Cyanidiales diversity in Yellowstone National Park.

The Cyanidiales are unicellular red algae that are unique among phototrophs. They thrive in acidic, moderately high-temperature habitats typically associated with geothermally active regions, although much remains to be learned about their distribution and diversity within such extreme environments. We focused on Yellowstone National Park (YNP), using culture-dependent efforts in combination with a park-wide environmental polymerase chain reaction (PCR) survey to examine Cyanidiales diversity and distribution in aqueous (i.e. submerged), soil and endolithic environments. Phylogenetic reconstruction of Cyanidiales biodiversity demonstrated the presence of Cyanidioschyzon and Galdieria lineages exhibiting distinct habitat preferences. Cyanidioschyzon was the only phylotype detected in aqueous environments, but was also prominent in moist soil and endolithic habitats, environments where this genus was thought to be scarce. Galdieria was found in soil and endolithic samples, but absent in aqueous habitats. Interestingly, Cyanidium could not be found in the surveys, suggesting this genus may be absent or rare in YNP. Direct microscopic counts and viable counts from soil samples collected along a moisture gradient were positively correlated with moisture content, providing the first in situ evidence that gravimetric moisture is an important environmental parameter controlling distribution of these algae.

A geothermal-linked biological oasis in Yellowstone Lake, Yellowstone National Park, Wyoming.

Hundreds of active and dormant geothermal vents have been located on the floor of Yellowstone Lake, although characterization of the associated biology (macro or micro) has been extremely limited. Herein, we describe an aquatic moss (Fontinalis) colony closely associated with vent emissions that considerably exceeded known temperature maxima for this plant. Vent waters were supersaturated with CO(2), likely accommodating a CO(2) compensation point that would be expected to be quite elevated under these conditions. The moss was colonized by metazoa, including the crustaceans Hyalella and Gammarus, a segmented worm in the Lumbriculidae family, and a flatworm specimen tentatively identified as Polycelis. The presence of these invertebrates suggest a highly localized food chain that derives from the presence of geothermal inputs and thus is analogous to the deep marine vents that support significant biodiversity.

Biogeographic and phylogenetic diversity of thermoacidophilic cyanidiales in Yellowstone National Park, Japan, and New Zealand.

Members of the rhodophytan order Cyanidiales are unique among phototrophs in their ability to live in extreme environments that combine low pH levels ( approximately 0.2 to 4.0) and moderately high temperatures of 40 to 56 degrees C. These unicellular algae occur in far-flung volcanic areas throughout the earth. Three genera (Cyanidium, Galdieria, and Cyanidioschyzon) are recognized. The phylogenetic diversity of culture isolates of the Cyanidiales from habitats throughout Yellowstone National Park (YNP), three areas in Japan, and seven regions in New Zealand was examined by using the chloroplast RuBisCO large subunit gene (rbcL) and the 18S rRNA gene. Based on the nucleotide sequences of both genes, the YNP isolates fall into two groups, one with high identity to Galdieria sulphuraria (type II) and another that is by far the most common and extensively distributed Yellowstone type (type IA). The latter is a spherical, walled cell that reproduces by internal divisions, with a subsequent release of smaller daughter cells. This type, nevertheless, shows a 99 to 100% identity to Cyanidioschyzon merolae (type IB), which lacks a wall, divides by "fission"-like cytokinesis into two daughter cells, and has less than 5% of the cell volume of type IA. The evolutionary and taxonomic ramifications of this disparity are discussed. Although the 18S rRNA and rbcL genes did not reveal diversity among the numerous isolates of type IA, chloroplast short sequence repeats did show some variation by location within YNP. In contrast, Japanese and New Zealand strains showed considerable diversity when we examined only the sequences of 18S and rbcL genes. Most exhibited identities closer to Galdieria maxima than to other strains, but these identities were commonly as low as 91 to 93%. Some of these Japanese and New Zealand strains probably represent undescribed species that diverged after long-term geographic isolation.

Molecular analysis of microbial community structure in an arsenite-oxidizing acidic thermal spring.

