Structure predictions suggest a partial conservation of RNA structure elements in coronavirus terminal genome regions. Here, we determined the structures of stem-loops (SL) 1 and 2 of two alphacoronaviruses, human coronavirus (HCoV) 229E and NL63, by RNA structure probing and studied the functional relevance of these putative cis-acting elements. HCoV-229E SL1 and SL2 mutants generated by reverse genetics were used to study the effects on viral replication of single-nucleotide substitutions predicted to destabilize the SL1 and SL2 structures. The data provide conclusive evidence for the critical role of SL1 and SL2 in HCoV-229E replication and, in some cases, revealed parallels with previously characterized betacoronavirus SL1 and SL2 elements. Also, we were able to rescue viable HCoV-229E mutants carrying replacements of SL2 with equivalent betacoronavirus structural elements. The data obtained in this study reveal a remarkable degree of structural and functional conservation of 5'-terminal RNA structural elements across coronavirus genus boundaries.

A 13.6-kilobase (kb) Sau3AI restriction endonuclease fragment of Clostridium acetobutylicum DNA cloned into pBR322 enabled Escherichia coli ato mutants to grow on butyrate as a sole carbon source (But').Complementation of the ato defect by the recombinant plasmid pJC6 was due to expression of the genes for phosphotransbutyrylase (PTB) and butyrate kinase (BK). Both genes were efficiently expressed in E. coli, as their products were readily detected by sodium dodecyl sulfate-poly-icrylamide gel electrophoresis of whole-cell extracts. PTB was found to have a polypeptide subunit molecular weight of approximately 31,000, while that of BK was approximately 39,000. Deletion analysis and TnS mutagenesis of plasmid pJC7 (a But' subclone containing a 4.4-kb Bamll fragment from the insert of pJC6) localized the PTB and BK genes within a region spanning approximately -2.9 kb. Preliminary evidence suggests that the two genes may form an operon that is transcribed as a single unit from a promoter of clostridial origin within the 4.4-kb insert of pJC7.The acetone-butanol fermentation of Clostridium acetobutylicum can be divided into two distinct phases. In the acidogenic phase, acetic and butyric acids are formed, with a concomitant drop in the pH of the medium to 4 to 4.5. As the culture enters the stationary phase of growth, there is a metabolic shift to solvent production (solventogenic phase). During this phase, growth slows as the acids present in the medium are reassimilated and metabolized to produce acetone, butanol, and ethanol. Eventually the high levels of solvents become toxic to the cells and growth ceases (6).Two enzymes, phosphotransbutyrylase (PTB) (EC 2.3.1.8) and butyrate kinase (EC 2.7.2.7), play a key role in the production of butyrate from butyryl coenzyme A (butyryl-CoA) during the acidogenic phase of growth in C. acetobutylicum. Both enzymes also play a major role in the energy metabolism of the organism, as ATP is produced during the conversion of butyryl-CoA to butyrate via the following mechanism (25):butyryl-CoA + Pi PTB butyryl-P + CoA butyryl-P + ADP butyrate kinase butyrate + ATP Although both of these enzymes are capable of catalyzing their respective reactions in the opposite direction, several studies have indicated that PTB and butyrate kinase are not responsible for the uptake of butyrate and its eventual conversion to butanol (12, 13). Hartmanis and Gatenbeck (12) reported that during the acidogenic phase, both PTB and butyrate kinase exhibit high specific activities. During solventogenesis, however, PTB activity showed a rapid decrease while the activity of butyrate kinase increased by as much as fivefold. Contrary to these reports, Weisenborn et

Twenty-two Bacillus spp. isolates from the rhizosphere of Phaseolus vulgaris 'Contender' were identified using Biolog™, gas chromatographic fatty acid methyl ester, and 23S rDNA analyses. Some of the Bacillus isolates produced significant amounts of the phytohormone indoleacetic acid (IAA) when grown in a liquid culture medium supplemented with 100 μg L-tryptophan/L; less IAA was produced in culture medium not supplemented with L-tryptophan. Thin-layer chromatography, high-performance liquid chromatography, gas chromatography – mass spectrometry, and the avena coleoptile bioassay were used to identify and quantify IAA produced by Bacillus isolates. Significant differences were observed in the amounts of IAA produced by different strains of Bacillus, with amounts varying from 0.40 to 4.88 μg/mL. α-Methyltryptophan-resistant mutants of Bacillus exhibited altered IAA production and excreted tryptophan into the growing medium. The IAA-producing Bacillus isolates promoted root growth and (or) nodulation when coinoculated with Rhizobium etli (TAL 182) on Phaseolus vulgaris 'Contender' under gnotobiotic conditions in growth chambers. Coinoculation resulted in increased nodule number, nodule fresh weight, nitrogenase activity, leghemoglobin content, and total soluble protein content in the root nodules of Phaseolus vulgaris. In contrast, coinoculation with α-methyltryptophan mutants resulted in decreased nodulation, indicating that Bacillus isolates have a direct effect on either the Rhizobium or the plant and the effect may not be singularly attributed to their ability to produce IAA in vitro.Key words: Bacillus, indoleacetic acid production, nodulation enhancement.

