- Volume 148, Issue 9, 2002

Proteome analysis of bacteria that can detoxify harmful organic compounds enables the discovery of enzymes involved in the biodegradation of these substances and proteins that protect the cell against poisoning. Exposure of Delftia acidovorans MC1 to 2,4-dichlorophenoxypropionic acid and its metabolites 2,4-dichlorophenol and 3,5-dichlorocatechol during growth on pyruvate as a source of carbon and energy induced several proteins. Contrary to the general hypothesis that lipophilic or reactive compounds induce heat shock or oxidative stress proteins, no induction of the GroEL, DnaK and AhpC proteins that were used as markers for the induction of heat shock and oxidative stress responses was observed. However, two chlorocatechol1,2-dioxygenases, identified by amino terminal sequence analysis, were induced. Both enzymes catalyse the conversion of 3,5-dichlorocatechol to 2,4-dichloro-cis,cis-muconate indicating that biodegradation is a major mechanism of resistance in the detoxifying bacterium D. acidovorans MC1.

To follow and model evolution of a microbial population in the chemostat, parameters are needed that give an indication of the absolute extent of evolution at a high resolution of time. In this study the evolution of the maximum specific growth rate (μmax) and the residual glucose concentration was followed for populations of Escherichia coli K-12 under glucose-limited conditions at dilution rates of 0·1 h−1, 0·3 h−1 and 0·53 h−1 during 500–700 h in continuous culture. Whereas μmax improved only during the initial 150 h, the residual glucose concentration decreased constantly during 500 h of cultivation and therefore served as a convenient parameter to monitor the evolution of a population at a high time resolution with respect to its affinity for the growth-limiting substrate. The evolution of residual glucose concentrations was reproducible in independent chemostats with a population size of 1011 cells, whereas no reproducibility was found in chemostats containing 107 cells. A model based on Monod kinetics assuming successive take-overs of mutants with improved kinetic parameters (primarily K s) was able to simulate the experimentally observed evolution of residual glucose concentrations. Similar values for the increase in glucose affinity of mutant phenotypes (K s(mutant) 0·6×K s(parent)) and similar mutation rates per cell per generation leading to these mutant phenotypes (1–5×10−7) were estimated in silico for all dilution rates. The model predicts a maximum rate of evolution at a dilution rate slightly below μmax/2. With increasing and decreasing dilution rates the evolution slows down, which also explains why in special cases a selection-driven evolution can exhibit apparent clock-like behaviour. The glucose affinity for WT cells was dependent on the dilution rate with highest values at dilution rates around μmax/2. Below 0·3 h−1 poorer affinity was mainly due to the effects of rpoS.

KaiA KaiB and KaiC are essential circadian clock proteins in the unicellular cyanobacterium Synechococcus elongatus PCC 7942. KaiA protein activates transcription of the kaiBC operon, which is believed to be a crucial step in the oscillating feedback loop of cyanobacteria. In this study, ∼∼400 mutations were introduced into kaiA by PCR-based mutagenesis, and rhythmic phenotypes of these mutants were studied by a bioluminescence reporter. In contrast to mutations in KaiB or KaiC, the vast majority of KaiA mutations extended the period and only rarely shortened it. The period could be extended to 35 h without lowering the mean or peak levels of kaiBC expression. However, several mutations resulted in low-amplitude oscillations or arrhythmia, which were accompanied by lowered kaiBC transcription. These results imply that the KaiA protein can change the period length of the circadian rhythm directly (through an unknown biochemical mechanism) or indirectly (by lowering kaiBC expression). Specific mutations of KaiA were identified in 34 mutants. While mutations mapped to various locations of the KaiA sequence, two clusters of period-altering mutations were found. This suggested that these regions are important domains of the KaiA protein for defining the period length. On the other hand, different sequences within KaiA to which arrhythmic mutations were mapped are important to enhance kaiBC expression.

The extremely radioresistant bacterium Deinococcus radiodurans encodes two genes that are homologous to those involved in bacterial lysine biosynthesis. In lysine biosynthesis, these genes are involved in the aminoadipate pathway and the diaminopimelate (DAP) pathway. DR1420 is homologous to lysZ, which is essential for bacterial lysine biosynthesis via the aminoadipate pathway, and DR1758 is homologous to lysA, which is essential for lysine biosynthesis via the DAP pathway. In this study, DR1420 and/or DR1758 were disrupted. Each disruptant of DR1420 and DR1758, and of DR1420 or DR1758 grew in a minimal medium, as did the wild-type. These results show that D. radiodurans performs lysine biosynthesis in a unique way.