Pseudomonas putida as saviour for troubled Synechococcus elongatus in a synthetic co-culture - interaction studies based on a multi-OMICs approach.

In their natural habitats, microbes rarely exist in isolation; instead, they thrive in consortia, where various interactions occur. In this study, a defined synthetic co-culture of the cyanobacterium S. elongatus cscB, which supplies sucrose to the heterotrophic P. putida cscRABY, is investigated to identify potential interactions. Initial experiments reveal a remarkable growth-promoting effect of the heterotrophic partner on the cyanobacterium, resulting in an up to 80% increase in the growth rate and enhanced photosynthetic capacity. Vice versa, the presence of the cyanobacterium has a neutral effect on P. putida cscRABY, highlighting the resilience of pseudomonads against stress and their potential as co-culture partners. Next, a suitable reference process reinforcing the growth-promoting effect is established in a parallel photobioreactor system, which sets the basis for the analysis of the co-culture at the transcriptome, proteome, and metabolome levels. In addition to several moderate changes, including alterations in the metabolism and stress response in both microbes, this comprehensive multi-OMICs approach strongly hints towards the exchange of further molecules beyond the unidirectional feeding with sucrose. Taken together, these findings provide valuable insights into the complex dynamics between both co-culture partners, indicating multi-level interactions, which can be employed for further streamlining of the co-cultivation system.

Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control.

For engineered microorganisms, the production of heterologous proteins that are often useless to host cells represents a burden on resources, which have to be shared with normal cellular processes. Within a certain metabolic leeway, this competitive process has no impact on growth. However, once this leeway, or free capacity, is fully utilized, the extra load becomes a metabolic burden that inhibits cellular processes and triggers a broad cellular response, reducing cell growth and often hindering the production of heterologous proteins. In this study, we sought to characterize the metabolic rearrangements occurring in the central metabolism of Pseudomonas putida at different levels of metabolic load. To this end, we constructed a P. putida KT2440 strain that expressed two genes encoding fluorescent proteins, one in the genome under constitutive expression to monitor the free capacity, and the other on an inducible plasmid to probe heterologous protein production. We found that metabolic fluxes are considerably reshuffled, especially at the level of periplasmic pathways, as soon as the metabolic load exceeds the free capacity. Heterologous protein production leads to the decoupling of anabolism and catabolism, resulting in large excess energy production relative to the requirements of protein biosynthesis. Finally, heterologous protein production was found to exert a stronger control on carbon fluxes than on energy fluxes, indicating that the flexible nature of P. putida's central metabolic network is solicited to sustain energy production.

Streamlining of a synthetic co-culture towards an individually controllable one-pot process for polyhydroxyalkanoate production from light and CO2.

Rationally designed synthetic microbial consortia carry a vast potential for biotechnological applications. The application of such a consortium in a bioprocess, however, requires tight and individual controllability of the involved microbes. Here, we present the streamlining of a co-cultivation process consisting of Synechococcus elongatus cscB and Pseudomonas putida for the production of polyhydroxyalkanoates (PHA) from light and CO2. First, the process was improved by employing P. putida cscRABY, a strain with a higher metabolic activity towards sucrose. Next, the individual controllability of the co-culture partners was addressed by providing different nitrogen sources, each exclusively available for one strain. By this, the growth rate of the co-culture partners could be regulated individually, and defined conditions could be set. The molC/molN ratio, a key value for PHA accumulation, was estimated from the experimental data, and the necessary feeding rates to obtain a specific ratio could be predicted. This information was then implemented in the co-cultivation process, following the concept of a DBTL-cycle. In total, the streamlining of the process resulted in an increased maximal PHA titer of 393 mg/L and a PHA production rate of 42.1 mg/(L•day).

Water potential governs the effector specificity of the transcriptional regulator XylR of Pseudomonas putida.

