Organization and sequence of histidine biosynthesis genes hisH, -A, -F, and -IE in Thermoanaerobacter ethanolicus.

Nucleotide sequence analysis of a 3.5-kb chromosomal fragment from the low G + C Gram-positive bacterium Thermoanaerobacter ethanolicus revealed a cluster of five contiguous open reading frames (ORFs) designated hisH, hisA, hisF, hisIE, and ORF5. The first four ORFs showed homology to genes of the histidine biosynthesis pathway, and ORF5 encoded a product with no significant similarities to polypeptides presently known. The hisH ORF was partial (truncated by cloning) and ORF5 was adjacent to xylF, which codes for a xylose-binding periplasmic protein. The five genes encoded putative proteins of >104, 237, 254, 216, and 169 amino acids, respectively. Amino acid sequence comparison of the four his gene products indicated closely related homologs in prokaryotes, varying from low G + C Gram-positive bacteria to archaea. This is the first report of his anabolic genes in a thermophilic anaerobic bacterium.

Characterization of the Euplotes crassus macronuclear rDNA and its potential as a DNA transformation vehicle.

We have cloned the macronuclear linear DNA molecule carrying the ribosomal RNA genes from the ciliated protozoan Euplotes crassus. DNA sequence analysis was carried out to locate coding regions and to determine whether sequences that have been mutated to confer antibiotic resistance are conserved in the E. crassus genes. The beginning and end of the primary transcript were mapped. In order to determine whether conserved sequences that might serve as replication origins were present, the 5' and 3' non-coding sequences from E. crassus were compared to the corresponding sequences from the macronuclear linear rDNA molecules from the following euplotid species: Euplotes vannus, Euplotes minuta, Euplotes raikovii and Euplotes rariseta. A DNA transformation construct was made by generating a putative anisomycin resistant mutation along with a mutation generating a restriction site polymorphism. Microinjection of the construct into the developing macronucleus of mated cells resulted in exconjugant cell lines with increased resistance to anisomycin. The injected rDNA with the restriction site polymorphism is detectable in the anisomycin resistant cells and appears to represent a minor fraction of the rDNA.

Xylose transport by the anaerobic thermophile thermoanaerobacter ethanolicus and the characterization of a D-xylose-binding protein

Thermoanaerobacter ethanolicus is a xylose-utilizing thermophilic anaerobe that produces considerable amounts of ethanol. A protein in xylose-growing cells was solubilized from cell membranes by extraction with octyl-beta-glucoside. Internal peptide sequencing revealed that the protein was the product of a gene, xylF, encoding a putative D-xylose-binding protein. Metabolic labeling with 14C palmitic acid suggested that this is a lipoprotein that is anchored to the cell membrane via a cysteine residue. Binding was highly specific for xylose as evident by the lack of competition by sugars with structures similar to xylose. The apparent Kd of the protein for xylose was approximately 1.5 &mgr;M, and this value was very similar to the affinity constant determined for xylose transport by whole cells at low substrate concentrations. Uptake experiments with cells also suggested the presence of a separate low-affinity system. Binding activity varied less than 20% over a pH range of 4-8, and the level of activity was virtually unaffected when temperature was varied between 40 degreesC and 80 degreesC. This is the first biochemical characterization of a D-xylose-binding protein from a thermophilic organism.

The D-xylose-binding protein, XylF, from Thermoanaerobacter ethanolicus 39E: cloning, molecular analysis, and expression of the structural gene.

