Activity of two catabolic enzymes of the phosphogluconate pathway in mesquite roots inoculated with Azospirillum brasilense Cd.

The mesquite amargo (Prosopis articulate), one of the main nurse trees of the Sonoran Desert in Mexico, is responsible for major, natural re-vegetation processes. It exudes gluconic acid in root exudates, a favorite carbon source for the plant growth-promoting bacterium Azospirillum brasilense. Two enzymes, gluconokinase (EC 2.7.1.12) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44), participating in the phosphogluconate pathway, are active in the bacteria. Bacterial 6-phosphogluconate dehydrogenase is a constitutive enzyme, while gluconokinase is induced upon exposure to gluconic acid. Both enzymes are active in young, non-inoculated mesquite seedlings growing under hydroponic conditions. When A. brasilense Cd bacteria are inoculated on the root system, the roots exhibit much higher activity of gluconokinase, but not 6-phosphogluconate dehydrogenase. Mesquite roots exhibit high levels of root colonization by the inoculating bacteria. At the same time, and also for plants growing under sand culture conditions, the seedlings grew taller, greener, had longer leaves, and were heavier.     

Microbial populations and activities in the rhizoplane of rock-weathering desert plants. I. Root colonization and weathering of igneous rocks

Dense layers of bacteria and fungi in the rhizoplane of three species of cactus (Pachycereus pringlei, Stenocereus thurberi, Opuntia cholla) and a wild fig tree (Ficus palmeri) growing in rocks devoid of soil were revealed by bright-field and fluorescence microscopy and field emission scanning electron microscopy. These desert plants are responsible for rock weathering in an ancient lava flow at La Purisima-San Isidro and in sedimentary rock in the Sierra de La Paz, both in Baja California Sur, Mexico. The dominant bacterial groups colonizing the rhizoplane were fluorescent pseudomonads and bacilli. Seven of these bacterial species were identified by the 16S rRNA molecular method. Unidentified fungal and actimomycete species were also present. Some of the root-colonizing microorganisms fixed in vitro N(2), produced volatile and non-volatile organic acids that subsequently reduced the pH of the rock medium in which the bacteria grew, and significantly dissolved insoluble phosphates, extrusive igneous rock, marble, and limestone. The bacteria were able to release significant amounts of useful minerals, such as P, K, Mg, Mn, Fe, Cu, and Zn from the rocks and were thermo-tolerant, halo-tolerant, and drought-tolerant. The microbial community survived in the rhizoplane of cacti during the annual 10-month dry season. This study indicates that rhizoplane bacteria on cacti roots in rock may be involved in chemical weathering in hot, subtropical deserts.     

Synergism between Phyllobacterium sp. (N(2)-fixer) and Bacillus licheniformis (P-solubilizer), both from a semiarid mangrove rhizosphere

Mangrove seedlings were treated with a mixture of two bacterial species, the slow-growing, N(2)-fixing bacterium Phyllobacterium sp. and the fast-growing, phosphate-solubilizing bacterium Bacillus licheniformis, both isolated from the rhizosphere from black, white, and red mangroves of a semiarid zone. Nitrogen fixation and phosphate solubilization increased when the mixture was used compared to the effects observed when adding individual cultures, notwithstanding that there was no increase in bacterial multiplication under these conditions. Inoculation of black mangrove seedlings in artificial seawater showed the mixture performed somewhat better than inoculation of the individual bacterium; more leaves were developed and higher levels of (15)N were incorporated into the leaves, although the total nitrogen level decreased. This study demonstrates that interactions between individual components of the rhizosphere of mangroves should be considered when evaluating these bacteria as plant growth promoters.     

Ultrastructure of interaction in alginate beads between the microalga Chlorella vulgaris with its natural associative bacterium Phyllobacterium myrsinacearum and with the plant growth-promoting bacterium Azospirillum brasilense

Chlorella vulgaris, a microalga often used in wastewater treatment, was coimmobilized and coincubated either with the plant growth-promoting bacterium Azospirillum brasilense, or with its natural associative bacterium Phyllobacterium myrsinacearum, in alginate beads designed for advanced wastewater treatment. Interactions between the microalga and each of the bacterial species were followed using transmission electron microscopy for 10 days. Initially, most of the small cavities within the beads were colonized by microcolonies of only one microorganism, regardless of the bacterial species cocultured with the microalga. Subsequently, the bacterial and microalgal microcolonies merged to form large, mixed colonies within the cavities. At this stage, the effect of bacterial association with the microalga differed depending on the bacterium present. Though the microalga entered a senescence phase in the presence of P. myrsinacearum, it remained in a growth phase in the presence of A. brasilense. This study suggests that there are commensal interactions between the microalga and the two plant associative bacteria, and that with time the bacterial species determined whether the outcome for the microalga is senescence or continuous multiplication.     

