Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Toluene degradation by Pseudomonas putida F1: genetic organization of the tod operon.

Pseudomonas putida PpF1 degrades toluene through cis-toluene dihydrodiol to 3-methylcatechol. The latter compound is metabolized through the well-established meta pathway for catechol degradation. The first four steps in the pathway involve the sequential action of toluene dioxygenase (todABC1C2), cis-toluene dihydrodiol dehydrogenase (todD), 3-methylcatechol 2,3-dioxygenase (todE), and 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase (todF). The genes for these enzymes form part of the tod operon which is responsible for the degradation of toluene by this organism. A combination of transposon mutagenesis of the PpF1 chromosome, as well as analysis of cloned chromosomal fragments, was used to determine the physical order of the genes in the tod operon. The genes were determined to be transcribed in the order todF, todC1, todC2, todB, todA, todD, todE.     

Toluene degradation by Pseudomonas putida F1: genetic organization of the tod operon

Pseudomonas putida PpF1 degrades toluene through cis-toluene dihydrodiol to 3-methylcatechol. The latter compound is metabolized through the well-established meta pathway for catechol degradation. The first four steps in the pathway involve the sequential action of toluene dioxygenase (todABC1C2), cis-toluene dihydrodiol dehydrogenase (todD), 3-methylcatechol 2,3-dioxygenase (todE), and 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase (todF). The genes for these enzymes form part of the tod operon which is responsible for the degradation of toluene by this organism. A combination of transposon mutagenesis of the PpF1 chromosome, as well as analysis of cloned chromosomal fragments, was used to determine the physical order of the genes in the tod operon. The genes were determined to be transcribed in the order todF, todC1, todC2, todB, todA, todD, todE.     

Identification of a Novel Dioxygenase Involved in Metabolism of o-Xylene, Toluene, and Ethylbenzene by Rhodococcus sp. Strain DK17

Rhodococcus sp. strain DK17 is able to grow on o-xylene, benzene, toluene, and ethylbenzene. DK17 harbors at least two megaplasmids, and the genes encoding the initial steps in alkylbenzene metabolism are present on the 330-kb pDK2. The genes encoding alkylbenzene degradation were cloned in a cosmid clone and sequenced completely to reveal 35 open reading frames (ORFs). Among the ORFs, we identified two nearly exact copies (one base difference) of genes encoding large and small subunits of an iron sulfur protein terminal oxygenase that are 6 kb apart from each other. Immediately downstream of one copy of the dioxygenase genes (akbA1_a and akbA2_a) is a gene encoding a dioxygenase ferredoxin component (akbA3), and downstream of the other copy (akbA1_b and akbA2_b) are genes putatively encoding a meta-cleavage pathway. RT-PCR experiments show that the two copies of the dioxygenase genes are operonic with the downstream putative catabolic genes and that both operons are induced by o-xylene. When expressed in Escherichia coli, AkbA1_a-AkbA2_a-AkbA3 transformed o-xylene into 2,3- and 3,4-dimethylphenol. These were apparently derived from an unstable o-xylene cis-3,4-dihydrodiol, which readily dehydrates. This indicates a single point of attack of the dioxygenase on the aromatic ring. In contrast, attack of AkbA1_a-AkbA2_a-AkbA3 on ethylbenzene resulted in the formation of two different cis-dihydrodiols resulting from an oxidation at the 2,3 and the 3,4 positions on the aromatic ring, respectively.     

Isolation and Characterization of Polycyclic Aromatic Hydrocarbon-Degrading Bacteria Associated with the Rhizosphere of Salt Marsh Plants

