Strategies of synthesis of aromatic polyketides using cyclohexa-1,4-and-1,3-dienes in Alder Rickett reactions

Cyclohexa-1, 4-dienes with appropriate substituents, obtained by birch reduction of the substituted benzene, react directly with derivatives of propiolic ester or aldchyde to yield aromatic polyketides. The following compounds have been synthesized; mycophenolic acid, nidulol methyl other, the root growth hormone 3, 5-dihydroxy-2-formyl-4-mythyl-benzoic acid, antibiotic DB 2073, the macrocyclic lactones lasiodiplodin and dihydrozearalenone and the biphenyl derivatives alternario and altenusin. Polyketide anthraquinones can be made from naphthoquinone precursors.

Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp. carotovora (Ecc) is a Gram-negative enterobacterium that causes soft-rot in potato and other crops. The main virulence determinants, the extracellular plant cell wall -degrading enzymes (PCWDEs), lead to plant tissue maceration. In order to establish a successful infection the production of PCWDEs are controlled by a complex regulatory network, including both specific and global activators and repressors. One of the most important virulence regulation systems in Ecc is mediated by quorum sensing (QS), which is a population density -dependent cell-to-cell communication mechanism used by many Gram-negative bacteria. In these bacteria N-acylhomoserine lactones (AHSL), act as diffusible signaling molecules enabling communication between bacterial cells. The AHSLs are structurally diverse and differ in their acyl chain length. This gives the bacteria signaling specificity and enables the recognition and communication within its own species. In order to detect and respond to the AHSLs the bacteria use QS regulators, LuxR-type proteins. The aim of this study was to get a deeper understanding of the Ecc QS system. In the first part of the study we showed that even different strains of Ecc use different dialects and of physiological concentrations, only the cognate AHSL with the correct acyl chain is recognized as a signal that can switch on virulence genes. The molecular basis of the substrate specificity of the AHSL synthase ExpI was investigated in order to recognize the acyl chain length specificity determinants of distinct AHSL synthases. Several critical residues that define the size of the substrate-binding pocket were identified. We demonstrated that in the ExpISCC1 mutations M127T and F69L are sufficient to change the N-3-oxohexanoyl-L-homoserine lactone producing ExpISCC1 to an N-3-oxooctanoyl-L-homoserine lactone (3-oxo-C8-HSL) producing enzyme. In the second study the means of sensing specificity and response to the AHSL signaling molecule were investigated. We demonstrated that the AHSL receptor ExpR1 of Ecc strain SCC3193 has strict specificity for the cognate AHSL 3-oxo-C8-HSL. In addition we identified a second AHSL receptor ExpR2 with a novel property to sense AHSLs with different acyl chain lengths. In the absence of AHSLs ExpR1 and ExpR2 were found to act synergistically to repress the virulence gene expression. This repression was shown to be released by addition of AHSLs and appears to be largely mediated by the global negative regulator RsmA. In the third study random transposon mutagenesis was used to widen the knowledge of the Ecc QS regulon. Two new QS-controlled target genes, encoding a DNA-binding regulator Hor and a plant ferredoxin-like protein FerE, were identified. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and as expression of PCWDE genes mediated by the RsmA repressor. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production, while FerE was shown to contribute to oxidative stress tolerance and in planta fitness of the bacteria. In addition our results suggest that QS is central to the control of oxidative stress tolerance in Ecc. In conclusion, these results indicate that Ecc strain SCC3193 is able to react and respond both to the cognate AHSL signal and the signals produced by other bacterial species, in order to control a wide variety of functions in the plant pathogen Ecc.

Evolutionary Significance of Self-acylation Property in Acyl Carrier Proteins

Acyl carrier protein is an integral component of many cellular metabolic processes. A number of studies have reported self-acylation behavior in acyl carrier proteins. Although AM exhibit high levels of similarity in their primary and tertiary structures, self-acylation behavior is restricted to only some ACPs that can be classified into two major families based on their function. The first family of ACPs is involved in polyketide biosynthesis, whereas the second family participates in fatty acid synthesis. Facilitated by the growing number of genome sequences available for analyses, large-scale phylogenetic studies were used in these studies to uncover as to how self-acylation behavior of acyl carrier proteins is linked with the evolution of metabolic pathways in organisms. These studies show that self-acylation behavior in acyl carrier proteins was lost during the course of evolution, with certain organisms and organelles viz. plastids, retaining it for specified functions. (C) 2009 IUBMB IUBMB Life, 61(8): 853-859, 2009

Self-acylation properties of type II fatty acid biosynthesis acyl carrier protein

