The Azospirillum brasilense type VI secretion system promotes cell aggregation, biocontrol protection against phytopathogens and attachment to the microalgae Chlorella sorokiniana.

The plant-growth-promoting bacterium Azospirillum brasilense is able to associate with the microalgae Chlorella sorokiniana. Attachment of A. brasilense increases the metabolic performances of the microalgae. Recent genome analyses have revealed that the A. brasilense Az39 genome contains two complete sets of genes encoding type VI secretion systems (T6SS), including the T6SS1 that is induced by the indole-3-acetic acid (IAA) phytohormone. The T6SS is a multiprotein machine, widespread in Gram-negative bacteria, that delivers protein effectors in both prokaryotic and eukaryotic cells. Here we show that the A. brasilense T6SS is required for Chlorella-Azospirillum synthetic mutualism. Our data demonstrate that the T6SS is an important determinant to promote production of lipids, carbohydrates and photosynthetic pigments by the microalgae. We further show that this is likely due to the role of the T6SS during the attachment stage and for the production of IAA phytohormones. Finally, we demonstrate that the A. brasilense T6SS provides antagonistic activities against a number of plant pathogens such as Agrobacterium, Pectobacterium, Dickeya and Ralstonia species in vitro, suggesting that, in addition to promoting growth, A. brasilense might confer T6SS-dependent bio-control protection to microalgae and plants against bacterial pathogens.

Early Changes in Nutritional Conditions Affect Formation of Synthetic Mutualism Between Chlorella sorokiniana and the Bacterium Azospirillum brasilense.

The effect of three different nutritional conditions during the initial 12 h of interaction between the microalgae Chlorella sorokiniana UTEX 2714 and the plant growth-promoting bacterium Azospirillum brasilense Cd on formation of synthetic mutualism was assessed by changes in population growth, production of signal molecules tryptophan and indole-3-acetic acid, starch accumulation, and patterns of cell aggregation. When the interaction was supported by a nutrient-rich medium, production of both signal molecules was detected, but not when this interaction began with nitrogen-free (N-free) or carbon-free (C-free) media. Overall, populations of bacteria and microalgae were larger when co-immobilized. However, the highest starch production was measured in C. sorokiniana immobilized alone and growing continuously in a C-free mineral medium. In this interaction, the initial nutritional condition influenced the time at which the highest accumulation of starch occurred in Chlorella, where the N-free medium induced faster starch production and the richer medium delayed its accumulation. Formation of aggregates made of microalgae and bacteria occurred in all nutritional conditions, with maximum at 83 h in mineral medium, and coincided with declining starch content. This study demonstrates that synthetic mutualism between C. sorokiniana and A. brasilense can be modulated by the initial nutritional condition, mainly by the presence or absence of nitrogen and carbon in the medium in which they are interacting.

Adaptation and tolerance mechanisms developed by mycorrhizal Bipinnula fimbriata plantlets (Orchidaceae) in a heavy metal-polluted ecosystem.

The adaptation and performance of orchid mycorrhizae in heavy metal-polluted soils have been poorly explored. In the present study, proteomic and metabolic approaches were used to detect physiological changes in orchid roots established in a heavy metal-polluted soil and to ascertain whether mycorrhizal fungi affect the metabolic responses of roots. Young Bipinnula fimbriata plantlets were established in control and heavy metal-polluted soils in a greenhouse. After 14 months, exudation of root organic acids, phenolics, percentage of mycorrhization, mineral content, and differential protein accumulation were measured. More root biomass, higher root colonization, and higher exudation rates of citrate, succinate, and malate were detected in roots growing in heavy metal-polluted soils. Higher accumulation of phosphorus and heavy metals was found inside mycorrhizal roots under metal stress. Under non-contaminated conditions, non-mycorrhizal root segments showed enhanced accumulation of proteins related to carbon metabolism and stress, whereas mycorrhizal root segments stimulated protein synthesis related to pathogen control, cytoskeleton modification, and sucrose metabolism. Under heavy metal stress, the proteome profile of non-mycorrhizal root segments indicates a lower induction of defense mechanisms, which, together with the stimulation of enzymes related to carotenoid biosynthesis and cell wall organization, may positively influence mycorrhizal fungi colonization. The results point to different metabolic strategies in mycorrhizal and non-mycorrhizal root segments that are exposed to heavy metal stress. The results indicate that root colonization by mycorrhizal fungi is stimulated to alleviate the negative effects of heavy metals in the orchids.

