Maximino Manzanera

Hydroxyectoine Is Superior to Trehalose for Anhydrobiotic Engineering of Pseudomonas putida KT2440

ABSTRACT Anhydrobiotic engineering aims to increase the level of desiccation tolerance in sensitive organisms to that observed in true anhydrobiotes. In addition to a suitable extracellular drying excipient, a key factor for anhydrobiotic engineering of gram-negative enterobacteria seems to be the generation of high intracellular concentrations of the nonreducing disaccharide trehalose, which can be achieved by osmotic induction. In the soil bacterium Pseudomonas putida KT2440, however, only limited amounts of trehalose are naturally accumulated in defined high-osmolarity medium, correlating with relatively poor survival of desiccated cultures. Based on the enterobacterial model, it was proposed that increasing intracellular trehalose concentration in P. putida KT2440 should improve survival. Using genetic engineering techniques, intracellular trehalose concentrations were obtained which were similar to or greater than those in enterobacteria, but this did not translate into improved desiccation tolerance. Therefore, at least for some populations of microorganisms, trehalose does not appear to provide full protection against desiccation damage, even when present at high concentrations both inside and outside the cell. For P. putida KT2440, it was shown that this was not due to a natural limit in desiccation tolerance since successful anhydrobiotic engineering was achieved by use of a different drying excipient, hydroxyectoine, with osmotically preconditioned bacteria for which 40 to 60% viability was maintained over extended periods (up to 42 days) in the dry state. Hydroxyectoine therefore has considerable potential for the improvement of desiccation tolerance in sensitive microorganisms, particularly for those recalcitrant to trehalose.

The XylS‐dependent Pm promoter is transcribed in vivo by RNA polymerase with σ32 or σ38 depending on the growth phase

The Pm promoter of the TOL plasmid of Pseudomonas putida is expressed at high level along the growth curve. This transcription is dependent on the positive regulator XylS activated by 3‐methylbenzoate. The sigma factor σ38 is required for expression in early stationary phase and thereafter. To test whether σ70 was involved in Pm transcription in exponential phase, we have followed mRNA synthesis in a rpoD thermosensitive strain. No difference in Pm transcription was found between the wild type and the thermosensitive strain at the restrictive temperature of 42°C, indicating that transcription was independent of the sigma factor σ70. However, basal levels of mRNA expression from Pm in this strain in exponential phase were more than twofold higher at 42°C, suggesting involvement of σ32 in Pm transcription. In a rpoH background, no expression of Pm took place in the exponential phase, whereas it increased during stationary phase, and in a rpoH rpoS double mutant no activity from the Pm promoter was detected along the growth curve. We have shown that the increase in the amount of σ32 factor necessary for transcription in exponential phase is provided through induction of the heat shock response by the presence of the effector 3‐methylbenzoate, which is also required for activation of the positive regulator XylS. We conclude that activation of Pm transcription is achieved through a switch between two stress‐responsive factors, σ32 in exponential phase and σ38 in stationary phase. In both cases, transcription is dependent on the activator XylS and presents the same transcription start point.

Microbial enzymatic activities in a pilot-scale MBR experimental plant under different working conditions.

Phosphatases, glucosidase, protease, esterase and dehydrogenase activities in a MBR (membrane bioreactor) system equipped with ultrafiltration membranes for the treatment of real urban wastewater were measured at different volatile suspended solid (VSS) concentrations, total suspended solid (TSS) concentrations, hydraulic retention times (HRT), temperatures and inflow rates. The results showed the capacity of the MBR system to remove COD and BOD(5) at TSS between 7200 and 13,300 mg/L; HRT values of 8.05 and 15.27 h; inflow rates of 14.67 and 27.81 L/h; and temperatures between 4 and 27 degrees C. The enzymatic activities are influenced by increases in VSS and TSS concentrations. These results suggest that the ability to get adapted to environmental changes of the bacterial populations and their microbial enzymatic activities is essential to understand the biological processes that occur in MBR systems and crucial for proper urban wastewater treatment when using MBR technologies.

