Study of PGM mutation mechanism in Yersinia pestis (plague pathogen) vaccine strain EV76

Yersinia pestis vaccine strain EV76 is a mutant of the virulent strain which has lost the pigmentation phenotype (Pgm+). This phenotype includes three characteristics: it absorbs pigments from agar media (Hms+), produces a siderophore yersiniabactin (Ybt+), and causes a lethal disease after subcutaneous inoculation of laboratory animals (Vir+). These characteristics are lost simultaneously after high frequency spontaneous deletion of 10 kB fragment of chromosomal DNA, termed the pgm locus. We compared the pgm locus-associated genetic and phenotypical properties of the vaccine strain with those of a typical Pgm- deletion mutant of a virulent strain. The results indicate that Pgm- phenotype of the vaccine strain results not from the deletion of the pgm locus, but from the insertion inactivation of the genes located in this locus. In contrast to the deletion mutant, the vaccine strain carries sequences detected by hybridization and PCR, which are complementary to the pgm locus genes. Moreover, the vaccine strain differed from the deletion mutant by a low level of Hms+ expression, a slower rate of cell death under iron-chelated conditions at 37 degrees C, "residual virulence" upon subcutaneous inoculation, and capacity to form revertants which restore the characteristics of Pgm+ phenotype after cell growth at 12 degrees C.     

Identification and cloning of a hemin storage locus involved in the pigmentation phenotype of Yersinia pestis.

The temperature-dependent absorption of sufficient exogenous hemin or Congo red to form pigmented colonies of Yersinia pestis has been termed the pigmentation phenotype (Pgm+). Spontaneous mutation to a Pgm- phenotype results in the loss of a number of divergent physiological characteristics, including the ability to store hemin and to bind Congo red at 26 degrees C. In this study, we generated and isolated transposon insertion mutants that are hemin storage negative (Hms-) and therefore unable to form pigmented colonies. These mutations are due to single mini-kan insertions within a 19.5-kilobase (kb) SalI fragment of chromosomal DNA. Restriction site analysis of eight mutants identified a minimum of six potentially different insertion sites spanning an approximately 10-kb hemin storage (hms) locus. The 19.5-kb SalI fragment (containing approximately 18 kb of Y. pestis DNA and the mini-kan insert) was cloned from one of these mutants, KIM6-2012. By using this cloned fragment as a DNA probe, the mechanism of spontaneous mutation to a Pgm- phenotype was identified as a massive deletion event. The deletion spans at least 18 kb of genomic DNA in spontaneous Pgm- mutants from nine separate strains of Y. pestis. DNA adjacent to the mini-kan insert was used to identify a clone containing a wild-type hms locus. A spontaneous Pgm- mutant of Y pestis KIM containing this clone exhibits an Hms+ phenotype. The hms::mini-kan mutations and cloned wild-type hms locus generated in this study will greatly aid in identifying the function of hemin storage in Y. pestis.     

Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element

The pigmentation (Pgm+) phenotype of Yersinia pestis encompasses a variety of different physiological traits, all of which are missing in Pgm- mutants. We have previously shown that loss of the Pgm+ phenotype is accompanied by the spontaneous deletion of at least 45 kb of chromosomal DNA, referred to as the pgm locus. Using chromosomal walking, we have now mapped the full extent of the pgm locus in Y. pestis strain KIM6+. Our results indicate that the locus spans 102 kb of DNA which is absent in the spontaneous Pgm- mutant, KIM6. Yersinia pseudotuberculosis PB1/0 contains sequences homologous to the entire pgm locus while only part of this region hybridized to Yersinia enterocolitica WA-LOX DNA. Restriction enzyme mapping and hybridization studies revealed the presence of a repetitive element at both ends of the pgm locus and in multiple copies elsewhere in the Y. pestis genome. This element may be responsible for generating the deletion.     

