Craig Daniels

Responses of Pseudomonas to small toxic molecules by a mosaic of domains

Transcriptional responses of microorganisms to environmental perturbations are broad and complex, and often involve regulatory cascades in which an interplay of regulatory factors trigger a specific expression program. Here, we describe how Pseudomonas responds to challenges from toxic chemicals, for which they use a dedicated program that subsequently confers resistance to these compounds. A mosaic of domains has been recruited to sense and metabolize these chemicals in order to obtain energy and carbon.

Domain cross-talk during effector binding to the multidrug binding TTGR regulator.

A major mechanism of antibiotic resistance in bacteria is the active extrusion of toxic compounds through membrane-bound efflux pumps. The TtgR protein represses transcription of ttgABC, a key efflux pump in Pseudomonas putida DOT-T1E capable of extruding antibiotics, solvents, and flavonoids. TtgR contains two distinct and overlapping ligand binding sites, one is broad and contains mainly hydrophobic residues, whereas the second is deep and contains polar residues. Mutants in the ligand binding pockets were generated and characterized using electrophoretic mobility shift assays, isothermal titration calorimetry, and promoter expression. Several mutants were affected in their response to effectors in vitro: mutants H70A, H72A, and R75A did not dissociate from promoter DNA in the presence of chloramphenicol. Other mutants exhibited altered binding to the operator: L66A and L66AV96A mutants bound 3- and 15-fold better than the native protein, whereas the H67A mutant bound with 3-fold lower affinity. In vivo expression assays using a fusion of the promoter of ttgA to lacZ and antibiotic tolerance correlated with the in vitro observations, namely that mutant H67A leads to increased basal expression levels and enhances antibiotic tolerance, whereas mutants L66A and L66AV96A exhibit lower basal expression levels and decreased resistance to antibiotics. The crystal structure of TtgR H67A was resolved. The data provide evidence for the inter-domain communication that is predicted to be required for the transmission of the effector binding signal to the DNA binding domain and provide important information to understand TtgR/DNA/effector interactions.

Functional analysis of new transporters involved in stress tolerance in Pseudomonas putida DOT-T1E.

Pseudomonas putida DOT-T1E is a highly solvent-tolerant strain. Although the main mechanism that confers solvent tolerance to the strain is the TtgGHI efflux pump, a number of other proteins are also involved in the response to toluene. Previous proteomic and transcriptomic analysis carried out in our lab with P. putida DOT-T1E, and the solvent-sensitive strain, P. putida KT2440, revealed several transporters that were induced in the presence of toluene. We prepared five mutants of the corresponding genes in P. putida DOT-T1E and analysed their phenotypes with respect to solvent tolerance, stress endurance and growth with different carbon, nitrogen and sulfur sources. The data clearly demonstrated that two transporters (Ttg2ABC and TtgK) are involved in multidrug resistance and toluene tolerance, whereas another (homologous to PP0219 of P. putida KT2440) is a sulfate/sulfite transporter. No clear function could be assigned to the other two transporters. Of the transporters shown to be involved in toluene tolerance, one (ttg2ABC) belongs to the ATP-Binding Cassette (ABC) family, and is involved in multidrug resistance in P. putida DOT-T1E, while the other belongs to the Major Facilitator Superfamily and exhibits homology to a putative transporter of the Bcr/CflA family that has not previously been reported to be involved in toluene tolerance.

New molecular tools for enhancing methane production, explaining thermodynamically limited lifestyles and other important biotechnological issues

a Planta, 14071, Cordoba, Spain. In this special issue a series of articles appear detailing methods of energy generation under different conditions; processes which are related to the future vision of microbes as a source of electric power (Lovley, 2009). Methanogenesis is identified as an important part of the biogeochemical carbon cycle in diverse anaerobic envi- ronments. Methane is an important fuel that can be gen- erated from several wastes in these anoxic environments and therefore, understanding how it is produced, which organisms produce it and the reactions required to achieve high yields is of major biotechnological interest. Alves and colleagues (2009) present a mini-review on the use of lipids as substrates for methane production. Conversion of lipids into methane is achieved by the syntrophic consortia of acetogenic bacteria and methano- genic archeae. The microbes involved are influenced by the nature of the incoming lipids and examples of different microbial communities when feeding reactors with oleate or palmitate are reviewed. Alves and colleagues (2009) point out that the number of acetogenic microorganisms that degrade butyrate or higher fatty acids is very low and includes microbes of the Syntrophaceae and Syntroph- omonadaceae families. These microbes live together with hydrogen-consuming methanogenic archeae and sulfate- reducing bacteria. The syntrophic cooperation seems to be optimal when microbes are organized as microcolo- nies, which is considered to favour interspecies hydrogen transfer. As in many biotechnological processes, lipid conversion into methane requires both the appropriate microbes and their performance optimization in the reac- tors, each of which still require some improvement. The authors note that previous failures to deal with high load lipids were related mainly to sludge flotation and biomass washout. To overcome these limitations the authors describe a new reactor concept which involves primary biomass retention through flotation and secondary biomass retention via settling. These reactors are being tested in scale and if, as expected, they solve the problem may soon come into industrial use. The seminal review of Bernhard Schinck (Schink, 1997)

