Identification and expression of the amphioxus Cks1 gene

A full-length Cks1 homologue gene, AmphiCks1, was identified in amphioxus, Branchiostoma belcheri tsingtauense. Sequence characteristics, phylogeny and patterns of expression during embryonic and larval development were established. The protein predicted from AmphiCks1 showed high sequence identity with vertebrate and invertebrate homologues. Protein structural studies and phylogenetic analysis suggested that Cks homologues are evolutionarily conserved. The AmphiCks1 transcript was detected in most early developmental stages by northern blotting and whole-mount in situ hybridization, suggesting a role for the gene in cell division. (c) 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

A transglutaminase from Chinese shrimp (Fenneropenaeus chinensis), full-length cDNA cloning, tissue localization and expression profile after challenge

Transglutaminase can catalyze the cross-linking reaction between soluble clotting protein molecules from the plasma for prevention of excess blood loss from a wound and obstructing micro-organisms from invading the wound in crustaceans. A novel transglutaminase (FcTG) gene was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 2972 bp, encoding 757 amino acids with a calculated molecular mass of 84.96 kDa and a theoretical isoelectric point of 5.61. FcTG contains a typical transglutaminase-like homologue (TGc domain: E-value = 1.94e-38). Three catalytic sites (Cys-324, His-391 and Asp-414) are present in this domain. The deduced amino acid sequence of FcTG showed high identity with black tiger shrimp TG, kuruma shrimp TG and crayfish TG. Transcripts of FcTG mRNA were mainly detected in gill, lymphoid organ and hemocytes by RT-PCR. RNA in situ hybridization further confirmed that FcTG was constitutively expressed in hemocytes both in the circulatory system and lymphoid organ. The variation of mRNA transcription level in hemocytes and lymphoid organ following injection of killed bacteria or infection with white spot syndrome virus (WSSV) was quantified by RT-PCR. The up-regulated expression of FcTG in shrimp lymphoid organ following injection of bacteria indicates that it is inducible and might be associated with bacterial challenge. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning and responsive expression to injury stimulus of a defender against cell death 1 (DAD1) gene from bay scallops Argopecten irradians

Apoptosis is an active process of cell death, which is an integral part of growth and development in multicellular organisms. The defender against cell death 1 (DAD1), the regulatory protein to inhibit the apoptosis process, was first cloned from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA end (RACE). The full-length cDNA of the A. irradians DAD1 was 607 bp, consist of a 5'-terminal untranslated region (UTR) of 63 bp, a 3'-terminal UTR of 205 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 339 bp. The deduced amino acid sequence of the A. irradians DAD1 showed 75.5% identity to Araneus ventricosus, 74.5% to Drosophila melanogaster, and 73.6% to Homo sapiens, Sus scrofa, Mesocricetus auratus, Rattus norvegicus and Mus musculus. Excluding the Saccharomyces cerevisiae DAD1 homologue, all animal DAD1 including A. irradians DAD1 homologue formed a subgroup and all plant DAD1 proteins formed another subgroup in the phylogenetic analysis. The A. irradians DAD1 was expressed in all examined tissues including adductor muscle, mantle, gills, digestive gland, gonad and hemolymph, suggesting that A. irradians DAD1 is expressed in most body tissues. Furthermore, the mRNA expression levels of A. irradians DAD1 gene of hemolymph were particularly high after injury, suggesting that the gene is responsive to injury stimuli.

