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1 Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, Bacterial Diseases Branch, Fort Collins, CO 80521, USA
2 Department of Microbiology and Molecular Genetics, Michigan State University, 2215 Biomedical Physical Sciences, East Lansing, MI 48824, USA
3 Department of Chemistry, University of Toledo, 2801 W. Bancroft Street, Toledo, OH 43606, USA
4 Department of Microbiology, The University of Chicago, 920 E. 58th Street, Chicago, IL 60637, USA
Correspondence
Robert R. Brubaker
t-rbruba{at}bsd.uchicago.edu
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). During subsequent evolution, the organism acquired a tropism for lymphatic tissue and gained unique LPS O-groups (Davies, 1961) and new enzymes including KatY (Garcia et al., 1999). Nevertheless, Y. pseudotuberculosis remains an enteropathogenic species although its incidence of soil-borne zoonotic infection is generally greater than that of Y. enterocolitica, which typically causes food-borne gastrointestinal disease in humans. Both of these patterns are in stark contrast to the epidemic habit of Yersinia pestis. Probably less than 20 000 years have elapsed since Y. pestis diverged from Y. pseudotuberculosis (Achtman et al., 1999), yet plague bacilli cause the most devastating acute infectious disease known to man as judged by its rapid onset, systemic pathology and extensive mortality. The brief existence of Y. pestis as a distinct species suggests that only a limited number of genetic changes have occurred that promote acute disease. This possibility was supported by comparative annotation of the Y. pestis and Y. pseudotuberculosis genomes (Chain et al., 2004, 2006; Deng et al., 2002; Parkhill et al., 2001). Results of these studies detected only a few Y. pestis-specific genes whereas about 13 % of the Y. pseudotuberculosis chromosome is now inactive in Y. pestis (Chain et al., 2004). These losses accrued as a consequence of chromosomal duplication, deletion, inversion, transposition, and the insertion of over 70 copies of IS elements (Chain et al., 2004, 2006; Deng et al., 2002; Parkhill et al., 2001). In addition, cells of typical Y. pestis as compared to Y. pseudotuberculosis possess an assumed missense mutation in glucose-6-phosphate dehydrogenase characterized by a single base transversion (T·A
C·G), causing replacement of active serine by proline at amino acid position 155 (Chain et al., 2004), and a proven missense mutation (Viola et al., 2008) in aspartate ammonia-lyase or aspartase (AspA) characterized by a single base transversion (G·CT·A), causing replacement of active valine by leucine (Chain et al., 2004). The existence of these various types of loss mutation accounts for most of the classic phenotypic differences used to distinguish Y. pestis from Y. pseudotuberculosis (Brubaker, 1991).
The few important Y. pestis-specific genes were acquired by lateral transfer as evidenced by carriage of two plasmids not shared with the enteropathogenic yersiniae (Ferber & Brubaker, 1981). These consist of 
10 kb pPCP encoding plasminogen activator (Pla), the bacteriocin pesticin, and its immunity protein (Ferber & Brubaker, 1981), and 100 kb pMT encoding capsular antigen fraction 1 (Caf1) and murine toxin (MT) (Kutyrev et al., 1986; Protsenko et al., 1983). Plague bacilli share a third 70 kb plasmid with the enteropathogenic yersiniae (termed pCD in Y. pestis and pYV in Y. pseudotuberculosis and Y. enterocolitica). The latter mediates a low calcium response (LCR) causing a temperature-dependent nutritional requirement in vitro for 2.5 mM Ca2+ (Kupferberg & Higuchi, 1958). If present, Ca2+ promotes vegetative growth at 37 °C while downregulating a host temperature-dependent pCD/pYV-encoded type III secretion system (T3SS) required for translocation of attendant virulence effectors. These determinants consist of yersiniae outer proteins (Yops) and LcrV, which collectively disrupt host cell scaffolding, cause apoptosis and repress inflammation (Heesemann et al., 2006). If Ca2+ is omitted from the environment in vitro, the organisms upregulate this T3SS and cease multiplication (Lcr+), although full-scale growth can still occur at mildly acidic pH provided that Na+ is also eliminated from the medium (Brubaker, 2007). The LCR is particularly stringent in Lcr+ cells of Y. pestis as judged by abrupt shutoff of growth following shift from 26 °C to 37 °C associated with secretion of L-aspartic acid (Brubaker, 2005, 2007). This phenomenon reflects loss of AspA activity (Dreyfus & Brubaker, 1978), where exit of metabolic L-aspartate likely contributes to the abrupt shutoff in growth of Lcr+ cells of Y. pestis. Since bacteriostasis as well as release of L-aspartate in Ca2+-deficient medium is dependent on the presence of Na+ (Brubaker, 2005, 2007; Fowler & Brubaker, 1994), the possibility exists that the latter cation causes both phenomena via its established role as porter for the translocation of L-aspartate via GltS (Deguchi et al., 1990). As noted below, the possibility also exists that missing Zwf activity (Mortlock, 1962; Mortlock & Brubaker, 1962) may also influence the LCR of Y. pestis during growth in vitro.
