Yersinia pseudotuberculosis blocks the phagosomal acidification of B10.A mouse macrophages through the inhibition of vacuolar H(+)-ATPase activity.

Yersinia pseudotuberculosis survived and multiplied in the phagosomes of B10.A mouse peritoneal macrophages. As one of the possible mechanisms for the bacteria's survival in the phagosomes, we demonstrated that live Y. pseudotuberculosis inhibited the phagosomal acidification; pH within phagosomes containing the live Y. pseudotuberculosis remained at about 6.0, whereas pH within phagosomes containing the dead Y. pseudotuberculosis fell to about 4. 5. This ability to inhibit intraphagosomal acidification was also shared by mutants lacking the 42 Md virulence plasmid, indicating that it is chromosomally encoded. The phagosomes containing dead bacteria raised the pH to 6.2 after the treatment of their macrophages with an inhibitor (bafilomycin A1) specific for V-ATPase. Although the amount of V-ATPase in the A and B subunits on the phagosomes was not significantly different between the live and dead bacteria infection, the phagosomes containing live bacteria had a 10-fold smaller V-ATPase activity than those containing the dead bacteria. These results indicated that the inhibition of phagosomal acidification by Y. pseudotuberculosis infection was due to the attenuation of V-ATPase activity, and not due to the exclusion of V-ATPase subunits from the phagosome membrane as found in Mycobacterium avium.

Detection and identification of Yersinia pestis by polymerase chain reaction (PCR) using multiplex primers.

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 (caf1)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.

[Possible control of capsule formation and intracellular synthesis of envelope antigen by each different plasmid].

Variants which lacked capsular envelopes on their cell surface were isolated from the culture of a highly virulent Yreka strain of Yersinia pestis grown in the presence of acridine orange, ethidium bromide or sodium dodecyl sulfate at incompletely growth-inhibitory concentrations. The variant could be divided into two types on the basis of the presence and the absence of intracellular envelope antigen. Both types of the variants lacked the 13 megadaltone (Md) plasmid. Thus, it may well be said that the 13 Md plasmid would play some decisive role in extracellular envelope formation and no concern with the synthesis of intracellular antigen. It was clarified that these characters were carried by each different gene. The intracellular envelope antigen synthesis could not be correlated with other plasmids isolated from Yreka strain, i.e., 7, 23, 44 and 59 Md plasmids. On the other hand, further treatment of the intracellular-positive variant with the above inhibitors resulted in the occurrence of the antigen-deficient type variant at a rate of 1-2%. The high frequency appearance of the variant by the plasmid-depleting agents might indicate possible presence of some yet unknown plasmid responsive to the intracellular synthesis of envelope antigen.

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.

Organization and transcription of the dnaA and dnaN genes of Escherichia coli.

The locations of the linked dnaA and dnaN genes of Escherichia coli in a specialized transducing lambda phage genome have been determined by electron microscopic heteroduplex analysis, using phages with deletions or insertions in the dnaA or dnaN gene. The transcription initiation sites for the dna genes were also localized by electron microscopic analysis of DNA-RBA heteroduplex molecules formed between the E. coli DNA fragment of the phage genome and the in vitro transcription products of the fragment. The dnaN gene was found to be transcribed in the same direction as the dnaA gene, and predominantly from the promoter of the dnaA gene.