Analysis of the expression and antioxidative property of a peroxiredoxin 6 from Scophthalmus maximus

Peroxiredoxins (Prxs) are a group of antioxidant proteins that protect cells from oxidative damage caused by various peroxides. To date, six different isoforms of peroxiredoxin (Prx1 to Prx6) have been identified, of which, Prx6 belongs to the 1-Cys Prx subfamily. Although Prx6 of several fish species have been reported at sequence level, there are very few documented studies on the potential function of fish Prx6. In this report, we describe the identification and analysis of a Prx6 homologue, SmPrx6, from turbot Scophthalmus maximus. The full length cDNA of SmPrx6 contains a 5'- untranslated region (UTR) of 60 bp, an open reading frame of 666 bp, and a 3'-UTR of 244 bp. The deduced amino acid sequence of SmPrx6 shares 81-87% overall identities with known fish Prx6. In silico analysis identified in SmPrx6 a conserved Prx6 catalytic motif, PVCTTE, and the catalytic triads putatively involved in peroxidase and phospholipase A2 activities. Expression of SmPrx6 was detected in most fish organs, with the highest expression levels found in blood and heart and the lowest level in spleen. Experimental challenges with bacterial pathogens and poly(I:C) upregulated SmPrx6 expression in liver and spleen in a manner that is dependent on the challenging agent and the tissue type. Treatment of cultured primary hepatocytes with H2O2 enhanced SmPrx6 expression in a dose-dependent manner. Recombinant SmPrx6 expressed in and purified from Escherichia coli exhibited thiol-dependent antioxidant activity and could protect cultured hepatocytes from H2O2-induced oxidative damage. Taken together, these results indicate that SmPrx6 is a Prx6 homologue with antioxidative property and is likely to be involved in both cellular maintenance and protective response during host immune defense against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.

Expression of Scophthalmus maximus CD83 correlates with bacterial infection and antigen stimulation

CD83 is a transmembrane glycoprotein of the immunoglobulin (Ig) superfamily and a surface marker for fully matured dendritic cells (DCs) in humans and mice. In teleosts, DC-like cells and their molecular markers are largely unknown. In this report, we described the identification and expressional analysis of a CD83 homologue, SmCD83, from turbot Scophthalmus maximus. The open reading frame of SmCD83 is 639 bp, which is preceded by a S'-untranslated region (UTR) of 87 bp and followed by a 3'-UTR of 1111 bp. The SmCD83 gene is 4716 bp in length, which contains five exons and four introns. The deduced amino acid sequence of SmCD83 shares 40-50% overall identities with the CD83 of several fish species. Like typical CD83, SmCD83 possesses an Ig-like extracellular domain, a transmembrane domain, and a cytoplasmic domain. The conserved disulfide bond-forming cysteine residues and the N-linked glycosylation sites that are preserved in CD83 are also found in SmCD83. Expressional analysis showed that constitutive expression of SmCD83 was high in gill, blood, spleen, muscle, and kidney and low in heart and liver. Bacterial infection and poly(I:C) treatment enhanced SmCD83 expression in kidney in time-dependent manners. Likewise, bacterial challenge caused significant induction of SmCD83 expression in cultured macrophages. Vaccination of turbot with a bacterin and a purified recombinant subunit vaccine-induced significant SmCD83 expression during the first week following vaccination. These results demonstrate that SmCD83 expression correlates with microbial challenge and antigen stimulation, which suggests the possibility that there may exist in turbot DC-like antigen-presenting cells that express SmCD83 upon activation by antigen uptake. In addition, these results also suggest that SmCD83 may serve as a marker for activated macrophages in turbot. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and molecular analysis of a stress-inducible Hsp70 from Sciaenops ocellatus