The Yfe system of Yersinia pestis transports iron and manganese and is required for full virulence of plague

Iron acquisition in Yersinia pestis is fundamental to the success of plague pathogenesis. We have previously identified an approximately 5.6 kb region (yfe) of Y. pestis genomic DNA, capable of restoring iron-deficient growth but not siderophore production to an Escherichia coli mutant (SAB11) incapable of synthesizing the siderophore, enterobactin. The yfe locus of Y. pestis, found in both pigmented (Pgm+) and nonpigmented (Pgm-) strains, comprises five genes arranged in two distinct operons (yfeA-D and yfeE ). The larger of these, yfeABCD, encodes an ABC transport system, whose expression is iron and Fur regulated and is repressed in cells grown in the presence of manganese. Cells from a Pgm-, Yfe- (DeltayfeAB ) mutant strain of Y. pestis exhibited reduced transport of both 55Fe and 54Mn. Furthermore, cells containing an intact yfe locus showed reduced 55Fe uptake when competing amounts of MnCl2 or ZnCl2 were present, whereas 54Mn uptake was inhibited by FeCl3 but not by ZnCl2. Similarly, yfe mutants of Y. pestis exhibited growth defects on media supplemented with the iron chelators 2,2'-dipyridyl or conalbumin. These growth defects were not relieved by supplementation with MnCl2. A ybt-, DeltayfeAB mutant of Y. pestis was completely avirulent in mice infected intravenously (LD50 > 1.7 x 107 cfu) compared with its parental ybt-, yfe+ strain, which had an LD50 of < 12. In addition, compared with its ybt+, yfe+ parent, a ybt+, DeltayfeAB mutant of Y. pestis had an approximately 100-fold increase in the LD50 from a subcutaneous route of infection. These data suggest that the Yfe and Ybt systems may function effectively to accumulate iron during different stages of the infectious process of bubonic plague.     

Plasmid-like properties of the four virulence-associated factors of Yersinia pestis

The virulence-associated factors of Yersinia pestis, which determine the abilities to produce pesticin I (Pst+), capsular fraction I antigen (Fra+), V and W antigen complex (Vwa+) and a cell-surface component for adsorption of exogenous pigments (Pgm+), were independently eliminated by cultivation of the cells in the presence of acridine orange, ethidium bromide or sodium dodecyl sulfate at a subinhibitory concentration. A virulent Y. pestis strain, Yreka, harbored at least five extrachromosomal DNA molecules of different sizes. In these molecules, a novel 13-megadalton DNA which was cured concomitantly with the elimination of the Fra factor was found, in addition to the known species of 7 and 44 megadaltons which were lost with the conversions to Pst- and Vwa-, respectively. Although the conversion to Pgm- could not be correlated with the lack of any proper extra-chromosomal DNA, the factor was transmitted to Pgm- cells with the aid of self-conjugative RP4 plasmid. The cells acquiring the Pgm factor regained virulence for mice.     

LcrG, a secreted protein involved in negative regulation of the low-calcium response in Yersinia pestis.

The purpose of this study was to define the function of LcrG, the product of the first gene in the lcrGVHyopBD operon of the low-Ca(2+)-response (LCR) virulence plasmid of Yersinia pestis. We created a Y. pestis strain having an in-frame deletion in lcrG. This nonpolar mutant had an abnormal LCR growth phenotype: it was unable to grow at 37 degrees C in the presence of 2.5 mM Ca2+ ("Ca2+ blind") but was able to grow at 37 degrees C when 18 mM ATP was present. At 37 degrees C it failed to downregulate the expression and secretion of its truncated product (LcrG), V antigen, and YopM. All of these mutant properties were complemented by plasmids carrying normal lcrG. However, a nonpolar lcrE mutation and an lcrH mutation (both also causing a Ca(2+)-blind phenotype) were not complemented in this way. The Y. pestis parent strain expressed LcrG at 37 degrees C in the presence and absence of Ca2+ and transported it to the medium when Ca2+ was absent. We identified two LCR-regulated loci, lcrD and yscDEF, required for this transport. Complementation analysis of the Y. pestis lcrR strain previously shown to lack the expression of LcrG showed that the loss of LcrG but not of LcrR caused the Ca(2+)-blind phenotype of that mutant. Taken together, the results show that LcrG is a negative regulator of the LCR, perhaps functioning in Ca2+ sensing along with LcrE.     

