Rapid spectrofluorometric screening of poly-hydroxyalkanoate-producing bacteria from microbial mats.

Microbial mat ecosystems are characterized by both seasonal and diel fluctuations in several physicochemical variables, so that resident microorganisms must frequently adapt to the changing conditions of their environment. It has been pointed out that, under stress conditions, bacterial cells with higher contents of poly-hydroxyalkanoates (PHA) survive longer than those with lower PHA content. In the present study, PHA-producing strains from Ebro Delta microbial mats were selected using the Nile red dying technique and the relative accumulation of PHA was monitored during further laboratory cultivation. The number of heterotrophic isolates in trypticase soy agar (TSA) was ca. 107 colony-forming units/g microbial mat. Of these, 100 randomly chosen colonies were replicated on mineral salt agar limited in nitrogen, and Nile red was added to the medium to detect PHA. Orange fluorescence, produced upon binding of the dye to polymer granules in the cell, was detected in approximately 10% of the replicated heterotrophic isolates. The kinetics of PHA accumulation in Pseudomonas putida, and P. oleovorans were compared with those of several of the environmental isolates spectrofluorometry. PHA accumulation, measured as relative fluorescence intensity, resulted in a steady-state concentration after 48 h of incubation in all strains assayed. At 72 h, the maximum fluorescence intensity of each strain incubated with glucose and fructose was usually similar. MAT-28 strain accumulated more PHA than the other isolates. The results show that data obtained from environmental isolates can highly improve studies based on modeling-simulation programs, and that microbial mats constitute an excellent source for the isolation of PHA-producing strains with industrial applications.     

A repressor protein,PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA

Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.     

红树林土壤中产聚羟基脂肪酸酯细菌的分离及其评估

[背景] 细菌能通过合成聚羟基脂肪酸酯（Polyhydroxyalknoates,PHA）在细胞内储存物质和能量,提高对环境的适应能力。在红树林中,由于土壤周期性受海水浸没,形成营养物质种类丰富和含量波动大的特殊生境,为细菌进化出特殊的PHA合成途径提供了条件。[目标] 为了增加对红树林产PHA细菌资源的了解,获得产PHA细菌,使用纯培养方法分离和鉴定细菌,并评估菌株的产PHA能力。[方法] 采集红树植物海桑根系和红树滩涂土壤样品,连续5周培养、分离纯化获得细菌菌株;通过16S rRNA基因相似性及系统进化分析鉴定细菌分类地位,利用PHA合成酶基因（phaC）鉴定细菌合成PHA的能力;通过基因组草图测序,分析细菌的phaC基因种类、代谢通路及系统进化关系;通过气相色谱分析细菌产PHA的累积量及组成。[结果] 从红树林土壤样品中分离得到97株细菌,其中13株带有phaC基因,包括坚强芽孢杆菌（Cytobacillus firmus）、弯曲芽孢杆菌（Bacillus flexus）、除烃海杆菌（Marinobacter hydrocarbonoclasticus）和酯香微杆菌（Microbacterium esteraromaticum）。B. flexus MN15-19以丙酮酸盐为碳源,可累积细胞干重11%的PHA,同时具有固碳功能的还原性三羧酸循环通路,有开发成为固碳产PHA工程菌株的潜力。酯香微杆菌可产PHA,但是其phaC基因结构特殊,基因组注释未能识别出任何已知phaC基因。[结论] 研究发现红树林土壤可培养细菌中存在未知的PHA合成途径,说明红树林生态系统中的细菌具有资源挖掘的重要价值。     

生产聚羟基脂肪酸酯的微生物细胞工厂

聚羟基脂肪酸酯(PHA)是一类由微生物合成的、生物可再生、生物可降解、具有多种材料学性能的高分子聚合物,在很多领域有着广泛的应用前景。以下从辅酶工程、代谢工程、微氧生产等方面综述了微生物法生产PHA的研究进展,并对利用PHA合成基因提高基因工程菌的代谢潜能进行了讨论。     

Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants - a review

The increasing effect of non-degradable plastic wastes is a growing concern. Polyhydroxyalkanoates (PHAs), macromolecule-polyesters naturally produced by many species of microorganisms, are being considered as a replacement for conventional plastics. Unlike petroleum-derived plastics that take several decades to degrade, PHAs can be completely bio-degraded within a year by a variety of microorganisms. This biodegradation results in carbon dioxide and water, which return to the environment. Attempts based on various methods have been undertaken for mass production of PHAs. Promising strategies involve genetic engineering of microorganisms and plants to introduce production pathways. This challenge requires the expression of several genes along with optimization of PHA synthesis in the host. Although excellent progress has been made in recombinant hosts, the barriers to obtaining high quantities of PHA at low cost still remain to be solved. The commercially viable production of PHA in crops, however, appears to be a realistic goal for the future.     