Hsp70 proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In this study, an Hsp70 homologue (SoHsp70) was identified from red drum Sciaenops ocellatus and analyzed at molecular level. The open reading frame of SoHsp70 is 1920 bp and intronless, with a 5'-untranslated region (UTR) of 399 bp and a 3'-UTR of 241 bp. The deduced amino acid sequence of SoHsp70 shares 84-92% overall identities with the Hsp70s of a number of fish species. In silico analysis identified in SoHsp70 three conserved Hsp70 domains involved in nucleotide and substrate binding. The coding sequence of SoHsp70 was subcloned into Escherichia coli, from which recombinant SoHsp70 was purified and, upon ATPase assay, found to exhibit apparent ATPase activity. Expressional analysis showed that constitutive expression of SoHsp70 was detectable in heart, liver, spleen, kidney, brain, blood, and gill. Experimental challenges with poly(I:C) and bacterial pathogens of Gram-positive and Gram-negative nature induced SoHsp70 expression in kidney to different levels. Stress-responsive analysis of SoHsp70 expression in primary cultures of red drum hepatocytes showed that acute heat shock treatment elicited a rapid induction of SoHsp70 expression which appeared after 10 min and 30 min of treatment. Exposure of hepatocytes separately to iron, copper, mercury, and hydrogen peroxide significantly unregulated SoHsp70 expression in time-dependent manners. Vaccination of red drum with a Streptococcus iniae bacterin was also found to induce SoHsp70 expression. Furthermore, recombinant SoHsp70 enhanced the immunoprotective effect of a subunit vaccine. Taken together, these results suggest that SoHsp70 is a stress-inducible protein that is likely to play a role in immunity and in coping with environmental and biological stresses. (C) 2010 Elsevier Ltd. All rights reserved.

Scophthalmus maximus cystatin B enhances head kidney macrophage-mediated bacterial killing

Cystatins form a large family of cysteine protease inhibitors found in a wide arrange of organisms. Studies have indicated that mammalian cystatins play important roles under both physiological and pathological conditions. However, much less is known about fish cystatins. In this report, we described the identification and analysis of a cystatin B homologue, SmCytB, from turbot Scophthalmus maximus. The open reading frame of SmCytB is 300 bp, which encodes a 99-residue protein that shares high levels of sequence identities with the cystatin B of a number of fish species and contains the conserved cysteine protease inhibitor motif of cystatin B. Constitutive expression of SmCytB is high in muscle, brain, heart and liver, and low in spleen. blood, gill and kidney. Bacterial infection upregulates SmCytB expression in kidney, spleen, liver and brain but not in muscle or heart. Functional analysis showed that recombinant SmCytB purified from Escherichia colt exhibits apparent cysteine protease inhibitor activity. Transient overexpression of SmCytB in head kidney macrophages enhances macrophage bactericidal activity probably through a nitric oxide-independent mechanism. These results indicate that SmCytB is involved in the immune defense of turbot against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and molecular analysis of a novel C-type lectin from Scophthalmus maximus

C-type lectins are calcium-dependent carbohydrate-binding proteins that play Important roles in innate immunity In this study, a C-type lectin homologue (SmLec1) was identified from turbot (Scophthalmus maximus) and analyzed at expression and functional levels. The open reading frame of SmLec1 is 504 bp, with a 5'-untranslated region (UTR) of 101 bp and a 3'-UTR of 164 bp The deduced amino acid sequence of SmLec1 shares 34%-38% overall identities with the C-type lectins of several fish species In silico analysis identified in SmLec1 conserved C-type lectin features, including a carbohydrate-recognition domain, four disulfide bond-forming cysteine residues, and the mannose-type carbohydrate-binding motif In addition, SmLec1 possesses a putative signal peptide sequence and is predicted to be localized in the extracellular. Expression of SmLec1 was highest in liver and responded positively to experimental challenges with fish pathogens Recombinant SmLec1 (rSmLec1) purified from yeast was able to agglutinate the Gram-negative fish pathogen Listonella anguillarum but not the Gram-positive pathogen Streptococcus uncle The agglutinating ability of rSmLec1 was abolished in the presence of mannose and ethylenediaminetetraacetic acid and by elevated temperature (65 degrees C) Further analysis showed that rSmLec1 could stimulate kidney lymphocyte proliferation and enhance the killing of bacterial pathogen by macrophages Taken together, these results suggest that SmLec1 is a unique mannose-binding C-type lectin that possesses apparent immunomodulating property and is likely to be involved in host defense against bacterial infection (C) 2010 Elsevier Ltd. All rights reserved

