Resuscitation of Viable But Not Culturable <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> 41 pRP4-<Emphasis Type="Italic">luc</Emphasis>: Effects of Oxygen and Host Plant

A plasmid-borne, firefly-derived, luciferase gene (luc) was inserted and stably inherited in Sinorhizobium meliloti 41 as a reporter gene. The strain obtained, S. meliloti 41/pRP4-luc, and its parental strain served as a model system for viable but not culturable (VBNC) resuscitation experiments in both in vitro and soil samples. Incubation under oxygen (O₂) concentrations varying from 1% to atmospheric levels did not result in resuscitation. A demonstration of recovery was attained through exposure to the appropriate concentrations of antibiotics, bacteriostatic chloramphenicol, and bactericidal ampicillin. The resuscitation ratio was 1 recovered VBNC cell in every 10⁵ 5-cyano-2,3-di-4-tolyl-tetrazolium chloride (CTC⁺) bacteria. Although isolated VBNC rhizobia were unable to nodulate Medicago sativa, which apparently did not enhance VBNC reversion, resuscitated bacteria maintained their symbiotic properties. Soil experiments showed that the lack of O₂ leads to onset of VBNC status as in liquid microcosm, but the number of recoverable and culturable cells decreased more drastically in soil.     

Introduction of a plant intron into the luciferase gene ofPhotinus pyralis

We describe a new luciferase reporter gene,luc ^INT, for early detection of luciferase activity inAgrobacterium transformation studies, and present improved techniques for the extraction of luciferase that decrease the time needed to quantitate luciferase activity. Theluc ^INT reporter gene combines the PIV2 intron fromGUS ^INT withluc ^*, the modified luciferase gene.luc ^INTis expressed in plant cells but not inAgrobacterium, allowing earlier detection of gene expression in the presence ofAgrobacterium during transformations in tobacco leaf discs. Stable expression levels ofluc ^INT andluc ^* in tobacco suspension cultures are compared for two different promoters. The nucleotide sequence data for the gene will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number U84006.     

Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters

We have tested the use of firefly luciferase for monitoring regulated symbiotic nitrogen fixation gene expression. Broad-host-range plasmids carrying translational fusions of Rhizobium meliloti nifH, fixA and nifA promoters were constructed. Despite low levels of promoter activity the absence of Escherichia coli endogenous luminescence and the high sensitivity of the bioluminescent assay for firefly luciferase allowed rapid screening for functional luciferase expression. Plasmids containing symbiotic promoter-luc fusions were established in R. meliloti. Luciferase activity was detected and measured in both vegetative and symbiotic cells giving comparable results with those obtained by beta-galactosidase assays. In addition, the luciferase assay was quicker, more sensitive and could be carried out with unrestricted cells. Furthermore, bioluminescence was high enough in alfalfa nodules containing nifH—luc fusion to be observed by a dark-adapted eye and photographed.     

Novel Cyanobacterial Biosensor for Detection of Herbicides

The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium, Synechocystis sp. strain PCC6803, was chromosomally marked with the luciferase gene luc (from the firefly Photinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of the luc gene during growth of Synechocystis sp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.     

Cloning and Identification of Conjugative Transfer Origins in the Rhizobium meliloti Genome

