Synthesis and degradation of FtsZ quantitatively predict the first cell division in starved bacteria.

In natural environments, microbes are typically non-dividing and gauge when nutrients permit division. Current models are phenomenological and specific to nutrient-rich, exponentially growing cells, thus cannot predict the first division under limiting nutrient availability. To assess this regime, we supplied starving Escherichia coli with glucose pulses at increasing frequencies. Real-time metabolomics and microfluidic single-cell microscopy revealed unexpected, rapid protein, and nucleic acid synthesis already from minuscule glucose pulses in non-dividing cells. Additionally, the lag time to first division shortened as pulsing frequency increased. We pinpointed division timing and dependence on nutrient frequency to the changing abundance of the division protein FtsZ. A dynamic, mechanistic model quantitatively relates lag time to FtsZ synthesis from nutrient pulses and FtsZ protease-dependent degradation. Lag time changed in model-congruent manners, when we experimentally modulated the synthesis or degradation of FtsZ. Thus, limiting abundance of FtsZ can quantitatively predict timing of the first cell division.

Capacity for instantaneous catabolism of preferred and non-preferred carbon sources in Escherichia coli and Bacillus subtilis.

Making the right choice for nutrient consumption in an ever-changing environment is a key factor for evolutionary success of bacteria. Here we investigate the regulatory mechanisms that enable dynamic adaptation between non-preferred and preferred carbon sources for the model Gram-negative and -positive species Escherichia coli and Bacillus subtilis, respectively. We focus on the ability for instantaneous catabolism of a gluconeogenic carbon source upon growth on a glycolytic carbon source and vice versa. By following isotopic tracer dynamics on a 1-2 minute scale, we show that flux reversal from the preferred glucose to non-preferred pyruvate as the sole carbon source is primarily transcriptionally regulated. In the opposite direction, however, E. coli can reverse its flux instantaneously by means of allosteric regulation, whereas in B. subtilis this flux reversal is transcriptionally regulated. Upon removal of transcriptional regulation, B. subtilis assumes the ability of instantaneous glucose catabolism. Using an approach that combines quantitative metabolomics and kinetic modelling, we then identify the additionally necessary key metabolite-enzyme interactions that implement the instantaneous flux reversal in the transcriptionally deregulated B. subtilis, and validate the most relevant allosteric interactions.

Bistability in a metabolic network underpins the de novo evolution of colony switching in Pseudomonas fluorescens.

Phenotype switching is commonly observed in nature. This prevalence has allowed the elucidation of a number of underlying molecular mechanisms. However, little is known about how phenotypic switches arise and function in their early evolutionary stages. The first opportunity to provide empirical insight was delivered by an experiment in which populations of the bacterium Pseudomonas fluorescens SBW25 evolved, de novo, the ability to switch between two colony phenotypes. Here we unravel the molecular mechanism behind colony switching, revealing how a single nucleotide change in a gene enmeshed in central metabolism (carB) generates such a striking phenotype. We show that colony switching is underpinned by ON/OFF expression of capsules consisting of a colanic acid-like polymer. We use molecular genetics, biochemical analyses, and experimental evolution to establish that capsule switching results from perturbation of the pyrimidine biosynthetic pathway. Of central importance is a bifurcation point at which uracil triphosphate is partitioned towards either nucleotide metabolism or polymer production. This bifurcation marks a cell-fate decision point whereby cells with relatively high pyrimidine levels favour nucleotide metabolism (capsule OFF), while cells with lower pyrimidine levels divert resources towards polymer biosynthesis (capsule ON). This decision point is present and functional in the wild-type strain. Finally, we present a simple mathematical model demonstrating that the molecular components of the decision point are capable of producing switching. Despite its simple mutational cause, the connection between genotype and phenotype is complex and multidimensional, offering a rare glimpse of how noise in regulatory networks can provide opportunity for evolution.

Performance of Chlorella sorokiniana under simulated extreme winter conditions.

High annual microalgae productivities can only be achieved if solar light is efficiently used through the different seasons. During winter the productivity is low because of the light and temperature conditions. The productivity and photosynthetic efficiency of Chlorella sorokiniana were assessed under the worst-case scenario found during winter time in Huelva, south of Spain. The maximum light intensity (800 µmol photons m(-2) s(-1)) and temperature (20°C) during winter were simulated in a lab-scale photobioreactor with a short light-path of 14 mm. Chemostat conditions were applied and the results were compared with a temperature-controlled situation at 38°C (optimal growth temperature for C. sorokiniana). When temperature was optimal the highest productivity was found at a dilution rate of 0.18 h(-1) (P(v) = 0.28 g Kg(-1) h(-1)), and the biomass yield on light energy was high (Y(x,E) = 1.2 g mol(-1) photons supplied). However, at suboptimal temperature, the specific growth rate of C. sorokiniana was surprisingly low, not being able to support continuous operation at a dilution rate higher than 0.02 h(-1). The slow metabolism under suboptimal temperature resulted in a decline of the light energy requirements of the cells. Consequently, the maximum winter irradiance was experienced as excessive, leading to a low photosynthetic efficiency and productivity (Y(x,E) = 0.5 g mol(-1) photons supplied, P(v) = 0.1 g Kg(-1) h(-1)). At suboptimal temperature a higher carotenoid-to-chlorophyll ratio was observed indicating the activation of light-dissipating processes. We conclude that temperature control and/or light dilution during winter time will enhance the productivity.

High stability and fast recovery of expression of the TOL plasmid-carried toluene catabolism genes of Pseudomonas putida mt-2 under conditions of oxygen limitation and oscillation.

Pseudomonas putida mt-2 harbors the TOL plasmid (pWWO), which contains the genes encoding the enzymes necessary to degrade toluene aerobically. The xyl genes are clustered in the upper operon and encode the enzymes of the upper pathway that degrade toluene to benzoate, while the genes encoding the enzymes of the lower pathway (meta-cleavage pathway) that are necessary for the conversion of benzoate to tricarboxylic acid cycle intermediates, are encoded in a separate operon. In this study, the effects of oxygen availability and oscillation on the expression of catabolic genes for enzymes involved in toluene degradation were studied by using P. putida mt-2 as model bacterium. Quantitative reverse transcription-PCR was used to detect and quantify the expression of the catabolic genes xylM (a key gene of the upper pathway) and xylE (a key gene of the lower pathway) in cultures of P. putida mt-2 that were grown with toluene as a carbon source. Toluene degradation was shown to have a direct dependency on oxygen concentration, where gene expression of xylM and xylE decreased due to oxygen depletion during degradation. Under oscillating oxygen concentrations, P. putida mt-2 induced or downregulated xylM and xylE genes according to the O2 availability in the media. During anoxic periods, P. putida mt-2 decreased the expression of xylM and xylE genes, while the expression of both xylM and xylE genes was immediately increased after oxygen became available again in the medium. These results suggest that oxygen is not only necessary as a cosubstrate for enzyme activity during the degradation of toluene but also that oxygen modulates the expression of the catabolic genes encoded by the TOL plasmid.