Purification and characterization of polygalacturonases produced by the hyphal fungus Aspergillus niger

Five endo-polygalacturonases (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) and one exo-polygalacturonase (poly(1,4-alpha-D-galacturonide) galacturonohydrolase, EC 3.2.1.67) were isolated from a commercial pectinase preparation derived from Aspergillus niger. All five endo-enzymes could be purified to homogeneity by affinity chromatography on cross-linked alginate, ion-exchange chromatography, chromatofocusing, and gel permeation chromatography. The exo-polygalacturonase was only partially purified but free from endo-polygalacturonase activity. The two most abundant endo-polygalacturonases (endo-I and endo-II), with molecular masses of 55 and 38 kDa, respectively, are quite different with respect to their isoelectric point, specific activity, mode of action on oligomeric substrates, and amino acid composition. The physicochemical properties of the other three endo-polygalacturonases (endo-IIIA, endo-IIIB, and endo-IV), present in low amounts, are quite similar to those of the endo-I type. The pH optima of all these endo-polygalacturonases are in the range of 4.3-4.9.     

Halobacterial flagellins are encoded by a multigene family. Characterization of five flagellin genes

Purified flagellar filaments of Halobacterium halobium contain three different protein species based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These proteins were designated as flagellins Fla I, Fla II, and Fla III and were characterized as sulfated glycoproteins with N-glycosidically linked oligosaccharides of the type GlcA-(1----4)-GlcA-(1----4)-GlcA-(1----4)-Glc. All halobacterial flagellin polypeptides are immunologically cross-reactive. A gene fragment of one flagellin was isolated in an expression vector using antibody probes. Using this gene fragment as probe, we identified, subcloned, and determined the nucleotide sequences of five different but highly homologous flagellin genes. Two flagellin (flg) genes are arranged tandemly at one locus (flg A1 and -2), and the other three in a tandem arrangement at a different locus (flg B1, -2, and -3), Two flg mRNAs were detected, one from the A genes and the other from the B genes. Based on immunological analysis, the products of the flg A1 and A2 are Fla II and Fla I, respectively.     

Short chain collagens in sponges are encoded by a family of closely related genes

Two previously described sponge cDNAs, EmC4 and C23, respectively encoding a short chain collagen and a fibrillar collagen, were used to characterize collagen gene families in a freshwater sponge. EmC4 detected several clones when used to screen a cDNA library. Two overlapping clones, EmC13 1 and 2, were sequenced and appeared highly homologous to EmC4. Contrarily to C23, EmC4 hybridized with 10-12 fragments of genomic DNA digested with restriction endonucleases and detected 10 times more positive clones than C23 when used to screen a genomic library. The genomic clone G41 contained two closely related genes, COLNF13, corresponding to EmC13 and COLNF6. Partial characterization of COLNF13 revealed two partial exons and four complete exons of 153, 219, 207, and 144 base pairs, with split glycine codons at their boundaries. The deduced encoded protein is a short chain collagen containing two uninterrupted collagenous domains of 66 and 171 amino acids and non-collagenous domains. A characterized 207-base pair exon of COLNF6 is 77% identical with the comparable COLNF13 exon. In situ hybridization using EmC4 cDNA and electron microscopy suggested that the cells expressing these genes were secreting spongin, a non-fibrillar, surface collagen of these sponges.     

The opacity proteins of Neisseria gonorrhoeae strain MS11 are encoded by a family of 11 complete genes

Variants of Neisseria gonorrhoeae MS11 show distinct colony morphologies because of the expression of a class of surface components called opacity (Opa, PII) proteins. Southern analyses combined with molecular cloning of genomic DNA from a single variant of MS11 has identified 11 opa genes contained in separate loci. These opa genes code for distinct opacity proteins which are distinguishable at their variable domains. The opa gene analyses were also extended to divergent variants of MS11. These studies have shown that, during in vitro and in vivo culture, 10 of the 11 opa genes did not undergo significant change in their primary sequence. However, in these variants, one gene (opaE) underwent non-reciprocal inter-opa recombinations to generate newer Opa variants. Phylogenic analysis of the opa gene sequences suggests that the opa gene family have evolved by a combination of gene duplication, gene replacement and partial inter-opa recombination events.     

Three polygalacturonases constitutively synthesized by Aspergillus alliaceus

Of three molecular forms of polygalacturonases synthesized by Aspergillus alliaceus on glucose media, two were exopolygalacturonases (exoPG₁ and exoPG₂) and one was an endopolygalacturonase (endoPG). Low-methoxylated beet pectin was the preferred substrate for the endoPG and exoPG₂ whereas pectic acid was the optimal substrate for exoPG₁. The highest activities of endoPG, exoPG₁ and exoPG₂ were at pH 5.5, 3.5, and 6.0 and at 35, 45 to 50 and 35°C, respectively. Disks of potato-tuber tissue were macerated by endoPG, but not by exoPG₁ or exoPG₂.The authors are with the Institute of Microbiology, Belarus Academy of Sciences, Zhodinskaya 2, 220141, Minsk, Belarus;     

Human cytoplasmic actin proteins are encoded by a multigene family

We characterized nine human actin genes that we isolated (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981) from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and alpha-, beta-, and gamma-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria we show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken beta-actin cDNA. We conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.     