A simple approach was used to identify Rhizobium meliloti DNA regions with the ability to convert a nontransmissible vector into a mobilizable plasmid, i.e., to contain origins of conjugative transfer (oriT, mob). RecA-defective R. meliloti merodiploid populations, where each individual contained a hybrid cosmid from an R. meliloti GR4 gene library, were used as donors en masse in conjugation with another R. meliloti recipient strain, selecting transconjugants for vector-encoded antibiotic resistance. Restriction analysis of cosmids isolated from individual transconjugants resulted in the identification of 11 nonoverlapping DNA regions containing potential oriTs. Individual hybrid cosmids were confirmed to be mobilized from the original recA donors at frequencies ranging from 10⁻² to 10⁻⁵ per recipient cell. DNA hybridization experiments showed that seven mob DNA regions correspond to plasmid replicons: four on symbiotic megaplasmid 1 (pSym1), one on pSym2, and another two on each of the two cryptic plasmids harbored by R. meliloti GR4. Another three mob clones could not be located to any plasmid and were therefore preliminarily assigned to the chromosome. With this strategy, we were able to characterize the oriT of the conjugative plasmid pRmeGR4a, which confirmed the reliability of the approach to select for oriTs. Moreover, transfer of the 11 mob cosmids from R. meliloti into Escherichia coli occurred at frequencies as high as 10⁻¹, demonstrating the R. meliloti gene transfer capacity is not limited to the family Rhizobiaceae. Our results show that the R. meliloti genome contains multiple oriTs that allow efficient DNA mobilization to rhizobia as well as to phylogenetically distant gram-negative bacteria.     

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO₂ evolution or as O₂ uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 10⁷ bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO₂ production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO₂, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.     

An improved bioluminescence‐based signaling assay for odor sensing with a yeast expressing a chimeric olfactory receptor

The goal of this work was to improve the bioluminescence‐based signaling assay system to create a practical application of a biomimetic odor sensor using an engineered yeast‐expressing olfactory receptors (ORs). Using the yeast endogenous pheromone receptor (Ste2p) as a model GPCR, we determined the suitable promoters for the firefly luciferase (luc) reporter and GPCR genes. Additionally, we deleted some genes to further improve the sensitivity of the luc reporter assay. By replacing the endogenous yeast G‐protein α‐subunit (Gpa1p) with the olfactory‐specific Gα_olf, the optimized yeast strain successfully transduced signal through both OR and yeast Ste2p. Our results will assist the development of a bioluminescence‐based odor‐sensing system using OR‐expressing yeast. Biotechnol. Bioeng. 2012; 109: 3143–3151. © 2012 Wiley Periodicals, Inc.     

Hydrogen Evolution from Alfalfa and Clover Nodules and Hydrogen Uptake by Free-Living Rhizobium meliloti

A series of Rhizobium meliloti and Rhizobium trifolii strains were used as inocula for alfalfa and clover, respectively, grown under bacteriologically controlled conditions. Replicate samples of nodules formed by each strain were assayed for rates of H₂ evolution in air, rates of H₂ evolution under Ar and O₂, and rates of C₂H₂ reduction. Nodules formed by all strains of R. meliloti and R. trifolii on their respective hosts lost at least 17% of the electron flow through nitrogenase as evolved H₂. The mean loss from alfalfa nodules formed by 19 R. meliloti strains was 25%, and the mean loss from clover nodules formed by seven R. trifolii strains was 35%. R. meliloti and R. trifolii strains also were cultured under conditions that were previously established for derepression of hydrogenase synthesis. Only strains 102F65 and 102F51 of R. meliloti showed measurable activity under free-living conditions. Bacteroids from nodules formed by the two strains showing hydrogenase activity under free-living conditions also oxidized H₂ at low rates. The specific activity of hydrogenase in bacteroids formed by either strain 102F65 or strain 102F51 of R. meliloti was less than 0.1% of the specific activity of the hydrogenase system in bacteroids formed by H₂ uptake-positive Rhizobium japonicum USDA 110, which has been investigated previously. R. meliloti and R. trifolii strains tested possessed insufficient hydrogenase to recycle a substantial proportion of the H₂ evolved from the nitrogenase reaction in nodules of their hosts. Additional research is needed, therefore, to develop strains of R. meliloti and R. trifolii that possess an adequate H₂-recycling system.     