Characterization of the norCBQD genes, encoding nitric oxide reductase, in the nitrogen fixing bacterium Bradyrhizobium japonicum.

The genes norCBQD that encode the bc-type nitric oxide reductase from Bradyrhizobium japonicum USDA110 have been isolated and characterized. norC and norB encode the cytochrome c-containing subunit II and cytochrome b-containing subunit I of nitric oxide reductase, respectively. norQ encodes a protein with an ATP/GTP-binding motif, and the predicted norD gene product shows similarity with NorD from other denitrifiers. Mutational analysis indicates that the two structural norC and norB genes are required for microaerobic growth under nitrate-respiring conditions. A mutant strain lacking a functional norC gene also lacked the 16 kDa c-type cytochrome that is normally detectable by haem-staining of proteins from membranes of microaerobically grown wild-type cells. Expression of a transcriptional fusion of the nor promoter region to the reporter gene lacZ (P(norC)-lacZ) was not detected in aerobically grown cells of USDA110, but the fusion was induced threefold when the cells were cultured under microaerobic conditions (1% O(2)) with either nitrite or nitric oxide, and about 18-fold when nitrate was the N oxide present in the medium. The P(norC)-lacZ fusion was not expressed in the B. japonicum fixK(2) mutant strain 9043, but complementation of the mutant with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. The promoter region of the norCBQD genes has been characterized by primer extension. A major transcript initiates 45.5 bp downstream of the centre of a putative binding site for the transcription factor FixK(2).

Cross-Regulation between a Novel Two-Component Signal Transduction System for Catabolism of Toluene in Pseudomonas mendocina and the TodST System from Pseudomonas putida

The tmoABCDEF genes encode the toluene-4-monooxygenase from Pseudomonas mendocina KR1. Upstream from the tmoA gene an open reading frame, tmoX, encoding a protein 83% identical to TodX (todX being the initial gene in the todXFC1C2BADEGIH operon from Pseudomonas putida DOT-T1E) was found. The tmoX gene is also the initial gene in the tmoXABCDEF gene cluster. The transcription initiation point from the tmoX promoter was mapped, and the sequence upstream revealed striking identity with the promoter of the tod operon of P. putida. The tod operon is regulated by a two-component signal transduction system encoded by the todST genes. Two novel genes from P. mendocina KR1, tmoST, were rescued by complementation of a P. putida DOT-T1E todST knockout mutant, whose gene products shared about 85% identity with TodS-TodT. We show that transcription from P(tmoX) and P(todX) can be mediated by TmoS-TmoT or TodS-TodT, in the presence of toluene, revealing cross-regulation between these two catabolic pathways.

Control of Expression of Divergent Pseudomonas putida put Promoters for Proline Catabolism

ABSTRACT Pseudomonas putida KT2440 uses proline as the sole C and N source. Utilization of this amino acid involves its uptake, which is mediated by the PutP protein, and its conversion into glutamate, mediated by the PutA protein. Sequence analysis revealed that theputA and putP genes are transcribed divergently. Expression from the putP and putAgenes was analyzed at the mRNA level in different host backgrounds in the absence and presence of proline. Expression from theput promoters was induced by proline. The transcription initiation points of the putP and putA genes were precisely mapped via primer extension, and sequence analysis of the upstream DNA region showed well-separated promoters for these two genes. The PutA protein acts as a repressor of put gene expression in P. putida because expression from theput promoters is constitutive in a host background with a knockout putA gene. This regulatory activity is independent of the catabolic activity of PutA, because we show that a point mutation (Glu896→Lys) that prevents catalytic activity allowed the protein to retain its regulatory activity. Expression from theput promoters in the presence of proline in aputA-proficient background requires a positive regulatory protein, still unidentified, whose expression seems to be ς54 dependent because the put genes were not expressed in a ς54-deficient background. Expression of the putA and putP genes was equally high in the presence of proline in ς38- and ihf-deficientP. putida backgrounds.

