Yersinia pestis strain Yreka was grown at 27 or 37°C, and the lipid A structures (lipid A-27°C and lipid A-37°C) of the respective lipopolysaccharides (LPS) were investigated by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Lipid A-27°C consisted of a mixture of tri-acyl, tetra-acyl, penta-acyl, and hexa-acyl lipid A's, of which tetra-acyl lipid A was most abundant. Lipid A-37°C consisted predominantly of tri-and tetra-acylated molecules, with only small amounts of penta-acyl lipid A; no hexa-acyl lipid A was detected. Furthermore, the amount of 4-amino-arabinose was substantially higher in lipid A-27°C than in lipid A-37°C. By use of mouse and human macrophage cell lines, the biological activities of the LPS and lipid A preparations were measured via their abilities to induce production of tumor necrosis factor alpha (TNF-␣). In both cell lines the LPS and the lipid A from bacteria grown at 27°C were stronger inducers of TNF-␣ than those from bacteria grown at 37°C. However, the difference in activity was more prominent in human macrophage cells. These results suggest that in order to reduce the activation of human macrophages, it may be more advantageous for Y. pestis to produce less-acylated lipid A at 37°C.Yersinia pestis was isolated as a causative agent of plague in Hong-Kong in 1894 independently by A. Yersin and S. Kitasato (3). Since then, this highly pathogenic bacterium has been investigated, and many virulence factors have been identified, including fraction 1 antigen, murine toxin, Yop proteins, pH 6 antigen, and iron acquisition systems (5, 28). Lipopolysaccharide (LPS) has also been studied for many decades as one of the virulence factors of Y. pestis, and its composition and endotoxic activity have been examined in earlier studies (1,40,41). By use of modern analytical methods, the LPS of Y. pestis was proven to be a rough type LPS without O-antigenic polysaccharide (7,8,25,29,30,35) that contains 3-hydroxy-myristic acid (3-OH-C 14:0 ) as a main fatty acid in the lipid A portion. However, there was a discrepancy in the amounts of the nonhydroxy-fatty acids found. Hartley et al. reported that nearly 90% of fatty acids in the lipid A of Y. pestis strain 195/R (a virulent strain) consisted of 3-OH-C 14:0 (14). On the other hand, considerable amounts of lauric acid (C 12:0 ), palmitic acid (C 16:0 ), and palmitoleic acid (C 16:1 ) were detected in the lipid A of Y. pestis strain EV40 (an avirulent strain) (4, 39). Using the same strain, Aussel et al. (2) proposed a hexa-acylated lipid A structure (four molecules of 3-OH-C 14:0 , one of C 12:0 , and one of C 16:1 ). In our preliminary experiment we isolated the lipid A from Y. pestis strain Yreka (virulent strain) grown at 37°C and found that it contained 3-OH-C 14:0 and only trace amounts of other fatty acids. A possible explanation of the controversy on fatty acids may be differences in strains or culture conditions. Darveau et al. (8) reported that the molecular size of the LPS from Y. pestis strain EV76 ...

A PCR method for detection of Yersinia pestis-virulence determinants by the use of multiplex primers was developed. Four pairs of oligonucleotide primers were designed from each gene of three kinds of virulent plasmids and a chromosomal DNA; 60-Md plasmid-located gene (cafl) encoding Y. pestis-specific capsular antigen fraction 1, a Y. pestis-specific region of a yopM gene encoded on 42-Md virulent plasmid, a plasminogen activator gene (pla) encoded on Y. pestis-specific 7-Md plasmid and an invasin protein gene (inv) encoded on chromosomal DNA. This multiplex-primer system was specific for the detection of Y. pestis among pathogenic Yersinia species and other enterobacteriaceae having antigens common to Y. pestis. Since this method is simple and safe, it will be useful to identify and confirm Y. pestis in cases of emergency and for the surveillance of epidemics.

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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 1 3-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 extrachromosomal DNA, the factor was transmitted to Pgm-cells with the aid of selfconjugative RP4 plasmid. The cells acquiring the Pgm factor regained virulence for mice.Virulence of Yersinia pestis is spontaneously attenuated at an unusually high frequency (5) . The conversion to avirulence is accompanied by loss of the ability to produce bacteriocinogenic pesticin I (Pst+), capsular fraction I antigen (Fra+), V and W antigen complex (Vwa+) or a cell-surface component permitting adsorption of certain exogenous pigments (Pgm+) (5, 6). It has been reported that the property of Pst+ was transmitted to nonpesticinogenic cells during conjugation directed by an E. coli R plasmid (12). Recently, it has been shown that Y. pestis cells harbor extrachromosomal DNA molecules whose existence is associated with Pst+ or Vwa+ (2, 9, 14). Portnoy et al (13) have identified a 47-megadalton plasmid participating in Vwa+. No plasmids responsible for Fra+ and Pgm+ have been found.The present study was undertaken to clarify plasmid-like properties of the virulence-associated factors of Fra, Pgm, Pst, and Vwa. We first investigated whether these factors could be eliminated from the virulent Yreka strain by culturing the cells in the presence of acridine orange, ethidium bromide or sodium dodecyl sulfate. These chemical agents are known to inhibit multiplication of certain plasmids preferentially to host cellular growth, resulting in curing of the plasmids from the cells 837

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