Pilus formation and protein secretion by the same machinery in Escherichia coli

The secreton (type II secretion) and type IV pilus biogenesis branches of the general secretory pathway in Gram-negative bacteria share many features that suggest a common evolutionary origin. Five components of the secreton, the pseudopilins, are similar to subunits of type IV pili. Here, we report that when the 15 genes encoding the pullulanase secreton of Klebsiella oxytoca were expressed on a high copy number plasmid in Escherichia coli, one pseudopilin, PulG, was assembled into pilus-like bundles. Assembly of the 'secreton pilus' required most but not all of the secreton components that are essential for pullulanase secretion, including some with no known homologues in type IV piliation machineries. Two other pseudopilins, pullulanase and two outer membrane-associated secreton components were not associated with pili. Thus, PulG is probably the major component of the pilus. Expression of a type IV pilin gene, the E.coli K-12 gene ppdD, led to secreton-dependent incorporation of PpdD pilin into pili without diminishing pullulanase secretion. This is the first demonstration that pseudopilins can be assembled into pilus-like structures.     

Disulfide bond formation in secreton component PulK provides a possible explanation for the role of DsbA in pullulanase secretion

When expressed in Escherichia coli, the 15 Klebsiella oxytoca pul genes that encode the so-called Pul secreton or type II secretion machinery promote pullulanase secretion and the assembly of one of the secreton components, PulG, into pili. Besides these pul genes, efficient pullulanase secretion also requires the host dsbA gene, encoding a periplasmic disulfide oxidoreductase, independently of disulfide bond formation in pullulanase itself. Two secreton components, the secretin pilot protein PulS and the minor pseudopilin PulK, were each shown to posses an intramolecular disulfide bond whose formation was catalyzed by DsbA. PulS was apparently destabilized by the absence of its disulfide bond, whereas PulK stability was not dramatically affected either by a dsbA mutation or by the removal of one of its cysteines. The pullulanase secretion defect in a dsbA mutant was rectified by overproduction of PulK, indicating reduced disulfide bond formation in PulK as the major cause of the secretion defect under the conditions tested (in which PulS is probably present in considerable excess of requirements). PulG pilus formation was independent of DsbA, probably because PulK is not needed for piliation.     

Structure and assembly of the pseudopilin PulG

The pseudopilin PulG is one of several essential components of the type II pullulanase secretion machinery (the Pul secreton) of the Gram-negative bacterium Klebsiella oxytoca. The sequence of the N-terminal 25 amino acids of the PulG precursor is hydrophobic and very similar to the corresponding region of type IV pilins. The structure of a truncated PulG (lacking the homologous region), as determined by X-ray crystallography, was found to include part of the long N-terminal alpha-helix and the four internal anti-parallel beta-strands that characterize type IV pilins, but PulG lacks the highly variable loop region with a disulphide bond that is found in the latter. When overproduced, PulG forms flexible pili whose structural features, as visualized by electron microscopy, are similar to those of bacterial type IV pili. The average helical repeat comprises 17 PulG subunits and four helical turns. Electron microscopy and molecular modelling show that PulG probably assembles into left-handed helical pili with the long N-terminal alpha-helix tightly packed in the centre of the pilus. As in the type IV pilins, the hydrophobic N-terminal part of the PulG alpha-helix is necessary for its assembly. Subtle sequence variations within this highly conserved segment seem to determine whether or not a type IV pilin can be assembled into pili by the Pul secreton.     

Type II protein secretion in Pseudomonas aeruginosa: the pseudopilus is a multifibrillar and adhesive structure

The type II secretion pathway of Pseudomonas aeruginosa is involved in the extracellular release of various toxins and hydrolytic enzymes such as exotoxin A and elastase. This pathway requires the function of a macromolecular complex called the Xcp secreton. The Xcp secreton shares many features with the machinery involved in type IV pilus assembly. More specifically, it involves the function of five pilin-like proteins, the XcpT-X pseudopilins. We show that, upon overexpression, the XcpT pseudopilin can be assembled in a pilus, which we call a type II pseudopilus. Image analysis and filtering of electron micrographs indicated that these appendages are composed of individual fibrils assembled together in a bundle structure. Our observations thus revealed that XcpT has properties similar to those of type IV pilin subunits. Interestingly, the assembly of the type II pseudopilus is not exclusively dependent on the Xcp machinery but can be supported by other similar machineries, such as the Pil (type IV pilus) and Hxc (type II secretion) systems of P. aeruginosa. In addition, heterologous pseudopilins can be assembled by P. aeruginosa into a type II pseudopilus. Finally, we showed that assembly of the type II pseudopilus confers increased bacterial adhesive capabilities. These observations confirmed the ability of pseudopilins to form a pilus structure and raise questions with respect to their function in terms of secretion and adhesion, two crucial biological processes in the course of bacterial infections.     

Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.     

Pseudopilin residue E5 is essential for recruitment by the type 2 secretion system assembly platform

Type II secretion systems (T2SSs) promote secretion of folded proteins playing important roles in nutrient acquisition, adaptation and virulence of Gram‐negative bacteria. Protein secretion is associated with the assembly of type 4 pilus (T4P)‐like fibres called pseudopili. Initially membrane embedded, pseudopilin and T4 pilin subunits share conserved transmembrane segments containing an invariant Glu residue at the fifth position, E5. Mutations of E5 in major T4 pilins and in PulG, the major pseudopilin of the Klebsiella T2SS abolish fibre assembly and function. Among the four minor pseudopilins, only PulH required E5 for secretion of pullulanase, the substrate of the Pul T2SS. Mass‐spectrometry analysis of pili resulting from the co‐assembly of PulG^E5A variant and PulG^WT ruled out an E5 role in pilin processing and N‐methylation. A bacterial two‐hybrid analysis revealed interactions of the full‐length pseudopilins PulG and PulH with the PulJ‐PulI‐PulK priming complex and with the assembly factors PulM and PulF. Remarkably, PulG^E5A and PulH^E5A variants were defective in interaction with PulM but not with PulF, and co‐purification experiments confirmed the E5‐dependent interaction between native PulM and PulG. These results reveal the role of E5 in a recruitment step critical for assembly of the functional T2SS, likely relevant to T4P assembly systems.     

Minor pseudopilin self-assembly primes type II secretion pseudopilus elongation

In Gram-negative bacteria, type II secretion systems (T2SS) assemble inner membrane proteins of the major pseudopilin PulG (GspG) family into periplasmic filaments, which could drive protein secretion in a piston-like manner. Three minor pseudopilins PulI, PulJ and PulK are essential for protein secretion in the Klebsiella oxytoca T2SS, but their molecular function is unknown. Here, we demonstrate that together these proteins prime pseudopilus assembly, without actively controlling its length or secretin channel opening. Using molecular dynamics, bacterial two-hybrid assays, cysteine crosslinking and functional analysis, we show that PulI and PulJ nucleate filament assembly by forming a staggered complex in the plasma membrane. Binding of PulK to this complex results in its partial extraction from the membrane and in a 1-nm shift between their transmembrane segments, equivalent to the major pseudopilin register in the assembled PulG filament. This promotes fully efficient pseudopilus assembly and protein secretion. Therefore, we propose that PulI, PulJ and PulK self-assembly is thermodynamically coupled to the initiation of pseudopilus assembly, possibly setting the assembly machinery in motion.     

Signal recognition particle-dependent inner membrane targeting of the PulG Pseudopilin component of a type II secretion system

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The beta-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.     

XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus

Pseudomonas aeruginosa is an opportunistic gram-negative pathogen equipped with multiple secretion systems. The type II secretion machinery (Xcp secreton) is involved in the release of toxins and enzymes. The Xcp secreton is a multiprotein complex, and most of its components share homology with proteins involved in type IV pili biogenesis. Among them, the XcpT-X pseudopilins possess characteristics of the major constituent of the type IV pili, the pilin PilA. We have shown previously that XcpT can be assembled in a multifibrillar structure that was called the pseudopilus. By using two different microscopic approaches, we show here that the pseudopili are preferentially isolated fibers rather than tight bundles. Moreover, none of the other four pseudopilins are able to form a pseudopilus, suggesting that the assembly of such a structure is a unique property of XcpT. Moreover, we show that 5 of the 12 Xcp proteins are not required for pseudopilus biogenesis, whereas they are for type II secretion. Most interestingly, we showed that one pseudopilin, XcpX, controls the assembly of XcpT into a pseudopilus. Indeed, when the number of XcpX subunits increases, the length of the pseudopilus decreases. Conversely, in the absence of XcpX, the pseudopilus length is abnormally long. Our results indicate that XcpT and XcpX directly interact with each other. Furthermore, this interaction induces a clear destabilization of XcpT. The interaction between XcpT and XcpX could be part of the molecular mechanism underlying the dynamic control of pseudopilus elongation, which could be crucial for type II-dependent protein secretion.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N and <Superscript>13</Superscript>C resonance assignments and secondary structure of PulG,the major pseudopilin from <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> type 2 secretion system

