Evaluation of distinct molecular architectures and coordinated regulation of the catabolic pathways of oestrogenic dioctyl phthalate isomers in Gordonia sp.

Bacterial strain GONU, belonging to the genus Gordonia, was isolated from a municipal waste-contaminated soil sample and was capable of utilizing an array of endocrine-disrupting phthalate diesters, including di-n-octyl phthalate (DnOP) and its isomer di(2-ethylhexyl) phthalate (DEHP), as the sole carbon and energy sources. The biochemical pathways of the degradation of DnOP and DEHP were evaluated in strain GONU by using a combination of various chromatographic, spectrometric and enzymatic analyses. Further, the upregulation of three different esterases (estG2, estG3 and estG5), a phthalic acid (PA)-metabolizing pht operon and a protocatechuic acid (PCA)-metabolizing pca operon were revealed based on de novo whole genome sequence information and substrate-induced protein profiling by LC-ESI-MS/MS analysis followed by differential gene expression by real-time PCR. Subsequently, functional characterization of the differentially upregulated esterases on the inducible hydrolytic metabolism of DnOP and DEHP revealed that EstG5 is involved in the hydrolysis of DnOP to PA, whereas EstG2 and EstG3 are involved in the metabolism of DEHP to PA. Finally, gene knockout experiments further validated the role of EstG2 and EstG5, and the present study deciphered the inducible regulation of the specific genes and operons in the assimilation of DOP isomers.

Biotransformation of 3-cyanopyridine to nicotinic acid using whole-cell nitrilase of Gordonia terrae mutant MN12.

In the present study, the Gordonia terrae was subjected to chemical mutagenesis using ethyl methane sulfonate (EMS) and methyl methane sulfonate (MMS), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 5-bromouracil (5-BU) and hydroxylamine with the aim of improving the catalytic efficiency of its nitrilase for conversion of 3-cyanopyridine to nicotinic acid. A mutant MN12 generated with MNNG exhibited increase in nitrilase activity from 0.5 U/mg dcw (dry cell weight) (in the wild G. terrae) to 1.33 U/mg dcw. Further optimizations of culture conditions using response surface methodology enhanced the enzyme production to 1.2-fold. Whole-cell catalysis was adopted for bench-scale synthesis of nicotinic acid, and 100% conversion of 100 mM 3-cyanopyridine was achieved in potassium phosphate buffer (0.1 M, pH 8.0) at 40 °C in 15 min. The whole-cell nitrilase of the mutant MN12 exhibited higher rate of product formation and volumetric productivity, i.e., 24.56 g/h/g dcw and 221 g/L as compared to 8.95 g/h/g dcw and 196.8 g/L of the wild G. terrae. The recovered product was confirmed by HPLC, FTIR and NMR analysis with high purity (> 99.9%). These results indicated that the mutant MN12 of G. terrae as whole-cell nitrilase is a very promising biocatalyst for the large-scale synthesis of nicotinic acid.

Comparative Genomics of Closely-Related Gordonia Cluster DR Bacteriophages.

Bacteriophages infecting bacteria of the genus Gordonia have increasingly gained interest in the scientific community for their diverse applications in agriculture, biotechnology, and medicine, ranging from biocontrol agents in wastewater management to the treatment of opportunistic pathogens in pulmonary disease patients. However, due to the time and costs associated with experimental isolation and cultivation, host ranges for many bacteriophages remain poorly characterized, hindering a more efficient usage of bacteriophages in these areas. Here, we perform a series of computational genomic inferences to predict the putative host ranges of all Gordonia cluster DR bacteriophages known to date. Our analyses suggest that BiggityBass (as well as several of its close relatives) is likely able to infect host bacteria from a wide range of genera-from Gordonia to Nocardia to Rhodococcus, making it a suitable candidate for future phage therapy and wastewater treatment strategies.

Pacemaker-induced endocarditis by Gordonia bronchialis.