Mycorrhizal compatibility and symbiotic seed germination of orchids from the Coastal Range and Andes in south central Chile.

Little is known about Orchidaceae plants in Chile and their mycorrhizal associations, a key issue for designing protective actions for endangered species. We investigated root fungi from seven terrestrial orchid species to identify potential mycorrhizal fungi. The main characteristics of Rhizoctonia-like fungi were observed under light microscopy, and isolates were identified through PCR-ITS sequencing. Molecular identification of fungal sequences showed a high diversity of fungi colonizing roots. Fungal ability to germinate seeds of different orchids was determined in symbiotic germination tests; 24 fungal groups were isolated, belonging to the genera Tulasnella, Ceratobasidium, and Thanatephorus. Furthermore, dark septate and other endophytic fungi were identified. The high number of Rhizoctonia-like fungi obtained from adult orchids from the Coastal mountain range suggests that, after germination, these orchids may complement their nutritional demands through mycoheterotrophy. Nonetheless, beneficial associations with other endophytic fungi may also co-exist. In this study, isolated mycorrhizal fungi had the ability to induce seed germination at different efficiencies and with low specificity. Germin ation rates were low, but protocorms continued to develop for 60 days. A Tulasnella sp. isolated from Chloraea gavilu was most effective to induce seed germination of different species. The dark septate endophytic (DSE) fungi did not show any effect on seed development; however, their widespread occurrence in some orchids suggests a putative role in plant establishment.

Accumulation of fatty acids in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoAcarboxylase, temperature, and co-immobilization with Azospirillum brasilense [corrected].

The relation between fatty acid accumulation, activity of acetyl-CoA carboxylase (ACC), and consequently lipid accumulation was studied in the microalgae Chlorella vulgaris co-immobilized with the plant growth-promoting bacterium Azospirillum brasilense under dark heterotrophic conditions with Na acetate as a carbon source. In C. vulgaris immobilized alone, cultivation experiments for 6 days showed that ACC activity is directly related to fatty acid accumulation, especially in the last 3 days. In co-immobilization experiments, A. brasilense exerted a significant positive effect over ACC activity, increased the quantity in all nine main fatty acids, increased total lipid accumulation in C. vulgaris, and mitigated negative effects of nonoptimal temperature for growth. No correlation between ACC activity and lipid accumulation in the cells was established for three different temperatures. This study demonstrated that the interaction between A. brasilense and C. vulgaris has a significant effect on fatty acid and lipid accumulation in the microalgae.

Biological deterioration of alginate beads containing immobilized microalgae and bacteria during tertiary wastewater treatment.

Secondary treatment of municipal wastewater affects the mechanical stability of polymer Ca-alginate beads containing the microalgae Chlorella vulgaris that are jointly immobilized with Azospirillum brasilense as treating agents whose presence do not affect bead stability. Nine strains of potential alginate-degrading bacteria were isolated from wastewater and identified, based on their nearly complete 16S rDNA sequence. Still, their population was relatively low. Attempts to enhance the strength of the beads, using different concentrations of alginate and CaCl2 or addition of either of three polymers (polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose), CaCO3, or SrCl2, failed. Beads lost their mechanical strength after 24 h of incubation but not the integrity of their shape for at least 96 h, a fact that sustained successful tertiary wastewater treatment for 48 h. In small bioreactors, removal of phosphorus was low under sterile conditions but high in unsterile wastewater. Alginate beads did not absorb PO4 (-3) in sterile wastewater, but in natural wastewater, they contained PO4 (-3). Consequently, PO4 (-3) content declined in the wastewater. A supplement of 10 % beads (w/v) was significantly more efficient in removing nutrients than 4 %, especially in a jointly immobilized treatment where >90 % of PO4 (-3) and >50 % ammonium were removed. Tertiary wastewater treatment in 25-L triangular, airlift, autotrophic bioreactors showed, as in small bioreactors, very similar nutrient removal patterns, decline in bead strength phenomena, and increase in total bacteria during the wastewater treatment only in the presence of the immobilized treatment agents. This study demonstrates that partial biological degradation of alginate beads occurred during tertiary wastewater treatment, but the beads survive long enough to permit efficient nutrient removal.

