From the phosphoenolpyruvate phosphotransferase system (PTS) 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 3 enzymes that form the so-called nitrogen-related PTS (PTSNtr), 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 (EIIANtr) in vivo. This gives rise to a complex actuator device where diverse physiological inputs are ultimately translated into phosphorylation or not of PtsN (EIIANtr) 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.This study was supported by the BIO Program of the Spanish Ministry of Science and Innovation, the ST-FLOW and ARYSIS Contracts of the EU, the ERANET-IB Program and the PROMT Project of the CAMPeer reviewe

Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTSFru), while the other (N-related PTS, or PTSNtr) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTSFru or PTSNtr genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIANtr) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIANtr protein of the PTSNtr thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology

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.This work has been funded by the German Federal Ministry of Education and Research (BMBF) through project HOBBIT (031B03)

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

Comparative analyses imply that the enigmatic sigma factor 54 is a central controller of the bacterial exterior

Contains fulltext : 95738.pdf (publisher's version ) (Open Access)BACKGROUND: Sigma-54 is a central regulator in many pathogenic bacteria and has been linked to a multitude of cellular processes like nitrogen assimilation and important functional traits such as motility, virulence, and biofilm formation. Until now it has remained obscure whether these phenomena and the control by Sigma-54 share an underlying theme. RESULTS: We have uncovered the commonality by performing a range of comparative genome analyses. A) The presence of Sigma-54 and its associated activators was determined for all sequenced prokaryotes. We observed a phylum-dependent distribution that is suggestive of an evolutionary relationship between Sigma-54 and lipopolysaccharide and flagellar biosynthesis. B) All Sigma-54 activators were identified and annotated. The relation with phosphotransfer-mediated signaling (TCS and PTS) and the transport and assimilation of carboxylates and nitrogen containing metabolites was substantiated. C) The function annotations, that were represented within the genomic context of all genes encoding Sigma-54, its activators and its promoters, were analyzed for intra-phylum representation and inter-phylum conservation. Promoters were localized using a straightforward scoring strategy that was formulated to identify similar motifs. We found clear highly-represented and conserved genetic associations with genes that concern the transport and biosynthesis of the metabolic intermediates of exopolysaccharides, flagella, lipids, lipopolysaccharides, lipoproteins and peptidoglycan. CONCLUSION: Our analyses directly implicate Sigma-54 as a central player in the control over the processes that involve the physical interaction of an organism with its environment like in the colonization of a host (virulence) or the formation of biofilm

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

Abstract 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)

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

Abstract 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.AK was funded in part by the FORSYS initiative from the German Federal Ministry of Education and Research (BMBF).Peer Reviewe

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