Microbial Degradation of Rubber: Actinobacteria.
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ABSTRACT:
SUBMITTER: Basik AA
PROVIDER: S-EPMC8235351 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Nocardia nova SH22a, a bacterium capable of degrading gutta-percha (GP) and natural rubber (NR), was used to investigate the GP degradation mechanism and the relations between the GP and NR degradation pathways. For this strain, a protocol of electroporation was systematically optimized, and an efficiency of up to 4.3 × 10(7) CFU per ?g of plasmid DNA was achieved. By applying this optimized protocol to N. nova SH22a, a Tn5096-based transposon mutagenesis library of this bacterium was constructed. Among about 12,000 apramycin-resistant transformants, we identified 76 stable mutants defective in GP or NR utilization. Whereas 10 mutants were specifically defective in GP utilization, the growth of the other 66 mutants was affected on both GP and NR. This indicated that the two degradation pathways are quite similar and share many common steps. The larger number of GP-degrading defective mutants could be explained in one of two ways: either (i) the GP pathway is more complex and harbors more specific steps or (ii) the steps for both pathways are almost identical, but in the case of GP degradation there are fewer enzymes involved in each step. The analysis of transposition loci and genetic studies on interesting genes confirmed the crucial role of an ?-methylacyl-coenzyme A racemase in the degradation of both GP and NR. We also demonstrated the probable involvement of enzymes participating in oxidoreduction reactions, ?-oxidation, and the synthesis of complex cell envelope lipids in the degradation of GP.
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Project description:The complete genome sequence of Nocardia nova SH22a was determined in light of the remarkable ability of rubber and gutta-percha (GP) degradation of this strain. The genome consists of a circular chromosome of 8,348,532 bp with a G+C content of 67.77% and 7,583 predicted protein-encoding genes. Functions were assigned to 72.45% of the coding sequences. Among them, a large number of genes probably involved in the metabolism of xenobiotics and hardly degradable compounds, as well as genes that participate in the synthesis of polyketide- and/or nonribosomal peptide-type secondary metabolites, were detected. Based on in silico analyses and experimental studies, such as transposon mutagenesis and directed gene deletion studies, the pathways of rubber and GP degradation were proposed and the relationship between both pathways was unraveled. The genes involved include, inter alia, genes participating in cell envelope synthesis (long-chain-fatty-acid-AMP ligase and arabinofuranosyltransferase), β-oxidation (α-methylacyl-coenzyme A [α-methylacyl-CoA] racemase), propionate catabolism (acyl-CoA carboxylase), gluconeogenesis (phosphoenolpyruvate carboxykinase), and transmembrane substrate uptake (Mce [mammalian cell entry] transporter). This study not only improves our insights into the mechanism of microbial degradation of rubber and GP but also expands our knowledge of the genus Nocardia regarding metabolic diversity.
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Project description:Polylactic acid (PLA) is one of the most commercially available and exploited bioplastics worldwide. It is an important renewable polymer for the replacement of petroleum-based plastic materials. They are both biodegradable and bio-based plastic. Microbial degrading activity is a desirable method for environmental safety and economic value for bioplastic waste managements. Members of the phylum actinobacteria are found to play an important role in PLA degradation. Most of the PLA degrading actinobacteria belong to the family Pseudonocardiaceae. Other taxa include members of the family Micromonosporaceae, Streptomycetaceae, Streptosporangiaceae, and Thermomonosporaceae. This mini-review aims to provide an overview on PLA degrading actinobacteria including their diversity and taxonomy, isolation and screening procedures and PLA degrading enzyme production from 1997 to 2019. Consideration is also given to where to sampling and how we might use these beneficial actinobacteria for PLA waste management.