The biodegradative capacity of bacteria in their natural habitats is affected by water availability. In this work, we have examined the activity and effector specificity of the transcriptional regulator XylR of the TOL plasmid pWW0 of Pseudomonas putida mt-2 for biodegradation of m-xylene when external water potential was manipulated with polyethylene glycol PEG8000. By using non-disruptive luxCDEAB reporter technology, we noticed that the promoter activated by XylR (Pu) restricted its activity and the regulator became more effector-specific towards head TOL substrates when cells were grown under water subsaturation. Such a tight specificity brought about by water limitation was relaxed when intracellular osmotic stress was counteracted by the external addition of the compatible solute glycine betaine. With these facts in hand, XylR variants isolated earlier as effector-specificity responders to the non-substrate 1,2,4-trichlorobenzene under high matric stress were re-examined and found to be unaffected by water potential in vivo. All these phenomena could be ultimately explained as the result of water potential-dependent conformational changes in the A domain of XylR and its effector-binding pocket, as suggested by AlphaFold prediction of protein structures. The consequences of this scenario for the evolution of specificities in regulators and the emergence of catabolic pathways are discussed.

Targeting Transcriptional and Translational Hindrances in a Modular T7RNAP Expression System in Engineered Pseudomonas putida.

The T7 RNA polymerase is considered one of the most popular tools for heterologous gene expression in the gold standard biotechnological host Escherichia coli. However, the exploitation of this tool in other prospective hosts, such as the biotechnologically relevant bacterium Pseudomonas putida, is still very scarce. The majority of the existing T7-based systems in P. putida show low expression strengths and possess only weak controllability. A fundamental understanding of these systems is necessary in order to design robust and predictable biotechnological processes. To fill this gap, we established and characterized a modular T7 RNA polymerase-based system for heterologous protein production in P. putida, using the enhanced Green Fluorescent Protein (eGFP) as an easy-to-quantify reporter protein. We have effectively targeted the limitations associated with the initial genetic setup of the system, such as slow growth and low protein production rates. By replacing the T7 phage-inherent TΦ terminator downstream of the heterologous gene with the synthetic tZ terminator, growth and protein production rates improved drastically, and the T7 RNA polymerase system reached a productivity level comparable to that of an intrinsic RNA polymerase-based system. Furthermore, we were able to show that the system was saturated with T7 RNA polymerase by applying a T7 RNA polymerase ribosome binding site library to tune heterologous protein production. This saturation indicates an essential role for the ribosome binding sites of the T7 RNA polymerase since, in an oversaturated system, cellular resources are lost to the synthesis of unnecessary T7 RNA polymerase. Eventually, we combined the experimental data into a model that can predict the eGFP production rate with respect to the relative strength of the ribosome binding sites upstream of the T7 gene.

Metabolic engineering of Halomonas elongata: Ectoine secretion is increased by demand and supply driven approaches.

The application of naturally-derived biomolecules in everyday products, replacing conventional synthetic manufacturing, is an ever-increasing market. An example of this is the compatible solute ectoine, which is contained in a plethora of treatment formulations for medicinal products and cosmetics. As of today, ectoine is produced in a scale of tons each year by the natural producer Halomonas elongata. In this work, we explore two complementary approaches to obtain genetically improved producer strains for ectoine production. We explore the effect of increased precursor supply (oxaloacetate) on ectoine production, as well as an implementation of increased ectoine demand through the overexpression of a transporter. Both approaches were implemented on an already genetically modified ectoine-excreting strain H. elongata KB2.13 (ΔteaABC ΔdoeA) and both led to new strains with higher ectoine excretion. The supply driven approach led to a 45% increase in ectoine titers in two different strains. This increase was attributed to the removal of phosphoenolpyruvate carboxykinase (PEPCK), which allowed the conversion of 17.9% of the glucose substrate to ectoine. For the demand driven approach, we investigated the potential of the TeaBC transmembrane proteins from the ectoine-specific Tripartite ATP-Independent Periplasmic (TRAP) transporter as export channels to improve ectoine excretion. In the absence of the substrate-binding protein TeaA, an overexpression of both subunits TeaBC facilitated a three-fold increased excretion rate of ectoine. Individually, the large subunit TeaC showed an approximately five times higher extracellular ectoine concentration per dry weight compared to TeaBC shortly after its expression was induced. However, the detrimental effect on growth and ectoine titer at the end of the process hints toward a negative impact of TeaC overexpression on membrane integrity and possibly leads to cell lysis. By using either strategy, the ectoine synthesis and excretion in H. elongata could be boosted drastically. The inherent complementary nature of these approaches point at a coordinated implementation of both as a promising strategy for future projects in Metabolic Engineering. Moreover, a wide variation of intracelllular ectoine levels was observed between the strains, which points at a major disruption of mechanisms responsible for ectoine regulation in strain KB2.13.

Adaptation to Varying Salinity in Halomonas elongata: Much More Than Ectoine Accumulation.