Restoration and recovery of hurricane-damaged mangroves using the knickpoint retreat effect and tides as dredging tools.

In 2001, a hurricane moved a large sand dune, blocking the sole outlet channel of a mangrove. In the absence of daily tidal flow, the two ponds containing the mangrove vegetation evaporated, the secondary drainage channels were lost, and a salt crust formed on the bed of the ponds. The mangrove lost most of its trees and the remaining suffered from osmotic shock that led to defoliation. Restoration involved creating a knickpoint retreat (waterfall retreat effect) and tidal flow as a dredging mechanism to restore the outlet and form secondary channels in the ponds. During a very low tide, we deepened the mouth of the outlet channel by 1 m below high tide level to form a small waterfall when high tides receded. During successive tides, this one-step knickpoint deteriorated and formed a series of low rapids. With a steep gradient, the rapids retreated upstream into the ponds, first reopening the outlet channel and then carving new secondary channels in the pond mud flat. The excavation process of the outlet channel was repeated three times and was sufficient to effectively improve the hydrology of the entire pond system; allowing adequate flooding and draining of the mangrove ponds. Hydrology analysis tested by the Engelund-Hansen sediment transport formula established that the output of sediment from the ecosystem is greater than the input of sand into the mangroves. This is keeping the main channel continuously open. After eight years, tidal flow continues to keep the channels open; the salt crust has disappeared; the trees have recovered, and a large area of new vegetation has emerged.

Alginate beads provide a beneficial physical barrier against native microorganisms in wastewater treated with immobilized bacteria and microalgae.

When the freshwater microalga Chlorella sorokiniana and the plant growth-promoting bacterium Azospirillum brasilense were deployed as free suspensions in unsterile, municipal wastewater for tertiary wastewater treatment, their population was significantly lower compared with their populations in sterile wastewater. At the same time, the numbers of natural microfauna and wastewater bacteria increased. Immobilization of C. sorokiniana and A. brasilense in small (2-4 mm in diameter), polymer Ca-alginate beads significantly enhanced their populations when these beads were suspended in normal wastewater. All microbial populations within and on the surface of the beads were evaluated by quantitative fluorescence in situ hybridization combined with scanning electron microscopy and direct measurements. Submerging immobilizing beads in wastewater created the following sequence of events: (a) a biofilm composed of wastewater bacteria and A. brasilense was created on the surface of the beads, (b) the bead inhibited penetration of outside organisms into the beads, (c) the bead inhibited liberation of the immobilized microorganisms into the wastewater, and (d) permitted an uninterrupted reduction of ammonium and phosphorus from the wastewater. This study demonstrated that wastewater microbial populations are responsible for decreasing populations of biological agents used for wastewater treatment and immobilization in alginate beads provided a protective environment for these agents to carry out uninterrupted tertiary wastewater treatment.

Restoration of eroded soil in the Sonoran Desert with native leguminous trees using plant growth-promoting microorganisms and limited amounts of compost and water.

Restoration of highly eroded desert land was attempted in the southern Sonoran Desert that had lost its natural capacity for self-revegetation. In six field experiments, the fields were planted with three native leguminous trees: mesquite amargo Prosopis articulata, and yellow and blue palo verde Parkinsonia microphylla and Parkinsonia florida. Restoration included inoculation with two of plant growth-promoting bacteria (PGPB; Azospirillum brasilense and Bacillus pumilus), native arbuscular mycorrhizal (AM) fungi, and small quantities of compost. Irrigation was applied, when necessary, to reach a rainy year (300 mm) of the area. The plots were maintained for 61 months. Survival of the trees was marginally affected by all supplements after 30 months, in the range of 60-90%. This variation depended on the plant species, where all young trees were established after 3 months. Plant density was a crucial variable and, in general, low plant density enhanced survival. High planting density was detrimental. Survival significantly declined in trees 61 months after planting. No general response of the trees to plant growth-promoting microorganisms and compost was found. Mesquite amargo and yellow palo verde responded well (height, number of branches, and diameter of the main stem) to inoculation with PGPB, AM fungi, and compost supplementation after three months of application. Fewer positive effects were recorded after 30 months. Blue palo verde did not respond to most treatments and had the lowest survival. Specific plant growth parameters were affected to varying degrees to inoculations or amendments, primarily depending on the tree species. Some combinations of tree/inoculant/amendment resulted in small negative effects or no response when measured after extended periods of time. Using native leguminous trees, this study demonstrated that restoration of severely eroded desert lands was possible.