栉孔扇贝CfLITAF基因的克隆与表达分析

关于无脊椎动物中是否存在细胞因子一直都存在着异议。从细胞因子本身入手，运用细胞生物学和免疫学手段，已经在软体动物、节肢动物和棘皮动物等无脊椎动物中证实了高等动物细胞因子样活性物质的存在，然而利用分子生物学手段至今没有得到任何核苷酸或蛋白序列信息，而从与细胞因子相关的分子如调控其表达的转录因子入手来研究无脊椎动物中的细胞因子样物质的存在，或许会得到意想不到的惊喜。 最近从人的单核细胞系THP-1中分离纯化出一种新型的转录因子，它可以被LPS诱导表达，产生的蛋白在TNF-α的表达过程中起着非常重要的作用，因而命名为LPS-induced TNF-α factor（LITAF）。随后该基因在小鼠和鸡中也被克隆分离出来，其编码的蛋白已经证实在TNF-α的表达过程中起着不可或缺的作用。迄今为止，在无脊椎动物中还没有相关同源基因的报道。 本研究采用大规模EST测序方法，结合锚定PCR技术从栉孔扇贝体内克隆到了高等动物LITAF基因的同源基因CfLITAF的全长cDNA序列。该cDNA全长为1240bp，其中5' 非编码区（UTR）为112 bp；开放阅读框（Open Reading Frame, ORF）包括450 bp，编码149个氨基酸残基，该多肽的理论分子量为16.08 kDa，等电点为6.77；3' UTR为678bp，包含一个poly A尾巴。SMART结构域预测发现CfLITAF蛋白含有一个保守的LITAF结构域。实时定量PCR检测CfLITAF基因在栉孔扇贝主要免疫器官中的表达分布情况，发现在所检测的六种组织血细胞、外套膜、肌肉、心、腮、性腺中均能检测到CfLITAF基因转录本的不同丰度的表达。血淋巴和肌肉中的表达量相差不大，而在心、外套膜、腮和性腺中的表达量要高于血淋巴和肌肉中的表达量，分别是血中表达量的2.45、2.82、9.23和24.93倍。 为了验证其表达量是否会受到LPS的诱导，利用QRT-PCR（quantitative real time PCR）检测了CfLITAF基因在受到LPS和PGN刺激后在血细胞中的表达规律。结果如下：在LPS刺激后3h，CfLITAF的表达量显著上调，达到了原初水平的1.5倍，随着时间的延长，其表达量逐渐恢复到刺激前水平；在用PGN刺激血细胞后的9个小时内均没有检测到CfLITAF基因mRNA表达量的显著变化。实验中所用血细胞为同一批原代培养的栉孔扇贝血淋巴细胞，细胞活力通过台盼蓝拒染实验测定。 CfLITAF基因的克隆与表达分析，不仅为进一步认识栉孔扇贝的免疫防御机制提供了实验参考，更重要的是为无脊椎动物中细胞因子样物质的研究提供了一个分子水平上的线索，为无脊椎动物中细胞因子样物质的研究奠定了理论基础。

