Artificial tripartite symbiosis involving a green alga (Chlamydomonas), a bacterium (Azotobacter) and a fungus (Alternaria): morphological and physiological characterization.

A long-living artificial tripartite symbiosis involving a green alga (Chlamydomonas), a bacterium (Azotobacter) and a fungus (Alternaria) was established on carbon- and nitrogen-free medium. The basis of the interdependence is the complementation of photosynthetic CO2 assimilation and atmospheric nitrogen fixation. Green color of the colonies indicated that the algal cells had enough nitrogen to synthesize chlorophylls. The chlorophyll content was nearly 40% of the control cells. The relatively high rate of photosynthetic oxygen evolution proved that nitrogen was effectively used for building up a well functioning photosynthetic apparatus. This was supported by the analysis of photosystems and ultrastructural investigations. In comparison with degreened algae cultured on nitrogen-free medium, the chloroplasts in the symbiont algal cells contained a well-developed, stacked thylakoid membrane system without extreme starch or lipid accumulation. The occurrence of the fungus in the association greatly increased the chlorophyll content. Far fewer types of amino acids were excreted by the tripartite cultures than by pure cultures. Cystathionine, which is a common intermediate in the sulfur-containing amino acid metabolism, was produced in high quantities by the tripartite symbiosis. This can mostly be attributed to the activity of the fungus.

Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble ligands.

Aromatic amino acids are believed to play a pivotal role in carbohydrate-binding proteins, by forming hydrophobic stacking interactions with the sugar rings of their target ligands. Family 10 cellulose-binding modules (CBM10s), present in a number of cellulases and xylanases expressed by Pseudomonas fluorescens subsp. cellulosa, contain two tyrosine and three tryptophan residues which are highly conserved. To investigate whether these amino acids play an important role in the interaction of CBM10 from P. fluorescens subsp. cellulosa xylanase A (Pf Xyn10A) with cellulose, each of these residues was changed to alanine in CBM10 expressed as a discrete module or fused to the catalytic domain of Pf Xyn10A (CBM10-CD), and the capacity of the mutant proteins of CBM10-CD to bind the polysaccharide was evaluated. The data showed that W22A, W24A, and Y8A bound very weakly to cellulose compared to the wild-type protein, while Y12A retained its capacity to interact with the glucose polymer. When the W7A mutation was introduced into CBM10 the protein domain did not accumulate in Escherichia coli. In contrast, the W7A mutant of CBM10-CD was efficiently expressed in E. coli, although the protein bound very weakly to cellulose. NMR spectra of wild-type CBM10, W22A, and W24A were very similar, suggesting that the mutations did not significantly affect the protein fold. Titration of wild-type CBM10, W22A, and W24A with N-bromosuccinimide indicated that Trp22 and Trp24 were on the surface of the protein, while Trp7 was buried. Collectively, these data indicate that Trp22, Trp24, and Tyr8 play a direct role in the binding of Pf Xyn10A CBM10 to cellulose. The results are discussed in the light of the three-dimensional structure of CBM10 [Raghothama, S., Simpson, P. J., Szabó, L., Nagy, T., Gilbert, H. J., and Williamson, M. P. (2000) Biochemistry 39, 978-984].

Interaction of Azospirillum lipoferum with wheat germ agglutinin stimulates nitrogen fixation.

In vitro, the nitrogen fixation capability of A. lipoferum is efficiently increased in the presence of wheat germ agglutinin (WGA). A putative WGA-binding receptor, a 32-kDa protein, was detected in the cell capsule. The stimulatory effect required N-acetyl-D-glucosamine dimer (GlcNAcdi) terminated sugar side chains of the receptor and was dependent on the number of GlcNAcdi links involved in receptor-WGA interface. Binding to the primary sugar binding sites on WGA had a larger stimulatory effect than binding to the secondary sites. The WGA-receptor complex generated stimulus led to elevated transcription of the nifH and nifA genes and of the glnBA gene cluster but not of the glnA gene from its own promoter. There may well be a signalling cascade contributing to the regulation of nitrogen fixation.

Phage abortive infection of Bacillus licheniformis ATCC 9800; identification of the abiBL11 gene and localisation and sequencing of its promoter region.