The ability to genetically alter the product-formation capabilities of Clostridium acetobutylicum is necessary for continued progress toward industrial production of the solvents butanol and acetone by fermentation. Batch fermentations at pH 4.5, 5.5, or 6.5 were conducted using C. acetobutylicum ATCC 824 (pFNK6). Plasmid pFNK6 contains a synthetic operon (the "ace operon") in which the three homologous acetone-formation genas (adc, ctfA, and ctfB) are transcribed from the adc promoter. The corresponding enzymes (acetoacetate decarboxylase and CoA-transferase) were best expressed in pH 4.5 fermentations. However, the highest levels of solvents were attained at pH 5.5. Relative to the plasmid-free control strain at pH 5.5, ATCC 824 (pFNK6) produced 95%, 37%, and 90% higher final concentrations of acetone, butanol, and ethanol, respectively; a 50% higher yield (g/g) of solvents on glucose; and a 22-fold lower mass of residual carboxylic acids. At all pH values, the acetone-formation enzymes were expressed earlier with ATCC 824 (pFNK6) than in control fermentations, leading to earlier induction of acetone formation. Furthermore, strain ATCC 824 (pFNK6) produced butanol significantly earlier in the fermentation and produced significant levels of solvents at pH 6.5. Only trace levels of solvents were produced by strain ATCC 824 at pH 6.5. Compared with ATCC 824, a plasmid-control strain containing a vector without the ace operon also produced higher levels of solvents [although lower than those of strain ATCC 824 (pFNK6)] and lower levels of acids. Strains containing plasmid-borne derivatives of the ace operon, in which either the acetoacetate decarboxylase or CoA-transferase alone were expressed at elevated levels, produced acids and solvents at levels similar to those of the plasmid-control strain.

In Clostridium acetobutylicum, conversion of butyraldehyde to butanol Is enzymatically achieved by butanol dehydrogenase (BDH). A C. acetobutyium gene that encodes this protein was identified by using an oligonucleotide designed on the basis of the N-terminal amino acid sequence of purified C. acetobutylicum NADH-dependent BDH. Enzyme assays of ceU extracts of Escherichia coli harboring the clostridial gene demonstrated 15-fold-higher NADH-dependent BDH activity than untransformed E. coli, as well as an additional NADPH-dependent BDH activity. Kinetic, sequence, and isoelectric focusing analyses suggest that the cloned clostridial DNA contains two or more distinct C. acetobutylicum enzymes with BDH activity.Production of butanol by the anaerobic bacterium Clostridium acetobutylicum is one of the oldest industrial fermentations using microorganisms. Because of renewed interest in butanol production for chemical feedstocks or fuel additives, much attention has been given to the molecular mechanisms whereby this fermentation is accomplished. In C. acetobutylicum, butyraldehyde is reduced to butanol by the enzyme butanol dehydrogenase (BDH) at the expense of a reduced NAD moiety. BDH is an inducible enzyme that reaches peak levels during the solventogenic stage (1). Confusion regarding the nature of the coenzyme required by BDH was widespread in early enzymatic investigations because of differences in the assay systems and strains employed (1,8,9,14,15). A new assay system (7) has made it clear that C. acetobutylicum contains at least two types of BDH which can be separated by ultracentrifugation. One type uses NADH as a cofactor, while the other enzyme is NADPH specific. The role(s) of the two enzymes in vivo has not been established. The pH optima of the two enzymes indicate that NADH-dependent BDH is more effective at the lower internal pH that exists in solvent-producing cells (7).The gene that encodes alcohol dehydrogenase (ADH) from C. acetobutylicum P262 was previously cloned by complementation of an Escherichia coli adh mutant (21). The enzyme was NADP dependent and had an apparent molecular weight of 43,000 (20). The enzyme was not very specific, utilizing butanol and ethanol nearly equally well, and was thus classified as an ADH. The solvent production ratio in C. acetobutylicum also makes it unlikely that this enzyme is responsible for most of the butanol production.Two distinct NADH-dependent BDH isozymes (BDHI and BDHII) have been purified to homogeneity (18, 19). Like the enzyme expressed from the cloned adh-l gene, they have subunit molecular masses of -42 kDa and a native molecular mass of -82 kDa (18, 19). However, one NADH-dependent enzyme (BDHII) was reported to have 46-fold-greater activity with butyraldehyde than with acetaldehyde and is 50-fold less active in the reverse direction (19). The other enzyme (BDHI) is only about twofold more active with butyraldehyde than with acetaldehyde. These enzymes were much more active at acidic pHs, with a maximum at pH 5.5 that dropped sharply to less than ...

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CoA-transferase [butyrate-acetoacetate CoA-transferase] [EC 2.8.3.9]) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The purification and properties of the enzyme have recently been described (D. P. Weisenborn, F. B. Rudolph, and E. T. Papoutsakis, Appl. Environ. Microbiol. 55:323-329, 1989). The genes encoding the two subunits of this enzyme have been cloned by using synthetic oligodeoxynucleotide probes designed from amino-terminal sequencing data from each subunit of the CoA-transferase. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was prepared and screened by using these probes. Subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defect in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of M, of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E. coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli. In the plasmid, however, transcription appears to be primarily from the lac promoter of the vector.

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an-2.1-kb EcoRIlBglll fragment. A polypeptide with a molecular weight of-28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although 3491