Electron microscopy (EM), denaturing gradient gel electrophoresis (DGGE) and 16S rDNA sequencing were used to examine the structure and diversity of microbial mats present in an acid-sulphate-chloride (pH 3.1) thermal (58-62 degrees C) spring in Norris Basin, Yellowstone National Park, WY, USA, exhibiting rapid rates of arsenite oxidation. Initial visual assessments, scanning EM and geochemical measurements revealed the presence of three distinct mat types. Analysis of 16S rDNA fragments with DGGE confirmed the presence of different bacterial and archaeal communities within these zones. Changes in the microbial community appeared to coincide with arsenite oxidation activity. Phylogenetic analysis of 1400 bp 16S rDNA sequences revealed that clone libraries prepared from both arsenic redox active and inactive bacterial communities were dominated by sequences phylogenetically related to Hydrogenobacter acidophilus and Desulphurella sp. The appearance of archaeal 16S rDNA sequences coincided with the start of arsenite oxidation, and sequences were obtained showing affiliation with both Crenarchaeota and Euryarchaeota. The majority of archaeal sequences were most similar to sequences obtained from marine hydrothermal vents and other acidic hot springs, although the level of similarity was typically just 90%. Arsenite oxidation in this system may result from the activities of these unknown archaeal taxa and/or the previously unreported arsenic redox activity of H. acidophilus- or Desulphurella-like organisms. If the latter, arsenite oxidation must be inhibited in the initial high-sulphide zone of the spring, where no change in the distribution of arsenite versus arsenate was observed.

Cloning and characterization of a second acid phosphatase from Sinorhizobium meliloti strain 104A14.

Sinorhizobium meliloti has two nonspecific periplasmic acid phosphatases. The NapD enzyme has been previously described, and a second acid phosphatase, NapE, is described in this report. NapE was partially purified from an S. meliloti napD mutant and characterized with respect to molecular mass and substrate range. As predicted from SDS-PAGE analysis, the subunit molecular mass of NapE is approximately 35.8 kDa and gel filtration experiments estimated the native molecular mass to be approximately 70 kDa, indicating that the active enzyme is a homodimer. NapE demonstrated significant activity with p-nitrophenyl phosphate, phenyl phosphate, and alpha-naphthyl-phosphate. The pH optimum was between 4.5 and 5.0. The gene encoding NapE was also sequenced and the inferred amino acid sequence from the predicted ORF was found to be 60% identical and 75% similar to that encoded by napD. An S. meliloti napE mutant was constructed and assessed for symbiotic competence. This mutant did not differ from the wild-type parent strain in nodulation and symbiotic efficiency.

Rapid oxidation of arsenite in a hot spring ecosystem, Yellowstone National Park.

Geothermal springs within Yellowstone National Park (YNP) often contain arsenic (As) at concentrations of 10-40 microM, levels that are considered toxic to many organisms. Arsenite (As(III)) is often the predominant valence state at the point of discharge but is rapidly oxidized to arsenate (As(V)) during transport in shallow surface water. The current study was designed to establish rates and possible mechanisms of As(III) oxidation and to characterize the geochemical environment associated with predominant microbial mats in a representative acid-sulfate-chloride (pH 3.1) thermal (58-62 degrees C) spring in Norris Basin, YNP. At the spring origin, total soluble As was predominantly As(III) at concentrations of 33 microM. No oxidation of As(III) was detected over the first 2.7 m downstream from the spring source, corresponding to an area dominated by a yellow filamentous S0-rich microbial mat However, rapid oxidation of As(III) to As(V) was observed between 2.7 and 5.6 m, corresponding to termination of the S0-rich mats, decreases in dissolved sulfide, and commencement of a brown Fe/As-rich mat. Rates of As(II) oxidation were estimated, yielding an apparent first-order rate constant of 1.2 min(-1) (half-life = 0.58 min). The oxidation of As(III) was shown to require live organisms present just prior to and within the Fe/As-rich mat. Complementary analytical tools used to characterize the brown mat revealed an As:Fe molar ratio of 0.7 and suggested that this filamentous microbial mat contains iron(III) oxyhydroxide coprecipitated with As(V). Results from the current work are the first to provide a comprehensive characterization of microbially mediated As(III) oxidation and the geochemical environments associated with microbial mats in acid-sulfate-chloride springs of YNP.

Factors affecting catalase expression in Pseudomonas aeruginosa biofilms and planktonic cells.

Previous work with Pseudomonas aeruginosa showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 microM as FeCl(3)) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.

Gene expression in Pseudomonas aeruginosa: evidence of iron override effects on quorum sensing and biofilm-specific gene regulation.

