Vinasse, a recalcitrant waste of the ethanol industry was employed for the production of polyhydroxyalkanoate (PHA) by the extremely halophilic archaeon, Haloarcula marismortui in shake flasks. The PHA was recovered by osmotic lysis of the cells and subsequent purification by sodium hypochlorite and organic solvents. Through UV-vis spectroscopy, differential scanning calorimetry, Fourier transform infrared, and nuclear magnetic resonance spectroscopy, the PHA was found to have characteristics very similar to that of the standard polyhydroxybutyrate (PHB) from Sigma. Inhibitory effect of polyphenols contained in vinasse was assessed by a quick and reliable cup-plate agar-diffusion method. Raw vinasse (10%) was utilized leading to accumulation of 23% PHA (of cell dry weight) and following an efficacious pre-treatment process through adsorption on activated carbon, 100% pre-treated vinasse could be utilized leading to 30% accumulation of PHB by H. marismortui. Maximum specific growth rate, specific production rate, and volumetric productivity attained using 10% raw vinasse were comparable to that obtained using a previously reported nutrient deficient medium (NDM), while the values with 100% pre-treated vinasse were higher than that determined using NDM medium. This is the first report of polyhydroxybutyrate production by a halophilic microorganism utilizing vinasse.

The endosomal compartment performs extensive sorting functions in most eukaryotes, some of which are accomplished with the help of the multivesicular body (MVB) sorting pathway. This pathway depends on the sequential action of complexes, termed the endosomal sorting complex required for transport (ESCRT). After successful sorting, the crucial step of recycling of the ESCRT complex components requires the activation of the AAA ATPase Vps4, and Did2/Vps46 plays an important role in this activation event. The endolysosomal system of the protozoan parasite Giardia lamblia appears to lack complexity, for instead of having distinct early endosomes, late endosomes and lysosomes, there are only peripheral vesicles (PVs) that are located close to the cell periphery. Additionally, comparative genomics studies predict the presence of only a subset of the ESCRT components in G. lamblia. Thus, it is possible that the MVB pathway is not functional in G. lamblia. To address this issue, the present study focused on the two putative orthologues of Did2/Vps46 of G. lamblia as their function is likely to be pivotal for a functional MVB sorting pathway. In spite of considerable sequence divergence, compared to other eukaryotic orthologues, the proteins encoded by both these genes have the ability to function as Did2/Vps46 in the context of the yeast ESCRT pathway. Furthermore, they also localized to the cellular periphery, where PVs are also located. Thus, this report is the first to provide experimental evidence indicating the presence of a functional ESCRT component in G. lamblia by characterizing the putative Did2/Vps46 orthologues.

BackgroundGiardia lamblia switches its lifecycle between trophozoite and cyst forms and the proteasome plays a pivotal role in this switching event. Compared to most model eukaryotes, the proteasome of this parasite has already been documented to have certain variations. This study was undertaken to characterize the ubiquitin receptor, GlRpn10, of the 19S regulatory particle of the Giardia proteasome and determine its cellular localization in trophozoites, encysting trophozoites and cysts.MethodSequence alignment and domain architecture analyses were performed to characterize GlRpn10. In vitro ubiquitin binding assay, functional complementation and biochemical studies verified the protein’s ability to function as ubiquitin receptor in the context of the yeast proteasome. Immunofluorescence localization was performed with antibody against GlRpn10 to determine its distribution in trophozoites, encysting trophozoites and cysts. Real-time PCR and Western blotting were performed to monitor the expression pattern of GlRpn10 during encystation.ResultGlRpn10 contained a functional ubiquitin interacting motif, which was capable of binding to ubiquitin. Although it contained a truncated VWA domain, it was still capable of partially complementing the function of the yeast Rpn10 orthologue. Apart from localizing to the nucleus and cytosol, GlRpn10 was also present at flagellar pores of trophozoites and this localization was microtubule-dependent. Although there was no change in the cellular levels of GlRpn10 during encystation, its selective distribution at the flagellar pores was absent.ConclusionGlRpn10 contains a noncanonical VWA domain that is partially functional in yeast. Besides the expected nuclear and cytosolic distribution, the protein displays microtubule-dependent flagellar pore localization in trophozoites. While the protein remained in the nucleus and cytosol in encysting trophozoites, it could no longer be detected at the flagellar pores. This absence at the flagellar pore regions in encysting trophozoites is likely to involve redistribution of the protein, rather than decreased gene expression or selective protein degradation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0737-1) contains supplementary material, which is available to authorized users.

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Giardia lamblia is the causative agent of the diarrheal disease giardiasis, against which only a limited number of drugs are currently available. Increasing reports of resistance to these drugs makes it necessary to identify new cellular targets for designing the next generation of anti-giardial drugs. Towards this goal, therapeutic agents that target the parasitic cellular machinery involved in the functioning of the unique microtubule-based cytoskeleton of the Giardia trophozoites are likely to be effective as microtubule function is not only important for the survival of trophozoites within the host, but also their extensive remodeling is necessary during the transition from trophozoites to cysts. Thus, drugs that affect microtubule remodeling have the potential to not only kill the disease-causing trophozoites, but also inhibit transmission of cysts in the community. Recent studies in other model organisms have indicated that the proteasome plays an integral role in the formation and remodeling of the microtubule-based cytoskeleton. This review draws attention to the various processes by which the giardial proteasome may impact the functioning of its microtubule cytoskeleton and highlights the possible differences of the parasitic proteasome and some of other cellular machinery involved in microtubule remodeling, compared to that of the higher eukaryotic host.