Bacteria use complex transporters to secrete functionally relevant proteins to the extracellular medium. The type 2 secretion system (T2SS) translocates folded proteins involved in bacterial nutrient acquisition, virulence and adaptation. The T2SS pseudopilus is a periplasmic filament, assembled by the polymerization of PulG subunits, the major pseudopilin. Pseudopilin proteins have a conserved N-terminal hydrophobic segment followed by a more variable C-terminal periplasmic and globular domain. To better understand the mechanism of assembly and function of the T2SS, we have been studying the structure and dynamics of PulG by NMR, as well as its interaction with other components of the secretion machinery. As a first step on this study, here we reported the chemical shift assignments of PulG C-terminal domain and its secondary structure prediction based on NMR data.     

Bacterial outer membrane ushers contain distinct targeting and assembly domains for pilus biogenesis

Biogenesis of a superfamily of surface structures by gram-negative bacteria requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway a periplasmic chaperone works together with an outer membrane usher to direct substrate folding, assembly, and secretion to the cell surface. We analyzed the structure and function of the PapC usher required for P pilus biogenesis by uropathogenic Escherichia coli. Structural analysis indicated PapC folds as a beta-barrel with short extracellular loops and extensive periplasmic domains. Several periplasmic regions were localized, including two domains containing conserved cysteine pairs. Functional analysis of deletion mutants revealed that the PapC C terminus was not required for insertion of the usher into the outer membrane or for proper folding. The usher C terminus was not necessary for interaction with chaperone-subunit complexes in vitro but was required for pilus biogenesis in vivo. Interestingly, coexpression of PapC C-terminal truncation mutants with the chromosomal fim gene cluster coding for type 1 pili allowed P pilus biogenesis in vivo. These studies suggest that chaperone-subunit complexes target an N-terminal domain of the usher and that subunit assembly into pili depends on a subsequent function provided by the usher C terminus.     

Outer membrane targeting, ultrastructure, and single molecule localization of the enteropathogenic Escherichia coli type IV pilus secretin BfpB

Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis.     

New tools in an old trade: CS1 pilus morphogenesis

CS1 pili serve as the prototype for a large class of serologically distinct pili associated with enterotoxigenic Escherichia coli that cause diarrhoea in humans. The four genes essential for CS1 pilus morphogenesis, cooB, A, C and D, are arranged in an operon and encode structural and assembly proteins unlike those of other pilus systems commonly associated with Gram-negative bacteria. CS1 pili are composed primarily of the major pilin subunit, CooA, which determines the serological type of the pilus. The major pilin subunit is assembled into pili by the proteins CooB, CooC and CooD. CooD is both a minor component found at the pilus tip and an essential assembly protein, whereas CooC is an outer membrane protein thought to be involved in pilin transport. CooB is a novel periplasmic chaperone-like protein that forms intermolecular complexes with and stabilizes the major and minor pilins. Unlike other pilin chaperones, CooB also stabilizes the outer membrane component of the assembly system, CooC. The proteins of CS1 pili have no significant homology to those of the well-characterized Pap (pyelonephritis-associated) pili and related systems, although most of the features of pilus morphogenesis are similar. Therefore, these appear to be among the rare cases of convergent evolution. Thus, for CS1 pili, enterotoxigenic E. coli use new protein 'tools' in the old 'trade' of forming functional pili.     

