Beatriz S. Méndez

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.

Poly(3-Hydroxybutyrate) Synthesis by Recombinant Escherichia coli arcA Mutants in Microaerobiosis

ABSTRACT We assessed the effects of different arcA mutations on poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli strains carrying the pha synthesis genes from Azotobacter sp. strain FA8. The arcA mutations used were an internal deletion and the arcA2 allele, a leaky mutation for some of the characteristics of the Arc phenotype which confers high respiratory capacity. PHB synthesis was not detected in the wild-type strain in shaken flask cultures under low-oxygen conditions, while ArcA mutants gave rise to polymer accumulation of up to 24% of their cell dry weight. When grown under microaerobic conditions in a bioreactor, the arcA deletion mutant reached a PHB content of 27% ± 2%. Under the same conditions, higher biomass and PHB concentrations were observed for the strain bearing the arcA2 allele, resulting in a PHB content of 35% ± 3%. This strain grew in a simple medium at a specific growth rate of 0.69 ± 0.07 h−1, whereas the deletion mutant needed several nutritional additives and showed a specific growth rate of 0.56 ± 0.06 h−1. The results presented here suggest that arcA mutations could play a role in heterologous PHB synthesis in microaerobiosis.

Pseudomonas extremaustralis sp. nov., a Poly(3-hydroxybutyrate) Producer Isolated from an Antarctic Environment

A Gram-negative, mobile, rod-shaped, non-spore-forming bacterium (strain 14-3T) was isolated from a temporary pond in Antarctica. On the basis of 16S rRNA gene sequence similarity, strain 14-3T was shown to belong to the genus Pseudomonas sensu stricto. Physiological and biochemical tests supported the phylogenetic affiliation. Strain 14-3T is closely related to Pseudomonas veronii DSM 11331T, sharing 99.7% sequence similarity. DNA–DNA hybridization experiments between the two strains showed only moderate reassociation similarity (35.1%). Tests for arginine dihydrolase and nitrate reduction were positive, while those for denitrification, indol production, glucose acidification, urease, ß-galactosidase, esculin, caseine and gelatin hydrolysis were negative. Growth of this bacterium occurred in a range from 4 to 37°C but not at 42°C. It accumulated poly(3-hydroxybutyrate) when grown on sodium octanoate medium. Strain 14-3T therefore represents the type strain of a new species, for which the name Pseudomonas extremaustralis sp. nov. is proposed. The type strain 14-3T has been deposited as DSM 17835T and as CIP 109839T.

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.

Poly(3-hydroxybutyrate) synthesis genes in Azotobacter sp. strain FA8.

ABSTRACT Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for β-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced intoRalstonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB.

Metabolic Flux Analysis of Escherichia coli creB and arcA Mutants Reveals Shared Control of Carbon Catabolism under Microaerobic Growth Conditions

Escherichia coli has several elaborate sensing mechanisms for response to availability of oxygen and other electron acceptors, as well as the carbon source in the surrounding environment. Among them, the CreBC and ArcAB two-component signal transduction systems are responsible for regulation of carbon source utilization and redox control in response to oxygen availability, respectively. We assessed the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions by means of (13)C-labeling experiments in chemostat cultures of a wild-type strain, DeltacreB and DeltaarcA single mutants, and a DeltacreB DeltaarcA double mutant. Continuous cultures were conducted at D = 0.1 h(-1) under carbon-limited conditions with restricted oxygen supply. Although all experimental strains metabolized glucose mainly through the Embden-Meyerhof-Parnas pathway, mutant strains had significantly lower fluxes in both the oxidative and the nonoxidative pentose phosphate pathways. Significant differences were also found at the pyruvate branching point. Both pyruvate-formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis from pyruvate, and their activity seemed to be modulated by both ArcAB and CreBC. Strains carrying the creB deletion showed a higher biomass yield on glucose compared to the wild-type strain and its DeltaarcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate shunt and lactate dehydrogenase were active mainly in the DeltaarcA strain. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in the mutant strains.

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides.

Aims:  Analysis of the physiology and metabolism of Escherichia coli arcA and creC mutants expressing a bifunctional alcohol‐acetaldehyde dehydrogenase from Leuconostoc mesenteroides growing on glycerol under oxygen‐restricted conditions. The effect of an ldhA mutation and different growth medium modifications was also assessed.

Escherichia coli arcA Mutants: Metabolic Profile Characterization of Microaerobic Cultures using Glycerol as a Carbon Source

ArcA is a global regulator that switches on the expression of fermentation genes and represses the aerobic pathways when Escherichia coli enters low oxygen growth conditions. The metabolic profile of E. coli CT1062 (ΔarcA)and CT1061 (arcA2) grown in microaerobiosis with glycerol as carbon source were determined and compared with E. coli K1060, the arcA+ parent strain. Both arcA mutants achieved higher biomass yields than the wild-type strain. The production of acetate, formate, lactate, pyruvate, succinate and ethanol were determined in the supernatants of cultures grown on glycerol under microaerobic conditions for 48 h. The yield of extracellular metabolites on glycerol showed lower acid and higher ethanol values for the mutants. The ethanol/acetate ratio was 0.87 for the parent strain, 2.01 for CT1062, and 12.51 for CT1061. Accordingly, the NADH/NAD+ ratios were 0.18, 0.63, and 0.97, respectively. The extracellular succinate yield followed a different pattern, with yield values of 0.164 for K1060, 0.442 for CT1062 and 0.214 for CT1061. The dissimilarities observed can be attributed to the different effects exerted by the deletion and point mutations in a global regulator.

A PCR-based method for the screening of bacterial strains with antifungal activity in suppressive soybean rhizosphere

Selection and evaluation of microbial strains for their antifungal activity in natural environments is time- and energy-consuming. We have adapted a PCR-based method to avoid these inconveniences. Soils that are naturally suppressive to plant disease were chosen as a source of antibiotic-producing bacteria. The screening was performed by means of PCR amplification using degenerate primers corresponding to peptide synthetase genes. Amplification fragments were obtained using template DNA from the rhizosphere of three different soybean fields. In order to assay their potential utility in pathogen control, several Bacillus strains were analysed for their in vitro antifungal activity by testing growth inhibition of Sclerotinia sclerotiorum. Four Bacillus sp. isolates gave a positive amplification signal, and three of them had an inhibitory effect on S. sclerotiorum growth, whereas two strains that failed to give an amplification signal did not inhibit fungal growth. These results show that PCR-based techniques could be useful to assess the presence of strains with potential use as biocontrol agents.

Making Green Polymers Even Greener:Towards Sustainable Production of Polyhydroxyalkanoates from Agroindustrial By-Products

Jose G. C. Gomez1, Beatriz S. Mendez2, Pablo I. Nikel2,3, M. Julia Pettinari2, Maria A. Prieto4 and Luiziana F. Silva1 1Institute of Biomedical Sciences, University of Sao Paulo 2Department of Biological Chemistry, Faculty of Sciences, University of Buenos Aires and National Council for Research (CONICET), 3Institute for Research in Biotechnology, University of San Martin, 4Department of Environmental Biology, Centro de Investigaciones Biologicas, 1Brazil 2,3Argentina 4Spain