Prior studies established that the Pseudomonas aeruginosa oxidative stress response is influenced by iron availability, whereas more recent evidence demonstrated that it was also controlled by quorum sensing (QS) regulatory circuitry. In the present study, sodA (encoding manganese-cofactored superoxide dismutase [Mn-SOD]) and Mn-SOD were used as a reporter gene and endogenous reporter enzyme, respectively, to reexamine control mechanisms that govern the oxidative stress response and to better understand how QS and a nutrient stress response interact or overlap in this bacterium. In cells grown in Trypticase soy broth (TSB), Mn-SOD was found in wild-type stationary-phase planktonic cells but not in a lasI or lasR mutant. However, Mn-SOD activity was completely suppressed in the wild-type strain when TSB was supplemented with iron. Reporter gene studies indicated that sodA transcription could be variably induced in iron-starved cells of all three strains, depending on growth stage. Iron starvation induction of sodA was greatest in the wild-type strain and least in the lasR mutant and was maximal in stationary-phase cells. Reporter experiments in the wild-type strain showed increased lasI::lacZ transcription in response to iron limitation, whereas the expression level in the las mutants was minimal and iron starvation induction of lasI::lacZ did not occur. Studies comparing Mn-SOD activity in P. aeruginosa biofilms and planktonic cultures were also initiated. In wild-type biofilms, Mn-SOD was not detected until after 6 days, although in iron-limited wild-type biofilms Mn-SOD was detected within the initial 24 h of biofilm establishment and formation. Unlike planktonic bacteria, Mn-SOD was constitutive in the lasI and lasR mutant biofilms but could be suppressed if the growth medium was amended with 25 microM ferric chloride. This study demonstrated that (i) the nutritional status of the cell must be taken into account when one is evaluating QS-based gene expression; (ii) in the biofilm mode of growth, QS may also have negative regulatory functions; (iii) QS-based gene regulation models based on studies with planktonic cells must be modified in order to explain biofilm gene expression behavior; and (iv) gene expression in biofilms is dynamic.

Microbial populations associated with the reduction and enhanced mobilization of arsenic in mine tailings.

Microbial reduction of arsenate [As(V)] to arsenite [As(III)] and the subsequent effects on As mobilization in contaminated mine tailings were studied under transport conditions. Molecular analysis of bacterial populations and traditional isolation techniques were used in conjunction with column experiments designed to observe relationships among pH (limed vs unlimed treatments), redox potential (Pt electrode), and mobilization of As. Liming increased pH values from approximately 4 to 8, resulting in a 5-fold increase in total As eluted from sterile columns. Elution of As from limed columns was further enhanced by microbial activity. As(III) was the predominant As species eluted from oxic, nonsterile columns. Conversely, in sterile treatments, As(V) was the predominant valence state in column effluent. Denaturing gradient gel electrophoresis coupled with sequence and phylogenetic analysis of 16S rRNA gene segments revealed that liming of the mine tailings stimulated specific Caulobacter-, Sphingomonas-, and Rhizobium-like populations. Pure culture isolates of these bacteria demonstrated the ability to rapidly reduce As(V) in aerated serum bottles. An intracellular As detoxification pathway was implicated in the reduction of As(V) by these isolates. These results indicate that microbial reduction of As(V) in As-contaminated soils may occur under aerobic conditions over relatively short time scales resulting in enhanced As mobilization.

The ++Sinorhizobium meliloti lon protease is involved in regulating exopolysaccharide synthesis and is required for nodulation of alfalfa.

While screening for Sinorhizobium meliloti Pho regulatory mutants, a transposon mutant was isolated that constitutively expressed higher levels of acid and alkaline phosphatase enzymes. This mutant was also found to form pseudonodules on alfalfa that were delayed in appearance relative to those formed by the wild-type strain, it contained few bacteroids, and it did not fix nitrogen. Sequence analysis of the transposon insertion site revealed the affected gene to have high homology to Lon proteases from a number of organisms. In minimal succinate medium, the mutant strain was found to grow more slowly, reach lower maximal optical density, and produce more extracellular polysaccharide (EPS) than the wild-type strain. The mutant fluoresced brightly on minimal succinate agar containing calcofluor (which binds to EPSI, a constitutively expressed succinoglycan), and gas chromotographic analysis of purified total EPS showed that the glucose-to-galactose ratio in the lon mutant total EPS was 5.0 +/- 0.2 (mean +/- standard error), whereas the glucose-to-galactose ratio in the wild-type strain was 7.1 +/- 0.5. These data suggested that in addition to EPSI, the lon mutant also constitutively synthesized EPSII, a galactoglucan which is the second major EPS known to be produced by S. meliloti, but typically is expressed only under conditions of phosphate limitation. (13)C nuclear magnetic resonance analysis showed no major differences between EPS purified from the mutant and wild-type strains. Normal growth, EPS production, and the symbiotic phenotype were restored in the mutant strain when the wild-type lon gene was present in trans. The results of this study suggest that the S. meliloti Lon protease is important for controlling turnover of a constitutively expressed protein(s) that, when unregulated, disrupts normal nodule formation and normal growth.