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Project description:Bacteria make a huge contribution to the purification of the environment from toxic stable pollutants of anthropogenic and natural origin due to the diversity of their enzyme systems. For example, the ability to decompose 3-chlorobenzoate (3CBA) by the four representative genera of Actinobacteria, such as Rhodococcus, Gordonia, Microbacterium, and Arthrobacter, was studied. In most cases, the formation of 4-chlorocatechol as the only key intermediate during the decomposition of 3CBA was observed. However, Rhodococcus opacus strain 1CP was an exception, whose cells decomposed 3CBA via both 3-chloro- and 4-chlorocatechol. The enzyme 3-Chlorobenzoate 1,2-dioxygenase (3CBDO) induced during the growth of these bacteria in the presence of 3CBA differed significantly in substrate specificity from the benzoate dioxygenases induced upon growth in the presence of benzoate. The R. opacus 6a strain was found to contain genes encoding chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and dienelactone hydrolase, whose nucleotide sequence was 100% consistent with the sequences of the corresponding genes encoding the enzymes of the modified 4-chlorocatechol ortho-cleavage pathway of the strain R. opacus 1CP. However, the gene encoding chloromuconolactone dehalogenase (clcF) was not found in the representatives of the actinomycete genera, including Gordonia and Arthrobacter. A linear mega-plasmid carrying 3-chlorocatechol degradation genes remained stable after maintaining the R. opacus 1CP strain on an agar-rich medium for 25 years. In general, a similar plasmid was absent in actinobacteria of other genera, as well as in closely related species of R. opacus 6a.
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Project description:Microbial catabolism-based grouping enables read-across of non-persistency for all constituents of hexyl glucoside and 2-ethylhexyl glucoside
| PRJEB38801 | ENA
Project description:A shotgun metagenome microarray was created and used to investigate gene transcription during vinyl chloride (VC) dechlorination by a microbial enrichment culture called KB1. The array was constructed by spotting genomic fragments amplified from short-insert libraries of KB1 metagenomic DNA. Subsequently, the microarrays were interrogated with RNA extracted from KB1 during VC dechlorination (VC+methanol), and in the absence of VC (methanol-only). The most differentially expressed spots, and spots with the highest intensities, were then chosen to be sequenced. Sequencing revealed that Dehalococcoides (Dhc) genes involved in transcription, translation and energy generation were up-regulated during VC degradation. Furthermore, the results indicated that the reductive dehalogenase homologous (RDH) gene KB1rdhA14 is the only RDH gene up-regulated upon VC degradation, and that multiple RDH genes were more highly transcribed in the absence of VC. Numerous hypothetical genes from Dehalococcoides were also more highly transcribed in methanol only treatments and indicate that many uncharacterized proteins are involved in cell maintenance in the absence of chlorinated substrates. Spots with genes from Spirochaetes, Chloroflexi, Geobacter, Methanogens and phage organisms were differentially expressed and sequencing provided information from these uncultivated organisms that can be used to design primers for more targeted studies. This array format is powerful, as it does not require a priori sequence knowledge. This study provides the first report of such arrays being used to investigate transcription in a mixed community, and shows that this array format can be used to screen metagenomic libraries for functionally important genes. 2 Biological replicate experimens conducted 1 month apart. In the first there were 2 dye-swapped duplicates (total 4) of VC+MeOH versus MeOH only. In the second experiment there was one set of dye swapped arrays. Thus 6 arrays were performed including biological replicates, dye swapped replicates and technical duplicates.
2010-01-26 | E-GEOD-20041 | biostudies-arrayexpress
Project description:Steroidal oestrogens (C18 ) are contaminants receiving increasing attention due to their endocrine-disrupting activities at sub-nanomolar concentrations. Although oestrogens can be eliminated through photodegradation, microbial function is critical for removing oestrogens from ecosystems devoid of sunlight exposure including activated sludge, soils and aquatic sediments. Actinobacteria were found to be key oestrogen degraders in manure-contaminated soils and estuarine sediments. Previously, we used the actinobacterium Rhodococcus sp. strain B50 as a model microorganism to identify two oxygenase genes, aedA and aedB, involved in the activation and subsequent cleavage of the estrogenic A-ring respectively. However, genes responsible for the downstream degradation of oestrogen A/B-rings remained completely unknown. In this study, we employed tiered comparative transcriptomics, gene disruption experiments and mass spectrometry-based metabolite profile analysis to identify oestrogen catabolic genes. We observed the up-regulation of thiolase-encoding aedF and aedK in the transcriptome of strain B50 grown with oestrone. Consistently, two downstream oestrogenic metabolites, 5-oxo-4-norestrogenic acid (C17 ) and 2,3,4-trinorestrogenic acid (C15 ), were accumulated in aedF- and aedK-disrupted strain B50 cultures. Disruption of fadD3 [3aα-H-4α(3'-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP)-coenzyme A-ligase gene] in strain B50 resulted in apparent HIP accumulation in oestrone-fed cultures, indicating the essential role of fadD3 in actinobacterial oestrogen degradation. In addition, we detected a unique meta-cleavage product, 4,5-seco-estrogenic acid (C18 ), during actinobacterial oestrogen degradation. Differentiating the oestrogenic metabolite profile and degradation genes of actinobacteria and proteobacteria enables the cost-effective and time-saving identification of potential oestrogen degraders in various ecosystems through liquid chromatography-mass spectrometry analysis and polymerase chain reaction-based functional assays.