The halophilic γ-proteobacterium Halomonas elongata DSM 2581 T thrives at salt concentrations well above 10 % NaCl (1.7 M NaCl). A well-known osmoregulatory mechanism is the accumulation of the compatible solute ectoine within the cell in response to osmotic stress. While ectoine accumulation is central to osmoregulation and promotes resistance to high salinity in halophilic bacteria, ectoine has this effect only to a much lesser extent in non-halophiles. We carried out transcriptome analysis of H. elongata grown on two different carbon sources (acetate or glucose), and low (0.17 M NaCl), medium (1 M), and high salinity (2 M) to identify additional mechanisms for adaptation to high saline environments. To avoid a methodological bias, the transcripts were evaluated by applying two methods, DESeq2 and Transcripts Per Million (TPM). The differentially transcribed genes in response to the available carbon sources and salt stress were then compared to the transcriptome profile of Chromohalobacter salexigens, a closely related moderate halophilic bacterium. Transcriptome profiling supports the notion that glucose is degraded via the cytoplasmic Entner-Doudoroff pathway, whereas the Embden-Meyerhoff-Parnas pathway is employed for gluconeogenesis. The machinery of oxidative phosphorylation in H. elongata and C. salexigens differs greatly from that of non-halophilic organisms, and electron flow can occur from quinone to oxygen along four alternative routes. Two of these pathways via cytochrome bo' and cytochrome bd quinol oxidases seem to be upregulated in salt stressed cells. Among the most highly regulated genes in H. elongata and C. salexigens are those encoding chemotaxis and motility proteins, with genes for chemotaxis and flagellar assembly severely downregulated at low salt concentrations. We also compared transcripts at low and high-salt stress (low growth rate) with transcripts at optimal salt concentration and found that the majority of regulated genes were down-regulated in stressed cells, including many genes involved in carbohydrate metabolism, while ribosome synthesis was up-regulated, which is in contrast to what is known from non-halophiles at slow growth. Finally, comparing the acidity of the cytoplasmic proteomes of non-halophiles, extreme halophiles and moderate halophiles suggests adaptation to an increased cytoplasmic ion concentration of H. elongata. Taken together, these results lead us to propose a model for salt tolerance in H. elongata where ion accumulation plays a greater role in salt tolerance than previously assumed.

Immobilization of PETase enzymes on magnetic iron oxide nanoparticles for the decomposition of microplastic PET.

Polyethylene terephthalate (PET) is responsible for a large amount of environmental contamination with microplastics. Based on its high affinity, the PET degrading enzyme PETase can be immobilized on superparamagnetic iron oxide nanoparticles through a His-tag. The His-tag increases enzyme stability, and allows magnetic separation for recovery. Multiple recycling steps are possible and microplastic particles can be decomposed depending on the PET's crystallinity. The separation or decomposition of PET allows for a sustainable way to remove microplastic from water.

Trehalose production by Cupriavidus necator from CO2 and hydrogen gas.

In this work, the hydrogen-oxidizing bacterium Cupriavidus necator H16 was engineered for trehalose production from gaseous substrates. First, it could be shown that C. necator is a natural producer of trehalose when stressed with sodium chloride. Bioinformatic investigations revealed a so far unknown mode of trehalose and glycogen metabolism in this organism. Next, it was found that expression of the sugar efflux transporter A (setA) from Escherichia coli lead to a trehalose leaky phenotype of C. necator. Finally, the strain was characterized under autotrophic conditions using a H2/CO2/O2-mixture and other substrates reaching titers of up to 0.47 g L-1 and yields of around 0.1 g g-1. Taken together, this process represents a new way to produce sugars with high areal efficiency. With further metabolic engineering, an application of this technology for the renewable production of trehalose and other sugars, as well as for the synthesis of 13C-labeled sugars seems promising.

Anaplerotic Pathways in Halomonas elongata: The Role of the Sodium Gradient.