Sugar (ribose), spice (peroxidase) and all things nice (laccase hair‐dyes)

The removal of pollutants from the environment has been declared a priority by a number of Environmental Protection Agencies (Roze et al., 2009). A great number of aerobic pathways have been deciphered and their relevance in microbiology and biotechnology has been reviewed several times (Garmendia et al., 2008; Siezen and Galardini, 2008; Govantes et al., 2008; Atlas and Bragg, 2009). In the area of biodegradation the role anaerobes and fungi play in removal of pollutants is of mounting interest. Microbial Biotechnology is publishing a number of new titles in this area, and here we have extracted some of the main conclusions. Tas and colleagues (2009) have dealt with mineralization of polychlorinated chemicals, which are harmful contaminants due to their persistence and their chronic toxicity to living organisms. Dehalococcoides spp. can anaerobically transform chlorinated xenobiotics to less‐ or even non‐noxious derivatives via reductive dechlorination. Tas and colleagues (2009) have reviewed the biology of this genus, focusing on its genetic peculiarities, its variability and, of course, its biodegradative properties. Dehalococcoides can replace chlorine by hydrogen atoms in recalcitrant halogenated compounds, using them as electron acceptors during anaerobic respiration. More than 100 16S rRNAs from environmental Dehalococcoides spp., are available, most of them corresponding to uncultured strains. In addition to the standard problems of cultivating anaerobic microbes, these coccoids usually grow in microbial communities where they can find a H2 supplier needed for thriving. The full genome sequences of several Dehalococcoides strains show that they have very small genomes which are highly similar. Moreover, they exhibit a large number of putative dehalogenase‐encoding genes (rdh), reaching up to 1.7% of the coding sequences in Dehalococcoides sp. Further work, combining transcriptional and proteomic techniques, will identify which proteins are really essential for the degradation of polychorinated xenobiotics. Jeon and colleagues (2009) also report in Microbial Biotechnology issues related with the attack of halogenated chemicals. They detail the discovery of four HAD (Halodehalogenases) defluorinases from different microbial genomes. Some of these dehalogenases have enhanced activities and this appears to arise from their sequence diversity (less than 30% sequence identity for HADs) (Prudnikova et al., 2009; Rye et al., 2009). The set of new dehalogenase were elucidated via biochemical characterization of 163 potential dehalogenases from the sequenced genomes of five common soil bacteria. Their discovery and characterization will be imperative to the future use of these enzymes in the biodegradation of halogenated chemicals. Another area of interest is the anaerobic degradation of monoaromatic compounds such as benzene, toluene, ethylbenzene and the xylene isomers (BTEX; Dou et al., 2008a; Wolicka et al., 2009). Anaerobic BTEX degradation has been shown to occur under denitrifying, sulfate‐reducing, iron‐reducing, manganese‐reducing and methanogenic conditions (Dou et al., 2008a,b; Barton and Fauque, 2009). These activities are of the relevance in removal of pollutants from contaminated aquifers and soils, and they are considered an important remediation strategy for hydrocarbon‐contaminated sites. New approaches based on isotopes are being taken, in fact, recently, compound‐specific isotope analysis was successfully used to distinguish between the effects of non‐degradative processes of mass loss such as sorption, volatilization, and dilution and those of biodegradation for aromatic hydrocarbons in the field (Fischer et al., 2008; Vogt et al., 2008). Compound‐specific isotope analysis is based on the fact that, in most chemical reactions, lighter isotopomers react faster than heavier ones, leading to a kinetic isotope effect. Herrmann and colleagues (2008) in Environmental Microbiology Reports, suggest that two‐dimensional isotope fractionation analyses are a valuable tool for identifying and monitoring anaerobic biodegradation of xylene isomers. They explored the carbon and hydrogen isotope fractionation of benzylsuccinate synthase (Bss)‐initiated degradation pathways for xylene isomers in order to obtain further information on the variability of isotope fractionation processes associated with Bss that might be important for the assessment of anaerobic degradation of xylene and toluene in the environment. The use of combined carbon and hydrogen isotope fractionation analyses may therefore be useful to monitor anaerobic xylene degradation at contaminated sites; this sort of technology will allow invaluable in situ monitoring of bioremediation processes.