Characterization of two inducible phosphate transport systems in Rhizobium tropici.

Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropici association is sensitive to the availability of phosphate (P(i)). To better understand phosphorus movement between the bacteroid and the host plant, P(i) transport was characterized in R. tropici. We observed two P(i) transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The K(m) and V(max) values for the low-affinity system were estimated to be 34 +/- 3 microM P(i) and 118 +/- 8 nmol of P(i) x min(-1) x mg (dry weight) of cells(-1), respectively, and the K(m) and V(max) values for the high-affinity system were 0.45 +/- 0.01 microM P(i) and 86 +/- 5 nmol of P(i) x min(-1) x mg (dry weight) of cells(-1), respectively. Both systems were inducible by P(i) starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but P(i) transport through both systems was eliminated by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide; the P(i) transport rate was correlated with the intracellular ATP concentration. Also, P(i) movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both P(i) transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with kdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.

Citrate synthase mutants of Sinorhizobium meliloti are ineffective and have altered cell surface polysaccharides.

The gltA gene, encoding Sinorhizobium meliloti 104A14 citrate synthase, was isolated by complementing an Escherichia coli gltA mutant. The S. meliloti gltA gene was mutated by inserting a kanamycin resistance gene and then using homologous recombination to replace the wild-type gltA with the gltA::kan allele. The resulting strain, CSDX1, was a glutamate auxotroph, and enzyme assays confirmed the absence of a requirement for glutamate. CSDX1 did not grow on succinate, malate, aspartate, pyruvate, or glucose. CSDX1 produced an unusual blue fluorescence on medium containing Calcofluor, which is different from the green fluorescence found with 104A14. High concentrations of arabinose (0.4%) or succinate (0. 2%) restored the green fluorescence to CSDX1. High-performance liquid chromatography analyses showed that CSDX1 produced partially succinylated succinoglycan. CSDX1 was able to form nodules on alfalfa, but these nodules were not able to fix nitrogen. The symbiotic defect of a citrate synthase mutant could thus be due to disruption of the infection process or to the lack of energy generated by the tricarboxylic acid cycle.

Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide.

Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm-grown Pseudomonas aeruginosa. P. aeruginosa QS requires two transcriptional activator proteins known as LasR and RhlR and their cognate autoinducers PAI-1 (N-(3-oxododecanoyl)-L-homoserine lactone) and PAI-2 (N-butyryl-L-homoserine lactone) respectively. This study provides evidence of QS control of genes essential for relieving oxidative stress. Mutants devoid of one or both autoinducers were more sensitive to hydrogen peroxide and phenazine methosulphate, and some PAI mutant strains also demonstrated decreased expression of two superoxide dismutases (SODs), Mn-SOD and Fe-SOD, and the major catalase, KatA. The expression of sodA (encoding Mn-SOD) was particularly dependent on PAI-1, whereas the influence of autoinducers on Fe-SOD and KatA levels was also apparent but not to the degree observed with Mn-SOD. beta-Galactosidase reporter fusion results were in agreement with these findings. Also, the addition of both PAIs to suspensions of the PAI-1/2-deficient double mutant partially restored KatA activity, while the addition of PAI-1 only was sufficient for full restoration of Mn-SOD activity. In biofilm studies, catalase activity in wild-type bacteria was significantly reduced relative to planktonic bacteria; catalase activity in the PAI mutants was reduced even further and consistent with relative differences observed between each strain grown planktonically. While wild-type and mutant biofilms contained less catalase activity, they were more resistant to hydrogen peroxide treatment than their respective planktonic counterparts. Also, while catalase was implicated as an important factor in biofilm resistance to hydrogen peroxide insult, other unknown factors seemed potentially important, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.

Pseudomonas aeruginosa biofilm sensitivity to biocides: use of hydrogen peroxide as model antimicrobial agent for examining resistance mechanisms.