Cells of Y. pseudotuberculosis actively invade host intestinal epithelial cells and typically downregulate the formation of a biofilm required by Y. pestis for blockage of fleas. Both of these properties have been lost by mutation in Y. pestis (Brubaker, 2004; Sun et al., 2008). It is also established that acquisition of MT via pMT facilitated colonization of the flea vector (Hinnebusch et al., 2002), thereby enabling the organisms to bypass natural environments by undergoing direct passage from one host to another. Little is known, however, about the sequence of these and other genetic changes that culminated in the ability of Y. pestis to express acute disease. For successful reliance upon transfer by fleabite, the organisms must kill their current rodent host in order to ensure that resident fleas disseminate in search of new hosts. Accordingly, mortality became an essential part of the life cycle of Y. pestis and selective pressure caused by the necessity for lethality has undoubtedly favoured subsequent increases in virulence. As a consequence, plague bacilli became efficient pathogens and have recently disseminated from ancient foci in central Asia to reservoirs throughout the world, where they now cause acute disease in a large number of animal species including man (Suntsov & Suntsova, 2008). The virulence of these potential ‘epidemic’ isolates contrasts sharply with ‘enzootic’ strains (including pestoides variants) of Y. pestis that remain contained within the original reservoirs where the species evolved (Anisimov et al., 2004; Martinevskii, 1969). Enzootic isolates are now classified according to subspecies designation (altaica, caucasica, hissarica, ulegeica and talassica; Anisimov et al., 2004) and commonly retain the normal ability of Y. pseudotuberculosis to ferment the sugars rhamnose and melibiose. They may also possess all known virulence factors of epidemic isolates. Nevertheless, enzootic strains are attenuated in many mammalian species, including guinea pigs and primates, but virulent in the rodent Superfamily Muroidea (Anisimov et al., 2004). Although pestoides isolates possess polymorphism in LcrV (Anisimov et al., 2007), a major pCD/pYV-encoded regulator, T3SS effector, and virulence factor, it seems unlikely that these differences influence biological activity (Abramov et al., 2007). The purposes of this report are to provide a description of ten typical enzootic isolates and to demonstrate that only three produce functional Zwf while, in stark contrast to epidemic isolates of Y. pestis, all express biologically active AspA.
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METHODS |
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). Plasmid profiles were characterized by electrophoresis in agarose (Kado & Liu, 1981). If plasmids or the Pgm locus were detected in original stock cultures, routine cultural precautions were undertaken to enrich their content in populations used for later experiments. Avirulent Lcr– derivatives (lacking pCD/pYV) were selected by plating and incubation on magnesium oxalate agar at 37 °C (Higuchi & Smith, 1961) to facilitate subsequent biochemical testing and enzymic analyses, although isolates containing pCD, if present, were used whenever possible to determine plasmid profiles (Fig. 1). Assurance that missing plasmids had not undergone integration into the chromosome was obtained by query of salient genes (pla, caf1 and lcrV) via PCR; retention of the temperature-dependent nutritional requirement for Ca2+ by strains containing pCD was verified by use of magnesium oxalate agar. Yersiniae grown to late-exponential phase at 37 °C in brain heart infusion broth (Beckton Dickinson) were used to express Caf1 and MT, which were determined by diffusion against monospecific polyclonal antisera in gels and by immunoblotting. Most of the pestoides strains used in this study were also described by Achtman et al. (2004).
