Influence of environmental parameters on pentachlorophenol biotransformation in soil by Lentinula edodes and Phanerochaete chrysosporium

 The influences of temperature, soil moisture potential and initial pH on the biotransformation of pentachlorophenol (PCP) by the lignicolous fungi Lentinula edodes and Phanerochaete chrysosporium were examined. At 10^°C, L. edodes was more effective in degrading PCP (P<0.05) than P. chrysosporium. At 15^°C similar results were obtained for the two fungi. The highest levels of degradation occurred for both fungi at 25^°C. With P. chrysosporium, the extent of PCP elimination was directly related to soil moisture content and optimal at approximately 47%. With L. edodes, in contrast, the process was inversely related to moisture content and maximal at 26%. The initial soil pH also had a marked influence, and pH 4.0 was optimal for both fungi. Received: 7 August 1995/Accepted: 22 August 1995     

Lipases of Fonsecaea pedrosoi and Phialophora verrucosa

Lipase activity was demonstrable titrimetrically in the culture filtrates of Fonsecaea pedrosoi and Phialophora verrucosa on the 6th day of incubation reaching a peak on the 15th and 12th days respectively for the two fungi. Purified lipases of F. pedrosoi and P. verrucosa, with specific activities of 36.0 and 39.4 fold increase respectively, were obtained by cold acetone extraction, gel filtration followed by ion exchange chromatography. The lipases had the same optimum pH (5.5) and temperature (35° C). The molecular weights of the lipases of F. pedrosoi and P. verrucosa, estimated by gel filtration on Sephadex G-100, were 25000 and 20000, respectively and the enzymes showed broad susbstrate specificity. The possible role of lipase in the pathogenesis of infection caused by the fungi is discussed.     

The Plasmid-Encoded Regulator Activates Factors Conferring Lysozyme Resistance on Enteropathogenic Escherichia coli Strains

We demonstrate that enhanced lysozyme resistance of enteropathogenic Escherichia coli requires the plasmid-encoded regulator, Per, and is mediated by factors outside the locus for enterocyte effacement. EspC, a Per-activated serine protease autotransporter protein, conferred enhanced resistance on nonpathogenic E. coli, and a second Per-regulated, espC-independent lysozyme resistance mechanism was identified.     

Purification, cloning, and sequencing of an enzyme mediating the reductive dechlorination of tetrachloroethylene (PCE) from Clostridium bifermentans DPH-1

An enzyme mediating the reductive dechlorination of tetrachloroethylene (PCE) from cell-free extracts of Clostridium bifermentans DPH-1 was purified, cloned, and sequenced. The enzyme catalyzed the reductive dechlorination of PCE to cis-1,2-dichloroethylene via trichloroethylene, at a Vmax and Km of 73 nmol/mg protein and 12 microM, respectively. Maximal activity was recorded at 35 degrees C and pH 7.5. Enzymatic activity was independent of metal ions but was oxygen sensitive. A mixture of propyl iodide and titanium citrate caused a light-reversible inhibition of enzymatic activity suggesting the involvement of a corrinoid cofactor. The molecular mass of the native enzyme was estimated to be approximately 70 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) revealed molecular masses of approximately 35 kDa and 35.7 kDa, respectively. A broad spectrum of chlorinated aliphatic compounds (PCE, trichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloropropane, and 1,1,2-trichloroethane) was degraded. With degenerate primers designed from the N-terminal sequence (27 amino acid residues), a partial sequence (81 bp) of the encoding gene was amplified by polymerase chain reaction (PCR) and sequenced. Southern analysis of C. bifermentans genomic DNA using the PCR product as a probe revealed restriction fragment bands. A 5.0 kb ClaI fragment, harboring the relevant gene (designated pceC) was cloned (pDEHAL5) and the complete nucleotide sequence of pceC was determined. The gene showed homology mainly with microbial membrane proteins and no homology with any known dehalogenase, suggesting a distinct PCE dehalogenase.     

In vitro dehalogenation of tetrachloroethylene (PCE) by cell-free extracts of Clostridium bifermentans DPH-1

Cell-free extracts of Clostridium bifermentans DPH-1 catalyzed tetrachloroethylene (PCE) dechlorination. PCE degradation was stimulated by addition of a variety of electron donors. Ethanol (0.61 mM) was the most effective electron donor for PCE dechlorination. Maximum activity was recorded at 30 degrees C and pH 7.5. Addition of NADH as a cofactor stimulated enzymatic activity but the activity was not stimulated by addition of metal ions. When the cell-free enzyme extract was incubated in the presence of titanium citrate as a reducing agent, the dehalogenase was rapidly inactivated by propyl iodide (0.5 mM). The activity of propyliodide-reacted enzyme was restored by illumination with a 250 W lamp. The dehalogenase activity was also inhibited by cyanide. The substrate spectrum of activity included trichloroethylene (TCE), cis-1,2-dichloroethylene (cDCE), trans-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloroethane, and 1,1,2-trichloroethane. The highest rate of degradation of the chlorinated aliphatic compounds was achieved with PCE, and PCE was principally degraded via TCE to cDCE. Results indicate that the dehalogenase could play a vital role in the breakdown of PCE as well as a variety of other chlorinated aliphatic compounds.     