Acyl carrier protein (ACIP) plays a central role in many metabolic processes inside the cell, and almost 4% of the total enzymes inside the cell require it as a cofactor. Here, we report self-acylation properties in ACPs from Plasmodium falciparum and Brassica napus that are essential components of type II fatty acid biosynthesis (FAS II), disproving the existing notion that this phenomenon is restricted only to ACPs involved in polyketide biosynthesis. We also provide strong evidence to suggest that catalytic self-acylation is intrinsic to the individual ACP. Mutational analysis of these ACPs revealed the key residue(s) involved in this phenomenon. We also demonstrate that these FAS 11 ACPs exhibit a high degree of selectivity for self-acylation employing only dicarboxylic acids as substrates. A plausible mechanism for the self-acylation reaction is also proposed.

A total synthesis of the epoxyquinone based antifungal natural product (±)-ambuic acid

A total synthesis of the recently isolated polyketide natural product (+/-)-ambuic acid has been accomplished from the readily available Diels-Alder adduct of cyclopentadiene and 2-allyl-p-benzoquinone through a simple sequence with sound stereocontrol. (c) 2005 Elsevier Ltd. All rights reserved.

Structural and Biochemical Bases for the Redox Sensitivity of Mycobacterium tuberculosis RslA

An effective transcriptional response to redox stimuli is of particular importance for Mycobacterium tuberculosis, as it adapts to the environment of host alveoli and macrophages. The M. tuberculosis a factor sigma(L) regulates the expression of genes involved in cell-wall and polyketide syntheses. sigma(L) interacts with the cytosolic anti-sigma domain of a membrane-associated protein, RslA. Here we demonstrate that RslA binds Zn2+ and can sequester sigma(L) in a reducing environment. In response to an oxidative stimulus, proximal cysteines in the CXXC motif of RslA form a disulfide bond, releasing bound Zn2+. This results in a substantial rearrangement of the sigma(L)/RslA complex, leading to an 8-fold decrease in the affinity of RslA for sigma(L). The crystal structure of the -35-element recognition domain of sigma(L), sigma(L)(4), bound to RslA reveals that RslA inactivates sigma(L) by sterically occluding promoter DNA and RNpolymerase binding sites. The crystal structure further reveals that the cysteine residues that coordinate Zn2+ in RslA are solvent exposed in the complex, thus providing a structural basis for the redox sensitivity of RslA. The biophysical parameters of sigma(L)/RslA interactions provide a template for understanding how variations in the rate of Zn2+ release and associated conformational changes could regulate the activity of a Zn2+-associated anti-sigma factor. (C) 2010 Elsevier Ltd. All rights reserved.

Uterine cervix nitric oxide and human papillomavirus infection in women

Most women acquire genital high risk human papillomavirus (HPV) infection during their lifetime, but seldom the infection persists and leads to cervical cancer. However, currently it is not possible to identify the women who will develop HPV mediated cervical cancer and this often results to large scale follow-up and overtreatment of the likely spontaneously regressing infection. Thus, it is important to obtain more information on the course of HPV and find markers that could help to identify HPV infected women in risk for progression of cervical lesions and ultimately cancer. Nitric oxide is a free radical gas that takes part both in immune responses and carcinogenesis. Nitric oxide is produced also by cervical cells and therefore, it is possible that cervical nitric oxide could affect also HPV infection. In the present study, including 801 women from the University of Helsinki between years of 2006 and 2011, association between HPV and cervical nitric oxide was evaluated. The levels of nitric oxide were measured as its metabolites nitrate and nitirite (NOx) by spectrophotometry and the expression of nitric oxide producing enzymes endothelial and inducible synthases (eNOS, iNOS) by Western blotting. Women infected with HPV had two-times higher cervical fluid NOx levels compared with non-infected ones. The expression levels of both eNOS and iNOS were higher in HPV-infected women compared with non-infected. Another sexually transmitted disease Chlamydia trachomatis that is an independent risk factor for cervical cancer was also accompanied with elevated NOx levels, whereas vaginal infections, bacterial vaginosis and candida, did not have any effect on NOx levels. The meaning of the elevated HPV related cervical nitric oxide was evaluated in a 12 months follow-up study. It was revealed that high baseline cervical fluid NOx levels favored HPV persistence with OR 4.1. However, low sensitivity (33%) and high false negative rate (67%) restrict the clinical use of the current NOx test. This study indicated that nitric oxide favors HPV persistence and thus it seems to be one of the cofactor associated with a risk of carcinogenesis.

Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity

2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4 A resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coil CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes. (C) 2010 Elsevier Inc. All rights reserved.

