Expression of the Escherichia coli pntA andpntB Genes, Encoding Nicotinamide Nucleotide Transhydrogenase, in Saccharomyces cerevisiae and Its Effect on Product Formation during Anaerobic Glucose Fermentation

1999 ◽  
Vol 65 (6) ◽  
pp. 2333-2340 ◽  
Author(s):  
Mikael Anderlund ◽  
Torben L. Nissen ◽  
Jens Nielsen ◽  
John Villadsen ◽  
Jan Rydström ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Anaerobic Fermentation ◽  
Product Formation ◽  
Physiological Effect ◽  
Glucose Fermentation ◽  
Genes Encoding ◽  
Membrane Bound ◽  
Glycerol Formation ◽  
High Level

ABSTRACT We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinantSaccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD+ in S. cerevisiaeand thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD+ by NADPH and by NADH in the presence of NADP+, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD+, NADPH, and NADP+were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD+.

Selective cloning of genes encoding RNase H from Salmonella typhimurium, Saccharomyces cerevisiae and Escherichia coli rnh mutant

1991 ◽  
Vol 227 (3) ◽  
pp. 438-445 ◽  
Author(s):  
Mitsuhiro Itaya ◽  
Dorothy McKelvin ◽  
Sunil K. Chatterjie ◽  
Robert J. Crouch
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Salmonella Typhimurium ◽  
Rnase H ◽  
Genes Encoding

scholarly journals Transcriptional control and essential roles of the Escherichia coli ccm gene products in formate-dependent nitrite reduction and cytochrome c synthesis

1998 ◽  
Vol 334 (2) ◽  
pp. 355-365 ◽  
Author(s):  
Sutipa TANAPONGPIPAT ◽  
Eleanor REID ◽  
Jeffrey A. COLE ◽  
Helen CROOKE
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Transcriptional Control ◽  
Regulatory System ◽  
Nitrite Reduction ◽  
Anaerobic Growth ◽  
Current View ◽  
Fnr Protein ◽  
Genes Encoding ◽  
Membrane Bound

The eight ccm genes located at minute 47 on the Escherichia coli chromosome, in the order ccmABCDEFGH, encode homologues of proteins which are essential for cytochrome c assembly in other bacteria. The ccm genes are immediately downstream from the napFDAGHBC genes encoding a periplasmic nitrate reductase. CcmH was previously shown to be essential for cytochrome c assembly. Deletion analysis and a two-plasmid strategy have now been used to demonstrate that CcmA, B, D, E, F and G are also essential for cytochrome c assembly, and hence for cytochrome-c-dependent nitrite reduction. The ccm genes are transcribed from a ccmA promoter located within the adjacent gene, napC, which is the structural gene for a 24 kDa membrane-bound c-type cytochrome, NapC. Transcription from this ccmA promoter is induced approximately 5-fold during anaerobic growth, independently of a functional Fnr protein: it is also not regulated by the ArcB–ArcA two-component regulatory system. The ccmA promoter is an example of the ‘extended -10 sequence ’ group of promoters with a TGX motif immediately upstream of the -10 sequence. Mutagenesis of the TG motif to TC, CT or CC resulted in loss of about 50% of the promoter activity. A weak second promoter is suggested to permit transcription of the downstream ccmEFGH genes in the absence of transcription readthrough from the upstream napF and ccmA promoters. The results are consistent with, but do not prove, the current view that CcmA, B, C and D are part of an essential haem transport mechanism, that CcmE, F and H are required for covalent haem attachment to cysteine-histidine motifs in cytochrome c apoproteins in the periplasm, and that CcmG is required for the reduction of cysteine residues on apocytochromes c in preparation for haem ligation.

Organization of genes encoding the L11, L1, L10, and L12 equivalent ribosomal proteins in eubacteria, archaebacteria, and eucaryotes

1989 ◽  
Vol 35 (1) ◽  
pp. 164-170 ◽  
Author(s):  
Lawrence C. Shimmin ◽  
C. Hunter Newton ◽  
Celia Ramirez ◽  
Janet Yee ◽  
Willa Lee Downing ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Sulfolobus Solfataricus ◽  
Mrna Translation ◽  
Restriction Fragments ◽  
E Coli ◽  
Transcription Pattern ◽  
Genes Encoding ◽  
Cytoplasmic Ribosomes