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) was estimated after incubation for 2 days at 26 °C on Congo red agar (Surgalla & Beesley, 1969). Pesticin was purified to near-homogeneity by the method of Hu & Brubaker (1974) and assayed at 37 °C for antibacterial activity on pesticin agar against the indicator organism under investigation (Brubaker & Surgalla, 1962). Sensitivity to pesticin was expressed in units, where one unit is defined as the reciprocal of the highest dilution providing detectable inhibition of growth. Production of pesticin was estimated qualitatively as ability of colonies to form zones of inhibition in overlayers of pesticin agar after seeding with cells of Y. pseudotuberculosis and incubation for 1 day at 37 °C (Brubaker & Surgalla, 1962). Pla was determined semiquantitatively by testing dilutions of bacteria washed in 0.033 M potassium phosphate buffer, pH 7.0, on fibrin plates (Astrup & Mullertz, 1952) as described previously (Beesley et al., 1967). Units of Pla were defined as the reciprocal of the highest dilution yielding fibrinolytic activity. These results and the original geographical origins of individual yersiniae are listed in Table 1.
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) containing L-threonine, L-methionine, L-phenylalanine, L-isoleucine and L-valine and modified by omission of glycine and D-lactate; this medium is sufficient for growth of typical epidemic isolates of Y. pestis at 26 °C.
Cell-free extracts.
Unless stated otherwise, bacteria used for preparation of cell-free extracts for enzyme analysis were always grown at 26 °C. The organisms were transferred from buffered glycerol stock cultures maintained at –20 °C (Beesley et al., 1967) to slopes of blood agar base (Becton Dickinson). After incubation for 48 h, the organisms were removed from the agar surface in sterile 0.033 M potassium phosphate buffer (pH 7.0) and inoculated at an optical density (620 nm) of 0.1 into 100 ml brain heart infusion broth (Beckton Dickinson) contained in a stoppered 1 l Erlenmeyer flask. This subculture was aerated overnight at 200 r.p.m. on a model G76 gyrotary water bath shaker (New Brunswick Scientific) and then used to inoculate second cultures of the same medium (200 ml per 2 l Erlenmeyer flasks), which were similarly aerated until the cultures entered the late-exponential growth phase. The bacteria were then harvested by centrifugation (10 000 g for 30 min), washed twice in 0.033 M potassium phosphate buffer (pH 7.0), suspended in 0.05 M Tris/HCl buffer (pH 8.0), and disrupted by passage through a French pressure cell (model FA030, Spectronic Unicam). Cell debris was then removed by centrifugation (10 000 g for 30 min) and the resulting extracts were dialysed overnight at 4 °C against 0.05 M Tris/HCl buffer (pH 7.5) before immediate use in enzyme assays.
Enzyme assays.
Zwf was determined by monitoring glucose-6-phosphate-dependent generation of NADPH by a minor modification of the assay described previously (Mortlock & Brubaker, 1962). The reaction mixture consisted of 100 µmol Tris/HCl (pH 8.0), 2.0 µmol MgCl2, 0.3 µmol NADP+ and 100 µmol glucose 6-phosphate (Sigma) in a volume of 2.9 ml contained within a quartz cuvette. The reaction was started by addition of 0.1 ml dialysed cell-free extract and increase of absorbance at 340 nm was monitored with a Beckman DU spectrophotometer (Beckman Coulter).
AspA was estimated by determining the L-aspartic acid-dependent release of 
with Nessler's reagent as described previously (Dreyfus & Brubaker, 1978). The reaction mixture consisted of 250 µmol Tris/HCl (pH 7.0), 5.0 µmol MgCl2 and dialysed cell-free extract in a total volume of 4.5 ml. The assay was started by addition of 250 µmol sodium L-aspartate in a volume of 0.5 ml and samples of 0.5 ml were removed at intervals and added to Eppendorf tubes containing 0.1 ml 1.5 M trichloroacetic acid. The tubes were then centrifuged at highest speed for 1 min in a model II microfuge (Beckman Coulter) and then 0.5 ml of clear supernatant fluid was carefully removed and added to a tube containing 8.5 ml distilled water. These samples received 1.0 ml Nessler's reagent and, after incubation for 10 min, were assayed for absorbance at 480 nm as described for determination of asparaginase (Yellin & Wriston, 1966). The resulting values were then compared against a standard curve prepared immediately before each determination composed of known concentrations of NH4Cl in samples of 10 ml containing the same concentrations of trichloroacetic acid and Nessler's reagent that were used to prepare samples for spectrophotometric analysis. In all cases, protein was determined by the Lowry method.
Pyrosequencing.