The Ralstonia eutropha PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the Ralstonia eutropha PhaR protein on the regulation of PhaP accumulation. R. eutropha strains with phaR, phaC, and/or phaP deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the phaC deletion strain accumulated no PhaP, as expected. In contrast, both the phaR and the phaR phaC deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the R. eutropha phaR, phaP, and PHA biosynthesis (phaCAB) genes into a heterologous system, Escherichia coli, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of phaR caused a decrease in PHA yields, and a phaR phaP deletion strain exhibited a more severe PHA defect than a phaP deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.     

Biodegradation of polyhydroxyalkanoic acids

Stimulated by the commercial availability of bacteriologically produced polyesters such as poly[(R)-3-hydroxybutyric acid], and encouraged by the discovery of new constituents of polyhydroxyalkanoic acids (PHA), a considerable body of knowledge on the metabolism of PHA in microorganisms has accumulated. The objective of this essay is to give an overview on the biodegradation of PHA. The following topics are discussed: (i) general considerations of PHA degradation, (ii) methods for identification and isolation of PHA-degrading microorganisms, (iii) characterization of PHA-degrading microorganisms, (iv) biochemical properties of PHA depolymerases, (v) mechanisms of PHA hydrolysis, (vi) regulation of PHA depolymerase synthesis, (vii) molecular biology of PHA depolymerases, (viii) influence of the physicochemical properties of PHA on its biodegradability, (ix) degradation of polyesters related to PHA, (x) biotechnological aspects of PHA and PHA depolymerases. Received: 28 May 1996 / Received revision: 5 August 1996 / Accepted: 12 August 1996     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

Involvement of polyhydroxyalkanoates in stress resistance of microbial cells: Biotechnological consequences and applications

Polyhydroxyalkanoates (PHA) are polyesters accumulated by numerous prokaryotes as storage materials; they attract attention as “green” alternatives to petrochemical plastics. Recent research has demonstrated that their biological role goes beyong their storage function, since they presence in cytoplasm enhances stress resistance of microorganisms. To address these complex functions, this review summarizes the protective effects of PHA for microrganisms; the involvement of PHA in stress resistance is discussed also from a praxis-oriented perspective. The review discourses the controlled application of stress to improve PHA productivity. Also the manifold advantages of using stress adapted microbes - extremophiles as PHA producers are discussed.     

Polyhydroxyalkanoate production by antarctic soil bacteria isolated from Casey Station and Signy Island

Polyhydroxyalkanoate (PHA) is a family of biopolymers produced by some bacteria and is accumulated intracellularly as carbon and energy storage material. Fifteen PHA-producing bacterial strains were identified from bacteria isolated from Antarctic soils collected around Casey Station (66°17'S, 110°32'E) and Signy Island (60°45'S, 45°36'W). Screening for PHA production was carried out by incubating the isolates in PHA production medium supplemented with 0.5% (w/v) sodium octanoate or glucose. 16S rRNA gene sequence analysis revealed that the isolated PHA-producing strains were mainly Pseudomonas spp. and a few were Janthinobacterium spp. All the isolated Pseudomonas strains were able to produce medium-chain-length (mcl) PHA using fatty acids as carbon source, while some could also produce mcl-PHA by using glucose. The Janthinobacterium strains could only utilize glucose to produce polyhydroxybutyrate (PHB). A Pseudomonas isolate, UMAB-40, accumulated PHA up to 48% cell dry mass when utilizing fatty acids as carbon source. This high accumulation occurred at between 5°C and 20°C, then decreased with increasing temperatures. Highly unsaturated mcl-PHA was produced by UMAB-40 from glucose. Such characteristics may be associated with the ability of UMAB-40 to survive in the cold.     

