Cells entering the VBNC state generally undergo a reduction in size. In the case of V. vulnificus, for example, whereas log phase (actively growing) cells might be 3 mm long, those in the VBNC state are typically 0.6 ^m in diameter. During this size reduction, significant changes in membrane structure, protein composition, ribosomal content, and possibly even DNA arrangement are experienced. Again using V. vulnificus as an example,1 we have found rapid and dramatic decreases in the levels of synthesis of DNA, RNA, and protein when these cells are exposed to a temperature downshift to 5°C (Fig. 1.3). However, such decreases do not mean that all synthesis has ceased. Indeed, protein synthesis appears to be essential for entry into this state, and under these conditions V. vulnificus produces some 40 new proteins not seen during growth at "normal" temperatures.7 At the same time, dramatic changes in membrane fatty acid composition,8 and decreases in nutrient transport and respiration rates have generally been reported to occur as cells enter this dormant state.
Cell wall synthesis, or at least metabolism of the constituents of these structures, also apparently continues, as addition of penicillin (an inhibitor of cell wall synthesis) to VBNC cells has generally been observed to result in rapid cell death.1
Most studies have observed that, if a cell entering the VBNC state harbors plasmids (extrachromosomal DNA elements which are able to control a variety of generally nonessential cell functions), then these plasmids are retained. This finding may prove to be highly relevant to the VBNC state of genetically modified cells released to the environment, as will be discussed later in this chapter. In contrast, it is becoming increasingly apparent that (possibly even major) changes in the cells' chromosomal DNA may be occurring as cells enter the VBNC state.1 This aspect may also be critical to release studies, as these changes bring into question the ability to employ such powerful molecular techniques as the polymerase chain reaction (PCR) to detect these otherwise undetectable cells. This concern is also dealt with later in this chapter.
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