PCR was performed using an iCycler real-time thermocycler (Bio-Rad). DNA templates consisted of 1 µl boiled lysis preparations or QIAamp DNA mini-preparations (Qiagen) of pure cultures grown on blood agar plates. Reactions consisted of 10 µl SYBR Green SuperMix (Bio-Rad), 5 mM MgCl2 and 10 pmol primers. The reaction volume was adjusted to 20 µl with sterile, nuclease-free sequencing buffer (Amresco). Primers (Table 2) were designed using the PSQ Assay Design program (Biotage) and synthesized by the Scientific Resources Program (CDC, Atlanta, GA). The reverse primer in each primer pair was biotinylated at its 5-prime end to facilitate streptavidin-mediated recovery of templates for subsequent pyrosequencing (Table 2). Amplification reactions were undertaken at 95 °C for 3 min followed by 45 cycles at 95 °C for 15 s and 60 °C for 30 s. PCR analysis and crossing threshold (Ct) value assignment was determined automatically by the iQ software (data not shown). Sequencing was performed on a PSQ96MA pyrosequencer (Biotage) according to the manufacturer's recommendations. Briefly, PCRs were adjusted to 40 µl with sterile MilliQ water (Millipore); a 40 µl slurry of streptavidin-coated Sepharose beads (Amersham Biosciences) in binding buffer (Biotage) was added to each sample. Reactions were mixed at 1400 r.p.m. for 10 min then bound to filter probes (Biotage) by vacuum aspiration. The probes were washed in 70 % ethanol, 0.2 M NaOH and wash buffer (Biotage) consecutively for 5 s each. The reaction mixes were eluted into a 96-well sequencing plate containing 40 µl aliquots of annealing buffer (Biotage) and sequencing primer (20 pmol). Sequencing reactions were heated at 80 °C for 2 min, and then allowed to cool to room temperature. The nucleotide dispensation orders were: zwf472-TAGCGATCTCAGTATACGATCAGTAGC, e-zwf472-GCGATCTCACGTGATACGATCAGTGCGATATCAC, aspA436-CACAGTATGCTACACGTCGCATGCGTCTAGC, e-aspA436-ACGTATGCTACACTGTCGCA, aspA1087-TACAGTCGATGAGCGTGATGTCAGCATGTGAGTCTATC, and e-aspA1087-CATCGATCGTCACTGAGCTGACTGCTCGCAGCATAG. All reactions were run in triplicate and pyrograms were analysed using the PSQ software (Biotage).
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RESULTS |
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. This information, as well as plasmid profiles, biochemical determinations and enzyme analyses, was used to combine organisms of similar habit in subsequent tables. Evidence is provided below in this context to reassign the pestoides J strain to the group of epidemic isolates and to retain the pestoides I strain within the group of enzootic isolates even though it failed to ferment rhamnose or melibiose.
Plasmid profiles
The plasmid profiles of enzootic pestoides variants were compared by agarose gel electrophoresis with those of a control isolate of epidemic Y. pestis CO92 and enteropathogenic Y. pseudotuberculosis TX83-0489 (Fig. 1). pMT appeared slightly larger in the pestoides A, B, C, D and Angola isolates as compared to the CO92 control (110 kb) due, at least in strain Angola, to two integrated copies of pPCP (NCBI accession NC_010158). As shown by separate measurements, pMT was further increased in size to 134 kb (pestoides E), 126 kb (pestoides F), and 118 kb (pestoides G). These three isolates entirely lacked pPCP whereas pCD and pMT were absent in the pestoides I and J isolates, respectively.
Nutritional requirements
A chemically defined solid medium containing the minimal nutritional requirements of epidemic Y. pestis was used to identify compounds necessary for growth of the pestoides isolates. Pestoides strains A, B, C, E and G required L-arginine (Table 3). L-Leucine was essential for growth of the pestoides A, B, C and D isolates and a need for other amino acids was occasionally observed; requirements for purines, pyrimidines, or vitamins were not detected.
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) yielded Pgm– mutants at high frequency as occurs in epidemic isolates of Y. pestis (Brubaker, 1969). Isolates lacking given plasmids also failed to express attendant virulence factors (Table 3) or encode salient genes detectable by PCR, emphasizing that these missing plasmids were indeed cured rather than integrated into the chromosome. Similarly, carriage of plasmids was associated with expression of known encoded virulence factors with the exception of the Angola isolate, which possessed pMT and produced MT (as detected by immunoassay) but not linked Caf1. In this context, it is significant that there were no matches against the completed Angola genome using caf1 from Y. pestis KIM as a query.