Salt tolerance in the γ-proteobacterium Halomonas elongata is linked to its ability to produce the compatible solute ectoine. The metabolism of ectoine production is of great interest since it can shed light on the biochemical basis of halotolerance as well as pave the way for the improvement of the biotechnological production of such compatible solute. Ectoine belongs to the biosynthetic family of aspartate-derived amino-acids. Aspartate is formed from oxaloacetate, thereby connecting ectoine production to the anaplerotic reactions that refill carbon into the tricarboxylic acid cycle (TCA cycle). This places a high demand on these reactions and creates the need to regulate them not only in response to growth but also in response to extracellular salt concentration. In this work, we combine modeling and experiments to analyze how these different needs shape the anaplerotic reactions in H. elongata. First, the stoichiometric and thermodynamic factors that condition the flux distributions are analyzed, then the optimal patterns of operation for oxaloacetate production are calculated. Finally, the phenotype of two deletion mutants lacking potentially relevant anaplerotic enzymes: phosphoenolpyruvate carboxylase (Ppc) and oxaloacetate decarboxylase (Oad) are experimentally characterized. The results show that the anaplerotic reactions in H. elongata are indeed subject to evolutionary pressures that differ from those faced by other gram-negative bacteria. Ectoine producing halophiles must meet a higher metabolic demand for oxaloacetate and the reliance of many marine bacteria on the Entner-Doudoroff pathway compromises the anaplerotic efficiency of Ppc, which is usually one of the main enzymes fulfilling this role. The anaplerotic flux in H. elongata is contributed not only by Ppc but also by Oad, an enzyme that has not yet been shown to play this role in vivo. Ppc is necessary for H. elongata to grow normally at low salt concentrations but it is not required to achieve near maximal growth rates as long as there is a steep sodium gradient. On the other hand, the lack of Oad presents serious difficulties to grow at high salt concentrations. This points to a shared role of these two enzymes in guaranteeing the supply of oxaloacetate for biosynthetic reactions.

A Nitrate-Blind P. putida Strain Boosts PHA Production in a Synthetic Mixed Culture.

One of the major challenges for the present and future generations is to find suitable substitutes for the fossil resources we rely on today. In this context, cyanobacterial carbohydrates have been discussed as an emerging renewable feedstock in industrial biotechnology for the production of fuels and chemicals. Based on this, we recently presented a synthetic bacterial co-culture for the production of medium-chain-length polyhydroxyalkanoates (PHAs) from CO2. This co-cultivation system is composed of two partner strains: Synechococcus elongatus cscB which fixes CO2, converts it to sucrose and exports it into the culture supernatant, and a Pseudomonas putida strain that metabolizes this sugar and accumulates PHAs in the cytoplasm. However, these biopolymers are preferably accumulated under conditions of nitrogen limitation, a situation difficult to achieve in a co-culture as the other partner, at best, should not perceive any limitation. In this article, we will present an approach to overcome this dilemma by uncoupling the PHA production from the presence of nitrate in the medium. This is achieved by the construction of a P. putida strain that is no longer able to grow with nitrate as nitrogen source -is thus nitrate blind, and able to grow with sucrose as carbon source. The deletion of the nasT gene encoding the response regulator of the NasS/NasT two-component system resulted in such a strain that has lost the ability use nitrate, but growth with ammonium was not affected. Subsequently, the nasT deletion was implemented in P. putida cscRABY, an efficient sucrose consuming strain. This genetic engineering approach introduced an artificial unilateral nitrogen limitation in the co-cultivation process, and the amount of PHA produced from light and CO2 was 8.8 fold increased to 14.8% of its CDW compared to the nitrate consuming reference strain. This nitrate blind strain, P. putidaΔnasT attTn7:cscRABY, is not only a valuable partner in the co-cultivation but additionally enables the use of other nitrate containing substrates for medium-chain-length PHA production, like for example waste-water.

Engineering sucrose metabolism in Pseudomonas putida highlights the importance of porins.

Using agricultural wastes as a substrate for biotechnological processes is of great interest in industrial biotechnology. A prerequisite for using these wastes is the ability of the industrially relevant microorganisms to metabolize the sugars present therein. Therefore, many metabolic engineering approaches are directed towards widening the substrate spectrum of the workhorses of industrial biotechnology like Escherichia coli, yeast or Pseudomonas putida. For instance, neither xylose or arabinose from cellulosic residues, nor sucrose, the main sugar in waste molasses, can be metabolized by most E. coli and P. putida wild types. We evaluated a new, so far uncharacterized gene cluster for sucrose metabolism from Pseudomonas protegens Pf-5 and showed that it enables P. putida to grow on sucrose as the sole carbon and energy source. Even when integrated into the genome of P. putida, the resulting strain grew on sucrose at rates similar to the rate of the wild type on glucose - making it the fastest growing, plasmid-free P. putida strain known so far using sucrose as substrate. Next, we elucidated the role of the porin, an orthologue of the sucrose porin ScrY, in the gene cluster and found that in P. putida, a porin is needed for sucrose transport across the outer membrane. Consequently, native porins were not sufficient to allow unlimited growth on sucrose. Therefore, we concluded that the outer membrane can be a considerable barrier for substrate transport, depending on strain, genotype and culture conditions, all of which should be taken into account in metabolic engineering approaches. We additionally showed the potential of the engineered P. putida strains by growing them on molasses with efficiencies twice as high as obtained with the wild-type P. putida. This can be seen as a further step towards the production of low-value chemicals and biofuels with P. putida from alternative and more affordable substrates in the future.