PURPOSE: Gordonia species are known to be opportunistic human pathogens causing secondary infections. We present the second case in the world of endocarditis caused by Gordonia bronchialis and a review of all the cases of endocarditis caused by Gordonia spp. METHODS: The identification was performed by matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing were performed to confirm the identification. Antimicrobial susceptibility was performed by MIC test Strip on Mueller-Hinton agar supplemented with 5% defibrinated sheep blood according to Clinical and Laboratory Standards Institute. RESULTS: Pacemaker-induced endocarditis due to Gordonia bronchialis infection was determined in an 88-year old woman. The patient was treated with ceftriaxone and ciprofloxacin until completing 6 weeks from the pacemaker explant with a good evolution. CONCLUSION: The case presented supports the pathogenic role of Gordonia bronchialis as an opportunistic pathogen and highlights the high risk of suffering infections caused by environmental bacteria.

Mechanistic Understanding of Gordonia sp. in Biodesulfurization of Organosulfur Compounds.

Although conventional oil refining process like hydrodesulfurization (HDS) is capable of removing sulfur compounds present in crude oil, it cannot desulfurize recalcitrant organosulfur compounds such as dibenzothiophenes (DBTs), benzothiophenes (BTs), etc. Biodesulfurization (BDS) is a process of selective removal of sulfur moieties from DBT or BT by desulfurizing microbes. Therefore, BDS can be used as a complementary and economically feasible technology to achieve deep desulfurization of crude oil without affecting the calorific value. In the recent past, members of biodesulfurizing actinomycete genus Gordonia, isolated from versatile environments like soil, activated sludge, human beings etc. have been greatly exploited in the field of petroleum refining technology. The bacterium Gordonia sp. is slightly acid-fast and has been used for unconventional but potential oil refining processes like BDS in petroleum refineries. Gordonia sp. is unique in a way, that it can desulfurize both aliphatic and aromatic organosulfurs without affecting the calorific value of hydrocarbon molecules. Till date, approximately six different species and nineteen strains of the genus Gordonia have been recognized for BDS activity. Various factors such as enzyme specificity, availability of essential cofactors, feedback inhibition, toxicity of organic pollutants and the oil-water separations limit the desulfurization rate of microbial biocatalyst and influence its commercial applications. The current review selectively highlights the role of this versatile genus in removing sulfur from fossil fuels, mechanisms and future prospects on sustainable environment friendly technologies for crude oil refining.

Microarray profiling and identification of core promoter sequence in Gordonia.

Gordonia are Gram-positive bacteria which have immense biotechnological potential. Genomes of several Gordonia spp. have been sequenced but a detailed analysis of the differentially expressed genes during growth, the promoters which drive their expression and the information on the core promoter sequence is lacking. Here, we report the identification of core promoter sequence in Gordonia sp. IITR100. The GC content of the promoters was found to be within a range of 62-65%. The 5'-UTR length in the genes was also analysed and about 56% promoters were found to have long 5'-UTR. The functionality of the promoters was validated by microarray profiling. Based on the differential expression of genes, two growth phase dependent promoters PdsbA and Pglx were isolated and analysed. They add to the existing repertoire of the promoters functional in both Gram-negative and Gram-positive bacteria. Our results suggest that the core promoter sequence identified is conserved in members of Gordonia spp. and is similar to that of other members of Actinobacteria.

Analysis of genome sequence and trehalose lipid production peculiarities of the thermotolerant Gordonia strain.

Gordoniae are one of the most promising hydrocarbon-oxidizing actinobacteria. Here we present the genome sequence analysis of thermotolerant strain Gordonia sp. 1D isolated from oil-refinery soil. It is capable of alkane consumption and biosurfactant production at temperatures of up to 50°C. Gordonia sp. 1D demonstrates maximum biosurfactant production when grown on hexadecane, and at 40°C it was slightly higher than at 27°C: 35 and 39 mN/m, respectively. For the first time, it was experimentally confirmed that the carbohydrate component of extracellular biosurfactants produced by strain 1D is trehalose. In addition, genes for the production of trehalose lipid biosurfactants were identified. The genetic determinants for two different pathways for trehalose synthesis were found. The strain carries genes otsA and otsB involved in de novo trehalose biosynthesis. Moreover, the genes treY and treZ responsible for trehalose biosynthesis from maltooligosaccharides and starch or glycogen were identified.

Characterization of DNA binding and ligand binding properties of the TetR family protein involved in regulation of dsz operon in Gordonia sp. IITR100.