Role of the Yersinia pestis yersiniabactin iron acquisition system in the incidence of flea-borne plague

Plague is a flea-borne zoonosis caused by the bacterium Yersinia pestis. Y. pestis mutants lacking the yersiniabactin (Ybt) siderophore-based iron transport system are avirulent when inoculated intradermally but fully virulent when inoculated intravenously in mice. Presumably, Ybt is required to provide sufficient iron at the peripheral injection site, suggesting that Ybt would be an essential virulence factor for flea-borne plague. Here, using a flea-to-mouse transmission model, we show that a Y. pestis strain lacking the Ybt system causes fatal plague at low incidence when transmitted by fleas. Bacteriology and histology analyses revealed that a Ybt-negative strain caused only primary septicemic plague and atypical bubonic plague instead of the typical bubonic form of disease. The results provide new evidence that primary septicemic plague is a distinct clinical entity and suggest that unusual forms of plague may be caused by atypical Y. pestis strains.     

High-Frequency RecA-Dependent and -Independent Mechanisms of Congo Red Binding Mutations in Yersinia pestis

Yersinia pestis, which causes bubonic and pneumonic plague, forms pigmented red colonies on Congo red (CR) dye agar. The hmsHFRS genes required for CR binding (Crb(+)) are genetically linked to virulence-associated genes encoding a siderophore uptake system. These genes are contained in a 102-kb chromosomal pgm locus that is lost in a high-frequency deletion event, resulting in loss of the Crb(+) phenotype. We constructed a recA mutant strain of Y. pestis KIM10+ (YPRA) to test whether the high frequency Crb mutants result from a RecA-mediated deletion of the IS100-flanked pgm locus. Two Pgm-associated phenotypes (Crb(+) and pesticin sensitivity [Pst(s)]) were used as markers for the presence of the pgm locus in the RecA(+) KIM10+ and RecA(-) YPRA strains. In KIM10+, both phenotypes were lost at a very high (2 x 10(-3)) frequency, due to the deletion of the entire pgm locus. In YPRA, the Crb(+) phenotype was still lost at a high frequency (4.5 x 10(-5)), although the loss of the Pst(s) phenotype occurred at spontaneous antibiotic resistance mutation frequencies (2 x 10(-7)). These RecA-independent Crb(-) mutants were caused by mutations in both the hmsHFRS locus and in a newly identified gene, hmsT. Nonpigmented Yersinia pseudotuberculosis and Escherichia coli strains transformed with both hmsT and hmsHFRS became Crb(+). This study demonstrates that in a laboratory culture, the Crb(+) phenotype is unstable, independent of the pgm locus deletion. We propose that a lack of selection for the CR-binding ability of Y. pestis in vitro may contribute to the mutation frequencies observed at the hmsHFRS and hmsT loci.     

Insights into Yersinia pestis biofilm development: topology and co-interaction of Hms inner membrane proteins involved in exopolysaccharide production

Primarily, three operons, hmsHFRS, hmsT and hmsP, are responsible for the development of a Yersinia pestis biofilm, which is essential for blockage-dependent transmission of plague from fleas to mammals. Here, using specific antibodies, a polymeric beta-1,6-N-acetyl-d-glucosamine-like polysaccharide was detected in the extracellular matrix of hmsHFRS-dependent Y. pestis biofilm. The production of this exopolysaccharide (EPS) was controlled by diguanylate cyclase HmsT and EAL domain phosphodiesterase HmsP, acting as positive and negative regulators respectively. Cellular compartmentalization of soluble segments of Hms inner membrane proteins, including the putative glycosyltransferase domain of HmsR, the diguanylate cyclase/GGDEF domain of HmsT and the phosphodiesterase/EAL domain of HmsP, was determined by a combination of topology prediction algorithms and construction of C-terminal translational fusions with beta-galactosidase and alkaline phosphatase. Multiple interactions of Hms inner membrane proteins were detected using bacterial cAMP based two-hybrid system. Biochemical analyses confirmed some of these protein-protein interactions. Our results indicate that synthesis and regulation of the Y. pestis biofilm EPS occurs in the cytoplasm by a proposed Hms enzymatic complex.     