An automated and parallelised DIY-dosing unit for individual and complex feeding profiles: Construction, validation and applications.

Since biotechnological research becomes more and more important for industrial applications, there is an increasing need for scalable and controllable laboratory procedures. A widely used approach in biotechnological research to improve the performance of a process is to vary the growth rates in order to find the right balance between growth and the production. This can be achieved by the application of a suitable feeding strategy. During this initial bioprocess development, it is beneficial to have at hand cheap and easy setups that work in parallel (e.g. in shaking flasks). Unfortunately, there is a gap between these easy setups and defined and controllable processes, which are necessary for up-scaling to an industrial relevant volume. One prerequisite to test and evaluate different process strategies apart from batch-mode is the availability of pump systems that allow for defined feeding profiles in shaking flasks. To our knowledge, there is no suitable dosing device on the market which fulfils the requirements of being cheap, precise, programmable, and parallelizable. Commercially available dosing units are either already integrated in bioreactors and therefore inflexible, or not programmable, or expensive, or a combination of those. Here, we present a LEGO-MINDSTORMS-based syringe pump, which has the potential of being widely used in daily laboratory routine due to its low price, programmability, and parallelisability. The acquisition costs do not exceed 350 € for up to four dosing units, that are independently controllable with one EV3 block. The system covers flow rates ranging from 0.7 µL min-1 up to 210 mL min-1 with a reliable flux. One dosing unit can convey at maximum a volume of 20 mL (using all 4 units even up to 80 mL in total) over the whole process time. The design of the dosing unit enables the user to perform experiments with up to four different growth rates in parallel (each measured in triplicates) per EV3-block used. We estimate, that the LEGO-MINDSTORMS-based dosing unit with 12 syringes in parallel is reducing the costs up to 50-fold compared to a trivial version of a commercial pump system (~1500 €) which fits the same requirements. Using the pump, we set the growth rates of a E. coli HMS174/DE3 culture to values between 0.1 and 0.4 h-1 with a standard deviation of at best 0.35% and an average discrepancy of 13.2%. Additionally, we determined the energy demand of a culture for the maintenance of the pTRA-51hd plasmid by quantifying the changes in biomass yield with different growth rates set. Around 25% of total substrate taken up is used for plasmid maintenance. To present possible applications and show the flexibility of the system, we applied a constant feed to perform microencapsulation of Pseudomonas putida and an individual dosing profile for the purification of a his-tagged eGFP via IMAC. This smart and versatile dosing unit, which is ready-to-use without any prior knowledge in electronics and control, is affordable for everyone and due to its flexibility and broad application range a valuable addition to the laboratory routine.

pTRA - A reporter system for monitoring the intracellular dynamics of gene expression.

The presence of standardised tools and methods to measure and represent accurately biological parts and functions is a prerequisite for successful metabolic engineering and crucial to understand and predict the behaviour of synthetic genetic circuits. Many synthetic gene networks are based on transcriptional circuits, thus information on transcriptional and translational activity is important for understanding and fine-tuning the synthetic function. To this end, we have developed a toolkit to analyse systematically the transcriptional and translational activity of a specific synthetic part in vivo. It is based on the plasmid pTRA and allows the assignment of specific transcriptional and translational outputs to the gene(s) of interest (GOI) and to compare different genetic setups. By this, the optimal combination of transcriptional strength and translational activity can be identified. The design is tested in a case study using the gene encoding the fluorescent mCherry protein as GOI. We show the intracellular dynamics of mRNA and protein formation and discuss the potential and shortcomings of the pTRA plasmid.

