An important link between the environment and the physiological state of bacteria is the regulation of the transcription of a large number of genes by global transcription factors. One of the global regulators, Fis (factor for inversion stimulation), is well studied in Escherichia coli, but the role of this protein in pseudomonads has only been examined briefly. According to studies in Enterobacteriaceae, Fis regulates positively the flagellar movement of bacteria. In pseudomonads, flagellar movement is an important trait for the colonization of plant roots. Therefore we were interested in the role of the Fis protein in Pseudomonas putida, especially the possible regulation of the colonization of plant roots. We observed that Fis reduced the migration of P. putida onto the apices of barley roots and thereby the competitiveness of bacteria on the roots. Moreover, we observed that overexpression of Fis drastically reduced swimming motility and facilitated P. putida biofilm formation, which could be the reason for the decreased migration of bacteria onto the root apices. It is possible that the elevated expression of Fis is important in the adaptation of P. putida during colonization of plant roots by promoting biofilm formation when the migration of bacteria is no longer favoured.

Stationary-phase mutations occur in populations of stressed, nongrowing, and slowly growing cells and allow mutant bacteria to overcome growth barriers. Mutational processes in starving cells are different from those occurring in growing bacteria. Here, we present evidence that changes in mutational processes also take place during starvation of bacteria. Our test system for selection of mutants based on creation of functional promoters for the transcriptional activation of the phenol degradation genes pheBA in starving Pseudomonas putida enables us to study base substitutions (C-to-A or G-to-T transversions), deletions, and insertions. We observed changes in the spectrum of promoter-creating mutations during prolonged starvation of Pseudomonas putida on phenol minimal plates. One particular C-to-A transversion was the prevailing mutation in starving cells. However, with increasing time of starvation, the importance of this mutation decreased but the percentage of other types of mutations, such as 2-to 3-bp deletions, increased. The rate of transversions was markedly elevated in the P. putida MutY-defective strain. The occurrence of 2-to 3-bp deletions required the stationaryphase sigma factor RpoS, which indicates that some mutagenic pathway is positively controlled by RpoS in P. putida.

Oxidative damage of DNA is a source of mutation in living cells. Although all organisms have evolved mechanisms of defense against oxidative damage, little is known about these mechanisms in nonenteric bacteria, including pseudomonads. Here we have studied the involvement of oxidized guanine (GO) repair enzymes and DNAprotecting enzyme Dps in the avoidance of mutations in starving Pseudomonas putida. Additionally, we examined possible connections between the oxidative damage of DNA and involvement of the error-prone DNA polymerase (Pol)V homologue RulAB in stationary-phase mutagenesis in P. putida. Our results demonstrated that the GO repair enzymes MutY, MutM, and MutT are involved in the prevention of base substitution mutations in carbonstarved P. putida. Interestingly, the antimutator effect of MutT was dependent on the growth phase of bacteria. Although the lack of MutT caused a strong mutator phenotype under carbon starvation conditions for bacteria, only a twofold increased effect on the frequency of mutations was observed for growing bacteria. This indicates that MutT has a backup system which efficiently complements the absence of this enzyme in actively growing cells. The knockout of MutM affected only the spectrum of mutations but did not change mutation frequency. Dps is known to protect DNA from oxidative damage. We found that dps-defective P. putida cells were more sensitive to sudden exposure to hydrogen peroxide than wild-type cells. At the same time, the absence of Dps did not affect the accumulation of mutations in populations of starved bacteria. Thus, it is possible that the protective role of Dps becomes essential for genome integrity only when bacteria are exposed to exogenous agents that lead to oxidative DNA damage but not under physiological conditions. Introduction of the Y family DNA polymerase PolV homologue rulAB into P. putida increased the proportion of A-to-C and A-to-G base substitutions among mutations, which occurred under starvation conditions. Since PolV is known to perform translesion synthesis past damaged bases in DNA (e.g., some oxidized forms of adenine), our results may imply that adenine oxidation products are also an important source of mutation in starving bacteria.Bacteria spend most of the time under starvation conditions, when their growth is inhibited due to a shortage of energy. Still, populations of stationary-phase bacteria undergo rapid evolution because of mutations. Mutagenesis occurring in resting cells is called adaptive mutation or stationary-phase mutation (22,23). Bacteria have several stress responses (including induction of the error-prone DNA polymerases carrying out translesion synthesis) that provide ways in which mutation rates can be increased in stationary-phase cells (for reviews, see references 24, 47, and 64). Oxidative DNA damage is also an important source of mutagenesis. It is known that the formation of 7,8-dihydro-8-oxo-2Ј-deoxyguanine (8-oxoG or GO) can give rise to stationary-phase mutations in Escherichia coli (13,14). Also, products...

RpoS is a bacterial sigma factor of RNA polymerase which is involved in the expression of a large number of genes to facilitate survival under starvation conditions and other stresses. The results of our study demonstrate that the frequency of emergence of base substitution mutants is significantly increased in long-term-starved populations of rpoS-deficient Pseudomonas putida cells. The increasing effect of the lack of RpoS on the mutation frequency became apparent in both a plasmid-based test system measuring Phe ؉ reversion and a chromosomal rpoB system detecting rifampin-resistant mutants. The elevated mutation frequency coincided with the death of about 95% of the cells in a population of rpoS-deficient P. putida. Artificial overexpression of superoxide dismutase or catalase in the rpoS-deficient strain restored the survival of cells and resulted in a decline in the mutation frequency. This indicated that, compared to wild-type bacteria, rpoS-deficient cells are less protected against damage caused by reactive oxygen species. 7,8-Dihydro-8-oxoguanine (GO) is known to be one of the most stable and frequent base modifications caused by oxygen radical attack on DNA. However, the spectrum of base substitution mutations characterized in rpoS-deficient P. putida was different from that in bacteria lacking the GO repair system: it was broader and more similar to that identified in the wild-type strain. Interestingly, the formation of large deletions was also accompanied by a lack of RpoS. Thus, the accumulation of DNA damage other than GO elevates the frequency of mutation in these bacteria. It is known that oxidative damage of proteins and membrane components, but not that of DNA, is a major reason for the death of cells. Since the increased mutation frequency was associated with a decline in the viability of bacteria, we suppose that the elevation of the mutation frequency in the surviving population of carbon-starved rpoS-deficient P. putida may be caused both by oxidative damage of DNA and enzymes involved in DNA replication and repair fidelity.Accumulation of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, and hydroxyl radicals leads to nucleic acid, protein, and cell membrane damage. ROS have been implicated in cancer, aging, and various diseases in humans but also in the death of microorganisms (53). Many microorganisms are continuously faced with ROS derived from different sources. For example, during infection, pathogenic bacteria are exposed to the exogenous oxidative stress that phagocytes use as a host defense mechanism (25, 47). Additionally, ROS are constantly generated as by-products of aerobic metabolism. To counteract oxidative stress, both prokaryotic and eukaryotic cells maintain inducible defense systems to detoxify oxidants and repair damage (14, 32). Gram-negative bacteria commonly synthesize both cytoplasmic and periplasmic isozymes of superoxide dismutases (SOD) to eliminate superoxide anions (40). Hydrogen peroxide is scavenged in most organisms by peroxidases an...

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