Pesticin
All pestoides strains harbouring pPCP produced similar amounts of the bacteriocin pesticin as judged by uniform zones of inhibition (8–10 mm) surrounding individual colonies overlayered with indicator cells of Y. pseudotuberculosis PB1. Isolates lacking pPCP (pestoides E, F and G) did not produce pesticin and were acutely sensitive to this bacteriocin as compared to pesticinogenic strains able to produce pPCP-encoded pesticin immunity protein (Table 3). Nevertheless, as reported by others (Anisimov et al., 2004), a few pesticinogenic pestoides variants exhibited modest sensitivity to pesticin.
Determinative properties
All tested pestoides isolates as well as epidemic strains of Y. pestis failed to hydrolyse urea or exhibit motility at 26 °C. In contrast, all of the pestoides strains except I resembled Y. pseudotuberculosis in that they fermented rhamnose and melibiose.
Glucose-6-phosphate dehydrogenase
Dialysed cell-free extracts of all tested epidemic Y. pestis controls lacked detectable Zwf activity as judged by direct enzymic assay whereas control extracts of enteropathogenic yersiniae were positive (Table 4). Only the pestoides E, F and G strains exhibited detectable Zwf. As reported previously, the only known difference between the enzymically active enzyme in Y. pseudotuberculosis and the inactive form in epidemic Y. pestis is the presence of serine instead of proline at amino acid position 155 (Chain et al., 2004). Results obtained by pyrosequencing (Table 4) demonstrated that all active enzymes, including those of the pestoides E, F and G isolates, contained serine (encoded by UCC) at position 155 whereas all inactive enzymes contained proline (encoded by CCC).
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). All of the enzootic variants of Y. pestis also expressed biologically functional AspA, although variation was observed in specific activity. The results of DNA sequencing verified that the codon for valine at amino acid position 363 of the active enzymes of Y. pseudotuberculosis was GUG and that this similarly encoded amino acid was present at the same position of the active enzymes from the pestoides A, B, C and D isolates. In contrast, position 363 of the enzymes from the pestoides E, F, G and I strains was occupied by serine (encoded by UCG) while that of the less active enzymes from the Angola and A16 isolates was phenylalanine (encoded by UUU). The distantly related Y. enterocolitica also contained valine at amino acid position 363 of fully active AspA but its codon was GUC.
AspA from strain Angola was purified to near homogeneity as described for AspA of Y. pseudotuberculosis and the corresponding CRIM (cross-reacting immunological material) of Y. pestis and subjected to similar kinetic analysis (Viola et al., 2008). The specific activity of the purified Angola AspA was 7.6 units mg–1, a 30-fold higher estimate than the nearly inactive Y. pestis CRIM but still more than 10-fold lower than the AspA of Y. pseudotuberculosis. The Angola enzyme had a comparable Km of 4.4±1.2 mM for L-aspartate but, similar to the Y. pestis CRIM, exhibited a reduced kcat (7.8±1.0 s–1). As a consequence, the kcat/Km of the Angola AspA was 1800±200 M–1 s–1, a value of only 4.2 % of the Y. pseudotuberculosis enzyme but still significantly more active than the Y. pestis CRIM (0.2 %) (Viola et al., 2008).
Amino acid position 146 of AspA consists of aspartic acid in Y. pseudotuberculosis (Chain et al., 2004) and Y. pestis strain CO92 (Parkhill et al., 2001) but is occupied by tyrosine in Y. pestis strain KIM (Deng et al., 2002). As judged by pyrosequencing, the codons of the aspartic acid and tyrosine residues at this location are GAU and UAU, respectively. Only the pestoides J and epidemic Camel isolates resembled Y. pestis KIM in encoding tyrosine at amino acid position 146; all remaining yersiniae that were evaluated expressed aspartic acid at this location. These results are consistent with the hypothesis that the transversion (G·CT·A) at amino acid 146 of AspA occurs only in a limited (biovar medievalis) clone of Middle Eastern origin.