Enantioselective Formal Synthesis of Palmerolide A

Enantioselective formal synthesis of macrolactone palmerolide A, a polyketide marine natural product, is described. Key strategies in the synthesis include the oxidative furan ring-opening of a chiral furyl carbinol for the installation of the 1,4-dienol core and a Jung nonaldol-aldol reaction for the dienamide core.

Phylogenetic Predictions on Grids

A phylogenetic or evolutionary tree is constructed from a set of species or DNA sequences and depicts the relatedness between the sequences. Predictions of future sequences in a phylogenetic tree are important for a variety of applications including drug discovery, pharmaceutical research and disease control. In this work, we predict future DNA sequences in a phylogenetic tree using cellular automata. Cellular automata are used for modeling neighbor-dependent mutations from an ancestor to a progeny in a branch of the phylogenetic tree. Since the number of possible ways of transformations from an ancestor to a progeny is huge, we use computational grids and middleware techniques to explore the large number of cellular automata rules used for the mutations. We use the popular and recurring neighbor-based transitions or mutations to predict the progeny sequences in the phylogenetic tree. We performed predictions for three types of sequences, namely, triose phosphate isomerase, pyruvate kinase, and polyketide synthase sequences, by obtaining cellular automata rules on a grid consisting of 29 machines in 4 clusters located in 4 countries, and compared the predictions of the sequences using our method with predictions by random methods. We found that in all cases, our method gave about 40% better predictions than the random methods.

Retrobiosynthetic Approach Delineates the Biosynthetic Pathway and the Structure of the Acyl Chain of Mycobacterial Glycopeptidolipids

Glycopeptidolipids (GPLs) are dominant cell surface molecules present in several non-tuberculous and opportunistic mycobacterial species. GPLs from Mycobacterium smegmatis are composed of a lipopeptide core unit consisting of a modified C-26-C-34 fatty acyl chain that is linked to a tetrapeptide (Phe-Thr-Ala-alaninol). The hydroxyl groups of threonine and terminal alaninol are further modified by glycosylations. Although chemical structures have been reported for 16 GPLs from diverse mycobacteria, there is still ambiguity in identifying the exact position of the hydroxyl group on the fatty acyl chain. Moreover, the enzymes involved in the biosynthesis of the fatty acyl component are unknown. In this study we show that a bimodular polyketide synthase in conjunction with a fatty acyl-AMP ligase dictates the synthesis of fatty acyl chain of GPL. Based on genetic, biochemical, and structural investigations, we determine that the hydroxyl group is present at the C-5 position of the fatty acyl component. Our retrobiosynthetic approach has provided a means to understand the biosynthesis of GPLs and also resolve the long-standing debate on the accurate structure of mycobacterial GPLs.

Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence.

The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (E-MSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in E-MSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic E-MSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, Mtb Delta whiB3 displayed intramacrophage survival defect, which can be rescued by pharmacological inhibition of phagosomal acidification. Last, Mtb Delta whiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.

Hiperagregabilidade plaquetária e depressão: é o óxido nítrico o mensageiro comum?