Photoautotrophic production of polyhydroxyalkanoates in a synthetic mixed culture of Synechococcus elongatus cscB and Pseudomonas putida cscAB.

BACKGROUND: One of the major challenges for the present and future generations is to find suitable substitutes for the fossil resources we rely on today. Cyanobacterial carbohydrates have been discussed as an emerging renewable feedstock in industrial biotechnology for the production of fuels and chemicals, showing promising production rates when compared to crop-based feedstock. However, intrinsic capacities of cyanobacteria to produce biotechnological compounds are limited and yields are low. RESULTS: Here, we present an approach to circumvent these problems by employing a synthetic bacterial co-culture for the carbon-neutral production of polyhydroxyalkanoates (PHAs) from CO2. The co-culture consists of two bio-modules: Bio-module I, in which the cyanobacterial strain Synechococcus elongatus cscB fixes CO2, converts it to sucrose, and exports it into the culture supernatant; and bio-module II, where this sugar serves as C-source for Pseudomonas putida cscAB and is converted to PHAs that are accumulated in the cytoplasm. By applying a nitrogen-limited process, we achieved a maximal PHA production rate of 23.8 mg/(L day) and a maximal titer of 156 mg/L. We will discuss the present shortcomings of the process and show the potential for future improvement. CONCLUSIONS: These results demonstrate the feasibility of mixed cultures of S. elongatus cscB and P. putida cscAB for PHA production, making room for the cornucopia of possible products that are described for P. putida. The construction of more efficient sucrose-utilizing P. putida phenotypes and the optimization of process conditions will increase yields and productivities and eventually close the gap in the contemporary process. In the long term, the co-culture may serve as a platform process, in which P. putida is used as a chassis for the implementation of synthetic metabolic pathways for biotechnological production of value-added products.

Metabolic engineering to expand the substrate spectrum of Pseudomonas putida toward sucrose.

Sucrose is an important disaccharide used as a substrate in many industrial applications. It is a major component of molasses, a cheap by-product of the sugar industry. Unfortunately, not all industrially relevant organisms, among them Pseudomonas putida, are capable of metabolizing sucrose. We chose a metabolic engineering approach to circumvent this blockage and equip P. putida with the activities necessary to consume sucrose. Therefore, we constructed a pair of broad-host range mini-transposons (pSST - sucrose splitting transposon), carrying either cscA, encoding an invertase able to split sucrose into glucose and fructose, or additionally cscB, encoding a sucrose permease. Introduction of cscA was sufficient to convey sucrose consumption and the additional presence of cscB had no further effect, though the sucrose permease was built and localized to the membrane. Sucrose was split extracellularly by the activity of the invertase CscA leaking out of the cell. The transposons were also used to confer sucrose consumption to Cupriavidus necator. Interestingly, in this strain, CscB acted as a glucose transporter, such that C. necator also gained the ability to grow on glucose. Thus, the pSST transposons are functional tools to extend the substrate spectrum of Gram-negative bacterial strains toward sucrose.

Osmoregulation in the Halophilic Bacterium Halomonas elongata: A Case Study for Integrative Systems Biology.

Halophilic bacteria use a variety of osmoregulatory methods, such as the accumulation of one or more compatible solutes. The wide diversity of compounds that can act as compatible solute complicates the task of understanding the different strategies that halophilic bacteria use to cope with salt. This is specially challenging when attempting to go beyond the pathway that produces a certain compatible solute towards an understanding of how the metabolic network as a whole addresses the problem. Metabolic reconstruction based on genomic data together with Flux Balance Analysis (FBA) is a promising tool to gain insight into this problem. However, as more of these reconstructions become available, it becomes clear that processes predicted by genome annotation may not reflect the processes that are active in vivo. As a case in point, E. coli is unable to grow aerobically on citrate in spite of having all the necessary genes to do it. It has also been shown that the realization of this genetic potential into an actual capability to metabolize citrate is an extremely unlikely event under normal evolutionary conditions. Moreover, many marine bacteria seem to have the same pathways to metabolize glucose but each species uses a different one. In this work, a metabolic network inferred from genomic annotation of the halophilic bacterium Halomonas elongata and proteomic profiling experiments are used as a starting point to motivate targeted experiments in order to find out some of the defining features of the osmoregulatory strategies of this bacterium. This new information is then used to refine the network in order to describe the actual capabilities of H. elongata, rather than its genetic potential.