Genetic analysis and simulation of the virulence of Yersinia pestis

The genetic analysis of Y. pestis virulence factors accomplished in the 358 strain isogenic system allowed us to determine a minimal set of known factors providing pathogenicity. The combination of chromosomal marker Pgm+ and calcium dependence plasmid (pCad) is shown to be sufficient for preserving the virulence of Y. pestis. Experimental modelling of virulence in this microorganism by the genetic exchange methods was carried out. The reduced virulence of the strains Pgm+ and pCad+ for guinea pigs was detected.     

Polyamines are essential for the formation of plague biofilm

We provide the first evidence for a link between polyamines and biofilm levels in Yersinia pestis, the causative agent of plague. Polyamine-deficient mutants of Y. pestis were generated with a single deletion in speA or speC and a double deletion mutant. The genes speA and speC code for the biosynthetic enzymes arginine decarboxylase and ornithine decarboxylase, respectively. The level of the polyamine putrescine compared to the parental speA+ speC+ strain (KIM6+) was depleted progressively, with the highest levels found in the Y. pestis DeltaspeC mutant (55% reduction), followed by the DeltaspeA mutant (95% reduction) and the DeltaspeA DeltaspeC mutant (>99% reduction). Spermidine, on the other hand, remained constant in the single mutants but was undetected in the double mutant. The growth rates of mutants with single deletions were not altered, while the DeltaspeA DeltaspeC mutant grew at 65% of the exponential growth rate of the speA+ speC+ strain. Biofilm levels were assayed by three independent measures: Congo red binding, crystal violet staining, and confocal laser scanning microscopy. The level of biofilm correlated to the level of putrescine as measured by high-performance liquid chromatography-mass spectrometry and as observed in a chemical complementation curve. Complementation of the DeltaspeA DeltaspeC mutant with speA showed nearly full recovery of biofilm to levels observed in the speA+ speC+ strain. Chemical complementation of the double mutant and recovery of the biofilm defect were only observed with the polyamine putrescine.     

Virulence-associated plasmids from Yersinia enterocolitica and Yersinia pestis.

A 44-megadalton plasmid associated with virulence and Ca2+ dependence from Yersinia enterocolitica 8081 was compared at the molecular level with a 47-megadalton plasmid associated with Ca2+ dependence from Yersinia pestis EV76. The plasmids were found to share 55% deoxyribonucleic acid sequence homology distributed over approximately 80% of the plasmid genomes. One region in which the plasmids differed was found to contain sequences concerned with essential plasmid functions. Forty-five mutants of Y. pestis were isolated which had spontaneously acquired the ability to grow on calcium-free medium (Ca2+ independence). Of these mutants, 21 were cured of their 47-megadalton plasmid, whereas the remaining had either suffered a major deletion of the plasmid or had a 2.2-kilobase insertion located in one of two adjacent BamHI restriction fragments encompassing approximately 9 kilobases. The inserted sequence was found at numerous sites on the Y. pestis chromosome and on all three plasmids in the strain and may represent a Y. pestis insertion sequence element.     

An endoplasmic reticulum-bound Ca(2+)/Mn(2+) pump,ECA1, supports plant growth and confers tolerance to Mn(2+) stress

Plants can grow in soils containing highly variable amounts of mineral nutrients, like Ca(2+) and Mn(2+), though the mechanisms of adaptation are poorly understood. Here, we report the first genetic study to determine in vivo functions of a Ca(2+) pump in plants. Homozygous mutants of Arabidopsis harboring a T-DNA disruption in ECA1 showed a 4-fold reduction in endoplasmic reticulum-type calcium pump activity. Surprisingly, the phenotype of mutant plants was indistinguishable from wild type when grown on standard nutrient medium containing 1.5 mM Ca(2+) and 50 microM Mn(2+). However, mutants grew poorly on medium with low Ca(2+) (0.2 mM) or high Mn(2+) (0.5 mM). On high Mn(2+), the mutants failed to elongate their root hairs, suggesting impairment in tip growth processes. Expression of the wild-type gene (CAMV35S::ECA1) reversed these conditional phenotypes. The activity of ECA1 was examined by expression in a yeast (Saccharomyces cerevisiae) mutant, K616, which harbors a deletion of its endogenous calcium pumps. In vitro assays demonstrated that Ca(2+), Mn(2+), and Zn(2+) stimulated formation of a phosphoenzyme intermediate, consistent with the translocation of these ions by the pump. ECA1 provided increased tolerance of yeast mutant to toxic levels of Mn(2+) (1 mM) and Zn(2+)(3 mM), consistent with removal of these ions from the cytoplasm. These results show that despite the potential redundancy of multiple Ca(2+) pumps and Ca(2+)/H(+) antiporters in Arabidopsis, pumping of Ca(2+) and Mn(2+) by ECA1 into the endoplasmic reticulum is required to support plant growth under conditions of Ca(2+) deficiency or Mn(2+) toxicity.     

