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. In the search for alternative methods of producing alcohol fuels, biodegradation of fibrous biomass by thermophilic anaerobic bacteria has been studied for the past 20 years (9, 13). Clostridium thermocellum has been the most extensively examined thermophile (6), but it does have limitations with regard to ethanol yield and its ability to use certain carbohydrate fractions (9). Therefore, some work has focused on the use of cocultures of thermophilic bacteria (4, 10). Although from the biotechnological standpoint it may be desirable to use cocultures, it is essential that pure cultures are available for study in order to elucidate the molecular regulation of genes encoding saccharolytic enzymes. However, culture purity can be a problem since cultures of cellulolytic thermophiles are prone to contamination by glycolytic, noncellulolytic thermophilic bacteria, including Clostridium thermosaccharolyticum-like bacteria (7). Several biochemical and physiological assays have been suggested to distinguish among closely related saccharolytic thermophiles and to verify the purity of C. thermocellum cultures (7). However, these assays (e.g., monitoring butyrate formation and growth on glucose at 72°C or 30°C and testing for proteolytic activity) are time consuming and may not be reliable when low levels of contaminants are present. Contrary to earlier findings that C. thermocellum JW20 was able to utilize xylose only after 2 to 5 weeks (7), we recently reported that this strain did not require long adaptation periods to grow on xylose (12). In fact, 95% xylose utilization within 12 h has been routinely observed in our laboratory. Based on this observation, we pursued a search for xylose utilization genes in this strain. Screening of a genomic library prepared from chromosomal DNA purified from the C. thermocellum JW20 culture resulted in the isolation of a genomic clone (5) containing a gene with a nucleotide sequence identical, except for four mismatches, to the xylose isomerase gene (xylA) from Thermoanaerobacter ethanolicus 39E (ATCC 33223) (3), formerly Clostridium thermohydrosulfuricum 39E. Since it is highly unlikely that two distinct bacterial species have virtually identical nucleotide sequences of a gene, we suspected that C. thermocellum JW20 was contaminated with a T. ethanolicus 39E-like organism. This observation, as well as the need to routinely assess culture purity, prompted us to design an assay based on DNA sequence specificity. In this report we describe

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.

The genes encoding xylose isomerase (xylA) and xylulose kinase (xylB) from the thermophilic anaerobeThermoanaerobacter 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 ofxylAB 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.

A xylose ABC (ATP-binding cassette) transport operon, xylFGH, was cloned from Thermoanaerobacter ethanolicus, a thermophilic ethanol-producing eubacterium. The cistrons code for a periplasmic D-xylose-binding protein (XylF, partial sequence of 250 amino acids), ATP-binding protein (XylG, 505 amino acids), and integral membrane protein (XylH, 388 amino acids). These results, together with previous work, indicate that duplicate copies of both xylF and xylH are present in the T. ethanolicus chromosome, suggesting ancient gene duplication or lateral gene transfer events. XylG resembles other eubacterial monosaccharide ABC-ATPases in that its two nucleotide-binding domains (NBDs) are highly homologous, yet significantly different with respect to putative catalytic residues. Unlike most other integral membrane ABC transport proteins, XylH apparently contains 11 or 12 transmembrane segments (TMS) and is similar to a small group of ABC permeases that defy the "2 x 6" helix paradigm. This is the first report of a monosaccharide ABC transport operon in a thermophilic anaerobic eubacterium.