Structural insight into the role of the ribosomal tunnel in cellular regulation

Nascent proteins emerge out of ribosomes through an exit tunnel, which was assumed to be a firmly built passive path. Recent biochemical results, however, indicate that the tunnel plays an active role in sequence-specific gating of nascent chains and in responding to cellular signals. Consistently, modulation of the tunnel shape, caused by the binding of the semi-synthetic macrolide troleandomycin to the large ribosomal subunit from Deinococcus radiodurans, was revealed crystallographically. The results provide insights into the tunnel dynamics at high resolution. Here we show that, in addition to the typical steric blockage of the ribosomal tunnel by macrolides, troleandomycin induces a conformational rearrangement in a wall constituent, protein L22, flipping the tip of its highly conserved beta-hairpin across the tunnel. On the basis of mutations that alleviate elongation arrest, the tunnel motion could be correlated with sequence discrimination and gating, suggesting that specific arrest motifs within nascent chain sequences may induce a similar gating mechanism.     

Ribosomal crystallography: peptide bond formation and its inhibition

Ribosomes, the universal cellular organelles catalyzing the translation of genetic code into proteins, are protein/RNA assemblies, of a molecular weight 2.5 mega Daltons or higher. They are built of two subunits that associate for performing protein biosynthesis. The large subunit creates the peptide bond and provides the path for emerging proteins. The small has key roles in initiating the process and controlling its fidelity. Crystallographic studies on complexes of the small and the large eubacterial ribosomal subunits with substrate analogs, antibiotics, and inhibitors confirmed that the ribosomal RNA governs most of its activities, and indicated that the main catalytic contribution of the ribosome is the precise positioning and alignment of its substrates, the tRNA molecules. A symmetry-related region of a significant size, containing about two hundred nucleotides, was revealed in all known structures of the large ribosomal subunit, despite the asymmetric nature of the ribosome. The symmetry rotation axis, identified in the middle of the peptide-bond formation site, coincides with the bond connecting the tRNA double-helical features with its single-stranded 3' end, which is the moiety carrying the amino acids. This thus implies sovereign movements of tRNA features and suggests that tRNA translocation involves a rotatory motion within the ribosomal active site. This motion is guided and anchored by ribosomal nucleotides belonging to the active site walls, and results in geometry suitable for peptide-bond formation with no significant rearrangements. The sole geometrical requirement for this proposed mechanism is that the initial P-site tRNA adopts the flipped orientation. The rotatory motion is the major component of unified machinery for peptide-bond formation, translocation, and nascent protein progression, since its spiral nature ensures the entrance of the nascent peptide into the ribosomal exit tunnel. This tunnel, assumed to be a passive path for the growing chains, was found to be involved dynamically in gating and discrimination.     

Hyaluronan is a key component in cryoprotection and formulation of targeted unilamellar liposomes

Lyophilized unilamellar liposomes (ULV), the dosage form of choice for shelf-life, revert upon reconstitution to the larger multilamellar liposomes (MLV), which is detrimental to the many carrier-mediated therapies that require small particles. High doses of sugars such as trehalose, sucrose and others, included in the original formulations for cryoprotection, were shown to prevent the conversion to MLV. In this study we set out to test whether hyaluronan (HA), the surface-bound ligand in our previously developed targeted bioadhesive liposomes (BAL), can also act as a cryoprotectant. The studies included structural and physicochemical characterization of original and reconstituted hyaluronan-ULV (HA-ULV). For each HA-ULV, similar regular ULV (RL-ULV) served as controls. Four properties were tested: particle size, zeta potential, encapsulation efficiency and half-life of drug release (tau(1/2)), for three drugs-chloramphenicol (CAM), vinblastine (VIN) and mitomycin C (MMC). Encapsulation efficiencies of the original systems were quite alike for similar RL-ULV and HA-ULV ranging from 25% to 70%. All systems acted as sustained-release drug depots, tau(1/2) ranging from 1.3 to 5.3 days. Drug species and lipid composition were the major determinants of encapsulation and release magnitudes. By all tests, as anticipated, lyophilization generated significant changes in the reconstituted RL-ULV: 17-fold increase in diameter; tripling of zeta potential; 25-60% drop in encapsulation efficiencies; 25-30% decrease in tau(1/2). In contrast, the reconstituted HA-ULV retained the same dimensions, zeta potentials, encapsulation efficiencies and tau(1/2) of the original systems. These data clearly show HA to be a cryoprotectant, adding another clinically relevant advantage to HA-BAL. We propose that, like the sugars, HA cryoprotects by providing substitute structure-stabilizing H-bonds.