Endophytic bacteria of Mammillaria fraileana, an endemic rock-colonizing cactus of the southern Sonoran Desert.

The small cactus Mammillaria fraileana is a pioneer rock-colonizing plant harboring endophytic bacteria with the potential for nitrogen fixation and rock weathering (phosphate solubilization and rock degradation). In seeds, only a combination of culture-independent methods, such as fluorescence in situ hybridization, scanning electron microscopy, and fluorescence vital staining, detected significant amounts of non-culturable, but living, endophytic bacteria distributed underneath the membrane covering the embryo, in the undifferentiated tissue of the embryo, and in the vascular tissue. Large populations of culturable endophytic bacteria were detected in stems and roots of wild plants colonizing rocks in the southern Sonoran Desert, but not in seeds. Among 14 endophytic bacterial isolates found in roots, four isolates were identified by full sequencing of their 16S rRNA gene. In vitro tests indicated that Azotobacter vinelandii M2Per is a potent nitrogen fixer. Solubilization of inorganic phosphate was exhibited by Pseudomonas putida M5TSA, Enterobacter sakazakii M2PFe, and Bacillus megaterium M1PCa, while A. vinelandii M2Per, P. putida M5TSA, and B. megaterium M1PCa weathered rock by reducing the size of rock particles, probably by changing the pH of the liquid media. Cultivated seedlings of M. fraileana, derived from disinfected seeds and inoculated with endophytic bacteria, showed re-colonization 105 days after inoculation. Their densities decreased from the root toward the stem and apical zones. Functional traits in planta of culturable and non-culturable endophytic bacteria in seeds remain unknown.

Growth of quailbush in acidic, metalliferous desert mine tailings: effect of Azospirillum brasilense Sp6 on biomass production and rhizosphere community structure.

Mine tailing deposits in semiarid and arid environments frequently remain devoid of vegetation due to the toxicity of the substrate and the absence of a diverse soil microbial community capable of supporting seed germination and plant growth. The contribution of the plant growth promoting bacterium (PGPB) Azospirillum brasilense Sp6 to the growth of quailbush in compost-amended, moderately acidic, high-metal content mine tailings using an irrigation-based reclamation strategy was examined along with its influence on the rhizosphere bacterial community. Sp6 inoculation resulted in a significant (2.2-fold) increase in plant biomass production. The data suggest that the inoculum successfully colonized the root surface and persisted throughout the 60-day experiment in both the rhizosphere, as demonstrated by excision and sequencing of the appropriate denaturing gradient gel electrophoresis (DGGE) band, and the rhizoplane, as indicated by fluorescent in situ hybridization of root surfaces. Changes in rhizosphere community structure in response to Sp6 inoculation were evaluated after 15, 30, and 60 days using DGGE analysis of 16S rRNA polymerase chain reaction amplicons. A comparison of DGGE profiles using canonical correspondence analysis revealed a significant treatment effect (Sp6-inoculated vs. uninoculated plants vs. unplanted) on bacterial community structure at 15, 30, and 60 days (p < 0.05). These data indicate that in an extremely stressed environment such as acid mine tailings, an inoculated plant growth promoting bacterium not only can persist and stimulate plant growth but also can directly or indirectly influence rhizobacterial community development.

Bacillus pumilus ES4: candidate plant growth-promoting bacterium to enhance establishment of plants in mine tailings.

Three plant growth-promoting bacteria (PGPB; Bacillus pumilus ES4, B. pumilus RIZO1, and Azospirillum brasilense Cd) were tested for their ability to enhance plant growth and development of the native Sonoran Desert shrub quailbush (Atriplex lentiformis) and for their effect on the native bacterial community in moderately acidic, high-metal content (AHMT) and in neutral, low metal content natural tailings (NLMT) in controlled greenhouse experiments. Inoculation of quailbush with all three PGPB significantly enhanced plant growth parameters, such as germination, root length, dry weight of shoots and roots, and root/shoot ratio in both types of tailings. The effect of inoculation on the indigenous bacterial community by the most successful PGPB Bacillus pumilus ES4 was evaluated by denaturating gradient gel electrophoresis (PCR-DGGE) fingerprinting and root colonization was followed by specific fluorescent in situ hybridization (FISH). Inoculation with this strain significantly changed the bacterial community over a period of 60 days. FISH analysis showed that the preferred site of colonization was the root tips and root elongation area. This study shows that inoculation of native perennial plants with PGPB can be used for developing technologies for phytostabilizing mine tailings.

