intraoperative factors (e.g., surgeon experience) associated with stricture have been identified as causal by prior studies. Isolating preoperative factors that may be causally related to stricture may aid in minimizing this complication. In their study, Perugini et al. showed preoperative hypertension and obstructive sleep apnea to be predictive of complications in general; yet, they cautioned that their findings might have been confounded by their high rate of stricture.20 By logistic regression analysis, our study found the variables of GERD and age to be significantly associated with a complication with one of the highest incidences—stricture. Our results are supportive of Perugini’s findings, in that we did not find the same preoperative predictors of complications (hypertension, obstructive sleep apnea) significantly associated with stricture and the overall rate was significantly less.
The odds of developing stricture in patients with preoperative GERD were found to be nearly two times higher than those for patients without GERD. GERD is a known inflammatory condition of the upper gastrointestinal tract that specifically affects the lower esophagus and fundus. If present, this inflammation may predispose patients to more vigorous scarring of the gastrojejunostomy. During the period of the study, we began testing patients preoperatively for H. pylori. Patients who tested positive were treated preoperatively. In general, the micropouch used to create the gastrojejunostomy has been assumed to be almost devoid of acid-producing cells, and the rate of ulceration at the anastomosis at the time of endoscopy is low. It is clear, though, that adding H2 antagonists for 30 days following surgery helps to decrease inflammation, and in addition, the polyglactin stitch may be less inflammatory in nature. Data were insufficient at study summary to demonstrate the statistical significance of administering H2 antagonists; however, we are encouraged by the trend of fewer strictures.
Finding that younger rather than older age was associated with the development of stricture seems clinically counterintuitive. One reason for this is that age affects the small vessels’ ability to provide necessary oxygenation to the anastomosis, and older patients are more likely overall to have diminished capability to oxygenate. In our center, maintenance on oxygen the night of surgery is standard; patients who require continuous positive airway pressure are started in the anesthesia recovery unit and are kept on supplemental oxygen until they can oxygenate in ambulating and resting modes at greater than 92%. However, as stated, in our first multivariate model, older age was shown to be slightly protective with respect to stricture. In the second multivariate model, designed to refine the interpretation of age effects on stricture, we found that the odds of developing stricture in those 35 years or younger were two times greater than those aged 36–45 years, and 2.32 times greater than those aged 46–55 years. The trend of age being protective against stricture leveled off and was not significant in those older than 55 years. More study of this finding is indicated.
Our original assumption that higher rates of central fat distribution in males might place greater tension on the anastomosis predisposing male patients, particularly those with higher BMIs, toward stricture, may be in error. The effect of increased central fat on the anastomosis may not be injurious if meticulous technique is used to ensure a tension-free anastomosis. Also, the ischemic effects of diabetes, sleep apnea, and chronic respiratory disease at the gastrojejunostomy site may be insufficient, in themselves, to cause stricture.
It has been well documented that operative technique can contribute to increased stricture rates. Previous studies have reported fewer strictures with hand-sewn anastomoses than with stapled ones. Some authors have demonstrated that the 25-mm EEA stapler may be associated with fewer strictures that the 21-mm EEA stapler; we have a 6.8% stricture rate with the 21-mm EEA stapler. Further studies of stapling methodologies are warranted.
It has been proposed that stricture rates may be lessened by the creation of a larger gastrojejunostomy anastomosis. The effect on the restrictive element of the bypass and subsequent effect on weight loss long term has not been evaluated. We avoided excessive dilation to allow the patient to maintain as much restriction as possible. Using a protocol of minimal dilation to between 12.5 and 15 mm resulted in only one perforation and acceptable weight loss. In our study and those of others,21,22 weight loss at 1 year is not hindered by dilation.
A limitation of this study is that is was retrospective. A prospective trial in which comorbidities were not controlled would not be feasible, although a prospective trial of H2 antagonists may be of value. It is difficult to discern the effect of absorbable suture on stricture rates vs the effect of H2 antagonists as their introduction to the protocol occurred in close proximity. The finding that readmissions within 30 days of surgery and postoperative peripheral neuropathy were statistically significant covariates of stricture most likely relates to the frequency of readmissions for peripheral neuropathy secondary to vitamin deficiencies following bariatric surgery.
The objective of this study was to reach back in the causal continuum, prior to the intraoperative effects of technique and surgeon experience, to identify other potentially salient contributors to postoperative stricture. This analysis of a large series of patients undergoing RYGB identified GERD and age as factors associated with gastrojejunostomy stricture.
Conclusions
Using absorbable suture at the gastrojejunostomy anastomosis appears to decrease stricture rates. As identified via multivariate logistic regression analysis, GERD and age are independent predictors of gastrojejunostomy stricture.

stricture
obesity
gastric bypass
gastrojejunostomy
bariatric surgery