Metabolic engineering, new antibiotics and biofilm viscoelasticity

In the following highlight we refer to a number of new advances in the field of Biotechnology that address issues relating to the synthesis of new antibiotics, new biocatalysts and matrices in biofilms.

Microbial Biotechnology from medicine to bacterial population dynamics.

In recent issues of Microbial Biotechnology there have been several exceptional articles ranging in topic from the antagonism of biofilm formation in pathogenic bacteria, and prebiotic selection of positive bacterial fermentations in the colon to the discovery of novel biotechnologically important enzymes encoded by bacteria of the deep‐sea floor.

The heat, drugs and knockout systems of Microbial Biotechnology

The current issue of Microbial Biotechnology completes the second volume of this journal that aims to collect the best fundamental science in the field of microbiology related to biotechnological applications. The journal has maintained a regular flow of articles and has published many extremely relevant articles in the field, some of which are being extensively cited; an acknowledgement of their high scientific value.

Environmental Microbiology meets Microbial Biotechnology

We would like to begin this editorial by paying a modest but sincere tribute to Professor Kenneth Timmis for his recent nomination as a member of the Royal Society of the United Kingdom. Kenneth Timmis has pioneered science fields related to plasmid replication, biodegradation, microbial ecology, biodiversity and most recently metagenomics. In addition, to these many relevant scientific contributions, his services to the scientific community have been of paramount importance at the level of training for new generations of scientists and promotion of excellence in science through his role in journal editorial. Initially, as Editor for the Journal of Bacteriology (1989–1997) and then with the creation of two journals; one, Environmental Microbiology, that 10 years after the first issue came to light is the fifth journal in the area of microbiology and probably the leading journal in the field of environmental microbiology; the other, Microbial Biotechnology, that reflects his vision of microbes as useful biotechnological tools. Although Microbial Biotechnology is in its early infancy it has already attracted the attention of a specialized audience. The title of this editorial also entertains the idea that the future of Microbial Biotechnology will not only be based on food, energy, pharmaceutical and clinical microbiology, but will bring into the equation the many relevant aspects of environmental microbiology which contribute to the diversity of processes biotechnology can offer to society. An excellent example of Environmental Microbiology meeting Microbial Biotechnology comes from the publications by Kamilova and colleagues (2008) and Pechy‐Tarr and colleagues (2008). These articles emphasize two very relevant features in environmental microbiology, namely, the identification of new genes that encode proteins with potential to control insect pests and the phytopathogen Fusarium. Biotechnological exploitation of biocontrol properties of microbes requires an in‐depth knowledge of microbial genetics and physiology and/or the dissection in the lab of these properties, as well as specific training in designing and performance of field trials in which to test the microbes’ new potential. Plant–microbe interactions constitute a fascinating field of inter‐kingdom communication, some novel aspects of how bacteria sense plant signals and vice versa have been summarized by van Dillewijn (2008) in his highlight article entitled: what gets turned on in the rhizosphere? The field of plant–microbe interactions is extremely important to biotechnology; for this reason Microbial Biotechnology will dedicate an entire special issue to this area, which is expected to be published mid‐2009 with the assistance of invited guest editors with expertise in a number of fields (see announcing flyer). A set of articles published in this first volume of Microbial Biotechnology have dealt with microbial biofuel production (Maeda et al., 2008; Vardar‐Schara et al., 2008; Wackett, 2008a,b). The scientific community recognizes the potential of hydrogen as an alternative fuel due to its higher energy content than fossil fuels, its renewable nature, and because its product of oxidation is water. These advantages were already envisaged some 25 years ago (Takakuwa et al., 1983); however, not until recently have social demands for a cleaner environment led to a renewed interest in the theme. A relevant question regarding hydrogen production is its production limits. Limitations can arise from physical constraints – which could be solved via improvements in process development and/or fermentor design, or from the thermodynamic limits that govern reactions that lead to hydrogen production. In relation to this issue, Veit and colleagues (2008) explore the thermodynamic aspects limiting hydrogen yield in microbial fermentations; key reactions NAD(P)H + H+ ↔ NAD(P)+ + H2 hypothetically achieve equilibrium at very low partial hydrogen pressure, and the authors probe this through