Comparative studies with tox plus and tox minus corynebacteriophages

The characteristics of nine inducible temperate corynebacteriophages designated alpha(tox+), beta(tox+), P(tox+), gamma(tox-), pi(tox+), K(tox-), rho(tox-), L(tox+), and delta(tox+) have been compared. Virion morphology and ability to recombine genetically with the well-studied phage beta(tox+) have been correlated with other properties of the phages, and the distribution of the genetic marker tox+ among related and relatively unrelated corynebacteriophages has been analyzed. The immunity specificity, host range, and plaque morphology of each phage were determined. The phages can be separated into five groups with different immunity specificities. Each type of host range previously recognized in mutants of phage beta(tox+) was present in one or more of the phages included in the present study, and the phages were found to produce plaques of several different morphological types. Representative phages with each of the five types of immunity specificity were further characterized with respect to virion morphology, ability to recombine with phage beta(tox+), latent period, average burst size, and neutralization by homologous and heterologous antiphage sera. All of these phages have polyhedral heads and long slender tails, but two distinct morphological types were distinguished by the sizes and proportions of the components of the virions. Only phages of the same morphological type as beta(tox+) were capable of genetic recombination with beta(tox+), but morphological similarity between phages was not sufficient to insure interfertility. The phages which recombined with beta(tox+) resembled one another in plaque morphology, latent period, and average burst size, whereas phages which failed to recombine with beta(tox+) differed in these characteristics. The phages capable of genetic recombination with beta(tox+) were found to differ from each other in immunity specificity, host range, neutralization by antiphage sera, and toxinogenicity. Thus, these latter characteristics are of limited value in establishing the extent of relatedness between corynebacteriophages. The genetic marker tox+ was not consistently correlated with any other property of the corynebacteriophages analyzed in this study. The most striking finding regarding the distribution of the tox+ marker is its presence both in beta(tox+) and delta(tox+), phages which fail to recombine genetically and which differ in virion morphology. The presence of the tox+ marker in genetically unrelated corynebacteriophages poses many questions concerning the origin(s) of tox+ and the evolution of the phage-host interactions which determine the ability of corynebacteria to synthesize diphtherial toxin.     

Isolation of anaerobic respiratory mutants of Shewannella putrefaciens and genetic analysis of mutants deficient in anaerobic growth on Fe3+.

A genetic approach was used to study (dissimilatory) ferric iron (Fe3+) reduction in Shewanella putrefaciens 200. Chemical mutagenesis procedures and two rapid plate assays were developed to facilitate the screening of Fe3+ reduction-deficient mutants. Sixty-two putative Fe3+ reduction-deficient mutants were identified, and each was subsequently tested for its ability to grow anaerobically on various compounds as sole terminal electron acceptors, including Fe3+, nitrate (NO3-), nitrite (NO2-), manganese oxide (Mn4+), sulfite (SO3(2-)), thiosulfate (S2O3(2-)), trimethylamine N-oxide, and fumarate. A broad spectrum of mutants deficient in anaerobic growth on one or more electron acceptors was identified. Nine of the 62 mutants (designated Fer mutants) were deficient only in anaerobic growth on Fe3+ and retained the ability to grow on all other electron acceptors. These results suggest that S. putrefaciens expresses at least one terminal Fe3+ reductase that is distinct from other terminal reductases coupled to anaerobic growth. The nine Fer mutants were conjugally mated with an S. putrefaciens genomic library harbored in Escherichia coli S17-1. Complemented S. putrefaciens transconjugants were identified by the acquired ability to grow anaerobically on Fe3+ as the sole terminal electron acceptor. All recombinant cosmids that conferred the Fer+ phenotype appeared to carry a common internal region.