FnrL and Three Dnr Regulators Are Used for the Metabolic Adaptation to Low Oxygen Tension in Dinoroseobacter shibae.

The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaerobic denitrification supplemented with aerobic anoxygenic photosynthesis for energy generation. The aerobic to anaerobic transition is controlled by four Fnr/Crp family regulators in a unique cascade-type regulatory network. FnrL is utilizing an oxygen-sensitive Fe-S cluster for oxygen sensing. Active FnrL is inducing most operons encoding the denitrification machinery and the corresponding heme biosynthesis. Activation of gene expression of the high oxygen affinity cbb3-type and repression of the low affinity aa3-type cytochrome c oxidase is mediated by FnrL. Five regulator genes including dnrE and dnrF are directly controlled by FnrL. Multiple genes of the universal stress protein (USP) and cold shock response are further FnrL targets. DnrD, most likely sensing NO via a heme cofactor, co-induces genes of denitrification, heme biosynthesis, and the regulator genes dnrE and dnrF. DnrE is controlling genes for a putative Na+/H+ antiporter, indicating a potential role of a Na+ gradient under anaerobic conditions. The formation of the electron donating primary dehydrogenases is coordinated by FnrL and DnrE. Many plasmid encoded genes were DnrE regulated. DnrF is controlling directly two regulator genes including the Fe-S cluster biosynthesis regulator iscR, genes of the electron transport chain and the glutathione metabolism. The genes for nitrate reductase and CO dehydrogenase are repressed by DnrD and DnrF. Both regulators in concert with FnrL are inducing the photosynthesis genes. One of the major denitrification operon control regions, the intergenic region between nirS and nosR2, contains one Fnr/Dnr binding site. Using regulator gene mutant strains, lacZ-reporter gene fusions in combination with promoter mutagenesis, the function of the single Fnr/Dnr binding site for FnrL-, DnrD-, and partly DnrF-dependent nirS and nosR2 transcriptional activation was shown. Overall, the unique regulatory network of the marine bacterium D. shibae for the transition from aerobic to anaerobic growth composed of four Crp/Fnr family regulators was elucidated.

A Precise Temperature-Responsive Bistable Switch Controlling Yersinia Virulence.

Different biomolecules have been identified in bacterial pathogens that sense changes in temperature and trigger expression of virulence programs upon host entry. However, the dynamics and quantitative outcome of this response in individual cells of a population, and how this influences pathogenicity are unknown. Here, we address these questions using a thermosensing virulence regulator of an intestinal pathogen (RovA of Yersinia pseudotuberculosis) as a model. We reveal that this regulator is part of a novel thermoresponsive bistable switch, which leads to high- and low-invasive subpopulations within a narrow temperature range. The temperature range in which bistability is observed is defined by the degradation and synthesis rate of the regulator, and is further adjustable via a nutrient-responsive regulator. The thermoresponsive switch is also characterized by a hysteretic behavior in which activation and deactivation occurred on vastly different time scales. Mathematical modeling accurately mirrored the experimental behavior and predicted that the thermoresponsiveness of this sophisticated bistable switch is mainly determined by the thermo-triggered increase of RovA proteolysis. We further observed RovA ON and OFF subpopulations of Y. pseudotuberculosis in the Peyer's patches and caecum of infected mice, and that changes in the RovA ON/OFF cell ratio reduce tissue colonization and overall virulence. This points to a bet-hedging strategy in which the thermoresponsive bistable switch plays a key role in adapting the bacteria to the fluctuating conditions encountered as they pass through the host's intestinal epithelium and suggests novel strategies for the development of antimicrobial therapies.

Modelling and analysis of a gene-regulatory feed-forward loop with basal expression of the second regulator.

Efficient adaptation strategies to changing environmental conditions are essential for bacteria to survive and grow. Fundamental restructuring of their metabolism is usually mediated by corresponding gene regulation. Here, often several different environmental stimuli have to be integrated into a reasonable, energy-efficient response. Fast fluctuations and overshooting have to be filtered out. The gene regulatory network for the anaerobic adaptation of the pathogenic bacterium Pseudomonas aeruginosa is organized as a feed-forward loop (FFL), which is a three-gene network motif composed of two transcription factors (Anr for oxygen, NarxL for nitrate) and one target (Nar for nitrate reductase). The upstream transcription factor (Anr) induces the downstream transcription factor (NarXL). Both regulators act together positively by inducing the target (Nar) via a direct and indirect regulation path (coherent type-1 FFL). Since full promoter activity is only achieved when both transcription factors are present the target operon is expressed with a delay. Thus, in response to environmental stimuli (oxygen, nitrate), signals are mediated and processed in a way that short pulses are filtered out. In this study we analyze a special kind of FFL called FFLk by means of a family of ordinary differential equation models. The secondary FFL regulator (NarXL) is expressed constitutively but further induced in the presence of the upstream stimuli. This FFL modification has substantial influence on the response time and cost-benefit ratio mediated by environmental fluctuations. In order to find conditions where this regulatory network motif might be beneficial, we analyzed various models and environments. We describe the observed evolutional advantage of FFLk and its role in environmental adaptation and pathogenicity.