中国明对虾Toll样受体基因的克隆与表达分析

对虾病害在世界范围内的广泛传播，给水产养殖和沿海农村经济造成了重大损失。自1993 年对虾白斑病暴发以来，中国明对虾的养殖一直一蹶不振。引起对虾大规模死亡的原因是多方面的，其主要原因是养殖环境恶化、对虾种质退化和抗病力下降。因此，深入开展对虾免疫机制研究，并在此基础上寻找对虾疾病防治的有效方法，改良种质和培育抗病品系，已成为对虾养殖业走可持续发展之路的当务之急。 Toll 样受体（Toll-like receptors, TLRs）家族是进化保守的哺乳动物模式识别蛋白（pattern recognition receptors, PRR），在先天免疫系统中起着非常重要的作用。本研究采用同源克隆和RACE（rapid amplification of cDNA ends）技术从中国明对虾中克隆到Toll 样受体同源基因，并将其命名为FcToll。它全长4115 bp，3’UTR 包含16 个poly A 尾巴，开放阅读框编码931 个氨基酸的多肽。预测的该多肽包含典型的Toll 样受体结构，分为胞外区、跨膜区和胞内区。其中胞外区有信号肽，有16 个富含亮氨酸的重复序列eucine-rich repeats, LRR），并含有2个LRR-C 末端基序和2 个LRR-N 末端基序；跨膜区是23 个氨基酸的一次跨膜结构域；胞内区是含有139 个氨基酸的TIR 结构域（Toll/Interleukin-1R）。克隆 发现FcToll 的基因组结构包含5 个外显子和4 个内含子。系统发生分析揭示FcToll归属于“昆虫型”的无脊椎动物Toll 样受体家族。组织分布研究发现FcToll 在中国明对虾中是组成型表达的，在淋巴器官中表达量较显著。分别利用不同病原体刺激健康的中国明对虾，Real-time PCR 发现该基因在刺激后表达水平呈现不同的表达谱：灭活鳗弧菌（Vibrio anguillarum）注射后5 小时，该基因表达显著 上调；而WSSV（white spot syndrome virus）注射后该基因表达则迅速下调，感染后23 小时内其表达水平均低于对应时间点的对照组。这就表明FcToll 可能参与中国明对虾的先天免疫防御，尤其可能参与入侵弧菌的免疫应答。

Sources and preservation of organic matter in Plum Island salt marsh sediments (MA, USA): long-chain n-alkanes and stable carbon isotope compositions

Elemental (TOC, TN, C/N) and stable carbon isotopic (delta(13)C) compositions and n-alkane (nC(16-38)) concentrations were measured for Spartina alterniflora, a C-4 marsh grass, Typha latifolia, a C-3 marsh grass, and three sediment cores collected from middle and upper estuarine sites from the Plum Island salt marshes. Our results indicated that the organic matter preserved in the sediments was highly affected by the marsh plants that dominated the sampling sites. delta(13)C values of organic matter preserved in the upper fresh water site sediment were more negative (-23.0+/-0.3) as affected by the C-3 plants than the values of organic matter preserved in the sediments of middle (-18.9+/-0.8) and mud flat sites (-19.4+/-0.1) as influenced mainly by the C4 marsh plants. The distribution of n-alkanes measured in all sediments showed similar patterns as those determined in the marsh grasses S. alterniflora and T. latifolia, and nC(21) to nC(33) long-chain n-alkanes were the major compounds determined in all sediment samples. The strong odd-to-even carbon numbered n-alkane predominance was found in all three sediments and nC(29) was the most abundant homologue in all samples measured. Both delta(13)C compositions of organic matter and n-alkane distributions in these sediments indicate that the marsh plants could contribute significant amount of organic matter preserved in Plum Island salt marsh sediments. This suggests that salt marshes play an important role in the cycling of nutrients and organic carbon in the estuary and adjacent coastal waters. (C) 2003 Elsevier Ltd. All rights reserved.

Análise preliminar de um processo para identificação e alinhamento de seqüências homólogas para proteínas com estrutura resolvida.

O objetivo deste trabalho é apresentar e fazer uma avaliação preliminar de um processo alternativo, denominado Sequences Homologue to the Query (Structure-having) Sequence-SH2Q, para elaboração de alinhamentos múltiplos semelhantes à aqueles relatados no HSSP. O processo aqui apresentado baseia-se em programas de domínio público para busca em bases de dados de sequências -Blast (Altschul et al., 1990, 1997) e para alinhamento múltiplo de sequências -ClustalW (Thompson et al., 1994) O critério para avaliação do mesmo é o grau de similaridade entre as medidas de Entropia Relativa, quando comparadas com os mesmos valores relatados pelo HSSP.