The biofilm mode of bacterial growth may be the preferred form of existence in nature. Because of the global impact of problematic biofilms, study of the mechanisms affording resistance to various biocides is of dire importance. Furthermore, understanding the physiological differences between biofilm and planktonic organisms ranks particularly high on the list of important and necessary research. Such contributions will only serve to broaden our knowledge base, especially regarding the development of better antimicrobials while also fine-tuning the use of current highly effective antimicrobials. Using H2O2 as a model oxidizing biocide, we demonstrate the marked resistance of biofilm bacteria relative to planktonic cells. Because many biocides are good oxidizing agents (e.g., H2O2, HOCl), understanding the mechanisms by which genes involved in combating oxidative stress are activated is important in determining the overall efficacy of such biocides. Future studies will focus on determining mechanisms of oxidative stress gene regulation in bacterial biofilms.

Protective role of catalase in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide.

The role of the two known catalases in Pseudomonas aeruginosa in protecting planktonic and biofilm cells against hydrogen peroxide (H(2)O(2)) was investigated. Planktonic cultures and biofilms formed by the wild-type strain PAO1 and the katA and katB catalase mutants were compared for their susceptibility to H(2)O(2). Over the course of 1 h, wild-type cell viability decreased steadily in planktonic cells exposed to a single dose of 50 mM H(2)O(2), whereas biofilm cell viability remained at approximately 90% when cells were exposed to a flowing stream of 50 mM H(2)O(2). The katB mutant, lacking the H(2)O(2)-inducible catalase KatB, was similar to the wild-type strain with respect to H(2)O(2) resistance. The katA mutant possessed undetectable catalase activity. Planktonic katA mutant cultures were hypersusceptible to a single dose of 50 mM H(2)O(2), while biofilms displayed a 10-fold reduction in the number of culturable cells after a 1-h exposure to 50 mM H(2)O(2). Catalase activity assays, activity stains in nondenaturing polyacrylamide gels, and lacZ reporter genes were used to characterize the oxidative stress responses of planktonic cultures and biofilms. Enzyme assays and catalase activity bands in nondenaturing polyacrylamide gels showed significant KatB catalase induction occurred in biofilms after a 20-min exposure to H(2)O(2), suggesting that biofilms were capable of a rapid adaptive response to the oxidant. Reporter gene data obtained with a katB::lacZ transcriptional reporter strain confirmed katB induction and that the increase in total cellular catalase activity was attributable to KatB. Biofilms upregulated the reporter in the constant presence of 50 mM H(2)O(2), while planktonic cells were overwhelmed by a single 50 mM dose and were unable to make detectable levels of beta-galactosidase. The results of this study demonstrated the following: the constitutively expressed KatA catalase is important for resistance of planktonic and biofilm P. aeruginosa to H(2)O(2), particularly at high H(2)O(2) concentrations; KatB is induced in both planktonic and biofilm cells in response to H(2)O(2) insult, but plays a relatively small role in biofilm resistance; and KatB is important to either planktonic cells or biofilm cells for acquired antioxidant resistance when initial levels of H(2)O(2) are sublethal.

Genes coding for phosphotransacetylase and acetate kinase in Sinorhizobium meliloti are in an operon that is inducible by phosphate stress and controlled by phoB.

Recent work in this laboratory has shown that the gene coding for acetate kinase (ackA) in Sinorhizobium meliloti is up-regulated in response to phosphate limitation. Characterization of the region surrounding ackA revealed that it is adjacent to pta, which codes for phosphotransacetylase, and that these two genes are part of an operon composed of at least two additional genes in the following order: an open reading frame (orfA), pta, ackA, and the partial sequence of a gene with an inferred peptide that has a high degree of homology to enoyl-ACP reductase (fabI). Experiments combining enzyme assays, a chromosomal lacZ::ackA transcriptional fusion, complementation analysis with cosmid subclones, and the creation of mutations in pta and ackA all indicated that the orfA-pta-ackA-fabI genes are cotranscribed in response to phosphate starvation. Primer extension was used to map the position of the phosphate starvation-inducible transcriptional start sites upstream of orfA. The start sites were found to be preceded by a sequence having similarity to PHO boxes from other phosphate-regulated genes in S. meliloti and to the consensus PHO box in Escherichia coli. Introduction of a phoB mutation in the wild-type strain eliminated elevated levels of acetate kinase and phosphotransacetylase activities in response to phosphate limitation and also eliminated the phosphate stress-induced up-regulation of the ackA::lacZ fusion. Mutations in either ackA alone or both pta and ackA did not affect the nodulation or nitrogen fixation phenotype of S. meliloti.