A depressão é uma doença grave que vem se tornando mais prevalente na população mundial e no Brasil. Segundo a Organização Mundial de Saúde (OMS), é a quarta doença mais incapacitante e estima-se que em 2020 ocupe o segundo lugar, ficando atrás apenas das doenças cardiovasculares (DCV), que são a principal causa de morte no mundo. O Transtorno depressivo maior (TDM) se caracteriza por humor deprimido, tristeza intensa ou desânimo ou perda de interesse ou de prazer por quase todas as atividades por, pelo menos, duas semanas. Além disso, tem um elevado índice de mortalidade cardiovascular, e esta associação parece ser multifatorial e altamente complexa, e ainda não está completamente elucidada. Recentes estudos sugerem que a ocorrência de aterotrombose e eventos cardiovasculares no TDM está associada a uma diminuição na biodisponibilidade do óxido nítrico (NO), um potente vasodilatador, anti-agregante plaquetário e neurotransmissor. O NO é um gás formado a partir da L-arginina, pela ação da família de enzimas NO sintases (NOS), e vai ocasionar um aumento de guanosina monofosfato cíclica (GMPc), que é posteriormente degradada pelas fosfodiesterases (PDE). A L-arginina participa em outras vias além da produção de NO, como a arginase. O estresse oxidativo também tem uma participação no desenvolvimento dos transtornos psiquiátricos e nas DCV, e pode reduzir a meia-vida do NO. O objetivo deste estudo é investigar a via NO-GMPc, o ciclo da uréia, marcadores de estresse oxidativo e de inflamação em plaquetas e a sua associação com a função plaquetária no TDM. Participaram da pesquisa nove pacientes com diagnóstico de depressão leve a moderada do Serviço de Psicologia Aplicada (SPA/UERJ) e onze indivíduos saudáveis pareados por idade como controles. Este projeto foi aprovado pelo Comitê de Ética e Pesquisa do Hospital Universitário Pedro Ernesto (1436-CEP/HUPE). A agregação plaquetária, a expressão e atividade da arginase II, a expressão da PDE 5, marcadores de estresse oxidativo (níveis de TBARS, carbonilação de proteínas, expressão da NADPH oxidase e da glutationa peroxidase (GPx) e atividade desta e da catalase (CAT), ambas enzimas anti-oxidantes) nas plaquetas e no soro, e o fibrinogênio sistêmico foram investigados. No presente estudo observou-se um aumento da agregação plaquetária induzida por ADP em pacientes com TDM comparados aos controles. Uma ativação da arginase II em plaquetas sem qualquer alteração na sua expressão foi demonstrada em pacientes com TDM. Além disso, um aumento na carbonilação de proteínas e na expressão de GPx, de NADPH e de PDE5 foi observado em plaquetas de pacientes com TDM. A produção de TBARS, a atividade de GPx e CAT nas plaquetas e no soro não foram afetados pelo TDM. Não houve diferença nos níveis de fibrinogênio entre pacientes com TDM e controles. A ativação da arginase, somada ao estresse oxidativo, reduziria a biodisponibilidade de NO levando à disfunção plaquetária nos pacientes com TDM. O presente estudo acrescenta dados importantes para a compreensão dos mecanismos celulares envolvidos na relação TDM e DCV. Além disso, abre caminho para a utilização de novas ferramentas farmacológicas, como os antioxidantes, para o tratamento do TDM.

A via L-argininaóxido nítrico, estresse oxidativo e ciclo da uréia na obesidade

A obesidade é um distúrbio metabólico de etiologia multifatorial e elevada prevalência no Brasil, que pode ser definida por um índice de massa corporal (peso em quilogramas dividido pela altura em metros ao quadrado) maior ou igual a 30 kg/m2, e que está associada de forma independente a um elevado risco de morbidade e mortalidade cardiovascular devido aos eventos aterotrombóticos. O óxido nítrico (NO), uma pequena molécula gasosa, é produzido através da conversão do aminoácido catiônico L-arginina em L-citrulina e NO em uma reação catalisada por uma família de enzimas denominadas NO-sintases (NOS), e funciona como um protetor cardiovascular modulando por exemplo o relaxamento do músculo liso vascular e a função plaquetária. O objetivo desta tese foi avaliar a via L-arginina-NO, bem como investigar a função plaquetária, o estresse oxidativo, e a atividade da arginase em pacientes com obesidade. O transporte de L-arginina, a produção de guanosina monofosfato cíclica (GMPc), a atividade e a expressão das isoformas da NOS (iNOS e eNOS), a atividade da arginase, o estresse oxidativo (produção de espécies reativas de oxigênio EROs; atividade da superóxido dismutase SOD; e atividade da catalase), bem como a função plaquetária foram medidos nas plaquetas dos pacientes com obesidade. Nas hemácias, foram medidos o transporte de L-arginina e a atividade da NOS e da arginase. Os níveis de aminoácidos e de marcadores inflamatórios (fibrinogênio e proteína C reativa) também foram medidos sistemicamente. Os resultados demonstram que o influxo de L-arginina via sistema y+L, a atividade da NOS e a produção de GMPc estão diminuídos nas plaquetas dos pacientes obesos em relação aos controles saudáveis, enquanto que não houve diferença na atividade da arginase. Além disso, a expressão das isoformas da NOS bem como a agregação plaquetária em plaquetas de pacientes com obesidade mostrou-se aumentada em relação aos controles. Nas hemácias destes pacientes, observou-se elevado influxo de L-arginina via sistema y+ e y+L e atividade da NOS, e nenhuma diferença na função da arginase. A concentração plasmática de L-arginina não foi afetada pela obesidade, mas já os marcadores inflamatórios estavam significativamente aumentados. A produção de EROs e a atividade da catalase nas plaquetas não estava alterada em pacientes com obesidade, enquanto que a atividade da SOD mostrou-se diminuida. Assim, apesar do aumento da produção de NO pelas hemácias, é possível que a baixa produção plaquetária de NO, além do estado inflamatório e um possível estresse oxidativo, estejam contribuindo para a elevada atividade plaquetária observada na obesidade. As descobertas aqui apresentadas contribuem para uma melhor compreensão dos eventos cardiovasculares presentes na obesidade.

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.