Sequence analysis and comparison of avocado fruit and bean abscission cellulases

A 1700 nucleotide cDNA clone for a bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase (endo-(1,4)-β-d-glucanase) has been identified and sequenced. This cDNA clone contains a 1485 nucleotide open reading frame which includes coding sequences for a putative signal peptide and mature protein. The nucleotide and deduced amino acid sequences for the bean abscission cellulase are compared to the previously reported sequences of an avocado fruit ripening cellulase. Optimal alignment of these sequences shows 64% and 50% identically matched nucleotides and amino acids, respectively. Analysis of the deduced amino acid sequences for the mature bean and avocado cellulases indicates that these two proteins share similar molecular weights, position of cysteine residues, and hydropathic character, but have very different isoelectric points and glycosylation. Genomic blot data suggest that the avocado fruit cellulase belongs to a small gene family, whereas the bean abscission cellulase appears to be encoded by a single gene or a few very closely related genes.     

Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1Tyr)

virR is the central regulatory locus required for coordinate temperature-regulated virulence gene expression in the human enteric pathogens of Shigella species. Detailed characterization of VirR+ clones revealed that virR consisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kb EcoRI-AccI DNA fragment from Shigella flexneri. Insertional inactivation of the virR ORF at a unique HpaI restriction site resulted in a loss of VirR+ activity. The virR ORF nucleotide sequence was virtually identical to the Escherichia coli hns gene, which encodes the histone-like protein, H-NS. Based on the predicted amino acid sequence of E. coli H-NS, only a single conservative base-pair change was identified in the virR gene. An additional clone, designated VirRP, which only partially complemented the virR mutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique from virR. Subclone mapping of this clone indicated that the VirRP phenotype was a result of the multiple copy expression of the S. flexneri gene for tRNA(Tyr). These data constitute the first direct genetic evidence that virR is an analogue of the E. coli hns gene, and suggest a model for temperature regulation of Shigella species virulence via the bacterial translational machinery.     

Identification of a matrix-associated region 5'' of an immunoglobulin heavy chain variable region gene.

In the accompanying report (C. F. Webb, C. Das, S. Eaton, K. Calame, and P. Tucker, Mol. Cell. Biol. 11:5197-5205, 1991), we characterize B-cell-specific protein-DNA interactions at -500 and -200 bp upstream of the mu immunoglobulin heavy chain promoter whose abundances were increased by interleukin-5 plus antigen. Because of the high A + T/G + C ratio of these sequences and the consistent findings by others that enhancer- and promoterlike regions are often located near matrix-associated regions, we asked whether these sequences might also be involved in binding to the nuclear matrix. Indeed, DNA fragments containing the -500 binding site were bound by nuclear matrix proteins. Furthermore, UV cross-linking studies showed that the DNA binding site for interleukin-5-plus-antigen-inducible proteins could also bind to proteins solubilized from the nuclear matrix. Nuclear matrix-associated sequences have also been demonstrated on either side of the intronic immunoglobulin heavy chain enhancer. Our data suggest a topological model by which interactions among proteins bound to the promoter and distal enhancer sequences might occur.     

Atypical mannolipids characterize Thy-1-negative lymphoma mutants.

Essentially all eukaryotic cells, including murine lymphomas, express surface proteins, such as Thy-1, which are anchored by a phosphoinositol mannolipid. Putative mannolipid anchor precursors can be detected in these cells. Six distinct Thy-1-negative lymphoma mutants lack complete mannolipids, and three mutants synthesize atypical mannolipids. The absence of complete mannolipids can account for the lack of expression of multiple mannolipid-anchored proteins and may also account for the lack of lipid anchoring in the human disease paroxysmal nocturnal hemoglobinuria. Structural information on the mannolipids of wild-type and mutant cells indicates that anchor biosynthesis in these cells may involve both transmembrane flip-flop of intermediates and a deacylation step.     

Acetonema longum gen.nov.sp.nov., an H2/CO2 acetogenic bacterium from the termite,Pterotermes occidentis

A previously undescribed, H₂-oxidizing CO₂-reducing acetogenic bacterium was isolated from gut contents of the wood-feeding termite, Pterotermes occidentis. Cells of representative strain APO-1 were strictly anaerobic, Gram-negative, endospore-forming motile rods which measured 0.30–0.40×6–60 m. Cells were catalase positive, oxidase negative, and had 51.5 mol percent G+C in their DNA. Optimum conditions for growth on H₂+CO₂ were at 30–33°C and pH (initial) 7.8, and under these conditions cells formed acetate according to the equation: 4 H₂+2 CO₂CH₃COOH+2 H₂O. Other energy sources supporting good growth of strain APO-1 included glucose, ribose, and various organic acids. Acetate and butyrate were major fermentation products from most organic compounds tested, however propionate, succinate, and 1,2-propanediol were also formed from some substrates. Based on comparative analysis of 16S rRNA nucleotide sequences, strain APO-1 was related to, but distinct from, members of the genus Sporomusa. Moreover, physiological and morphological differences between strain APO-1 and the six known species of Sporomusa were significant. Consequently, it is proposed herewith that a new genus, Acetonema, be established with strain APO-1 as the type strain of the new species, Acetonema longum. A. longum may contribute to the nutrition of P. occidentis by forming acetate, propionate and butyrate, compounds which are important carbon and energy sources for termites.     

Clostridium mayombei sp. nov., an H2/CO2 acetogenic bacterium from the gut of the African soil-feeding termite,Cubitermes speciosus

Clostridium mayombei sp. nov., a previously undescribed H₂-oxidizing CO₂-reducing acetogenic bacterium, was isolated from gut contents of the African soilfeeding termite, Cubitermes speciosus. Cells were anaerobic, Gram positive, catalase and oxidase negative, endospore-forming motile rods which measured 1×2 – 6 m and which had a DNA base composition of 25.6 mol% G+C (strain SFC-5). Optimum conditions for growth on H₂+CO₂ were at 33°C and pH 7.3, and under these conditions cells produced acetate according to the equation: 4 H₂+2 CO₂CH₃COOH+2 H₂O. Other substrates supporting good growth included carbohydrates (e.g. glucose, xylose, starch), sugar alcohols, and organic and amino acids, and with these substrates acetate was almost always the principle fermentation product. Comparative analysis of 16S rRNA nucleotide sequences confirmed that C. mayombei was closely related to various members of the genus Clostridium. However, morphological and physiological differences between C. mayombei and other homoacetogenic clostridia were deemed significant enough to warrant creation of a new taxon. Results are discussed in light of the diversity of H₂/CO₂ acetogens recently isolated from various termites, and in terms of the relative importance of H₂/CO₂ acetogenesis to termite nutrition.