14 C-acetate incorporation and nucleic acid synthesis in human lymphocytes stimulated by PHA and tuberculin in vitro

Studies on the timing of incorporation of labeled acetate in relationship to other cellular events in phytohemagglutinin (PHA)-treated lymphocytes have suggested that acetylation of nuclear histones may constitute an important regulatory mechanism for gene activation. In the present investigation, it was shown that PHA stimulation of lymphocytes from a tuberculin-positive patient caused an early increased incorporation of ¹⁴C-acetate prior to RNA and DNA synthesis. Lymphocytes from the same patient, however, repeatedly showed no increased incorporation of ¹⁴C-acetate following exposure to the sensitizing antigen, tuberculin (PPD), even though RNA and DNA synthesis were markedly stimulated. These results suggest that regulatory mechanisms of DNA template activity other than acetylation may be operative in sensitized lymphocytes responding to specific antigen. One possible explanation for the differences in ¹⁴C-acetate incorporation is that the increased uptake of acetate exhibited by PHA-treated cells is an effect related to nonspecific membrane changes caused by the PHA. If this is the case, then template regulation in PHA and antigen-stimulated lymphocytes may be achieved via similar but yet to be defined mechanisms.     

Post-translational control of the long and winding road to cholesterol

The synthesis of cholesterol requires more than 20 enzymes, many of which are intricately regulated. Post-translational control of these enzymes provides a rapid means for modifying flux through the pathway. So far, several enzymes have been shown to be rapidly degraded through the ubiquitin–proteasome pathway in response to cholesterol and other sterol intermediates. Additionally, several enzymes have their activity altered through phosphorylation mechanisms. Most work has focused on the two rate-limiting enzymes: 3-hydroxy-3-methylglutaryl CoA reductase and squalene monooxygenase. Here, we review current literature in the area to define some common themes in the regulation of the entire cholesterol synthesis pathway. We highlight the rich variety of inputs controlling each enzyme, discuss the interplay that exists between regulatory mechanisms, and summarize findings that reveal an intricately coordinated network of regulation along the cholesterol synthesis pathway. We provide a roadmap for future research into the post-translational control of cholesterol synthesis, and no doubt the road ahead will reveal further twists and turns for this fascinating pathway crucial for human health and disease.     

PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail)

Protein phosphatase 2A (PP2A), a major phospho-serine/threonine phosphatase, is conserved throughout eukaryotes. It dephosphorylates a plethora of cellular proteins, including kinases and other signaling molecules involved in cell division, gene regulation, protein synthesis and cytoskeleton organization. PP2A enzymes typically exist as heterotrimers comprising catalytic C-, structural A- and regulatory B-type subunits. The B-type subunits function as targeting and substrate-specificity factors; hence, holoenzyme assembly with the appropriate B-type subunit is crucial for PP2A specificity and regulation. Recently, several biochemical and structural determinants have been described that affect PP2A holoenzyme assembly. Moreover, the effects of specific post-translational modifications of the C-terminal tail of the catalytic subunit indicate that a 'code' might regulate dynamic exchange of regulatory B-type subunits, thus affecting the specificity of PP2A.     

Rapid genetic characterization of poly(hydroxyalkanoate) synthase and its applications

Microorganisms containing short-chain-length (scl-) or medium-chain-length (mcl-) poly(hydroxyalkanoates) (PHAs) are commonly screened by applying rapid staining methods using lipophilic reagents. These methods provide powerful means for general screening of organisms actively producing and accumulating PHAs. The Southern blot hybridization method additionally allows the identification of potential PHA-producing microorganisms. Polymerase chain reaction (PCR)-based detection methods further afford rapid and sensitive means to screen for PHA biosynthesis genes. Specific PCR assays had been developed for the simultaneous or individual detection of the class II mcl-PHA synthase genes of Pseudomonas. The amplicons (approximately 0.54 kb) can be directly sequenced or used as probes for hybridization studies. The sequence information can further be used to initiate chromosome walking for an eventual cloning of the complete PHA biosynthesis operon. In addition, the amplification pattern and sequence data can be used to differentiate subgroups of organisms, as demonstrated for P. corrugata and P. mediterranea. Other researchers reported PCR methods for the detection of scl-PHA synthase genes and those of Bacillus spp., thus greatly expanding the types of PHA synthase gene and the organisms that can be characterized by this approach. The vast sequence information obtainable through PCR-based studies of various PHA synthase operons should facilitate the identification or construction of new PHA synthases capable of synthesizing novel PHAs.     