Gordonia sp. IITR100 is a biodesulfurizing bacterium which can metabolize dibenzothiophene (DBT) to 2 hydroxybiphenyl in four steps via the 4S pathway. The genes involved in the metabolism are present in the form of an operon, dszABC, which gets activated by a TetR family protein. Here, we report the detailed characterization of the DNA binding and ligand binding property of the TetR family protein. The protein was found to be conserved across other desulfurizing organisms. The protein was purified and was found to exist as dimer. The presence of ligand binding site was identified by docking studies and the structural changes in the protein upon ligand binding were determined by CD spectroscopy and tryptophan fluorescence. Further, it was determined that this protein binds to an imperfect palindromic DNA sequence present in the dsz promoter DNA. Binding to the DNA also changes conformation of the protein.

Biodegradation of Structurally Diverse Phthalate Esters by a Newly Identified Esterase with Catalytic Activity toward Di(2-ethylhexyl) Phthalate.

Herein, we report a double enzyme system to degrade 12 phthalate esters (PAEs), particularly bulky PAEs, such as the widely used bis(2-ethylhexyl) phthalate (DEHP), in a one-pot cascade process. A PAE-degrading bacterium, Gordonia sp. strain 5F, was isolated from soil polluted with plastic waste. From this strain, a novel esterase (GoEst15) and a mono(2-ethylhexyl) phthalate hydrolase (GoEstM1) were identified by homology-based cloning. GoEst15 showed broad substrate specificity, hydrolyzing DEHP and 10 other PAEs to monoalkyl phthalates, which were further degraded by GoEstM1 to phthalic acid. GoEst15 and GoEstM1 were heterologously coexpressed in Escherichia coli BL21 (DE3), which could then completely degrade 12 PAEs (5 mM), within 1 and 24 h for small and bulky substrates, respectively. To our knowledge, GoEst15 is the first DEHP hydrolase with a known protein sequence, which will enable protein engineering to enhance its catalytic performance in the future.

Identification of LcpRBA3(2), a novel regulator of lcp expression in Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) is a rubber-degrading actinomycete that harbors one gene coding for a latex clearing protein (lcpA3(2)). Within the genome of S. coelicolor A3(2), we identified a gene coding for a novel protein of the TetR family (LcpRBA3(2)) downstream of lcpA3(2) and demonstrated its binding upstream of lcpA3(2). This indicates a role of LcpRBA3(2) in the regulation of lcp expression. LcpRBA3(2) shows no homology to LcpRVH2, a putative regulator of lcp expression in Gordonia polyisoprenivorans VH2. Additionally, LcpRVH2 homologs did not occur in the genome of S. coelicolor A3(2). Reverse transcriptase (RT) experiments showed that the expression of lcpA3(2) and lcpRBA3(2) is induced with poly(cis-1,4-isoprene) as sole carbon source. For further experiments, we heterologously expressed lcpRBA3(2) in Escherichia coli, purified the protein, and subsequently verified a binding of LcpRBA3(2) upstream of lcpA3(2). The operator site was examined by a DNase I footprinting assay: it comprises 31 bp and exhibits an inverted repeat of nine bases for the putative binding region. Interestingly, two N-terminal DNA-binding HTH domains of the TetR-type (PF00440) were identified within the sequence of LcpRBA3(2). The native molecular weight of LcpRBA3(2) was determined as 44 kDa by size exclusion chromatography which correlates to the molecular weight of a monomer. Normally, proteins of the TetR family occur as dimers so that the monomeric state is a novelty. Furthermore, LcpRBA3(2) homologs were identified in silico in several Lcp-containing actinomycetes, suspecting a conserved regulation mechanism. Apparently, the expression of lcps is regulated either by an LcpRB or by an LcpR.

Enhanced plasmid-mediated bioaugmentation of RDX-contaminated matrices in column studies using donor strain Gordonia sp. KTR9.

Horizontal gene transfer (HGT) is the lateral movement of genetic material between organisms. The RDX explosive-degrading bacterium Gordonia sp. KTR9 has been shown previously to transfer the pGKT2 plasmid containing the RDX degradative genes (xplAB) by HGT. Overall, fitness costs to the transconjugants to maintain pGKT2 was determined through growth and survivability assessments. Rhodococcus jostii RHA1 transconjugants demonstrated a fitness cost while other strains showed minimal cost. Biogeochemical parameters that stimulate HGT of pGKT2 were evaluated in soil slurry mating experiments and the absence of nitrogen was found to increase HGT events three orders of magnitude. Experiments evaluating RDX degradation in flow-through soil columns containing mating pairs showed 20% greater degradation than columns with only the donor KTR9 strain. Understanding the factors governing HGT will benefit bioaugmentation efforts where beneficial bacteria with transferrable traits could be used to more efficiently degrade contaminants through gene transfer to native populations.

