The penicillin tolerance of ammo acid-deprived re/A+ Escherichia coli is attributed to the stringent response. The β-lactam-induced lysis of amino acid-deprived bacteria resulting from relaxation of the stringent response was inhibited by cerulenin, or by glycerol deprivation in the case of a gpsA mutant (defective in the biosynthetic snglycerol 3-phosphate dehydrogenase). Therefore, β-lactam-induced lysis of amino acid-deprived cells was dependent on phospholipid synthesis. Both the priming and the lysis induction stages of β-lactam-induced lysis were shown to require phospholipid synthesis. It has been known for some time that phospholipid synthesis is inhibited by the stringent reponse. These results indicate that the inhibition of peptidoglycan synthesis and the induction of penicillin tolerance during the stringent response are both secondary consequences of the inhibition of phospholipid synthesis. Direct experimental evidence is presented for the first time indicating that the penicillin tolerance of amino acid-deprived E coli was directly attributable to action of guanosine 3',5'-bispyrophosphate (ppGpp) and not to some other effect of amino acid deprivation. The overproduction of ppGpp resulted in the inhibition of peptidoglycan and phospholipid synthesis and in penicillin tolerance. Penicillin tolerance and the inhibition of peptidoglycan synthesis were both suppressed when ppGpp accumulation was prevented by treatment of the bacteria with chloramphenicol, an inhibitor of ppGpp synthetase I activation. Glycerol-3-phosphate acyltransferase, the product of plsB gene, was recently identified as the main site of ppGpp inhibition in phospholipid synthesis. The overexpression of the cloned plsB gene reversed the penicillin tolerance conferred by ppGpp accumulation. This also indicates that the membrane-associated events in peptidoglycan metabolism were dependent on ongoing phospholipid synthesis. Interestingly , treatment with β-lactam antibiotics by itself induced re/A-dependent ppGpp accumulation, but the maximum levels attained were insufficient to confer penicillin tolerance. It was al so demonstrated that penicillin tolerance was induced when phospholipid synthesis was inhibited in normal growing E. coli. This penicillin tolerance was not the result a simple inhibition of growth or a decrease in the membrane levels of individual phospholipids (e.g., acidic phospholipids), but rather the direct result of the inhibition of net phospholipid synthesis. A number of factors that interfere with β-lactam-induced lysis were investigated. (i) It was demonstrated that de-energization of the E. coli cytoplasmic membrane resulted in penicillin tolerance due to the inhibition of both the priming and the lysis induction stages. (ii) Inhibition of protein synthesis in the absence of the stringent response promoted both the priming and the lysis induction stages resulting in a faster onset of β-lactam-induced lysis. (iii) The temperature sensitivity of β-lactam-induced lysis in amino acid-deprived E. coli was re-investigated. Penicillin tolerance resulting from a temperature up-shift was not due to the induction of the heat-shock response, as previously reported, but from a reversible inhibition of unidentified thermosensitive enzyme(s) involved in the lysis induction stage. / Graduate

For a sustainable future, there is a call to increase the market share of bio-based technologies and materials. Microbial-based technologies have the potential and the ability to contribute substantively on many levels to global efforts to achieve sustainability. Development and utilization of microbial technologies is, however, an extensive process involving numerous steps, including the discovery of novel technologies and the development of industrially viable production systems. In the presented thesis, individual steps of microbial biotechnology development were addressed. In the first part of the study, a variety of methodological approaches were employed in order to study the effect of the anthropogenic activity (i.e., decades lasting production of penicillin G) on the structure of soil microbial communities. Moreover, both cultivable and non-cultivable fractions of populations were subjected to functional screening in order to unravel the biotechnological potential of the microorganisms in terms of production of enzymes involved in biotransformation of beta-lactam antibiotics: penicillin G acylase (PGA) and alpha amino acid ester hydrolase (AEH). Our results indicated that the impacted communities harbour a microbial community with increased diversity and richness. However, on the...