Expression and regulation of phosphate stress inducible genes in Sinorhizobium meliloti.

Sinorhizobium meliloti 104A14 was mutated with transposon Tn5B22, which creates lacZ transcriptional fusions when inserted in the correct orientation relative to the promoter. This promoter reporter allowed us to identify six phosphate stress inducible (psi) genes in S. meliloti that are up-regulated in response to inorganic phosphate (Pi) starvation. The transposon and flanking DNA were cloned from each psi::Tn5B22 reporter mutant and the junction DNA sequenced. High identity/similarity of the inferred peptides with those in major data bases allowed identification of the following genes: dnaK, expC, pssB, ackA, vipC, and prkA. The prkA homolog was also found to be up-regulated in response to carbon starvation and when nitrate replaced ammonium as the nitrogen source. Through allele replacement techniques, PhoB- mutants were generated for the expC, ackA, vipC, and pssB reporter strains. Loss of a functional PhoB resulted in the absence of Pi-sensitive induction in all four genes. These experiments suggest the Pho regulon in S. meliloti includes genes that presumably are not directly linked to Pi acquisition or assimilation. The psi strains were tested for their symbiotic properties under growth conditions that were Pi-limiting or Pi-nonlimiting for the host plant. All were Nod+ and Fix+ except the reporter strain of dnaK transcription, which was less effective than the wild-type strain under both P treatments, indicating DnaK is required for optimum symbiotic function.

Cloning and characterization of a Rhizobium meliloti nonspecific acid phosphatase.

Nodulated legumes require high levels of phosphorus for optimal symbiotic performance. However, the basis for this elevated phosphorus requirement is poorly understood, and very little information regarding bacteroid phosphorus metabolism is available. To develop an understanding of the relative importance of organic and inorganic phosphorus sources for bacteroids, we investigated phosphatase activity in Rhizobium meliloti. An R. meliloti plasmid library clone that complemented an Escherichia coli phosphatase mutant was isolated, and the clone was sequenced. The complementing fragment contained a 337-amino-acid open reading frame that has a potential leader sequence and processing sites characteristic of periplasmic proteins. The phosphatase activity was located in the periplasm of R. meliloti and of E. coli containing the cloned gene. The subunit molecular mass of the cloned phosphatase was 33 kDa, and gel filtration indicated the active enzyme was a 66-kDa homodimer. Lack of substrate specificity suggests the cloned gene, napD, encodes a nonspecific acid phosphatase with a pH optimum of approximately 6.5. An R. meliloti napD transposon-insertion mutant was constructed, and its symbiotic phenotype was determined to be Fix+ regardless of the level of phosphorus provided to the host plant.

P Metabolism in the Bean-Rhizobium tropici Symbiosis.

Nodulated legumes require more P than legumes growing on mineral nitrogen, but little is known about the basis for the higher P requirement. Experiments were conducted to determine how Rhizobium tropici responds to P limitation and to understand how P is partitioned between the symbionts under conditions of adequate or limiting P. Free-living R. tropici responds to P stress by increasing P transport capacity and inducing both an acid and an alkaline phosphatase. This P-stress response occurs when the medium P concentration decreases below 1 [mu]M. Both P-stress-inducible phosphatases are found in bacteroids taken from plants growing with adequate P, suggesting that P levels in the symbiosome space is low enough to induce the expression of these enzymes. Bacteroid alkaline phosphatase-specific activity was highest during vegetative growth of the bean plant, but decreased approximately 75% during the host reproductive stages. In hydroponic experiments 32P-tracer studies showed that in vivo rates of P accumulation were significantly higher in bacteroids from P-limited plants compared with those from plants that had been supplied with adequate P. In contrast, label accumulation in leaves was greatest in plants grown with adequate P.

Regulation of the Phosphate Stress Response in Rhizobium meliloti by PhoB.

Alkaline phosphatase activity and phosphate transport rates in Rhizobium meliloti increased significantly when medium phosphate levels decreased to approximately 10 (mu)M. Both responses were abolished in a Tn5:: phoB mutant, but the mutant could be complemented by a plasmid that contained cloned R. meliloti phoB. The PhoB(sup-) mutant had a normal symbiosis phenotype under growth conditions that supplied either limiting or nonlimiting levels of phosphate to the host plant Medicago sativa, suggesting that induction of genes by PhoB was not required for normal symbiotic function.