Thermophilic microbial degradation of polyethylene succinate

This paper examined the biodegradability of a new aliphatic polyester, polyethylene succinate (PES), at a high incubation temperature of 50°C. The distribution and population of total colonies and of PES degrading micro organisms on polymer-emulsified agar plates were determined using the plate count and clear zone methods. The PES-decomposers were present in six of 10 soil samples and the total number ranged from 2.0×104 to 2.2×106 c.f.u./g of samples. Degrading microorganisms constituted between 20 and 80% of the total colonies on PES–agar plates. A single PES-degrading strain, TT96, was isolated and tested for its biodegrading capacity on PES powder and on other aliphatic polyesters: poly(beta-hydroxybutyrate) (PHB), polycaprolactone (PCL), poly(butylene succinate) (PBS), and poly(L-lactide) (PLA). Degraded films of PES and PBS were presented and compared using scanning electron microscopy. Strain TT96 was able to create clear zones on all the polymers used, except on PHB-agar plates. Liquid culture test after 2 weeks showed that TT96 completely degraded PCL powder but had very little activity on other samples. Scanning electron micrograph confirmed the microbial attack of TT96 on PES and PBS films. PES film surfaces were degraded more uniformly compared to PBS films which were decomposed only in some parts.     

Pollen development and tube growth are affected in the symbiotic mutant of Lotus japonicus, crinkle

The symbiotic mutant of Lotus japonicus, crinkle (crk), exhibits abnormal nodulation and other alterations in the root hairs, trichomes, and seedpods. Defective nodulation in crk mutant is due to the arrested infection thread growth from the epidermis into the cortex. Here, we describe that crk is also affected in male fertility that causes the production of small pods with few seeds. Under in vitro conditions, pollen germination and tube growth were markedly reduced in the crk mutant. A swollen tip phenotype with disorganized filamentous actin (F-actin) was observed in the mutant pollen tubes after prolonged in vitro culture. During pollen development, the striking difference noted in the mutant was the small size of the microspores that remained spherical. Histological examination of ovule development, as well as outcrosses of the mutant as female to wild type as male, showed no evidence of abnormality in the female gametophyte development. Based on these findings, the Crk gene, aside from its role in the infection process during nodulation, is also involved in male gametophyte development and function. Therefore, this gene represents a connection between nodule symbiosis, polar tip growth, and other plant developmental processes.     

crinkle,a novel symbiotic mutant that affects the infection thread growth and alters the root hair,trichome, and seed development in Lotus japonicus

To elucidate the mechanisms involved in Rhizobium-legume symbiosis, we examined a novel symbiotic mutant, crinkle (Ljsym79), from the model legume Lotus japonicus. On nitrogen-starved medium, crinkle mutants inoculated with the symbiont bacterium Mesorhizobium loti MAFF 303099 showed severe nitrogen deficiency symptoms. This mutant was characterized by the production of many bumps and small, white, uninfected nodule-like structures. Few nodules were pale-pink and irregularly shaped with nitrogen-fixing bacteroids and expressing leghemoglobin mRNA. Morphological analysis of infected roots showed that nodulation in crinkle mutants is blocked at the stage of the infection process. Confocal microscopy and histological examination of crinkle nodules revealed that infection threads were arrested upon penetrating the epidermal cells. Starch accumulation in uninfected cells and undeveloped vascular bundles were also noted in crinkle nodules. Results suggest that the Crinkle gene controls the infection process that is crucial during the early stage of nodule organogenesis. Aside from the symbiotic phenotypes, crinkle mutants also developed morphological alterations, such as crinkly or wavy trichomes, short seedpods with aborted embryos, and swollen root hairs. crinkle is therefore required for symbiotic nodule development and for other aspects of plant development.     

New nodulation mutants responsible for infection thread development in Lotus japonicus

Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.     

Phytoene production utilizing the isoprenoid biosynthesis capacity of Thermococcus kodakarensis

Phytoene (C₄₀H₆₄) is an isoprenoid and a precursor of various carotenoids which are of industrial value. Archaea can be considered to exhibit a relatively large capacity to produce isoprenoids, as they are components of their membrane lipids. Here, we aimed to produce isoprenoids such as phytoene in the hyperthermophilic archaeon Thermococcus kodakarensis. T. kodakarensis harbors a prenyltransferase gene involved in the biosynthesis of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which are precursors of squalene and phytoene, respectively. However, homologs of squalene synthase and phytoene synthase, which catalyze their condensation reactions, are not found on the genome. Therefore, a squalene/phytoene synthase homolog from an acidothermophilic archaeon Sulfolobus acidocaldarius, Saci_1734, was introduced into the T. kodakarensis chromosome under the control of a strong promoter. Production of the Saci_1734 protein was confirmed in this strain, and the generation of phytoene was detected (0.08–0.75 mg L⁻¹ medium). We then carried out genetic engineering in order to increase the phytoene production yield. Disruption of an acetyl-CoA synthetase I gene involved in hydrolyzing acetyl-CoA, the precursor of phytoene, together with the introduction of a second copy of Saci_1734 led to a 3.4-fold enhancement in phytoene production.

     

Thermophilic microbial degradation of polyethylene succinate

This paper examined the biodegradability of a new aliphatic polyester, polyethylene succinate (PES), at a high incubation temperature of 50°C. The distribution and population of total colonies and of PES degrading micro organisms on polymer-emulsified agar plates were determined using the plate count and clear zone methods. The PES-decomposers were present in six of 10 soil samples and the total number ranged from 2.0×104 to 2.2×106 c.f.u./g of samples. Degrading microorganisms constituted between 20 and 80% of the total colonies on PES–agar plates. A single PES-degrading strain, TT96, was isolated and tested for its biodegrading capacity on PES powder and on other aliphatic polyesters: poly(beta-hydroxybutyrate) (PHB), polycaprolactone (PCL), poly(butylene succinate) (PBS), and poly(L-lactide) (PLA). Degraded films of PES and PBS were presented and compared using scanning electron microscopy. Strain TT96 was able to create clear zones on all the polymers used, except on PHB-agar plates. Liquid culture test after 2 weeks showed that TT96 completely degraded PCL powder but had very little activity on other samples. Scanning electron micrograph confirmed the microbial attack of TT96 on PES and PBS films. PES film surfaces were degraded more uniformly compared to PBS films which were decomposed only in some parts.     

Microbial degradation of poly-β-hydroxybutyrate using landfill soils

The population of poly-β-hydroxybutyrate-degrading microorganisms and the biodegradation of PHB in local landfill soils were examined in vitro and in vivo. Forty-two PHB-degraders consisting of 12 bacteria, 25 actinomycetes and 5 moulds were isolated. The total PHB-degraders averaged 4.7 × 10⁷ and 20 × 10⁴ colony forming units (cfu)/g for San Mateo wet and dry soils, respectively, and 2.3 × 10⁷ and 8.5 × 10⁴ cfu/g for Carmona wet and dry samples, respectively. The PHB-degraders formed 0–59% of the total microbial population in San Mateo and 8–42% in Carmona. Complete (100%) degradation of PHB powder was observed for Chryseomonas-27 and Aspergillus-39 on day 5 in shake flask culture and for Streptomyces-4 on day 7. Burial test in landfill soils showed a 90–91% weight loss of PHB film strips within four weeks; the weight loss of polypropylene film strips was up to 0.12% only. Scanning electron micrographs of degraded films revealed the attachment of microbial cells and fungal mycelium and spores on the surfaces. Holes and cavities were also noted due to the microbial degradation processes.