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DISCUSSION |
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) and 10–5 (Brubaker, 1969), respectively, and that Caf1 is also lost at high frequency (Burrows, 1957; Burrows & Bacon, 1958). The possibility therefore exists that these determinants were initially present upon primary isolation but lost during subsequent storage. In this regard, pCD is absent in pestoides I, and pestoides J lacked pMT, which encodes MT and Caf1. Cells of the Angola strain carried pMT but failed to express detectable Caf1 as judged by immunoassay, real-time PCR and query versus caf1 of Y. pestis KIM. Deficiency of Caf1 is sufficient to cause reduced virulence in guinea pigs (Burrows, 1957); these phenomena alone may have prompted inclusion of the Angola and pestoides J strains among the enzootic variants. The pestoides E, F and G isolates lacked pPCP encoding plasminogen activator, a known invasive virulence factor of epidemic Y. pestis (Brubaker et al., 1965; Lähteenmäki et al., 1998; Sodeinde et al., 1992). However, this deficiency in the pestoides F isolate did not influence virulence in mice by peripheral routes of injection (Worsham & Hunter, 1998; Worsham & Roy, 2003) although the absence of plasminogen activator might account for attenuation in hosts other than Muridae. The pestoides E, F and G isolates also carried oversize pMT plasmids similar to a pestoides strain from the Caucasus (Golubov et al., 2004).
The mutation frequency to Pgm– provided another criterion that distinguished tested enzootic and epidemic isolates of Y. pestis. The high mutation rate to pesticin resistance in epidemic Pgm+ yersiniae lacking pPCP (Brubaker, 1969) reflects the fact that the 100 kb deletable Pgm region (Lucier & Brubaker, 1992) is flanked by polar IS elements that undergo reciprocal recombination, thereby favouring deletion of the entire sequence (Fetherston et al., 1992; Fetherston & Perry, 1994). This event eliminates the Hms+ locus responsible for pigment binding and biofilm production (Kirillina et al., 2004; Perry et al., 2001), the yersiniabactin operon including the pesticin/yersiniabactin receptor (Fetherston et al., 1995), and ripABC required for growth in macrophages activated post-infection (Pujol et al., 2005). Yersiniae that undergo this high-frequency deletion therefore lose the linked pesticin receptor (Fetherston et al., 1992; Fetherston & Perry, 1994) and are thus resistant to pesticin. Similarly, yersiniae and other enterobacteriaceae that otherwise lack this receptor typically exhibit little or no sensitivity to pesticin (Buchrieser et al., 1999). Typical pestoides isolates, however, were found by Zudina (2000) to possess only one flanking IS element and are therefore incapable of yielding Pgm isolates at high frequency. Mutation to Pgm in our pestoides strains also occurred at low frequency and this event was ostensibly a point mutation as judged by retention of other elements of the Pgm locus.
Results of further study showed that some of the pestoides isolates possessed nutritional requirements not shared by typical epidemic strains of Y. pestis. Further effort will be required to determine the missing enzymes accounting for these requirements but it may be significant that certain arginine auxotrophs of Y. pestis are known to be attenuated in guinea pigs but not mice (Brubaker, 1972). Most isolates considered here were typical of those described in the literature in that they fermented rhamnose and melibiose but lacked other obvious properties of Y. pseudotuberculosis. A major exception to this rule was the pestoides I isolate, which did not ferment either one of these carbohydrates. Considered together, our characterization of this collection as it presently exists is generally consistent with previous reports of enzootic variants (Achtman et al., 2004; Anisimov et al., 2004; Garcia et al., 2007; Golubov et al., 2004) and supports consideration of the pestoides A, B and C isolates as Y. pestis subspecies altaici, D as hissarica, and E, F and G as caucasia (Anisimov et al., 2004).
The pestoides J isolate was an authentic epidemic isolate as judged by typical loss of zwf and aspA. This strain was also atypical in that it is negative for both glycerol fermentation and nitrate reduction and therefore does not correspond to any of Devignat's biovars (Devignat, 1951). However, like the Middle Eastern epidemic KIM and Camel strains (biovar medievalis), pestoides J encodes a tyrosine residue rather than aspartic acid at amino acid position 146 of AspA, suggesting the same clonal origin.
The Zwf determinant
The pestoides E, F and G strains contained serine at amino acid position 155 of Zwf and thus expressed full biological activity. These isolates are therefore more closely related to their progenitor (Y. pseudotuberculosis) than are the remaining pestoides strains which, like epidemic Y. pestis, contained inactive proline at this location. The position of these three strains in the initial phase of the evolutionary pathway leading to epidemic Y. pestis is also supported by the absence of pPCP and carriage of atypically large and probably more primitive pMT (Golubov et al., 2004).