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from contaminated estuarine sediment and salt marsh rhizosphere by enrichment using either naphthalene, phenanthrene, or biphenyl as the sole source of carbon and energy. Pasteurization of samples prior to enrichment resulted in isolation of gram-positive, spore-forming bacteria. The isolates were characterized using a variety of phenotypic, morphologic, and molecular properties. Identification of the isolates based on their fatty acid profiles and partial 16S rRNA gene sequences assigned them to three main bacterial groups: gram-negative pseudomonads; gram-positive, non-spore-forming nocardioforms; and the gram-positive, spore-forming group, Paenibacillus. Genomic digest patterns of all isolates were used to determine unique isolates, and representatives from each bacterial group were chosen for further investigation. Southern hybridization was performed using genes for PAH degradation from Pseudomonas putida NCIB 9816-4, Comamonas testosteroni GZ42, Sphingomonas yanoikuyae B1, and Mycobacterium sp. strain PY01. None of the isolates from the three groups showed homology to the B1 genes, only two nocardioform isolates showed homology to the PY01 genes, and only members of the pseudomonad group showed homology to the NCIB 9816-4 or GZ42 probes. The Paenibacillus isolates showed no homology to any of the tested gene probes, indicating the possibility of novel genes for PAH degradation. Pure culture substrate utilization experiments using several selected isolates from each of the three groups showed that the phenanthrene-enriched isolates are able to utilize a greater number of PAHs than are the naphthalene-enriched isolates. Inoculating two of the gram-positive isolates to a marine sediment slurry spiked with a mixture of PAHs (naphthalene, fluorene, phenanthrene, and pyrene) and biphenyl resulted in rapid transformation of pyrene, in addition to the two- and three-ringed PAHs and biphenyl. This study indicates that the rhizosphere of salt marsh plants contains a diverse population of PAH-degrading bacteria, and the use of plant-associated microorganisms has the potential for bioremediation of contaminated sediments.     

Dimerization of Receptor Protein-Tyrosine Phosphatase alpha in living cells

Background  

Dimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined.     

Freshwater crayfish invasions in South Africa: past,present and potential future

Freshwater crayfish invasions have been studied around the world, but less so in Africa, a continent devoid of native freshwater crayfish. The present study reviews historical and current information on alien freshwater crayfish species introduced into South Africa and aims to indicate which areas are at risk from invasion. As is the case elsewhere, South Africans have shown a keen interest in both farming and keeping freshwater crayfish as pets, which has resulted in Cherax cainii, Cherax destructor, Cherax quadricarinatus and Procambarus clarkii being introduced to the country. There is evidence of successful establishment in the wild for C. quadricarinatus and P. clarkii in different parts of the country. Species distribution models suggest that the eastern part of the country and parts of the Eastern and Western Cape are at higher risk of invasion. At present, illegal translocations represent the most likely pathway of crayfish spread in South Africa. A continued risk of invasion by freshwater crayfish species in South Africa is highlighted, which reinforces the need for more research, as well as for strong mitigation measures, such as stronger policing of existing regulations, management or eradication where feasible and public education.     

The PagN protein of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium is an adhesin and invasin

Background  

The pagN gene of Salmonella enterica serovar Typhimurium is a PhoP-regulated gene that is up-regulated during growth within macrophages and in vivo in murine models of infection. The PagN protein displays similarity to the Hek and Tia invasins/adhesins of Escherichia coli. Thus far no function has been ascribed to the PagN protein.     

Putative tumor suppressor cytoglobin promotes aryl hydrocarbon receptor ligand–mediated triple negative breast cancer cell death

Nearly 40 000 women die annually from breast cancer in the United States. Clinically available targeted breast cancer therapy is largely ineffective in triple negative breast cancer (TNBC), characterized by tumors that lack expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2). TNBC is associated with a poor prognosis. Previous reports show that aryl hydrocarbon receptor (AhR) partial agonist 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) selectively inhibits the growth of breast cancer cells, including those of the TNBC subtype. We previously demonstrated that 5F 203 induced the expression of putative tumor suppressor gene cytoglobin (CYGB) in breast cancer cells. In the current study, we determined that 5F 203 induces apoptosis and caspase-3 activation in MDA-MB-468 TNBC cells and in T47D ER⁺ PR ⁺ Her2 ⁻ breast cancer cells. We also show that caspases and CYGB promote 5F 203–mediated apoptosis in MDA-MB-468 cells. 5F 203 induced lysosomal membrane permeabilization (LMP) and cathepsin B release in MDA-MB-468 and T47D cells. In addition, silencing CYGB attenuated the ability of 5F 203 to induce caspase-3/-7 activation, proapoptotic gene expression, LMP, and cathepsin B release in MDA-MB-468 cells. Moreover, 5F 203 induced CYGB protein expression, proapoptotic protein expression, and caspase-3 cleavage in MDA-MB-468 cells and in MDA-MB-468 xenograft tumors grown orthotopically in athymic mice. These data provide a basis for the development of AhR ligands with the potential to restore CYGB expression as a novel strategy to treat TNBC.