Plastic Encapsulation of Stabilized Escherichia coli and Pseudomonas putida

ABSTRACT Escherichia coli and Pseudomonas putida dried in hydroxyectoine or trehalose are shown to be highly resistant to the organic solvents chloroform and acetone, and consequently, they can be encapsulated in a viable form in solid plastic materials. Bacteria are recovered by rehydration after physical disruption of the plastic. P. putida incorporated into a plastic coating of maize seeds was shown to colonize roots efficiently after germination.

Biodiesel: An Alternative Fuel

Biodiesel is an alternative energy source and could be a substitute for petroleum-based diesel fuel. To be a viable alternative, a biofuel should provide a net energy gain, have environmental benefits, be economically competitive, and be producible in large quantities without reducing food supplies. Most of the sources, methods and apparatus to produce biodiesel are reviewed here. Some of the patents propose the use of oils and fats of animal or vegetal origin and other kind of sources. Many others focus on the methods for the production or oxidation stability of the biofuel in order to make its production economically competitive. Several apparatus comprising reactors and refineries are also presented. This review article summarizes recent and important patents relating to the production of biodiesel to make its production a viable alternative.

Arthrobacter siccitolerans sp. nov., a highly desiccation-tolerant, xeroprotectant-producing strain isolated from dry soil.

A novel desiccation-tolerant, xeroprotectant-producing bacterium, designated strain 4J27T, was isolated from a Nerium oleander rhizosphere subjected to seasonal drought in Granada, Spain. Phylogenetic analysis based on 16S rRNA gene sequencing placed the isolate within the genus Arthrobacter, its closest relative being Arthrobacter phenanthrenivorans Shep3 DSM 18606T, with which it showed 99.23 % 16S rRNA gene sequence similarity. DNA–DNA hybridization measurements showed less than 25 % relatedness between strain 4J27T and Arthrobacter phenanthrenivorans DSM 18606T. The DNA base composition of strain 4J27T was 65.3 mol%. The main fatty acids were anteiso C15 : 0, anteiso C17 : 0, C16 : 0 and iso C16 : 0 and the major menaquinone was MK-9 (H2). The peptidoglycan type was A3α with an l-Lys–l-Ser–l-Thr–l-Ala interpeptide bridge. The bacterium tested positive for catalase activity and negative for oxidase activity. Phylogenetic, chemotaxonomic and phenotypic analyses indicated that the desiccation-tolerant strain 4J27T represents a novel species within the genus Arthrobacter, for which the name Arthrobacter siccitolerans is proposed. The type strain is 4J27T ( = CECT 8257T = LMG 27359T).

Mutational analysis of the highly conserved C-terminal residues of the XylS protein, a member of the AraC family of transcriptional regulators

The XylS protein of the TOL plasmid of Pseudomonas putida belongs to the so‐called AraC/XylS family of regulators, that includes more than 100 different bacterial proteins. A conserved stretch of about 100 amino acids is present at the C‐terminal end. This conserved region is believed to contain seven α‐helices, including two helix‐turn‐helix (HTH) DNA binding motifs (α2‐T‐α3 and α5‐Tα‐6), connected by a linker α‐helix (α4), and two flanking α‐helices (α1 and α7). The second HTH motif is the region with the highest homology in the proteins of the family, with certain residues showing almost 90% identity. We have constructed XylS single mutants in the most conserved residues and have analysed their ability to stimulate transcription from its cognate promoter, Pm, fused to ′lacZ. The analysis revealed that mutations in the α5‐helix conserved residues had little effect on the XylS transcriptional activity, whereas the distribution of polarity in the α6‐helix was important for the activity. The strongest effect of the mutations was observed in conserved residues located outside the DNA binding domain, namely, Gly‐290 in the turn between the two helices, Pro‐309 located downstream of α6, and Leu‐313, in the small last helix α7, that seems to play an important role in the activation of RNA‐polymerase. Our analysis shows that conservation of amino acids in the family reflects structural requirements rather than functionality in specific DNA interactions.