IDENTIFICATION AND CHARACTERIZATION OF THE YERSINIA-ENTEROCOLITICA GSRA GENE, WHICH PROTECTIVELY RESPONDS TO INTRACELLULAR STRESS-INDUCED BY MACROPHAGE PHAGOCYTOSIS AND TO EXTRACELLULAR ENVIRONMENTAL-STRESS

Yersinia enterocolitica is able to resist the microbicidal mechanisms of macrophages and to grow within phagocytic cells. Some bacteria incl uding Y. enterocolitica have been shown to respond to the hostile envi ronment in macrophages by producing a set of stress proteins which are also induced by environmental stresses. To understand the role of str ess proteins in intracellular survival of bacteria, we identified and cloned a Y. enterocolitica gene, called gsrA (global stress requiremen t). The gsrA gene was identified because its insertional inactivation by a transposon resulted in the inability of the organism to grow at a n elevated temperature and to survive within macrophages after phagocy tosis. The gsrA gene mas sequenced and shown to encode a basic, 49,500 -Da protein. The GsrA protein shows significant amino acid sequence ho molog to the HtrA stress protein which was originally identified in Es cherichia coli. Furthermore, the genetically defined Y. enterocolitica gsrA mutant was constructed and characterized. The insertional mutati on of gsrA resulted in inhibition of growth at temperatures above 39 d egrees C and greatly increased susceptibility to oxidative and osmotic stresses. The mutant additionally lost the ability to survive and rep licate within macrophages. These results, taken together, indicate tha t the gsrA gene is an essential component of the protection mechanism employed by Y. enterocolitica, allowing it to respond to the intracell ular stress in macrophages as well as extracellular environmental stre ss.