the design and thermodynamic analysis of a synthetic NAD(P)H:H2 pathway in Escherichia coli BL21 (DE3). The system consists of a ferredoxin‐dependent hydrogenase, ferredoxin as electron acceptor/donor intermediate, and a NAD(P)H:ferredoxin oxidoreductase. Results revealed that in both cases these pathways are influenced by partial headspace hydrogen pressure under closed bath conditions and that the NADPH:H2 system allows higher hydrogen accumulation than the NADH:H2 pathway. Novel molecules are the panacea for survival of large and small companies in the Biotech area. New methods that lead to more efficient synthesis of added value molecules are needed. This may involve the discovery of new molecules or the continuous improvement of a process through the identification of new enzymes. Both aspects are under consideration in this issue. Thiwthong and colleagues (2008) report on new aldehyde dehydrogenases for the synthesis of glyoxylic acid, an important compound in the pharmaceutical industry. They have purified and characterized two aldehyde oxidases, F10 and F13, from Pseudomonas sp. MX‐058. Both catalyse the oxidation of glyoxal to glyoxilic acid, and their kinetic properties hold potential for being economically and industrially exploitable. The authors claim these novel enzymes overcame limitations found earlier with aldehyde dehydrogenases from different eukaryotes. A unique feature is that the quaternary structure of the F13 enzyme revealed it to be heteropentameric. Expression of heterologous genes for use in a number of biotechnological applications has been the subject of intense research over the last 20 years (for a review see Bertram and Hillen, 2008). In this issue Fisher and colleagues (2008) systematically examined the twin‐arginine translocation system for secretion of heterologous proteins. The system is superior in yield and specific activity for secreted proteins than the ‘classical’ SEC pathway. One of the beauties of the system is that only properly folded proteins are exported to the periplasm; a screening process that guarantees correct protein properties. Medical and pharmaceutical industries are searching new cell targets for drug discovery. Glutamate racemase, a member of the cofactor‐independent, two thiol‐based family of amino acid racemases, has been implicated in maintaining sufficient d‐glutamate pool levels required for peptidoglycan cross‐linking; which in turn is of critical importance for bacterial growth. Fisher (2008) reviews the history of this enzyme, the recent biochemical and structural characterization of several isoenzymes and how a set of new inhibitors have been found. This research is of upmost significance for the pharmaceutical sector because peptidoglycan synthesis has long been an important antimicrobial drug target. Continuing on the theme of biotechnological applications of medical importance is the article published by Barbuddhe and Chakraborty (2008). Here the authors give a comprehensive review of the current and potential uses for the human pathogen Listeria monocytogenes in biotechnology. Because L. monocytogenes is capable of subverting host cell function and is able to survive and replicate within numerous eukaryotic cells during the infection process, it is an attractive delivery vehicle for use in both clinical and biotechnological applications. In fact Listeria are already being used to facilitate heterologous antigen presentation on the surface of antigen presenting cells and the Listeriolysin O protein has been used to assist DNA delivery by cell transfection for use in cell biology studies. The authors also consider the potential use of Listeria in the development of novel vaccine and drug delivery systems by exploiting their sophisticated approach to infection. Undoubtedly, future research allowing the development of these so‐called ‘patho‐biotechnology’ approaches will be of great interest over the coming years. Biosensors have also been the subject of mini‐review in Microbial Biotechnology, and a model for internal calibration of biosensors was recently published (Wackwitz et al., 2008). In the current issue a type of cell‐based biosensor is reported that permits one to distinguish pathogenic from non‐pathogenic strains of Bacillus cereus, a technology that holds promise for detection of pathogenic bacteria in food (Hutchison et al., 2008). The whole‐cell sensor system consists of erytrophore cells of Betta splendeus. The extension of the system to detect other pathogens is of major interest and of great importance in food biosafety.

Extrusion Pumps for Hydrocarbons: An Efficient Evolutionary Strategy to Confer Resistance to Hydrocarbons

Efflux pumps of the RND family are primarily involved in the extrusion of hydrocarbons. These pumps, specific to gram-negative bacteria, are composed of three components. Two components are transmembrane proteins located in the inner and outer membrane whereas the third one spans the periplasm connecting the other two subunits. The large part of information available on RND pumps is related to their capacity to extrude antibiotics. Structural data indicate that substrate binding may occur preferentially in the periplasm at the inner membrane protein.