The stringent response of marine bacteria - assessment of (p)ppGpp accumulation upon stress conditions.

Microorganisms are particularly adapted to alterations in their environment. One of the global regulatory mechanisms involved in these adaptations is the stringent response. The unusual nucleotides, guanosine penta and tetraphosphates, (p)ppGpp act as alarmones of this response, heralding nutrient limitation and stressors. Marine bacteria encounter numerous stresses of sparse nutrient supplies and changes in physicochemical conditions. The aim of this work was to assess whether the stress conditions common in marine environment can induce the stringent response and what is a kinetic of this process. The representative bacterial species, Shewanella baltica, Acinetobacter johnsonii, Vibrio harveyi, and Escherichia coli were subjected to a variety of stressors. We analyzed the kinetics of (p)ppGpp synthesis by labeling in vivo nucleotides and analysis by thin layer chromatography. The (p)ppGpp accumulation followed the elevated temperature and amino acid starvation for all bacteria tested. The carbon and nitrogen limitation resulted in the response limited to V. harveyi and S. baltica. The DNA damaging agents induced the (p)ppGpp production in all strains, while osmotic stress did not result in significant alarmone synthesis. The representative marine bacteria species were shown to induce with varying extent the stringent response upon the onset of stress and limitation conditions. Importantly, the in vivo labeling and subsequent separation of the nucleotides by thin layer chromatography serves as a valid method for the analysis of the stringent response and (p)ppGpp accumulation in environmental bacteria.

The dual role of DksA protein in the regulation of Escherichia coli pArgX promoter.

Gene expression regulation by the stringent response effector, ppGpp, is facilitated by DksA protein; however DksA and ppGpp can play independent roles in transcription. In Escherichia coli, the pArgX promoter which initiates the transcription of four tRNA genes was shown to be inhibited by ppGpp. Our studies on the role of DksA in pArgX regulation revealed that it can stimulate transcription by increasing the binding of RNA polymerase to the promoter and the productive transcription complex formation. However, when DksA is present together with ppGpp a severe down-regulation of promoter activity is observed. Our results indicate that DksA facilitates the effects of ppGpp to drive formation of inactive dead-end complexes formed by RNA polymerase at the ArgX promoter. In vivo, ppGpp-mediated regulation of pArgX transcription is dependent on DksA activity. The potential mechanisms of opposing pArgX regulation by ppGpp and DksA are discussed. pArgX is the first reported example of the promoter stimulated by DksA and inhibited by ppGpp in vitro when an overall inhibition occurs in the presence of both regulators. A dual role is thus proposed for DksA in the regulation of the pArgX promoter activity.

Transformation of Shewanella baltica with ColE1-like and P1 plasmids and their maintenance during bacterial growth in cultures.

The presence of natural plasmids has been reported for many Shewanella isolates. However, knowledge about plasmid replication origin and segregation mechanisms is not extensive for this genus. Shewanella baltica is an important species in the marine environment due to its denitrification ability in oxygen-deficient zones and the potential role in bioremediation processes. However, no information about possible use of plasmid vectors in this species has been reported to date. Here we report that plasmids with ColE1-type and plasmid P1 origin can transform S. baltica and replicate in this bacterium. Without the antibiotic selection pressure plasmid maintenance is less efficient than in Escherichia coli. Nevertheless, cultivation of S. baltica in the presence of appropriate antibiotics caused relatively stable maintenance of ColE1-like and P1-derived plasmids. This indicates that plasmid-based genetic manipulations and gene transfer in S. baltica are possible.