From the phosphoenolpyruvate phosphotransferase system to selfish metabolism: a story retraced in Pseudomonas putida.

Although DNA is the ultimate repository of biological information, deployment of its instructions is constrained by the metabolic and physiological status of the cell. To this end, bacteria have evolved intricate devices that connect exogenous signals (e.g. nutrients, physicochemical conditions) with endogenous conditions (metabolic fluxes, biochemical networks) that coordinately influence expression or performance of a large number of cellular functions. The phosphoenolpyruvate:carbohydrate-phosphotransferase system (PTS) is a bacterial multi-protein phosphorylation chain which computes extracellular (e.g. sugars) and intracellular (e.g. phosphoenolpyruvate, nitrogen) signals and translates them into post-translational regulation of target activities through protein-protein interactions. The PTS of Pseudomonas putida KT2440 encompasses one complete sugar (fructose)-related system and the three enzymes that form the so-called nitrogen-related PTS (PTS(N) (tr) ), which lacks connection to transport of substrates. These two PTS branches cross-talk to each other, as the product of the fruB gene (a polyprotein EI-HPr-EIIA) can phosphorylate PtsN (EIIA(N) (tr) ) in vivo. This gives rise to a complex actuator device where diverse physiological inputs are ultimately translated into phosphorylation or not of PtsN (EIIA(N) (tr) ) which, in turn, checks the activity of key metabolic and regulatory proteins. Such a control of bacterial physiology highlights the prominence of biochemical homeostasis over genetic ruling -and not vice versa.

Cra regulates the cross-talk between the two branches of the phosphoenolpyruvate : phosphotransferase system of Pseudomonas putida.

The gene that encodes the catabolite repressor/activator, Cra (FruR), of Pseudomonas putida is divergent from the fruBKA operon for the uptake of fructose via the phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS(Fru)). The expression of the fru cluster has been studied in cells growing on substrates that change the intracellular concentrations of fructose-1-P (F1P), the principal metabolic intermediate that counteracts the DNA-binding ability of Cra on an upstream operator. While the levels of the regulator were not affected by any of the growth conditions tested, the transcription of fruB was stimulated by fructose but not by the gluconeogenic substrate, succinate. The analysis of the P(fruB) promoter activity in a strain lacking the Cra protein and the determination of key metabolites revealed that this regulator represses the expression of PTS(Fru) in a fashion that is dependent on the endogenous concentrations of F1P. Because FruB (i.e. the EI-HPr-EIIA(Fru) polyprotein) can deliver a high-energy phosphate to the EIIA(Ntr) (PtsN) enzyme of the PTS(Ntr) branch, the cross-talk between the two phosphotransferase systems was examined under metabolic regimes that allowed for the high or low transcription of the fruBKA operon. While fructose caused cross-talk, succinate prevented it almost completely. Furthermore, PtsN phosphorylation by FruB occurred in a Δcra mutant regardless of growth conditions. These results traced the occurrence of the cross-talk to intracellular pools of Cra effectors, in particular F1P. The Cra/F1P duo seems to not only control the expression of the PTS(Fru) but also checks the activity of the PTS(Ntr) in vivo.

Modeling and analysis of flux distributions in the two branches of the phosphotransferase system in Pseudomonas putida.

BACKGROUND: Signal transduction plays a fundamental role in the understanding of cellular physiology. The bacterial phosphotransferase system (PTS) together with the PEP/pyruvate node in central metabolism represents a signaling unit that acts as a sensory element and measures the activity of the central metabolism. Pseudomonas putida possesses two PTS branches, the C-branch (PTSFru) and a second branch (PTSNtr), which communicate with each other by phosphate exchange. Recent experimental results showed a cross talk between the two branches. However, the functional role of the crosstalk remains open. RESULTS: A mathematical model was set up to describe the available data of the state of phosphorylation of PtsN, one of the PTS proteins, for different environmental conditions and different strain variants. Additionally, data from flux balance analysis was used to determine some of the kinetic parameters of the involved reactions. Based on the calculated and estimated parameters, the flux distribution during growth of the wild type strain on fructose could be determined. CONCLUSION: Our calculations show that during growth of the wild type strain on the PTS substrate fructose, the major part of the phosphoryl groups is provided by the second branch of the PTS. This theoretical finding indicates a new role of the second branch of the PTS and will serve as a basis for further experimental studies.