Cloning and characterization of Thiobacillus ferrooxidans genes involved in sulfur assimilation
The sulfate activating locus from Thiobacillus ferrooxidans has been cloned and expressed in Escherichia coli. The genes encoding ATP sulfurylase (ATP: sulfate adenylyl transferase, EC 2.7.7.4) and APS kinase (ARP: adenosine 5'-phosphosulfate 3'-phopho transferase, EC 2.7.1.25) have been isolated from a T. ferrooxidans genomic library constructed in pBR322. The approach used involved the utilization of mutants of E. coli defective in specific steps of the sulfur assimilatory pathway to isolate and identify, by genetic complementation, the corresponding genes and gene products in T. ferrooxidans. In E. coli, the genes of sulfur assimilation are found in a locus mapping at 59 minutes of the chromosome. Included here are a cluster of genes organized in an operon designated cysDCHIJ. The proteins encoded by these genes include: ATP sulfurylase, APS kinase, PAPS reductase and sulfite reductase respectively. A plasmid designated pTFcys13 which contains a 4.7 kb T. ferrooxidans insert was found to complement both cysD{sup {minus}} and cysC{sup {minus}} E. coli mutants. pTFcyc13 did not complement E. coli mutants carrying either cysI, cysJ or cycH defective genes. The genetic organization of the sulfate reducing locus has been inferred from deletion analysis of the insert in pTFcys13 coupled to complementation studies. Most deletions obtained led to a complete loss of ATP sulfurylase activity pointing to the possibility that more than one gene may encode this enzyme and thus, to its probable multimeric nature. The multimeric make-up of the sulfurylase was verified by in vitro transcription-translation studies with the cycD-complementing plasmid that indicated that enzyme is made up of two non-identical subunits with an estimated size of 36 and 62 kD. 24 refs., 7 figs., 5 tabs.
- Research Organization:
- Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
- Sponsoring Organization:
- DOE/ER
- DOE Contract Number:
- W-7405-ENG-48
- OSTI ID:
- 5212524
- Report Number(s):
- UCRL-102413; CONF-8908195-1; ON: DE90005190
- Resource Relation:
- Conference: Biohydro-metallurgy '89, Jackson Hole, WY (USA), 13-18 Aug 1989
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
THIOBACILLUS FERROXIDANS
GENETIC ENGINEERING
BACTERIOPHAGES
CLONING
DESULFURIZATION
GENES
GENETICS
MICROORGANISMS
ORGANIC SULFUR COMPOUNDS
PLASMIDS
REDUCTION
SULFUR COMPOUNDS
BACILLUS
BACTERIA
BIOLOGY
CELL CONSTITUENTS
CHEMICAL REACTIONS
ORGANIC COMPOUNDS
PARASITES
SULFUR-OXIDIZING BACTERIA
VIRUSES
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