Jimena A. Ruiz

Polyhydroxyalkanoate degradation is associated with nucleotide accumulation and enhances stress resistance and survival of Pseudomonas oleovorans in natural water microcosms

ABSTRACT Pseudomonas oleovorans GPo1 and its polyhydroxyalkanoic acid (PHA) depolymerization-minus mutant, GPo500 phaZ, residing in natural water microcosms, were utilized to asses the effect of PHA availability on survival and resistance to stress agents. The wild-type strain showed increased survival compared to the PHA depolymerase-minus strain. The appearance of a round cellular shape, characteristic of bacteria growing under starvation conditions, was delayed in the wild type in comparison to the mutant strain. Percent survival at the end of ethanol and heat challenges was always higher in GPo1 than in GPo500. Based on these results and on early experiments (H. Hippe, Arch. Mikrobiol. 56:248–277, 1967) that suggested an association of PHA utilization with respiration and oxidative phosphorylation, we investigated the association between PHA degradation and nucleotide accumulation. ATP and guanosine tetraphosphate (ppGpp) production was analyzed under culture conditions leading to PHA depolymerization. A rise in the ATP and ppGpp levels appeared concomitant with PHA degradation, while this phenomenon was not observed in the mutant strain unable to degrade the polymer. Complementation of the phaZ mutation restored the wild-type phenotype.

A Polyhydroxybutyrate-Producing Pseudomonas sp. Isolated from Antarctic Environments with High Stress Resistance

Pseudomonas sp. 14-3, a strain that accumulates large quantities of polyhydroxybutyrate (PHB) when grown on octanoate, was isolated from Antarctic environments. This isolate was characterized on the basis of phenotypic features and partial sequencing of its 16S ribosomal RNA gene. Pseudomonas sp. 14-3 showed increased tolerance to both thermal and oxidative stress compared with three other Pseudomonas species. Stress tolerance of Pseudomonas sp. 14-3 was analyzed in polyhydroxyalkanoate accumulating and non-accumulating conditions, and increased levels of stress resistance were observed when PHB was produced. Pseudomonas sp. 14-3 was isolated from Antarctic regions, a habitat normally exposed to extreme conditions. An association between high PHB accumulation and high stress resistance in bacteria adapted to extreme environments is suggested.

rpoS Gene Expression in Carbon-Starved Cultures of the Polyhydroxyalkanoate-Accumulating Species Pseudomonas oleovorans

The expression of the rpoS gene during PHA depolymerization was monitored in Pseudomonas oleovorans GPo1 and its mutant defective in PHA degradation by analyzing the tolerance to oxidative and thermal stresses and the RpoS intracellular content. An increase in the tolerance to H2O2 and heat shock was observed coincidentally with PHA degradation. Western blotting experiments performed in carbon-starved cultures showed that the RpoS levels were higher in the wild type than in the mutant strain. Complementation of the phaZ mutation restores the wild-type RpoS levels. These results suggest a probable association between PHA depolymerization and the stress tolerance phenotype controlled by RpoS.

Survival of poly-3-hydroxybutyrate-producing bacteria in soil microcosms

Bacillus megaterium is a potential bioremediation and biocontrol agent. The accumulation of reserve polymers, such as poly-3-hydroxybutyrate (PHB), increases survival of B. megaterium in water. We used wild-type strains of this species and mutant strains deficient in PHB synthesis in soil microcosms for testing the hypothesis that differences in survival capabilities and spore quality between strains is maintained in heterogeneous environments enriched with organic matter. No differences in survival between strains, nor a decrease in bacterial cell numbers were observed in sterile soil microcosms. In non-sterile soil, the total cell number (vegetative cells plus spores) of the PHB wild-type strain was 3.5 times higher than that of the PHB-negative mutant. We suggest that for predictive purposes, validation of survival in a variety of conditions is necessary.

Escherichia coli redox mutants as microbial cell factories for the synthesis of reduced biochemicals

Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative aerobe Escherichia coli, the microbial cell factory par excellence, has elaborate sensing and signal transduction mechanisms that respond to the availability of electron acceptors and alternative carbon sources in the surrounding environment. In particular, the ArcBA and CreBC two-component signal transduction systems are largely responsible for the metabolic regulation of redox control in response to O2 availability and carbon source utilization, respectively. Significant advances in the understanding of the biochemical, genetic, and physiological duties of these regulatory systems have been achieved in recent years. This situation allowed to rationally-design novel engineering approaches that ensure optimal carbon and energy flows within central metabolism, as well as to manipulate redox homeostasis, in order to optimize the production of industrially-relevant metabolites. In particular, metabolic flux analysis provided new clues to understand the metabolic regulation mediated by the ArcBA and CreBC systems. Genetic manipulation of these regulators proved useful for designing microbial cells factories tailored for the synthesis of reduced biochemicals with added value, such as poly(3-hydroxybutyrate), under conditions with restricted O2 supply. This network-wide strategy is in contrast with traditional metabolic engineering approaches, that entail direct modification of the pathway(s) at stake, and opens new avenues for the targeted modulation of central catabolic pathways at the transcriptional level.