Joint immobilization of plant growth-promoting bacteria and green microalgae in alginate beads as an experimental model for studying plant-bacterium interactions.

A simple, quantitative experimental model, offering a convenient and basic approach to studies of plant-bacterium interactions, is proposed. This involves immobilizing a unicellular, freshwater microalga, a Chlorella species, serving as the plant, with a plant growth-promoting bacterium, an Azospirillum species, in small alginate beads to allow close interaction and to avoid external interference from bacterial contaminants.

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.

Chlorella sorokiniana UTEX 2805, a heat and intense, sunlight-tolerant microalga with potential for removing ammonium from wastewater.

In the summer of 2003, a microalga strain was isolated from a massive green microalgae bloom in wastewater stabilization ponds at the treatment facility of La Paz, B.C.S., Mexico. Prevailing environmental conditions were air temperatures over 40 degrees C, water temperature of 37 degrees C, and insolation of up to 2400 micromol m2 s(-1) at midday for several hours at the water surface for four months. The microalga was identified as Chlorella sorokiniana Shih. et Krauss, based on sequencing its entire 18S rRNA gene. In a controlled photo-bioreactor, this strain can grow to high population densities in synthetic wastewater at temperatures of 40-42 degrees C and light intensity of 2500 micromol m2 s(-1) for 5h daily and efficiently remove ammonium from the wastewater under these conditions better than under normal lower temperature (28 degrees C) and lower light intensity (60 micromol m2 s(-1)). When co-immobilized with the bacterium Azospirillum brasilense that promotes growth of microalgae, the population of microalga grew faster and removed even more ammonium. Under exposure to extreme growth conditions, the quantity of four photosynthetic pigments increased in the co-immobilized cultures. This strain of microalga has potential as a wastewater treatment agent under extreme conditions of temperature and light intensity.

Immobilization of microalgae cells in alginate facilitates isolation of DNA and RNA.

Isolation of nucleic acids from Chlorella is difficult, given the chemically complex nature of their cell walls and variable production of metabolites. Immobilization of microalgae in polymers adds additional difficulty. Here, we modified, amended, and standardized methods for isolation of nucleic acids and compared the yield of DNA and RNA from free-living and encapsulated microalgae C. sorokiniana. Isolation of nucleic acids from immobilized cells required two steps in dissolving the alginate matrix, releasing the cells, and mechanical disruption with glass beads. For DNA extraction, we used modified versions of a commercial kit along with the hexadecyltrimethylammonium bromide (CTAB) method. For RNA extraction, we used the commercial TRI reagent procedure and the CTAB-dithiotreitol method. Quantity and quality of nucleic acids in extracts varied with growth conditions, isolation procedures, and time of incubation of the original culture. There were consistently higher amounts of DNA and RNA in extracts from immobilized cells. Quantitatively, the modified procedure with the commercial Promega kit was the most reliable procedure for isolating DNA and a modified commercial TRI reagent procedure was the choice for isolating RNA. All four procedures eliminated proteins efficiently and had low levels of contamination from residual polysaccharides from the matrices and/or metabolites naturally produced by the microalgae. All DNA extracts under both growth conditions, time of incubation, and two isolation methods successfully amplified the 18S ribosomal RNA by PCR and quantitative reverse transcription (RT-qPCR).

Enhanced performance of the microalga Chlorella sorokiniana remotely induced by the plant growth-promoting bacteria Azospirillum brasilense and Bacillus pumilus.

