Glutamine synthetase from an alkalophilic Bacillus No. 170 was purified to homogeneity from cells grown in synthetic medium at pH 9.0. The purified enzyme had a molecular weight of 600, 000 and a subunit size of 50, 000, similar to the enzymes from Bacillus subtilis and Bacillus cereus. Either Mg2+ or Mn2+ activated the enzyme; however, the highest activity was obtained when 20mM Mg2+ with 0.2mM Mn2+ were added to the assay mixture. The peculiar divalent cation dependency of the glutamine synthetase was similar to that of other Bacillus enzymes but differed from that of the enteric bacteria enzymes. The kinetic properties of the enzyme, such as optimum pH, Km for each substrate, Vmax values, and inhibition by glutamine, were similar to those in the case of other Bacillus enzymes. The enzyme from this alkalophilic Bacillus was modified by iodoacetoamide or the ATP analog, 5′-p-fluorosulfonyl benzoyladenosine (FSBA). Only Mg2+-dependent activity was decreased by iodoacetamide, whereas each of the Mn2+-, Mg2+-, and Mg2+ plus Mn2+-dependent activities was decreased with FSBA. The sequence of the NH2-terminal 20 amino acids of alkalophilic Bacillus glutamine synthetase was similar to those of other Bacillus enzymes.
Measurement of chitinase activity in crude homogenates of Candidaalbicans with the artificial substrates 4-methylumbelliferyl β-D-N, N′, N"-triacetylchitotrioside and 4-methylumbelliferyl-β-D-N, N′-diacetylchitobioside (MU-[GlcNAc]3, 2) showed that both chitobiase and chitinase were involved in the liberation of the fluorophore methylumbelliferone (MU). The substrate MU-[GlcNAc]3 appeared to be digested by means of two separate mechanisms, resulting in a lag-phase in the time curve of the MU-release, and thus making this release non-linear with time. The first mechanism is the action of an endochitinase that released MU in one step; this reaction could be inhibited by allosamidin. The second probable mechanism involves the digestion of the substrate by the stepwise release of the outer two G1cNAc residues by a chitobiase, followed either by a third chitobiase-catalyzed release of the final GlcNAc or by the action of a β-N-acetylhexosaminidase, that is also present in C. albicans. Hydrolysis of the substrate MU-[GlcNAc]2 was not inhibited by allosamidin, suggesting that the two GlcNAc residues in this substrate are probably released in two steps, either by the action of chitobiase alone, or by the successive action of chitobiase (step 1) and β-N-acetylhexosaminidase (step 2). Our results show that the activities of the individual enzymes involved in chitin degradation cannot be determined on the basis of MU release in unfractionated homogenates of C. albicans.
The presence or absence of cellulose-binding proteins (CBPs) in the cell lysate of 31 strains representing 17 species of rumen bacteria was examined. Both carboxymethylcellulose (CMC) and sodium dodecyl sulfate (SDS) were used to elute CBPs of these bacteria from cellulose. For 16 strains classified into 4 species of Fibrobacter succinogenes, Fibrobacterintestinalis, Ruminococcus flavefaciens and Eubacterium cellulosolvens, the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the CMC-eluted CBPs were identical with those of the SDS-eluted CBPs. For 4 strains representing 4 species of Veillonella parvula, Megasphaera elsdenii, Prevotella ruminicola subsp. ruminicola and Eubacterium ruminantium, the SDS-PAGE patterns of both the CMC-eluted CBPs and the SDS-eluted CBPs were different. In the case of 3 strains representing Ruminococcus bromii, Butyrivibrio fibrisolvens and Ruminobacter amylophilus, CBPs were eluted only with SDS from cellulose, but not with CMC. On the other hand, none of the other strains tested contained a detectable amount of CBPs. Western immunoblot analysis indicated that CBP (120kDa) of both the 10 strains of F. succinogenes and 1 strain of F. intestinalis was antigenically identical with CBP1 of F. succinogenes S85, and CBPs of other bacteria did not cross-react with rabbit antiserum to CBP1.
The isolate BPR 2001, a potent cellulose producer in agitation cultures, was examined to determine its taxonomic characteristics. The isolate BPR 2001 was characterized by the formation of dihydroxyacetone from glycerol and by ubiquinone-10. The DNA homologies of the isolate BPR 2001 were estimated to be low (15.6 and 0.3%, respectively), when hybridized with the DNAs of the type strains of Acetobacter hansenii and Acetobacter pasteurianus. However, the DNA of the isolate BPR 2001 hybridized with that of the type strain of Acetobacter xylinum to some extent (58.2%). These data indicate that the isolate BPR 2001 should not be classified as A. hansenii, but as a subspecies in A. xylinum. The isolate BPR 2001 was distinguished phenotypically from the type strain of A. xylinum by growing on ethanol, glycerol, dulcitol, and sucrose, by acid formation from D-galactose, glycerol, meso-erythritol, sucrose, and trehalose, and by oxidation of glycerol, D-mannitol, and lactose. We here propose a new subspecies, Acetobacter xylinum subsp. sucrofermentans as a potent cellulose producer.
Promotion of plant growth with plant growth-promoting rhizobacteria (PGPR) is sometimes related to disease suppression and induction of general defense responses in plants inoculated with such bacteria. Induced disease suppression was observed after seed bacterization in a bean (Phaseolus vulgaris) challenged with Pseudomonas syringae pv. phaseolicola in greenhouse conditions, both in terms of symptom expression and bacterial viable counts in the leaves and in the intercellular washing fluids (IWF) from the leaves. Comparison of in vitro growth of the pathogenic strain in IWF from protected and unprotected plants suggested that the inhibitory activity against pathogen establishment lies partly in the IWF of bacterized plants 10 days after challenge inoculation. SDS-polyacrylamide gel electrophoresis revealed that the relative levels of proteins had increased in the leaf extracts of protected plants. A correlation was thus found between the reduction in symptom expression and a lower bacterial population in the leaves and a general rise in protein content in the IWF. Poor establishment of the test pathogen in the bean inoculated with PGPR strain Pseudomonas fluorescens, S97 may also involve accumulation of certain phenolic compounds.
The effect of Tween 80 on the cellular fatty acid spectra of different strains of two closely related Lactobacillus species, Lactobacillus buchneri and Lactobacillus brevis, was studied. When cultivated in MRS-broth containing Tween 80 all L. buchneri strains contained two different cyclopropane fatty acids, dihydrosterculic and lactobacillic acid, while L. brevis strains contained only dihydrosterculic acid. In contrast, when the cells were cultivated in a modified MRS-broth without Tween 80, only lactobacillic acid was found in the bacteria except in one L. brevis strain that did not contain either lactobacillic acid or dihydrosterculic acid. This L. brevis strain was in contrast to most Lactobacillus strains unable to cyclisize cis-vaccenic acid (cis-11-octadecenoic acid) produced by its own biosynthetic machinery. Still it was able to cyclisize oleic acid (cis-9-octadecenoic acid) supplemented by its growth medium.
Complete sequences of 16S rRNA encoding genes from extreme halophiles Halobacterium saccharovorum, Halobacterium lacusprofundi, and Halobacterium distributum were determined. The polar lipids, particularly the glycolipids, of these and 20 other isolates were also analyzed. Based on both genetic and chemotaxonomic data, the following two novel genera are proposed: Halorubrobacterium (comprising Halorubrobacteriumsaccharovorum, Halorubrobacterium sodomense, Halorubrobacteriumlacusprofundi, Halorubrobacterium distributum, and Halorubrobacteriumcoriensis) and Natrialba (comprising Natrialba asiatica). Evidence for a third new genus is also presented.