The alternative sigma factor, σS, affects polyhydroxyalkanoate metabolism in Pseudomonas putida

To determine whether the stationary sigma factor, sigma(S), influences polyhydroxyalkanoate metabolism in Pseudomonas putida KT2440, an rpoS-negative mutant was constructed to evaluate polyhydroxyalkanoate accumulation and expression of a translational fusion to the promoter region of the genes that code for polyhydroxyalkanoate synthase 1 (phaC1) and polyhydroxyalkanoate depolymerase (phaZ). By comparison with the wild-type, the rpoS mutant showed a higher polyhydroxyalkanoate degradation rate and increased expression of the translational fusion during the stationary growth phase. These results suggest that sigma(S) might control the genes involved in polyhydroxyalkanoate metabolism, possibly in an indirect manner. In addition, survival and oxidative stress assays performed under polyhydroxyalkanoate- and nonpolyhydroxyalkanoate- accumulating conditions demonstrated that the accumulated polyhydroxyalkanoate increased the survival and stress tolerance of the rpoS mutant. According to this, polyhydroxyalkanoate accumulation would help cells to overcome the adverse conditions encountered during the stationary phase in the strain that lacks RpoS.

ArcA redox mutants as a source of reduced bioproducts.

Escherichia coli and other facultative anaerobes can adapt their metabolism according to oxygen availability by means of aerobic and anaerobic respiration and fermentation. ArcAB is a two-component signal transduction system that controls, at the transcriptional level, the choice of energy generation pathway according to the intracellular redox state. High throughput studies on different redox regulator mutants, involving transcriptome analysis, RT-PCR and phenotypic arrays enabled the elucidation of a repertoire of operons coordinated by ArcA which extended beyond respiration control including, among others, those which code for survival, chromosome replication and degradation of fatty acids. Flux analysis by 13C labeling provided new clues to the understanding of the distribution of metabolites mediated by ArcAB. The genetic manipulation of this regulator proved to be useful for the generation of reduced products of commercial value.

Manipulation of the Anoxic Metabolism in Escherichia coli by ArcB Deletion Variants in the ArcBA Two-Component System

ABSTRACT Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative anaerobe Escherichia coli has elaborate sensing and signal transduction mechanisms for redox control in response to the availability of O2 and other electron acceptors. The ArcBA two-component system consists of ArcB, a membrane-associated sensor kinase, and ArcA, the cognate response regulator. The tripartite hybrid kinase ArcB possesses a transmembrane, a PAS, a primary transmitter (H1), a receiver (D1), and a phosphotransfer (H2) domain. Metabolic fluxes were compared under anoxic conditions in a wild-type E. coli strain, its ΔarcB derivative, and two partial arcB deletion mutants in which ArcB lacked either the H1 domain or the PAS-H1-D1 domains. These analyses revealed that elimination of different segments in ArcB determines a distinctive distribution of d-glucose catabolic fluxes, different from that observed in the ΔarcB background. Metabolite profiles, enzyme activity levels, and gene expression patterns were also investigated in these strains. Relevant alterations were observed at the P-enol-pyruvate/pyruvate and acetyl coenzyme A metabolic nodes, and the formation of reduced fermentation metabolites, such as succinate, d-lactate, and ethanol, was favored in the mutant strains to different extents compared to the wild-type strain. These phenotypic traits were associated with altered levels of the enzymatic activities operating at these nodes, as well as with elevated NADH/NAD+ ratios. Thus, targeted modification of global regulators to obtain different metabolic flux distributions under anoxic conditions is emerging as an attractive tool for metabolic engineering purposes.

High polyhydroxybutyrate production in Pseudomonas extremaustralis is associated with differential expression of horizontally acquired and core genome polyhydroxyalkanoate synthase genes.

Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA) infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA). All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI) associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC) in comparison with the mclPHA core genome genes (phaC1 and phaC2) indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases.

A novel Burkholderia ambifaria strain able to degrade the mycotoxin fusaric acid and to inhibit Fusarium spp. growth

Fusaric acid (FA) is a fungal metabolite produced by several Fusarium species responsible for wilts and root rot diseases of a great variety of plants. Bacillus spp. and Pseudomonas spp. have been considered as promising biocontrol agents against phytopathogenic Fusarium spp., however it has been demonstrated that FA negatively affects growth and production of some antibiotics in these bacteria. Thus, the capability to degrade FA would be a desirable characteristic in bacterial biocontrol agents of Fusarium wilt. Taking this into account, bacteria isolated from the rhizosphere of barley were screened for their ability to use FA as sole carbon and energy source. One strain that fulfilled this requirement was identified according to sequence analysis of 16S rRNA, gyrB and recA genes as Burkholderia ambifaria. This strain, designated T16, was able to grow with FA as sole carbon, nitrogen and energy source and also showed the ability to detoxify FA in barley seedlings. This bacterium also exhibited higher growth rate, higher cell densities, longer survival, higher levels of indole-3-acetic acid (IAA) production, enhanced biofilm formation and increased resistance to different antibiotics when cultivated in Luria Bertani medium at pH 5.3 compared to pH 7.3. Furthermore, B. ambifaria T16 showed distinctive plant growth-promoting features, such as siderophore production, phosphate-solubilization, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, in vitro antagonism against Fusarium spp. and improvement of grain yield when inoculated to barley plants grown under greenhouse conditions. This strain might serve as a new source of metabolites or genes for the development of novel FA-detoxification systems.