RinA controls phage-mediated packaging and transfer of virulence genes in gram-positive bacteria

Phage-mediated transfer of microbial genetic elements plays a crucial role in bacterial life style and evolution. In this study, we identify the RinA family of phage-encoded proteins as activators required for transcription of the late operon in a large group of temperate staphylococcal phages. RinA binds to a tightly regulated promoter region, situated upstream of the terS gene, that controls expression of the morphogenetic and lysis modules of the phage, activating their transcription. As expected, rinA deletion eliminated formation of functional phage particles and significantly decreased the transfer of phage and pathogenicity island encoded virulence factors. A genetic analysis of the late promoter region showed that a fragment of 272 bp contains both the promoter and the region necessary for activation by RinA. In addition, we demonstrated that RinA is the only phage-encoded protein required for the activation of this promoter region. This region was shown to be divergent among different phages. Consequently, phages with divergent promoter regions carried allelic variants of the RinA protein, which specifically recognize its own promoter sequence. Finally, most Gram-postive bacteria carry bacteriophages encoding RinA homologue proteins. Characterization of several of these proteins demonstrated that control by RinA of the phage-mediated packaging and transfer of virulence factor is a conserved mechanism regulating horizontal gene transfer.

The identification and characterisation of Rab4 interacting proteins

Rab4 is a member of the Rab superfamily of small GTPases. It is localized to the early sorting endosome and plays a role in regulating the transport from this compartment to the recycling and degradative pathways. In order to further our understanding of the role Rab4 plays in endocytosis, a yeast two-hybrid screen was performed to identify putative Rab4 effectors. A constitutively active mutant of Rab4, Rab4Q67L, when used as bait to screen a HeLa cDNA library, identified a novel 80kDa protein that interacted with Rab4-GTP. This protein was called Rab Coupling Protein (RCP). RCP interacts preferentially with the GTP-bound form of Rab4. Subsequent work demonstrated that RCP also interacts with Rab11, and that this interaction is not nucleotide-depenedent. RCP is predominantly membrane-bound and localised to the perinuclear recycling compartment. Expression of a truncation mutant of RCP, that contains the Rab binding domain, in HeLa cells, results in the formation of an extensive tubular network that can be labelled with transferrin. These tubules are derived from the recycling compartment since they are inaccessible to transferrin when the ligand is internalised at 18oC. The truncation mutant-induced morphology can be rescued by overexpression of active Rab11, but not active Rab4. This suggests that RCP functions between Rab4 and Rab11 in the receptor recycling pathway, and may act as a ‘molecular bridge’ between these two sequentially acting small GTPases. Quantitative assays demonstrated that overexpression of the truncation mutant results in a dramatic inhibition in the rate of receptor recycling. Database analysis revealed that RCP belongs to a family of Rab interacting proteins, each characterised by a carboxy-terminal coiled-coil domain and an amino-terminal phospholipid-binding domain. KIAA0941, an RCP homologue, interacts with Rab11, but not with Rab4. Overexpression of its Rab binding domain also results in a tubular network, however, this tubulation cannot be rescued by active Rab11.

Investigation of polyhydroxyalkanoate production by an activated sludge microbial consortium treating artificial dairy wastewater