Nisin biosynthesis genes are encoded by a novel conjugative transposon

Summary Genes for biosynthesis of the lactococcal peptide antibiotic nisin were shown to be encoded by a novel chromosomally located transposon Tn5301. The element is 70 kb in size and lacks inverted repeats at its termini. Although a copy of the insertion sequence IS904 is located near to one end, this did not appear to be involved in the transposition process. The integrated element is flanked by the directly repeated sequence 5-TTTTTG-3. Analysis of ten independent transconjugants revealed that Tn5301 integration is site-specific; two chromosomal targets were identified and shown to have some sequence homology. The element shares features with the Tn916 family of conjugative transposons and with Tn554 but is also exhibits some unique properties. Tn5301 is thus considered to be the prototype of a novel class of conjugative transposon.     

Reduction of cycloalkanones by Aspergillus niger.

The mould Aspergillus niger was grown in a carefully established culture medium containing one of the following test substrates: cyclopentanone, -hexanone, -heptanone, and their 2-substituted methyl derivatives. Growth curves, glucose consumption curves and curves showing the course of the oxido-reduction reaction are given.     

Ochratoxin A production by strains of Aspergillus niger var. niger.

In a survey of the occurrence of ochratoxin A (OA)-positive strains isolated from feedstuffs, two of the 19 isolates of Aspergillus niger var. niger that were studied produced OA in 2% yeast extract-15% sucrose broth and in corn cultures. This is the first report of production of OA by this species.     

Expression profiling of pectinolytic genes from Aspergillus niger

The expression of 26 pectinolytic genes from Aspergillus niger was studied in a wild type strain and a CreA derepressed strain, under 16 different growth conditions, to obtain an expression profile for each gene. These expression profiles were then submitted to cluster analysis to identify subsets of genes with similar expression profiles. With the exception of the feruloyl esterase encoding genes, all genes were expressed in the presence of D-galacturonic acid, polygalacturonate, and/or sugar beet pectin. Despite this general observation five distinct groups of genes were identified. The major group consisted of 12 genes of which the corresponding enzymes act on the pectin backbone and for which the expression, in general, is higher after 8 and 24 h of incubation, than after 2 or 4 h. Two other groups of genes encoding pectin main chain acting enzymes were detected. Two additional groups contained genes encoding L-arabinose and D-galactose releasing enzymes, and ferulic acid releasing enzymes, respectively. The genes encoding beta-galactosidase and the L-arabinose releasing enzymes were not only expressed in the presence of D-galacturonic acid, but also in the presence of L-arabinose, suggesting that they are under the control of two regulatory systems. Similarly, the rhamnogalacturonan acetylesterase encoding gene was not only expressed in the presence of D-galacturonic acid, polygalacturonate and sugar beet pectin, but also in the presence of L-rhamnose. The data presented provides indications for a general pectinolytic regulatory system responding to D-galacturonic acid or a metabolite derived from it. In addition, subsets of pectinolytic genes are expressed in response to the presence of L-arabinose, L-rhamnose or ferulic acid.     

Multiple lobster tubulin isoforms are encoded by a simple gene family

Microtubule proteins isolated from pleopod tegumental gland (PTG) tissue of the American lobster, Homarus americanus, reveal a complex tubulin (Tub) profile. To determine whether Tub heterogeneity in PTG is due to expression of a large tub gene family or the result of post-translational modification, a PTG cDNA library was constructed. Clones coding for both α- and β-Tub were isolated, sequenced and found to contain open reading frames (ORFs) of 451 amino acids (aa). Alignments reveal phylogenetic clustering with other arthropods and identify unique changes in primary structure which may have functional significance. These clones, when used to probe restriction enzyme-digested lobster genomic DNA in transfer-hybridization experiments, revealed a simple banding pattern indicating a lobster tub gene family of limited complexity. Lobsters appear to make use of a small tub gene family and fulfill the varied functional requirements imposed upon cellular microtubules through post-translational modifications of relatively few gene products.     

The Aspergillus niger acuA and acuB genes correspond to the facA and facB genes in Aspergillus nidulans

Mutants in Aspergillus niger unable to grow on acetate as a sole carbon source were previously isolated by resistance to 1.2% propionate medium containing 0.1% glucose. AcuA mutants lacked acetyl-CoA synthetase (ACS) activity and acuB mutants lacked both ACS and isocitrate lyase activity. An acuA mutant was transformed to the acu+ phenotype with a clone of ACS (facA) from Aspergillus nidulans. The acuB mutant was transformed with the A. niger facB clone which has been identified by cross-hybridisation of an A. nidulans facB clone. These results confirm that acuA in A. niger is the gene for ACS and acuB is analogous to the A. nidulans facB regulatory gene.     

The occurrence of a phosphorylated glycosphingolipid in Aspergillus niger.

A novel type of water-soluble phosphorylated glycosphingolipid was isolated from Aspergillus niger by a simple procedure involving precipitation, DEAE-cellulose chromatography and preparative t.l.c. Besides ceramide and phosphorus it contains inositol, galactose, mannose and small amounts of glucosamine.     

Halobacterial flagellins are encoded by a multigene family. Identification of all five gene products

Flagellins of Halobacterium halobium are encoded in five different but homologous genes. Flagellins isolated from purified flagella were digested and the resulting peptides sequenced. The amino acid sequence data obtained prove that all five gene products are expressed and integrated into the flagellar bundle.     

黑曲霉产木聚糖酶发酵条件的研究

正交设计试验结果表明,黑曲霉（Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ ｍ１２）产木聚糖酶活力达７６．６０ｕ／ｍｌ,合适的产酶发酵条件如下,培养基（ｇ／Ｌ）：麸皮４０,尿素６．６７,ＫＨ２ＰＯ４ １．０,ＭｇＳＯ４．７Ｈ２Ｏ０．５,ＮａＣｌ０．３,Ｔｗｅｅｎ－８０ ３．０,ＣａＣＯ３ ２．０,２８℃,１２０ｒ／ｍｉｎ水浴振荡培养５．５ｄ。