The virulent bacteriophage BL11 infects almost all Bacillus licheniformis strains tested, including the industrial bacitracin-producing B. licheniformis 19. B. licheniformis ATCC 9800, however, was virtually insensitive to phage BL11 infection, and all of the few surviving progeny phages proved to be mutants. The phage-resistance mechanism was neither inhibition of adsorption, nor restriction or exclusion provided by a resident prophage, but was, instead, of another type. Phage BL11 adsorbed well on to ATCC 9800 cells, its DNA was injected, but replication of phage DNA was inhibited and the infected cells died. Thus, the mechanism of phage resistance was identified as abortive infection (AbiBL11). The so-called abiBL11 gene was identified on the chromosome of strain ATCC 9800 by Tn917PF1 transposon mutagenesis. Part of the abiBL11 gene from the phage-sensitive ATCC 9800::Tn917PFI was cloned. Gene-disruption analysis, based on Campbell-type integration, showed that a 0.3-kb EcoRI fragment contained the 5' end of abiBL11. The promoter region of abiBL11 was identified using promoter- and terminator-probe plasmids. The deduced sequence (206 amino acids) of the N-terminal part of abiBL11 showed no significant homology to known abortive-infection genes, but did show homology to a Saccharomyces cerevisiae gene coding for a serine/threonine protein kinase (RCK1).

Cloning and expression of a beta-1,4-endoglucanase gene from Cellulomonas sp. CelB7 in Escherichia coli; purification and characterization of the recombinant enzyme.

A gene library of a newly isolated Cellulomonas sp. strain was constructed in Escherichia coli and clones were screened for endoglucanase activity using dye-labelled carboxymethylcellulose. Seventeen clones were isolated that carried DNA inserts coding for endoglucanase enzymes. Of the 17 clones, one carrying the gene cegA, was further characterized. The recombinant endoglucanase was purified by FPLC. The endoglucanase was active against carboxymethylcellulose, lichenin and also degraded crystalline cellulose and birchwood xylan. The molecular mass of the enzyme (36 kDa), and its pH (7.4) and temperature (35 degrees C) optima were determined.

The conserved noncatalytic 40-residue sequence in cellulases and hemicellulases from anaerobic fungi functions as a protein docking domain.

Two cDNAs, designated xynA and manA, encoding xylanase A (XYLA) and mannanase A (MANA), respectively, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Piromyces. XYLA and MANA displayed properties typical of endo-beta 1,4-xylanases and mannanases, respectively. Neither enzyme hydrolyzed cellulosic substrates. The nucleotide sequences of xynA and manA revealed open reading frames of 1875 and 1818 base pairs, respectively, coding for proteins of M(r) 68,049 (XYLA) and 68,055 (MANA). The deduced primary structure of MANA revealed a 458-amino acid sequence that exhibited identity with Bacillus and Pseudomonas fluorescens subsp. cellulosa mannanases belonging to glycosyl hydrolase Family 26. A 40-residue reiterated sequence, which was homologous to duplicated noncatalytic domains previously observed in Neocallimastix patriciarum xylanase A and endoglucanase B, was located at the C terminus of MANA. XYLA contained two regions that exhibited sequence identity with the catalytic domains of glycosyl hydrolase Family 11 xylanases and were separated by a duplicated 40-residue sequence that exhibited strong homology to the C terminus of MANA. Analysis of truncated derivatives of MANA confirmed that the N-terminal 458-residue sequence constituted the catalytic domain, while the C-terminal domain was not essential for the retention of catalytic activity. Similar deletion analysis of XYLA showed that the C-terminal catalytic domain homologue exhibited catalytic activity, but the corresponding putative N-terminal catalytic domain did not function as a xylanase. Fusion of the reiterated noncatalytic 40-residue sequence conserved in XYLA and MANA to glutathione S-transferase, generated a hybrid protein that did not associate with cellulose, but bound to 97- and 116-kDa polypeptides that are components of the multienzyme cellulase-hemicellulase complexes of Piromyces and Neocallimastix patriciarum, respectively. The role of this domain in the assembly of the enzyme complex is discussed.