Immediately downstream from the Thermoanaerobacter ethanolicus xylAB operon, comprising genes that encode D-xylose isomerase and D-xylulose kinase, lies a 1,101-bp open reading frame that exhibits 61% amino acid sequence identity to the Escherichia coli D-xylose binding periplasmic receptor, XylF, a component of the high-affinity binding-protein-dependent D-xylose transport. The 25-residue N-terminal fragment of the deduced T. ethanolicus XylF has typical features of bacterial leader peptides. The C-terminal portion of this leader sequence matches the cleavage consensus for lipoproteins and is followed by a 22-residue putative linker sequence rich in serine, threonine, and asparagine. The putative mature 341-amino-acid-residue XylF (calculated molecular mass of 37,069 Da) appears to be a lipoprotein attached to the cell membrane via a lipid anchor covalently linked to the N-terminal cysteine, as demonstrated by metabolic labelling of the recombinant XylF with [14C]palmitate. The induced E. coli avidly bound D-[14C]xylose, yielding additional evidence that T. ethanolicus XylF is the D-xylose-binding protein. On the basis of sequence comparison of XylFs to other monosaccharide-binding proteins, we propose that the sequence signature of binding proteins specific for hexoses and pentoses be refined as (KDQ)(LIVFAG)3IX3(DN)(SGP)X3(GS)X(LIVA) 2X2A. Transcription of the monocistronic 1.3-kb xylF mRNA is inducible by xylose and unaffected by glucose. Primer extension analysis indicated that xylF transcription initiates from two +1 sites, both situated within the xylAB operon. Unlike in similar transport systems in other bacteria, the genes specifying the membrane components (e.g., ATP-binding protein and permease) of the high-affinity D-xylose uptake system are not located in the vicinity of xylF in T. ethanolicus. This is the first report of a gene encoding a xylose-binding protein in a gram-positive or thermophilic bacterium.

Cloning and characterization of transcription of the xylAB operon in Thermoanaerobacter ethanolicus.

The genes encoding xylose isomerase (xylA) and xylulose kinase (xylB) from the thermophilic anaerobe Thermoanaerobacter ethanolicus were found to constitute an operon with the transcription initiation site 169 nucleotides upstream from the previously assigned (K. Dekker, H. Yamagata, K. Sakaguchi, and S. Udaka, Agric. Biol. Chem. 55:221-227, 1991) promoter region. The bicistronic xylAB mRNA was processed by cleavage within the 5'-terminal portion of the XylB-coding sequence. Transcription of xylAB was induced in the presence of xylose, and, unlike in all other xylose-utilizing bacteria studied, was not repressed by glucose. The existence of putative xyl operator sequences suggested that xylose utilization is controlled by a repressor-operator mechanism. The T. ethanolicus xylB gene coded for a 500-amino-acid-residue protein with a deduced amino acid sequence highly homologous to those of other XylBs. This is the first report of an xylB nucleotide sequence and an xyLAB operon from a thermophilic anaerobic bacterium.

Clostridium thermocellum JW20 (ATCC 31549) is a coculture with Thermoanaerobacter ethanolicus.

A PCR assay based on 16S rRNA sequence differences among four thermophilic anaerobic bacterial strains was used to demonstrate contamination of Clostridium thermocellum JW20 (ATCC 31549) with a Thermoanaerobacter ethanolicus strain. Therefore, we suggest that interpretation of experimental results with C. thermocellum JW20 be viewed with caution.

Sequence of the macronuclear DNA encoding large subunit ribosomal protein 29 (L29) in Euplotes crassus and cycloheximide sensitivity.

As a first step towards developing a DNA transformation method for the ciliated protozoan Euplotes crassus we determined the minimum inhibitory concentration (MIC) for cell division in the presence of cycloheximide (Chx) for several cell lines and the range of Chx sensitivity for 106 different progeny cell lines derived by mating two lines. All of the cell lines are highly sensitive to Chx. Progeny cell lines show a wider range of sensitivities than the parental lines. Because site-directed mutagenesis of the RPL29 gene encoding the large subunit ribosomal protein 29 (L29) has been used to generate a Chx-resistance marker (ChxR) for another ciliate, Tetrahymena thermophila [Yao and Yao, Proc. Natl. Acad. Sci. USA 88 (1991) 9493-9497], we isolated and sequenced the entire E. crassus macronuclear DNA carrying RPL29. The encoded peptide is 52-73% identical in sequence to L29 sequences from organisms ranging from T. thermophila and Saccharomyces cerevisiae to mouse. In E. crassus, the codon that has been mutated to confer Chx resistance in both S. cerevisiae and T. thermophila already encodes the amino-acid residue of one of the mutant forms identified in these other organisms. Thus, E. crassus RPL29 is not a convenient source of a selectable marker. Notable features of the macronuclear DNA carrying RPL29 are its extremely short non-coding regions and a TAG stop codon.