| S-EPMC8913865 | biostudies-literature
Project description:A shotgun metagenome microarray was created and used to investigate gene transcription during vinyl chloride (VC) dechlorination by a microbial enrichment culture called KB1. The array was constructed by spotting genomic fragments amplified from short-insert libraries of KB1 metagenomic DNA. Subsequently, the microarrays were interrogated with RNA extracted from KB1 during VC dechlorination (VC+methanol), and in the absence of VC (methanol-only). The most differentially expressed spots, and spots with the highest intensities, were then chosen to be sequenced. Sequencing revealed that Dehalococcoides (Dhc) genes involved in transcription, translation and energy generation were up-regulated during VC degradation. Furthermore, the results indicated that the reductive dehalogenase homologous (RDH) gene KB1rdhA14 is the only RDH gene up-regulated upon VC degradation, and that multiple RDH genes were more highly transcribed in the absence of VC. Numerous hypothetical genes from Dehalococcoides were also more highly transcribed in methanol only treatments and indicate that many uncharacterized proteins are involved in cell maintenance in the absence of chlorinated substrates. Spots with genes from Spirochaetes, Chloroflexi, Geobacter, Methanogens and phage organisms were differentially expressed and sequencing provided information from these uncultivated organisms that can be used to design primers for more targeted studies. This array format is powerful, as it does not require a priori sequence knowledge. This study provides the first report of such arrays being used to investigate transcription in a mixed community, and shows that this array format can be used to screen metagenomic libraries for functionally important genes.
2010-01-27 | GSE20041 | GEO
Project description:A Gram-positive rubber-degrading bacterium, Actinoplanes sp. strain OR16 (strain NBRC 114529), is able to grow on agar plates containing natural and synthetic rubber as the sole sources of carbon and energy. When this strain was grown on natural rubber latex overlay agar plates, translucent halos around the cells were observed. To identify the natural rubber degradation genes and other features of its metabolism, its complete genome sequence was determined. The genome of OR16 consists of 9,293,892 bp and comprises one circular chromosome (GenBank accession number AP019371.1) with a G + C content of 70.3%. The genome contains 8238 protein-coding and 18 rRNA genes. A homology search of the genome sequence revealed that three genes (lcp1, lcp2, and lcp3) are homologous to an extracellular latex-clearing protein (Lcp) of Streptomyces sp. K30. RT-PCR analysis revealed that lcp1 and lcp2 seem to constitute an operon. Purified lcp gene products have oxygen consumption activity toward natural rubber latex, suggesting that all these genes encode rubber-degrading enzymes in OR16. Quantitative reverse transcription-PCR analysis indicated that the transcription of these genes is induced during the growth of OR16 on natural rubber. The genes located adjacent to lcp1 and lcp3, which code for a TetR/AcrR-type transcriptional regulator, can bind to the promoter regions of these lcp genes. It is suggested that the putative regulators play a role in regulating the transcription of the lcp genes. These results strongly suggested that three lcp genes are required for the utilization of natural rubber in strain OR16. Key Points • The complete genome sequence of Actinoplanes sp. strain OR16 was determined. • Three lcp genes which are involved in the natural rubber degradation in OR16 were identified. • Transcription of these lcp genes is induced during utilization of rubber in OR16. • Two regulators, which bind to the promoter regions of lcp, were determined.
| S-EPMC7413915 | biostudies-literature