Microbial communities related to volatile organic compound emission in automobile air conditioning units.

During operation of mobile air conditioning (MAC) systems in automobiles, malodours can occur. We studied the microbial communities found on contaminated heat exchanger fins of 45 evaporators from car MAC systems which were operated in seven different regions of the world and identified corresponding volatile organic compounds. Collected biofilms were examined by scanning electron microscopy and fluorescent in situ hybridization. The detected bacteria were loosely attached to the metal surface. Further analyses of the bacteria using PCR-based single-strand conformation polymorphism and sequencing of isolated 16S rRNA gene fragments identified highly divergent microbial communities with multiple members of the Alphaproteobacteriales, Methylobacteria were the prevalent bacteria. In addition, Sphingomonadales, Burkholderiales, Bacillales, Alcanivorax spp. and Stenotrophomonas spp. were found among many others depending on the location the evaporators were operated. Interestingly, typical pathogenic bacteria related to air conditioning systems including Legionella spp. were not found. In order to determine the nature of the chemical compounds produced by the bacteria, the volatile organic compounds were examined by closed loop stripping analysis and identified by combined gas chromatography/mass spectrometry. Sulphur compounds, i.e. di-, tri- and multiple sulphides, acetylthiazole, aromatic compounds and diverse substituted pyrazines were detected. Mathematical clustering of the determined microbial community structures against their origin identified a European/American/Arabic cluster versus two mainly tropical Asian clusters. Interestingly, clustering of the determined volatiles against the origin of the corresponding MAC revealed a highly similar pattern. A close relationship of microbial community structure and resulting malodours to the climate and air quality at the location of MAC operation was concluded.

Construction and characterization of nitrate and nitrite respiring Pseudomonas putida KT2440 strains for anoxic biotechnical applications.

Pseudomonas putida KT2440 is frequently used in biotechnical research and applications due to its metabolic versatility and organic solvent resistance. A major drawback for a broad application is the inability of the bacterium to survive and grow under anoxic conditions, which prohibits the production of oxygen-sensitive proteins and metabolites. To develop a P. putida strain, which is able to survive under anoxic conditions, the enzymatic systems of anaerobic nitrate and nitrite respiration were introduced into KT2440. For this purpose, two cosmids encoding all structural, maturation and regulatory genes for P. aeruginosa nitrate reductase (pNAR) and nitrite- and nitric oxide reductase (pNIR-NOR) were stably maintained in P. putida KT2440. Transcriptome analyses revealed expression of the encoded nar, nir and nor operons and accessory genes under anoxic conditions. The produced enzyme systems efficiently reduced nitrate or nitrite, respectively, sustaining anaerobic life of recombinant KT2440. Interestingly, anaerobic life of P. putida induced genes involved in arginine-fermentation and genes encoding a putative copper stress resistance operon.

InFiRe -- a novel computational method for the identification of insertion sites in transposon mutagenized bacterial genomes.

MOTIVATION: InFiRe, Insertion Finder via Restriction digest, is a novel software tool that allows for the computational identification of transposon insertion sites in known bacterial genome sequences after transposon mutagenesis experiments. The approach is based on the fact that restriction endonuclease digestions of bacterial DNA yield a unique pattern of DNA fragments with defined sizes. Transposon insertion changes the size of the hosting DNA fragment by a known number of base pairs. The exact size of this fragment can be determined by Southern blot hybridization. Subsequently, the position of insertion can be identified with computational analysis. The outlined method provides a solid basis for the establishment of a new high-throughput technology. AVAILABILITY AND IMPLEMENTATION: The software is freely available on our web server at www.infire.tu-bs.de. The algorithm was implemented in the statistical programming language R. For the most flexible use, InFiRe is provided in two different versions. A web interface offers the convenient use in a web browser. In addition, the software and source code is freely available for download as R-packages on our website. CONTACT: m.steinert@tu-bs.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.