LcpRVH2 - regulating the expression of latex-clearing proteins in Gordonia polyisoprenivorans VH2.

Gordonia polyisoprenivorans VH2 harbours two latex clearing proteins, which are responsible for the cleavage of poly(cis-1,4-isoprene) into oligoisoprenes, thereby allowing growth in presence of, e.g. natural rubber. A gene coding for a putative regulator of the TetR-family (lcpRVH2) is located 131 bp upstream of lcp1VH2. We heterologously expressed lcpRVH2 in Escherichia coli, and purified and characterized the protein with respect to its ability to bind to the operator region of lcp1VH2. LcpRVH2 forms a dimer in its native state. The size of the dimer was determined to be 52.7 kDa by size exclusion chromatography, whereas the calculated size of a monomer was 24.1 kDa. Electrophoretic mobility shift assays (EMSAs) with the purified protein revealed a shift upon binding to the intergenic region between lcpRVH2 and lcp1VH2. Within this region, an inverted repeat was identified in silico, probably being the binding site of LcpRVH2. This binding sequence was confirmed by a DNase I footprinting assay. A shift also occurred in EMSAs with this 44 bp sequence only. Interestingly, no regulator was detected upstream of the second lcp (lcp2VH2). Therefore, we performed EMSA studies with LcpRVH2 and the putative operator region upstream of lcp2VH2, and discovered by DNase I footprinting another binding sequence upstream of lcp2VH2. Hence, we concluded that LcpRVH2 binds the operator region of both lcps and, most likely, regulates their expression in G. polyisoprenivorans VH2.

Characterization and genomic analysis of highly efficient thermotolerant oil-degrading bacterium Gordonia sp. 1D.

A thermotolerant bacterial strain 1D isolated from refinery oil-contaminated soil was identified as Gordonia sp. based on the analysis of 16S rRNA and gyrB gene sequences. The strain was found to utilize crude oil, diesel fuel, and a wide spectrum of alkanes at temperatures up to 50 °C. Strain 1D is the first representative of Gordonia amicalis capable of utilizing alkanes of chain length up to С36 at a temperature of 45-50 °C. The degree of crude oil degradation by Gordonia sp. 1D at 45 °C was 38% in liquid medium and 40% in soil (with regard to abiotic loss). There are no examples of so effective hydrocarbon-oxidizing thermotolerant Gordonia in the world literature. The 1D genome analysis revealed the presence of two alkane hydroxylase gene clusters, genes of dibenzothiophene cleavage, and the cleavage of salicylate and gentisate - naphthalene metabolism intermediates. The highly efficient thermotolerant strain Gordonia sp. 1D can be used in remediation of oil-contaminated soils in hot climates.

Synthesis of polyhydroxyalkanoates through the biodegradation of poly(cis-1,4-isoprene) rubber.

The search of alternative substrates for the synthesis of polyhydroxyalkanoates (PHA) has become an important factor in order to decrease the production costs. Therefore, the use of industrial by-products or waste materials as carbon and energy sources for different PHA-producing microorganisms has been evaluated during the last decades. Recombinant strains of Gordonia polyisoprenivorans VH2 harboring plasmid pAK68, which contains phaCAB from Ralstonia eutropha and plasmid pAK71 comprising phaC1 from Pseudomonas aeruginosa were evaluated for PHA production. Cultivations were performed in shake flasks, using different carbon sources under an N-starvation condition. Having in consideration the rubber degrading capability of the actinomycete, poly(cis-1,4-isoprene) was utilized as sole carbon source. After twenty days of cultivation the PHA content was analyzed using GC-MS. In cultures of G. polyisoprenivorans harboring pAK68, the detection of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) monomer units indicated the accumulation of the copolyester poly(3HB-co-3HV). This study proposes a recycling method for rubber waste through its biotransformation into bioplastic.