Cellulose is the most abundant renewable carbon resource on earth and is an indispensable raw material for the wood, paper, and textile industries. A model system to study the mechanism of cellulose biogenesis is the bacterium Acetobacter xylinum which produces pure cellulose as an extracellular product. It was from this organism that in vitro preparations which possessed high levels of cellulose synthase activity were first obtained in both membranous and soluble forms. We recently demonstrated that this activity is subject to a complex multi-component regulatory system, in which the synthase is directly affected by an unusual cyclic nucleotide activator enzymatically formed from GTP, and indirectly by a Ca (2+) -sensitive phosphodiesterase which degrades the activator. The cellulose synthase activator (CSA) has now been identified as bis-(3' 5')-cyclic diguanylic acid (5'G3'p5'G3'p) on the basis of mass spectroscopic data, nuclear magnetic resonance analysis and comparison with chemically synthesized material. We also report here on intermediary steps in the synthesis and degradation of this novel circular dinucleotide, which have been integrated into a model for the regulation of cellulose synthesis.

Immobilized penicillin acylase is a biocatalyst suitable for the kinetically controlled industrial synthesis of semi-synthetic antibiotics in aqueous environments. Amoxicillin and ampicillin are obtained by condensing 6-aminopenicillanic acid with the amide or ester of d-(−)-4-hydroxyphenylglycine and d-(−)-phenylglycine, respectively. Similarly, the cephalosporin antibiotics cefadroxil and cephalexin can be obtained from 7-aminodesacetoxycephalosporanic acid.

small bacteriocin was isolated from the culture broth of the gram-negative bacterium Rhizobium leguminosarum, which forms symbiotic nitrogen-fixing root nodules on a number of leguminous plants. The structure of the molecule was elucidated by spectroscopic methods and identified as N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone. The absolute configuration of both asymmetric carbon atoms in the molecule was determined by the use of the chiral solvating agents S-(؉)-and R-(؊)-2,2,2-trifluoro-1-(9-anthryl)-ethanol. small bacteriocin is structurally related to the quorum sensing co-transcription factors for genes from other bacteria such as Vibrio fischeri, Pseudomonas aeruginosa, Erwinia carotovora, and Agrobacterium tumefaciens which are involved in animal-microbe or plant-microbe interactions. The mechanism of regulation of such interactions by this kind of co-transcription factors is still unknown in R. leguminosarum.small bacteriocin (small) is produced by strains of all three biovars of Rhizobium leguminosarum and inhibits the growth of R. leguminosarum bv. viciae 248 and several other strains of this species which, like strain 248, contain a self-transmissible plasmid (12,17). Two genes located close to the transfer (tra) genes (17) on the Sym plasmid pRL1JI of strain 248 are responsible for the fact that this strain does not produce small (rps [repression production small] gene) and that it is sensitive to small (sbs [small bacteriocin sensitivity] gene) (12,17). When strain 248 is cured of its Sym plasmid, pRL1JI (as, for example, in strain RBL1390), it is insensitive to small and also produces small (12, 17). To our knowledge, small is produced only by R. leguminosarum strains, and in nonproducing strains of this species, a gene for small production like that in strain 248 is present (12,17). Therefore, the presence of small is considered a characteristic of this species. Since another typical property of this species is symbiotic root nodule formation on certain leguminous plants, it is possible that both properties are related. However, a strain with a Tn5 insertion in the small gene(s) could induce formation of normal root nodules, which shows that the small gene is not required for root nodule formation (16). However, this does not exclude an ecological link between small production and the interaction of Rhizobium spp. with plants, since other genes may complement the lost function or the function is not essential. In order to detect its biological significance, small was extracted from the bacterial culture medium with chloroform (16) and identified chemically. The small molecule appeared to contain an N-acyl homoserine lactone structure, as known from quorum sensing signal molecules, which function as co-transcription factors in bacteria that often interact with higher organisms (reviewed by Fuqua et al. [10]). The implications of this finding are discussed. MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains used in this study were the small-sensitive strain...

Bleomycin is an antitumor agent whose activity has long been thought to derive from its ability to degrade DNA. Recent findings suggest that cellular RNA may be a therapeutically relevant locus. At micromolar concentrations, Fe(II)-bleomycin readily cleaved a Bacilus subtilis tRNAHis precursor in a highly selective fashion, but Escherichia coli tRNATYr precursor was largely unaffected even under more forcing conditions. Other substrates included an RNA transcript encoding a large segment of the reverse transcriptase from human immunodeficiency virus 1. RNA cleavage was oxidative, %10-fold more selective than DNA cleavage, and largely unaffected by nonsubstrate RNAs. RNA sequence analysis suggested recognition of RNA tertiary structure, rather than recognition of specific sequences; subsets of nucleotides at the junction of single-and double-stranded regions were especially susceptible to cleavage. The ready accessibility of cellular RNAs to xenobiotic agents, the high selectivity of bleomycin action on RNAs, and the paucity ofmechanisms for RNA repair suggest that RNA may be a therapeutically relevant target for bleomycin.The bleomycins (BLMs) are antitumor antibiotics believed to function by DNA degradation (1, 2). DNA degradation is sequence-selective, occurring primarily at a subset of all 5'-GT-3' and 5'-GC-3' sequences (3, 4). A wealth of information exists concerning the mechanism of BLM-mediated DNA degradation in cell-free (1, 2, 5) and cellular (5, 6) systems.In contrast, few studies have dealt with RNA as a substrate for BLM. Further, in spite of the compartmentalization of DNA and RNA in nucleated cells, most studies of RNA cleavage have been carried out with DNA present. For example, BLM degraded only the poly(dT) strand of a poly(rA)-poly(dT) hybrid (7,8). Hori (9) showed that relaxation of simian virus 40 form I DNA was hardly affected by even a large excess of Escherichia coli tRNA. Those experiments that employed RNA alone were carried out in the absence of added metal ions (10, 11). Magliozzo et al. (12) have reported degradation of yeast tRNAPhe by high concentrations of Fe(II)-BLM; product bands corresponding to <5-10% of the starting tRNA were observed by methylene blue staining of a polyacrylamide gel. Thin layer chromatographic analysis indicated products that comigrated with adenine and uracil.Although none ofthe foregoing studies suggested that RNA might constitute an efficient substrate for BLM, ongoing studies in this laboratory of BLM-mediated DNA oligonucleotide degradation have demonstrated that alteration of DNA conformation can significantly change the pattern and extent of cleavage normally obtained with B-form DNA (13,14). On the chance that the limited RNA cleavage observed (vide supra) might actually have resulted from highly efficient cleavage at a few conformationally unique sites, we studied several RNAs as substrates. Presently, we show that Fe(II)-BLM does mediate RNA degradation and that the process is highly site-selective.

No abstract