The general secretory pathway of Klebsiella oxytoca: no evidence for relocalization or assembly of pilin-like PulG protein into a multiprotein complex

It has been proposed that the four type IV pilin-like proteins that are required for extracellular protein secretion by the general secretory pathway (GSP) might assemble into a trans-periplasm complex resembling a type IV pilus. To test this idea, we examined the subcellular distribution and oligomeric state of PulG, one of the type IV pilin-like proteins required for pullulanase secretion in Klebsiella oxytoca. Fractionation of Escherichia coli cells carrying a single copy of each pul gene showed that PulG protein was located in two distinct envelope fractions corresponding to the outer and cytoplasmic membranes. The protein was partially released by treating the membranes with Triton X-100 + EDTA or at high pH, but not by Triton X-100 atone or by 8M urea, 6M guanidine hydrochloride or 1 M NaCl. Like type IV pilins, non-sedimentable PuiG that had been released from the membranes at high pH could be sedimented by centrifugation when the pH was lowered. Treatment of whole cells, sphaeroplasts or isolated membranes with a cleavable cross-linking agent produced mainly PulG homodimers. Previous studies showed that both PulO, which cleaves and N-methylates the PulG precursor, and PulE, a putative ATP-binding protein, share extensive sequence identity with proteins known to be required for type IV pilus processing and assembly. However, mutations which disrupted either pulE or pulO, or indeed the complete absence of all other components of the pullulanase secretion apparatus, had little or no effect on any of the properties of PulG protein described above. We conclude that there is no evidence that PulG protein assembles into a stable multiprotein complex or that processing of the PulG precursor causes a detectable change in its subcellular distribution.     

Assembly of complex organelles: pilus biogenesis in gram-negative bacteria as a model system

Pathogenic bacteria assemble a variety of adhesive structures on their surface for attachment to host cells. Some of these structures are quite complex. For example, the hair-like organelles known as pili or fimbriae are generally composed of several components and often exhibit composite morphologies. In gram-negative bacteria assembly of pili requires that the subunits cross the cytoplasmic membrane, fold correctly in the periplasm, target to the outer membrane, assemble into an ordered structure, and cross the outer membrane to the cell surface. Thus, pilus biogenesis provides a model for a number of basic biological problems including protein folding, trafficking, secretion, and the ordered assembly of proteins into complex structures. P pilus biogenesis represents one of the best-understood pilus systems. P pili are produced by 80-90% of all pyelonephritic Escherichia coli and are a major virulence determinant for urinary tract infections. Two specialized assembly factors known as the periplasmic chaperone and outer membrane usher are required for P pilus assembly. A chaperone/usher pathway is now known to be required for the biogenesis of more than 30 different adhesive structures in diverse gram-negative pathogenic bacteria. Elucidation of the chaperone/usher pathway was brought about through a powerful combination of molecular, biochemical, and biophysical techniques. This review discusses these approaches as they relate to pilus assembly, with an emphasis on newer techniques.     

Uptake of extracellular DNA: Competence induced pili in natural transformation of Streptococcus pneumoniae

Transport of DNA across bacterial membranes involves complex DNA uptake systems. In Gram‐positive bacteria, the DNA uptake machinery shares fundamental similarities with type IV pili and type II secretion systems. Although dedicated pilus structures, such as type IV pili in Gram‐negative bacteria, are necessary for efficient DNA uptake, the role of similar structures in Gram‐positive bacteria is just beginning to emerge. Recently two essentially very different pilus structures composed of the same major pilin protein ComGC were proposed to be involved in transformation of the Gram‐positive bacterium Streptococcus pneumoniae – one is a long, thin, type IV pilus‐like fiber with DNA binding capacity and the other one is a pilus structure that was thicker, much shorter and not able to bind DNA. Here we discuss how competence induced pili, either by pilus retraction or by a transient pilus‐related opening in the cell wall, may mediate DNA uptake in S. pneumoniae.     

Analysis of the requirements for pilus biogenesis at the outer membrane usher and the function of the usher C-terminus

Uropathogenic strains of Escherichia coli assemble type 1 and P pili to colonize the bladder and kidney respectively. These pili are prototype structures assembled by the chaperone/usher secretion pathway. In this pathway, a periplasmic chaperone works together with an outer membrane (OM) usher to control the folding of pilus subunits, their assembly into a pilus fibre and secretion of the fibre to the cell surface. The usher serves as the assembly and secretion platform in the OM. The usher has distinct functional domains, with the N-terminus providing the initial targeting site for chaperone-subunit complexes and the C-terminus required for subsequent stages of pilus biogenesis. In this study, we investigated the molecular interactions occurring at the usher during pilus biogenesis and the function of the usher C-terminus. We provide genetic and biochemical evidence that the usher functions as a complex in the OM and that interaction of the pilus adhesin with the usher is critical to prime the usher for pilus biogenesis. Analysis of C-terminal truncation and substitution mutants of the P pilus usher PapC demonstrated that the C-terminus is required for proper binding of chaperone-subunit complexes to the usher and plays an important role in assembly of complete pili.     