Archaebacterial and eucaryotic cytoplasmic ribosomes contain proteins equivalent to the L11, L1, L10, and L12 proteins of the eubacterium Escherichia coli. In E. coli the genes encoding these ribosomal proteins are clustered, cotranscribed, and autogenously regulated at the level of mRNA translation. Genomic restriction fragments encoding the L11e, L1e, L10e, and L12e (equivalent) proteins from two divergent archaebacteria, Halobacterium cutirubrum and Sulfolobus solfataricus, and the L10e and L12e proteins from the eucaryote Saccharomyces cerevisiae have been cloned, sequenced, and analyzed. In the archaebacteria, as in eubacteria, the four genes are clustered and the L11e, L1e, L 10e, and L12e order is maintained. The transcription pattern of the H. cutirubrum cluster is different from the E. coli pattern and the flanking genes on either side of the tetragenic clusters in E. coli, H. cutirubrum, and Sulfolobus solfataricus are all unrelated to each other. In the eucaryote Saccharomyces cerevisiae there is a single L10e gene and four separate L12e genes that are designated L12eIA, L12eIB, L12eIIA, and L12eIIB. These five genes are not closely linked and each is transcribed as a monocistronic mRNA; the L10e, L12eIA, L12eIB, and the L12eIIA genes are contiguous and uninterrupted, whereas the L12eIIB gene is interrupted by a 301 nucleotide long intron located between codons 38 and 39.Key words: archaebacteria, ribosome, Halobacterium, Sulfolobus.

scholarly journals Engineering of Artificial Plant Cytochrome P450 Enzymes for Synthesis of Isoflavones by Escherichia coli

2007 ◽  
Vol 73 (22) ◽  
pp. 7246-7251 ◽  
Author(s):  
Effendi Leonard ◽  
Mattheos A. G. Koffas
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Cytochrome P450 Enzymes ◽  
E Coli ◽  
Recombinant Production ◽  
Membrane Bound ◽  
High Level ◽  
Membrane Recognition ◽  
In Vivo Synthesis

ABSTRACT Engineered microbes are becoming increasingly important as recombinant production platforms. However, the nonfunctionality of membrane-bound cytochrome P450 enzymes precludes the use of industrially relevant prokaryotes such as Escherichia coli for high-level in vivo synthesis of many functional plant-derived compounds. We describe the design of a series of artificial isoflavone synthases that allowed the robust production of plant estrogen pharmaceuticals by E. coli. Through this methodology, a plant P450 construct was assembled to mimic the architecture of a self-sufficient bacterial P450 and contained tailor-made membrane recognition signals. The specific in vivo production catalyzed by one identified chimera was up to 20-fold higher than that achieved by the native enzyme expressed in a eukaryotic host and up to 10-fold higher than production by plants. This novel biological device is a strategy for the utilization of laboratory bacteria to robustly manufacture high-value plant P450 products.

scholarly journals High level, context dependent misincorporation of lysine for arginine in Saccharomyces cerevisiae al homeodomain expressed in Escherichia coli

1998 ◽  
Vol 7 (2) ◽  
pp. 500-503 ◽  
Author(s):  
Michael D. Forman ◽  
Robert F. Stack ◽  
Paul S. Masters ◽  
Charles R. Hauer ◽  
Susan M. Baxter
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Context Dependent ◽  
High Level

Enteropathogenic Escherichia coli type III effectors alter cytoskeletal function and signalling in Saccharomyces cerevisiae

Microbiology ◽  
2005 ◽  
Vol 151 (9) ◽  
pp. 2933-2945 ◽  
Author(s):  
Isabel Rodríguez-Escudero ◽  
Philip R. Hardwidge ◽  
César Nombela ◽  
Víctor J. Cid ◽  
B. Brett Finlay ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Effector Proteins ◽  
Type Iii Effectors ◽  
Type Iii ◽  
Genes Encoding ◽  
Cytoskeletal Function ◽  
Enterocyte Effacement ◽  
Functional Analyses ◽  
Map Kinase Pathways

Enteropathogenic Escherichia coli (EPEC) strains cause attaching/effacing lesions in enterocytes through the development of actin-supported pedestals at the site of bacterial adhesion. Pathogenesis requires a type III secretion system (TTSS), which injects into the host cell the intimin receptor, Tir, as well as other effectors called Esps (Escherichia secreted proteins). The genes encoding TTSS structural components and Esps are found within a pathogenicity island called the locus of enterocyte effacement (LEE). This paper describes the application of Saccharomyces cerevisiae as a model to probe the functions of LEE-encoded genes. In a systematic approach, the LEE-encoded translocator and effector proteins were endogenously expressed in yeast and their effects on cell growth, cytoskeletal function and signalling pathways were studied. EspD, EspG and Map inhibited growth by depolarizing the actin cortical cytoskeleton, whereas EspF expression altered the septin cytoskeleton. Specific yeast MAP kinase pathways were activated by EspF, EspG, EspH and Map. The yeast system was used to define functional domains in Map by expressing truncated versions; it was concluded that the C-terminal region of the protein is necessary for actin disruption and toxicity, but not for mitochondrial localization. The utility of the yeast model for functional analyses of EPEC pathogenesis is discussed.

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