Zwf– mutants of Escherichia coli exhibit a silent phenotype that requires concomitant loss of transketolase or transaldolase in the absence of pentose before growth becomes limited (Fraenkel, 1968). Transketolase and transaldolase are functional in both Y. pseudotuberculosis (Brubaker, 1968) and Y. pestis (Mortlock, 1962); furthermore, exogenous pentose is probably always available to Y. pestis during its closed life cycle. In this context, catabolism of gluconate, arabinose and ribose undergoes significant upregulation at 37 °C in cells of the epidemic strain KIM (Motin et al., 2004), further emphasizing the dispensable nature of biosynthetic Zwf. On the other hand, Zwf is a major supplier of NADPH to the bacterial cell and a deficiency of this intermediate could have serious consequences during expression of the LCR. It would therefore be interesting to determine if cells of the Zwf+ pestoides isolates are resistant to the D-glucose-dependent lysis in synthetic medium reported for epidemic strains of Y. pestis (Brownlow & Wessman, 1960). These findings emphasize that loss of Zwf is an obvious early milestone in the emergence of Y. pestis from Y. pseudotuberculosis.
The AspA determinant
It is remarkable that all of the tested pestoides isolates expressed at least partially active AspA. This phenomenon resulted from either retention of valine at amino acid number 363 (pestoides A, B, C and D) or intragenic suppressor mutations causing introduction at that position of serine (pestoides E, F, G and I) or phenylalanine (Angola and A16). The specific activity of crude AspA from strain Angola was unusually low, due perhaps to the presence of a second missense mutation in this enzyme at amino acid position 378 causing replacement of isoleucine with threonine (NCBI accession no. NC_010159.1). Further study will be necessary to determine if the marginal activity of AspA in strain Angola reflects this additional amino acid substitution. In any event, the reduced activity detected for the crude extract of the Angola enzyme remained proportional to that observed for the purified cloned enzyme. The basis of this decrease, as in the case of the essentially inert AspA of Y. pestis caused by replacement of leucine for valine at position 363 (Viola et al., 2008), was a markedly reduced kcat but little change in the Km for aspartic acid. This finding is in accord with the previous suggestion that mutations at position 363 indirectly inhibit enzyme activity by altering the position of catalytic residues Glu334 and Gln191, thereby disrupting their contributions to turnover (Viola et al., 2008).
Elimination of AspA, unlike Zwf, may have untoward consequences for the host. As already noted, this mutation accentuates the pCD/pYV-dependent nutritional requirement for Ca2+ at 37 °C and promotes excretion of L-aspartic acid at the expense of metabolic L-glutamate (Brubaker, 2005; Dreyfus & Brubaker, 1978). This event, should it occur in vivo, could be deleterious by altering the equilibrium of amino acid pools, thereby reducing host adenylate energy charge. It is significant that the aspA of the attenuated microtus biovar encodes valine at amino acid position 363, indicating that AspA is active in these organisms; cells of this enzootic isolate are also attenuated in man (Fan et al., 1995) but are fully virulent in mice (Zhou et al., 2004). AspA is widespread among saprophytic bacteria but was neither required for virulence of salmonellae (Tchawa Yimga et al., 2006) nor detected in the genomes of other facultative intracellular parasites including Mycobacterium tuberculosis (Fleischmann et al., 2002) and Francisella tularensis (Larsson et al., 2005) or obligate intracellular rickettsiae (Andersson et al., 1998). The latter, like epidemic isolates of Y. pestis, also convert exogenous L-glutamate to L-aspartate, which is released into the culture medium (Bovarnick & Miller, 1950).
Some of the enzootic strains used in this study possessed mutations that would cause attenuation of even epidemic isolates in guinea pigs. Formal proof demonstrating that mutational loss of AspA activity in epidemic isolates of Y. pestis and the consequences of this loss are directly associated with lethality in guinea pigs (and humans) will require additional effort involving exchange of aspA. The present study, however, provides convincing evidence that functional AspA is at least a biomarker for attenuation.
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ACKNOWLEDGEMENTS |
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Edited by: P. van der Ley
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Received 10 June 2008;
revised 29 September 2008;
accepted 1 October 2008.
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