The usher N terminus is the initial targeting site for chaperone-subunit complexes and participates in subsequent pilus biogenesis events

Pilus biogenesis on the surface of uropathogenic Escherichia coli requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway, periplasmic chaperone-subunit complexes target an outer membrane (OM) usher for subunit assembly into pili and secretion to the cell surface. The molecular mechanisms of protein secretion across the OM are not well understood. Mutagenesis of the P pilus usher PapC and the type 1 pilus usher FimD was undertaken to elucidate the initial stages of pilus biogenesis at the OM. Deletion of residues 2 to 11 of the mature PapC N terminus abolished the targeting of the usher by chaperone-subunit complexes and rendered PapC nonfunctional for pilus biogenesis. Similarly, an intact FimD N terminus was required for chaperone-subunit binding and pilus biogenesis. Analysis of PapC-FimD chimeras and N-terminal fragments of PapC localized the chaperone-subunit targeting domain to the first 124 residues of PapC. Single alanine substitution mutations were made in this domain that blocked pilus biogenesis but did not affect targeting of chaperone-subunit complexes. Thus, the usher N terminus does not function simply as a static binding site for chaperone-subunit complexes but also participates in subsequent pilus assembly events.     

Deciphering the Xcp Pseudomonas aeruginosa type II secretion machinery through multiple interactions with substrates

The type II secretion system enables gram-negative bacteria to secrete exoproteins into the extracellular milieu. We performed biophysical and biochemical experiments to identify systematic interactions between Pseudomonas aeruginosa Xcp type II secretion system components and their substrates. We observed that three Xcp components, XcpP(C), the secretin XcpQ(D), and the pseudopilus tip, directly and specifically interact with secreted exoproteins. We established that XcpP(C), in addition to its interaction with the substrate, likely shields the entire periplasmic portion of the secreton. It can therefore be considered as the recruiter of the machinery. Moreover, the direct interaction observed between the substrate and the pseudopilus tip validates the piston model hypothesis, in which the pseudopilus pushes the substrate through the secretin pore during the secretion process. All together, our results allowed us to propose a model of the different consecutive steps followed by the substrate during the type II secretion process.     

A second prepilin peptidase gene in Escherichia coli K-12

Escherichia coli K-12 strains grown at 37°C or 42°C, but not at 30°C, process the precursors of the Neisseria gonorrhoeae type IV pilin PilE and the Klebsiella oxytoca type IV pseudopilin PulG in a manner reminiscent of the prepilin peptidase-dependent processing of these proteins that occurs in these bacteria. Processing of prePulG in Escherichia coli requires a glycine at position −1, as does processing by the cognate prepilin peptidase (PulO), and is unaffected by mutations that inactivate several non-specific proteases. These data suggested that E . coli K-12 has a functional prepilin peptidase, despite the fact that it does not itself appear to express either type IV pilin or pseudopilin genes under the conditions that allow prePilE and prePulG processing. The E . coli K-12 genome contains two genes encoding proteins with significant sequence similarity to prepilin peptidases: gspO at minute 74.5 and pppA (f310c) at minute 67 on the genetic map. We have previously obtained evidence that gspO encodes an active enzyme but is not transcribed. pppA was cloned and shown to code for a functional prepilin peptidase capable of processing typical prepilin peptidase substrates. Inactivation of pppA eliminated the endogenous, thermoinducible prepilin peptidase activity. PppA was able to replace PulO prepilin peptidase in a pullulanase secretion system reconstituted in E . coli when expressed from high-copy-number plasmids but not when present in a single chromosomal copy. The analysis of pppA–lacZ fusions indicated that pppA expression was very low and regulated by the growth temperature at the level of translation, in agreement with the observed temperature dependence of PppA activity. Polymerase chain reaction and Southern hybridization analyses revealed the presence of the pppA gene in 12 out of 15 E . coli isolates.