Recent Progress in Polyhydroxyalkanoates‐Based Copolymers for Biomedical Applications

In recent years, naturally biodegradable polyhydroxyalkanoate (PHA) monopolymers have become focus of public attentions due to their good biocompatibility. However, due to its poor mechanical properties, high production costs, and limited functionality, its applications in materials, energy, and biomedical applications are greatly limited. In recent years, researchers have found that PHA copolymers have better thermal properties, mechanical processability, and physicochemical properties relative to their homopolymers. This review summarizes the synthesis of PHA copolymers by the latest biosynthetic and chemical modification methods. The modified PHA copolymer could greatly reduce the production cost with elevated mechanical or physicochemical properties, which can further meet the practical needs of various fields. This review further summarizes the broad applications of modified PHA copolymers in biomedical applications, which might shred lights on their commercial applications.     

Screening for MCL-PHA-Producing Fluorescent Pseudomonads and Comparison of MCL-PHA Production Under Iso-osmotic Conditions Induced by PEG and NaCl

The medium chain length polyhydroxyalkanoates (MCL-PHA) have attracted much attention from academic and industrial communities for their interesting applications in medical field. The aim of this study was to screen high MCL-PHA-producing fluorescent pseudomonads, and to compare the effect of osmotic stress generated by NaCl (ionic) and polyethylene glycol (PEG, non-ionic inert polymer) on PHA production. A total of 50 fluorescent pseudomonads isolated from rhizospheric soil were screened for PHA production by Sudan Black staining. Out of all the PHA-producing isolates only five were MCL-PHA producers as detected by MCL-PCR. Isolate Bar1 identified as Pseudomonas fluorescens by 16S rRNA gene sequencing was selected for further analysis due to its high MCL-PHA production ability. The iso-osmotic stress generated by NaCl and PEG-6000 showed 5.75- and 3.19-fold enhanced production of PHA at ?2 bar osmotic potential, over control (0 bar), respectively. There was 1.8-fold enhanced production of PHA at ?2 bar osmotic stress induced by NaCl over PEG. PEG reduces availability of water to microorganisms without reducing exogenously provided nutrients which appear to be responsible for its down performance over NaCl. The FTIR analysis of PHA sample purified from cells showed strong marker bands near 1742, 2870, 1170, 1099, and 2926 cm^?1, corresponding to MCL-PHA. The study reported that supplementation of NaCl (electrolyte) in growth media enhances the production of MCL-PHA which can be very useful for its industrial production.     

Quorum sensing regulation in <Emphasis Type="Italic">Pseudomonas</Emphasis>

Expression of many bacterial genes is regulated in a cell density-dependent manner via small signal molecules known as autoinducers; this type of regulation is termed quorum sensing (QS). The QS systems that employ N-acyl-homoserine lactones (HSLs) are best un derstood in Gram-negative bacteria. QS regulates expression of various genes, including the genes responsible for the production of virulence factors, synthesis of exoenzymes and antibiotics, antagonistic properties of bacteria, etc. The QS systems of the genus Pseudomonas are linked to other global regulatory networks of the cell, and their functions are controlled by numerous additional regulatory factors. Such regulators and the QS systems together form an intricate multifactorial cascade regulatory network. The review considers the QS systems of several Pseudomonas species, their interaction with other regulatory systems, and their roles in the regulation of cell processes.     

厌氧发酵液中溶解性有机物对活性污泥合成聚羟基脂肪酸酯影响的研究进展

利用活性污泥微生物将剩余污泥发酵液中的挥发性脂肪酸(Volatile fatty acids,VFAs)转化为聚羟基脂肪酸酯(Polyhydroxyalkanoates,PHA)是目前环境生物技术领域的研究热点.但针对发酵液中非VFAs物质(主要是溶解性有机物,Dissolved organic matter,DOM)...