Biomedical subjects
Publications and source records attributed to M J Grossman.
Dibenzothiophene (DBT), and in particular substituted DBTs, are resistant to hydrodesulfurization (HDS) and can persist in fuels even after aggressive HDS treatment. Treatment by Rhodococcus sp. strain ECRD-1 of a middle distillate oil whose sulfur content was virtually all substituted DBTs produced extensive desulfurization and a sulfur level of 56 ppm.
Rhodococcus sp. strain ECRD-1 was evaluated for its ability to desulfurize a 232 to 343 degrees C middle-distillate (diesel range) fraction of Oregon basin (OB) crude oil. OB oil was provided as the sole source of sulfur in batch cultures, and the extent of desulfurization and the chemical fate of the residual sulfur in the oil after treatment were determined. Gas chromatography (GC), flame ionization detection, and GC sulfur chemiluminesce detection analysis were used to qualitatively evaluate the effect of Rhodococcus sp. strain ECRD-1 treatment on the hydrocarbon and sulfur content of the oil, respectively. Total sulfur was determined by combustion of samples and measurement of released sulfur dioxide by infrared absorption. Up to 30% of the total sulfur in the middle distillate cut was removed, and compounds across the entire boiling range of the oil were affected. Sulfur K-edge X-ray absorption-edge spectroscopy was used to examine the chemical state of the sulfur remaining in the treated OB oil. Approximately equal amounts of thiophenic and sulfidic sulfur compounds were removed by ECRD-1 treatment, and over 50% of the sulfur remaining after treatment was in an oxidized form. The presence of partially oxidized sulfur compounds indicates that these compounds were en route to desulfurization. Overall, more than two-thirds of the sulfur had been removed or oxidized by the microbial treatment.
The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp. strain X309 was localized to a 4-kb KpnI fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS-, and the chloramphenicol resistance (Cmr) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus, rendering it sensitive to the presence of sucrose.
The cloned sulfur oxidation (desulfurization) genes (sox) for dibenzothiophene (DBT) from the prototype Rhodococcus sp. strain IGTS8 were used in Southern hybridization and PCR experiments to establish the DNA relatedness in six new rhodococcal isolates which are capable of utilizing DBT as a sole sulfur source for growth. The ability of these strains to desulfurize appears to be an exclusive property of a 4-kb gene locus on a large plasmid of ca. 150 kb in IGTS8 and ca. 100 kb in the other strains. Besides a difference in plasmid profile, IGTS8 is distinguishable from the other strains in at least the copy number of the insertion sequence IS1166, which is associated with the sox genes.
Dibenzothiophenes (DBTs) bearing alkyl substitutions adjacent to the sulfur atom, such as 4,6-diethyldibenzothiophene (4,6-DEDBT), are referred to as sterically hindered with regard to access to the sulfur moiety. By using enrichment cultures with 4,6-DEDBT as the sole sulfur source, bacterial isolates which selectively remove sulfur from sterically hindered DBTs were obtained. The isolates were tentatively identified as Arthrobacter species. 4,6-DEDBT sulfone was shown to be an intermediate in the 4,6-DEDBT desulfurization pathway, and 2-hydroxy-3,3(prm1)-diethylbiphenyl (HDEBP) was identified as the sulfur-free end product.
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Ferritin is the iron-storage protein of eukaryotic organisms. The nucleotide sequence encoding Azotobacter vinelandii bacterioferritin, a hemoprotein, was determined. The deduced amino acid sequence reveals a high degree of identity with Escherichia coli bacterioferritin and a striking similarity to eukaryotic ferritins. Moreover, derivation of a global alignment shows that virtually all key residues specifying the unique structural motifs of eukaryotic ferritin are conserved or conservatively substituted in the A. vinelandii sequence. The alignment suggests specific methionine residues as heme-binding ligands in bacterioferritins. The overall sequence similarity with conservation of key structural residues implies that all ferritins form a unified family of proteins. The results implicate ferritins as proteins potentially common to all aerobic organisms and as such useful in taxonomic classification, evolutionary analysis, and environmental monitoring.
The effort of clean up the T/V Exxon Valdez oil spill in Prince William Sound, Alaska, included the use of fertilizers to accelerate natural microbial degradation of stranded oil. A program to monitor various environmental parameters associated with this technique took place during the summer of 1990. Microbiological assays for numbers of heterotrophic and oil-degrading microbes and their hydrocarbon mineralization potentials were performed in support of this program. Fertilizer addition resulted in higher hexadecane and phenanthrene mineralization potentials on treated plots than on untreated reference plots. Microbial numbers in treated and reference surface sediments were not significantly different immediately after the first nutrient application in May 1990. However, subsurface sediments from treated plots had higher numbers of hydrocarbon degraders than did reference sediments shortly after treatment. The second application of fertilizer, later in summer, resulted in surface and subsurface increases in numbers of hydrocarbon degraders with respect to reference sediments at two of the three study sites. Elevated mineralization potentials, coupled with increased numbers of hydrocarbon degraders, indicated that natural hydrocarbon biodegradation was enhanced. However, these microbiological measurements alone are not sufficient to determine in situ rates of crude oil biodegradation.
BlaI repressor for the beta-lactamase gene (blaP) of Bacillus licheniformis 749, was shown to repress expression of blaP and of the repressor gene (blaI), using the purified protein in a DNA-directed in vitro translation assay. Binding of BlaI to the promoter regions of blaP and blaI was examined by equilibrium and competitive binding assays. BlaI binds to the blaP promoter with an equal or only slightly higher affinity than to the blaI promoter. DNase I footprinting shows that BlaI binds directly to the blaP and blaI promoters, such that RNA polymerase binding and/or transcript elongation would be blocked.
The location of the repressor gene, blaI, for the beta-lactamase gene blaP of Bacillus licheniformis 749, on the 5' side of blaP, was confirmed by sequencing the bla region of the constitutive mutant 749/C. An amber stop codon, likely to result in a nonfunctional truncated repressor, was found at codon 32 of the 128 codon blaI open reading frame (ORF) located 5' to blaP. In order to study the DNA binding activity of the repressor, the structural gene for blaI, from strain 749, with its ribosome binding site was expressed using a two plasmid T7 RNA polymerase/promotor system (S. Tabor and C. C. Richardson. Proc. Natl. Acad. Sci. 82, 1074-1078 (1985). Heat induction of this system in Escherichia coli K38 resulted in the production of BlaI as 5-10% of the soluble cell protein. Repressor protein was then purified by ammonium sulfate fractionation and cation exchange chromatography. The sequence of the N-terminal 28 amino acid residues was determined and was as predicted from the DNA. Binding of BlaI to DNA was detected by the slower migration of protein DNA complexes during polyacrylamide gel electrophoresis. BlaI was shown to selectively bind DNA fragments carrying the promoter regions of blaI and blaP.
At the time of laparoscopic examination performed for the evaluation of infertility, 55 patients with hydrosalpinges were identified. Perihepatic "violin-string" adhesions suggestive of a prior episode of Fitz-Hugh-Curtis syndrome were seen in 15 of these patients. Of 13 patients with proximal tubal obstruction and 7 patients with periadnexal adhesions and patent fallopian tubes, none of whom had surgical procedures or endometriosis, none were noted to have perihepatic adhesions. No patients with perihepatic adhesions had symptoms referable to the liver.