Project description:Metaproteomic analysis of air particulate matter provides information about the properties of bioaerosols in the atmosphere and their influence on climate and public health. In this work, a new method for the extraction and analysis of proteins in airborne particulate matter from quartz microfiber filters was developed. Different protein extraction procedures were tested in order to select the best extraction protocol in terms of protein recovery. The optimized method was tested for extraction of proteins from spores of ubiquitous bacteria species and used for the first time for the metaproteomics characterization of filters from work environment. In particular, ambient aerosol samples were collected in different working environments, i.e. a composting plant, wastewater treatment plant and agricultural holding. One-hundred seventy-nine, 15, 205 and 444 proteins were successfully identified in composting plant, wastewater treatment plant, and agricultural holding, respectively. All identified proteins were mainly originate from fungi, bacteria and plants which is in line with the major categories of primary biological aerosol particles. The paper is the first metaproteomic study applied to bioaereosol samples collected in occupationally relevant environmental sites providing interesting information on the composting, wastewater treatment and feed blending processes. Significance This manuscript describes the metaproteomic analysis of aerosol samples collected in work enviroments. This is a novel use of aereosol samples and is needed as there is no really comprehensive way of analysing aereosol samples from a metaproteomic point of view. This paper could help to advance methods for metaproteomic analysis of bioaersols, specifically by comparing protein extraction protocols and pairing the best performing extraction protocol with a gel-free protein separation procedure applied for the first time for analysis of bioaerosol samples. The obtained data showed as bioaerosol was essentially made of fungi, bacteria and plant proteins, many of which could be associated to possible aerosolisation and could be a major health concern for workers on site and to the populations residing in neighbouring area.

Project description:Transcriptomics analysis of biopolymer (medium chain length polyhydroxyalkanoate) producing strain P.putida LS46 cultured with biodiesel derived waste carbon sources: studies of cellular adaptation to the industrial waste streams and metabolic profiling under the polymer producing conditions. We are reporting RNAseq analysis data here as part of our multi-level Omics study of medium chain length polyhydroxyalkanoate (mcl-PHA) producing strain P.putida LS46 culture with biodiesel derived waste glycerol and waste fatty acids. The data presented here will be used in two separate manuscripts. The objectives of this study are a): to evaluate cellular responses of P.putida LS46 under industrial waste stream. b): to study gene expression profile under two selected mcl-PHA producing conditions of P.putida LS46. Comparative multi-level Omics study: for objective a): Exponential P.putida LS46 cell from waste glycerol culture compared against reagent grade pure glycerol culture. For objective b): Two mcl-PHA producing conditions, namely stationary phase waste glycerol culture and exponential phase waste fatty acid culture of P.putida LS46, were compared against exponential phase waste glycerol culture of P.putida LS46. Major results from objective a): The waste glycerol substrate induced expression of a large number of genes putatively involved in heavy metal tolerance, including three gene clusters: a putative cusABC transcript unit and two copies of copAB, which are usually involved in copper resistance and tolerance to other monovalent heavy metals. A local gene relocation was observed in cluster 1 consisting cusABC and copAB relative to the KT2440 type strain according to the phylogenetic and gene neighbourhood analyses on various P. putida strains. P. putida LS46 also contains 11 putative MerR family regulators, which sense various environmental stimuli including heavy metals. MerR-1 is an ortholog of the copper response regulator of other gram-negative bacteria, and was highly up-regulated in waste glycerol cultures. Finally, a number of genes involved in cell responses to high extra-cellular Na+ concentrations, and genes of the fatty acid beta-oxidation pathway were up-regulated in waste glycerol cultures Major results from objective b): Regardless to the type of substrates, up-regulation of two mcl-PHA synthase (PhaC1 and PhaC2), and two phasin proteins (PhaF and PhaI) are the most common genotype under mcl-PHA production conditions. PhaG and possible PhaJ4 connect fatty acid de novo synthesis to mcl-PHA in waste glycerol culture. Interestingly, expression of gene, fabZ, in production of unsaturated fatty acid from fatty acid de novo synthesis was only observed in waste glycerol culture. On the other hand, PhaJ1 and PhaJ4 derived mcl-PHA production via fatty acid beta-oxidation was observed under waste fatty acid culture. These results would help to explain observed different production kinetics and monomer distribution of the polymer. Although under active mcl-PHA production condition, depression on the expression of glpF genes in glycerol transportation system prevent further channelling extra-cellular glycerol into the cell. Waste glycerol culture also triggers trahalose synthesis pathway, a potential competing pathway during mcl-PHA synthesizing. In waste fatty acid culture, the intermediates (acyl-CoA and 3-hydroxyacyl-CoA) of fatty acid beta-oxidation were used for mcl-PHA production and were also likely hydrolysed to their free acid forms via an up-regulated thioesteras coding gene, tesA. Acetyl-CoA cleaved from the pathway was clearly channeled into glyoxylate shut for C2 carbon assimilation over spillage as CO2 through TCA cycle or used in fatty acid biosynthesis pathway. In total 4 sampling points, namely exponential phase of pure glycerol, waste glycerol and waste free fatty acids cultures, and stationary phase of waste glycerol culture. For each sampling point, 2 biological replicates were taken. (Thus 8 samples in total)

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