Petrochemical plastics/polymers are a common feature of day to day living as they occur in packaging, furniture, mobile phones, computers, construction equipment etc. However, these materials are produced from non-renewable materials and are resistant to microbial degradation in the environment. Considerable research has therefore been carried out into the production of sustainable, biodegradable polymers, amenable to microbial catabolism to CO2 and H2O. A key group of microbial polyesters, widely considered as optimal replacement polymers, are the Polyhydroxyalkaonates (PHAs). Primary research in this area has focused on using recombinant pure cultures to optimise PHA yields, however, despite considerable success, the high costs of pure culture fermentation have thus far hindered the commercial viability of PHAs thus produced. In more recent years work has begun to focus on mixed cultures for the optimisation of PHA production, with waste incorporations offering optimal production cost reductions. The scale of dairy processing in Ireland, and the high organic load wastewaters generated, represent an excellent potential substrate for bioconversion to PHAs in a mixed culture system. The current study sought to investigate the potential for such bioconversion in a laboratory scale biological system and to establish key operational and microbial characteristics of same. Two sequencing batch reactors were set up and operated along the lines of an enhanced biological phosphate removal (EBPR) system, which has PHA accumulation as a key step within repeated rounds of anaerobic/aerobic cycling. Influents to the reactors varied only in the carbon sources provided. Reactor 1 received artificial wastewater with acetate alone, which is known to be readily converted to PHA in the anaerobic step of EBPR. Reactor 2 wastewater influent contained acetate and skim milk to imitate a dairy processing effluent. Chemical monitoring of nutrient remediation within the reactors as continuously applied and EBPR consistent performances observed. Qualitative analysis of the sludge was carried out using fluorescence microscopy with Nile Blue A lipophillic stain and PHA production was confirmed in both reactors. Quantitative analysis via HPLC detection of crotonic acid derivatives revealed the fluorescence to be short chain length Polyhydroxybutyrate, with biomass dry weight accumulations of 11% and 13% being observed in reactors 1 and 2, respectively. Gas Chromatography-Mass Spectrometry for medium chain length methyl ester derivatives revealed the presence of hydroxyoctanoic, -decanoic and -dodecanoic acids in reactor 1. Similar analyses in reactor 2 revealed monomers of 3-hydroxydodecenoic and 3-hydroxytetradecanoic acids. Investigation of the microbial ecology of both reactors as conducted in an attempt to identify key species potentially contributing to reactor performance. Culture dependent investigations indicated that quite different communities were present in both reactors. Reactor 1 isolates demonstrated the following species distributions Pseudomonas (82%), Delftia acidovorans (3%), Acinetobacter sp. (5%) Aminobacter sp., (3%) Bacillus sp. (3%), Thauera sp., (3%) and Cytophaga sp. (3%). Relative species distributions among reactor 2 profiled isolates were more evenly distributed between Pseudoxanthomonas (32%), Thauera sp (24%), Acinetobacter (24%), Citrobacter sp (8%), Lactococcus lactis (5%), Lysinibacillus (5%) and Elizabethkingia (2%). In both reactors Gammaproteobacteria dominated the cultured isolates. Culture independent 16S rRNA gene analyses revealed differing profiles for both reactors. Reactor 1 clone distribution was as follows; Zooglea resiniphila (83%), Zooglea oryzae (2%), Pedobacter composti (5%), Neissericeae sp. (2%) Rhodobacter sp. (2%), Runella defluvii (3%) and Streptococcus sp. (3%). RFLP based species distribution among the reactor 2 clones was as follows; Runella defluvii (50%), Zoogloea oryzae (20%), Flavobacterium sp. (9%), Simplicispira sp. (6%), Uncultured Sphingobacteria sp. (6%), Arcicella (6%) and Leadbetterella bysophila (3%). Betaproteobacteria dominated the 16S rRNA gene clones identified in both reactors. FISH analysis with Nile Blue dual staining resolved these divergent findings, identifying the Betaproteobacteria as dominant PHA accumulators within the reactor sludges, although species/strain specific allocations could not be made. GC analysis of the sludge had indicated the presence of both medium chain length as well short chain length PHAs accumulating in both reactors. In addition the cultured isolates from the reactors had been identified previously as mcl and scl PHA producers, respectively. Characterisations of the PHA monomer profiles of the individual isolates were therefore performed to screen for potential novel scl-mcl PHAs. Nitrogen limitation driven PHA accumulation in E2 minimal media revealed a greater propensity among isoates for mcl-pHA production. HPLC analysis indicated that PHB production was not a major feature of the reactor isolates and this was supported by the low presence of scl phaC1 genes among PCR screened isolates. A high percentage distribution of phaC2 mcl-PHA synthase genes was recorded, with the majority sharing high percentage homology with class II synthases from Pseudomonas sp. The common presence of a phaC2 homologue was not reflected in the production of a common polymer. Considerable variation was noted in both the monomer composition and ratios following GC analysis. While co-polymer production could not be demonstrated, potentially novel synthase substrate specificities were noted which could be exploited further in the future.