Comparative Genomics and Characterization of Hybrid Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains

Background

Shigatoxigenic Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) cause serious foodborne infections in humans. These two pathogroups are defined based on the pathogroup-associated virulence genes: stx encoding Shiga toxin (Stx) for STEC and elt encoding heat-labile and/or est encoding heat-stable enterotoxin (ST) for ETEC. The study investigated the genomics of STEC/ETEC hybrid strains to determine their phylogenetic position among E. coli and to define the virulence genes they harbor.

Methods

The whole genomes of three STEC/ETEC strains possessing both stx and est genes were sequenced using PacBio RS sequencer. Two of the strains were isolated from the patients, one with hemolytic uremic syndrome, and one with diarrhea. The third strain was of bovine origin. Core genome analysis of the shared chromosomal genes and comparison with E. coli and Shigella spp. reference genomes was performed to determine the phylogenetic position of the STEC/ETEC strains. In addition, a set of virulence genes and ETEC colonization factors were extracted from the genomes. The production of Stx and ST were studied.

Results

The human STEC/ETEC strains clustered with strains representing ETEC, STEC, enteroaggregative E. coli, and commensal and laboratory-adapted E. coli. However, the bovine STEC/ETEC strain formed a remote cluster with two STECs of bovine origin. All three STEC/ETEC strains harbored several other virulence genes, apart from stx and est, and lacked ETEC colonization factors. Two STEC/ETEC strains produced both toxins and one strain Stx only.

Conclusions

This study shows that pathogroup-associated virulence genes of different E. coli can co-exist in strains originating from different phylogenetic lineages. The possibility of virulence genes to be associated with several E. coli pathogroups should be taken into account in strain typing and in epidemiological surveillance. Development of novel hybrid E. coli strains may cause a new public health risk, which challenges the traditional diagnostics of E. coli infections.     

Protein Profiling in Hepatocellular Carcinoma by Label-Free Quantitative Proteomics in Two West African Populations

Background

Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC.

Methods

Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis.

Results

Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages.

Conclusions

The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose definitive cut-offs and combinations for evaluation of performance.     

A second large plasmid encodes conjugative transfer and antimicrobial resistance in O119:H2 and some typical O111 enteropathogenic Escherichia coli strains

A novel and functional conjugative transfer system identified in O119:H2 enteropathogenic Escherichia coli (EPEC) strain MB80 by subtractive hybridization is encoded on a large multidrug resistance plasmid, distinct from the well-described EPEC adherence factor (EAF) plasmid. Variants of the MB80 conjugative resistance plasmid were identified in other EPEC strains, including the prototypical O111:NM strain B171, from which the EAF plasmid has been sequenced. This separate large plasmid and the selective advantage that it confers in the antibiotic era have been overlooked because it comigrates with the virulence plasmid on conventional gels.     

Biodegradation of methyl tertiary butyl ether (MTBE) by a bacterial enrichment consortia and its monoculture isolates

Methyl tertiary butyl ether (MTBE), an important gasoline additive, is a recalcitrant compound posing serious environmental health problems. In this study, MTBE-degrading bacteria were enriched from five environmental samples. Enrichments from Stewart Lake sediments and an MTBE contaminated soil displayed the highest rate of MTBE removal; 29.6 and 27.8% respectively, in 28 days. A total of 12 bacterial monocultures isolated from enrichment cultures were screened for MTBE degradation in liquid cultures. In a nutrient-limited medium containing MTBE as the sole source of carbon and energy, the highest rate of MTBE elimination was achieved with IsoSL1, which degraded 30.6 and 50.2% in 14 and 28 days, respectively. In a nutrient-rich medium containing ethanol and yeast extract, the bacterium (Iso2A) substantially removed MTBE (20.3 and 28.1% removal in 14 and 28 days, respectively). Based upon analysis of the 16s rRNA gene sequence and data base comparison, IsoSL1 and Iso2A were identified as a Streptomyces sp. and Sphingomonas sp., respectively. The Streptomyces sp. is a new genera of bacteria degrading MTBE and could be useful for MTBE bioremediation.