Remote effects (occurring without physical contact) of two plant growth-promoting bacteria (PGPB) Azospirillum brasilense Cd and Bacilus pumilus ES4 on growth of the green microalga Chlorella sorokiniana UTEX 2714 were studied. The two PGPB remotely enhanced the growth of the microalga, up to six-fold, and its cell volume by about three-fold. In addition to phenotypic changes, both bacteria remotely induced increases in the amounts of total lipids, total carbohydrates, and chlorophyll a in the cells of the microalga, indicating an alteration of the microalga's physiology. The two bacteria produced large amounts of volatile compounds, including CO2, and the known plant growth-promoting volatile 2,3-butanediol and acetoin. Several other volatiles having biological functions in other organisms, as well as numerous volatile compounds with undefined biological roles, were detected. Together, these bacteria-derived volatiles can positively affect growth and metabolic parameters in green microalgae without physical attachment of the bacteria to the microalgae. This is a new paradigm on how PGPB promote growth of microalgae which may serve to improve performance of Chlorella spp. for biotechnological applications.

Seasonal seawater temperature as the major determinant for populations of culturable bacteria in the sediments of an intact mangrove in an arid region.

Mangroves are highly productive marine ecosystems where bacteria (culturable and non-culturable) actively participate in biomineralization of organic matter and biotransformation of minerals. This study explores spatial and seasonal fluctuations of culturable heterotrophic bacteria and Vibrio spp. in the sediments of an intact mangrove ecosystem and determines the dominant environmental factors that govern these fluctuations. Sediment samples were collected monthly from three stations in the mangroves of Laguna de Balandra, Baja California Sur, Mexico, through an annual cycle. Physicochemical parameters included seawater temperature, salinity, and concentration of dissolved oxygen. Viable counts of culturable heterotrophic bacteria and Vibrio spp. were made. In one sample (March 2003), nutrient concentrations (ammonium, nitrites, nitrates, and phosphates), organic matter, pH and sediment texture were also determined. General cluster analyses, analysis of variance of specific variables, and several principal component analyses demonstrated that seawater temperature is the principal determinant of seasonal distribution of culturable heterotrophic bacteria and Vibrio spp. in mangrove sediments. Soil texture, concentration of nutrients, and organic matter concentration contribute to heterogenicity to a lesser extent. A large spatial variation in bacterial populations was observed over short distances ( approximately 1 m) in sampling areas within the same site. These analyses show that the culturable bacterial distribution in sediments of mangroves has high spatial and temporal heterogeneity.

Pleiotropic physiological effects in the plant growth-promoting bacterium Azospirillum brasilense following chromosomal labeling in the clpX gene.

Azospirillum brasilense 8-I was chromosomally labeled with green fluorescent protein (gfp) genes, using either the native promoterless gfp gene or the mutant gfpmut2 gene under the transcriptional control of the neomycin phosphate transferase (npt2) promoter inserted into Tn5 suicide plasmid vectors. One A. brasilense exconjugant, showing a steady and strong fluorescence following irradiation with 365-nm UV light was characterized in detail. This strain, A. brasilense 8-I-gfp showed increased N(2)-fixation of approximately threefold, up to a twofold increase in exopolysaccharide production, and a significant decrease in indole-3-acetic acid and poly-beta-hydroxybutyrate production over the parental strain. Sequence analysis showed that the Tn5 carrying the gfp gene was inserted in the clpX gene encoding a heat-shock protein. This data is consistent with a model in which the observed physiological changes are a consequence of pleiotropic changes that occur as a consequence of impaired heat shock (stress) protein synthesis. In summary, (i) chromosomally labelled Azospirillum brasilense was obtained carrying either native or mutant gfp genes, (ii) Pleiotropic physiological effects were caused by disruption of the clpX gene as the consequence of the insertion, (iii) a new indole-3-acetic acid-attenuated mutant of A. brasilense producing only 0.25% of the indole-3-acetic acid produced by the wild-type is presented.

Image analysis for quantification of bacterial rock weathering.

A fast, quantitative image analysis technique was developed to assess potential rock weathering by bacteria. The technique is based on reduction in the surface area of rock particles and counting the relative increase in the number of small particles in ground rock slurries. This was done by recording changes in ground rock samples with an electronic image analyzing process. The slurries were previously amended with three carbon sources, ground to a uniform particle size and incubated with rock weathering bacteria for 28 days. The technique was developed and tested, using two rock-weathering bacteria Pseudomonas putida R-20 and Azospirillum brasilense Cd on marble, granite, apatite, quartz, limestone, and volcanic rock as substrates. The image analyzer processed large number of particles (10(7)-10(8) per sample), so that the weathering capacity of bacteria can be detected.