A quick and efficient approach for gene silencing by using triple putative microRNA-based short hairpin RNAs

The RNA interference (RNAi) technique has been widely used in gene function studies. It is typical to screen for effective siRNAs by knocking down targeted genes since a single gene can be suppressed by several siRNAs to varying degrees. The miRNA-based short hairpin RNA (shRNA) is a natural inducer of RNAi and has been used in siRNA expression strategies. We investigated the potential application of multiple putative microRNA-based shRNAs for gene silencing and studied the inhibition efficiency of exogenous GFP and firefly luciferase (luc) by triple human mir155-based shRNA expression vectors. A total of three candidate siRNA sequences targeted against GFP or luc were selected based on an online prediction program. Single and triple miRNA-155-based shRNAs targeted against GFP or luc were transfected into HEK293 cells mediated by the pcDNA₃ vector with an RNA polymerase II-type CMV (cytomegalovirus) promoter. Comparisons with negative control shRNAs revealed that GFP levels were markedly reduced by the triple miRNA-155-based GFP shRNA by fluorescent microscopy. Consistent results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-based GFP shRNA significantly suppressed GFP expression (P < 0.01), without significant differences from the most effective single miRNA-155-based GFP shRNA (P > 0.05). Results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-based luc shRNA significantly suppressed luc expression as the most effective single miRNA-155-based luc shRNA (P < 0.05). These studies demonstrated the gene silencing efficiency mediated by the triple putative miRNA-155-based shRNAs. This suggested that multiple miRNA-based shRNAs are quick and valuable strategies for gene silencing.     

In Vivo Bioluminescence Imaging for the Study of Intestinal Colonization by Escherichia coli in Mice

Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R² = 0.98) or transcutaneously in the abdominal region of whole animals (R² = 0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2ΩP_amiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.Among the wide variety of bacteria that colonize the gastrointestinal tracts of mammals, Escherichia coli is the most abundant facultative anaerobe of the human intestinal microflora. Aside from being part of the normal flora, E. coli is also a versatile organism capable of causing a variety of intestinal and extraintestinal diseases (18). The mechanisms that allow commensal E. coli to colonize the intestine and survive successfully in this niche remain poorly characterized. Conventional mice display natural resistance to colonization by commensal E. coli, but oral administration of streptomycin, which alters the intestinal microflora, allows colonization of the mouse large intestine by this species (25). The streptomycin-treated mouse model has been used extensively to study the factors of gram-negative bacteria implicated in the intestinal colonization process. However, this model is limited to the viable plate counts of bacteria in the feces and misses some critical information, such as the kinetics of colonization, the fate of the bacterial cells across the digestive tract, and the site of colonization. A better understanding of colonization would be facilitated by direct in vivo follow-up of this process.Bioluminescence imaging (BLI) technology is emerging as a powerful tool for the study of a wide range of biological processes in live animals, including real-time monitoring of infections (16). Bioluminescence systems emit visible light due to the luciferase-mediated oxidation of a luciferin substrate. A variety of luciferin-luciferase systems with different peak emissions have been identified in nature from numerous species (14). The luciferase of the soil bacterium Photorhabdus luminescens has been expressed successfully in gram-negative and gram-positive bacteria. This system emits blue-green light, with an emission maximum of approximately 490 nm, and does not require the addition of an exogenous substrate since the luciferase operon contains the genes required for synthesis of the substrate. Therefore, this luciferase has been used extensively to monitor bacterial infections in the living mouse. One of the first investigations with Salmonella enterica serovar Typhimurium transformed with the lux operon of P. luminescens evaluated the tissue distribution and the virulence of various S. Typhimurium strains (9). Subsequent modification of the lux operon led to the generation of highly bioluminescent Staphylococcus aureus and allowed the monitoring of infections due to this species in living mice (11). The modified lux operon was engineered into a lux-kan transposon cassette for chromosomal integration in gram-positive bacteria, such as S. aureus, Streptococcus pneumoniae, group A Streptococcus, and Listeria monocytogenes (16). Replication of L. monocytogenes in the lumen of the gall bladder was demonstrated for the first time by BLI (13).Bioluminescent E. coli was used in the neutropenic mouse thigh model of infection to evaluate the in vivo activity of antimicrobial agents (29). Bioluminescence was as indicative of therapeutic efficacy as CFU counts but, in addition, allowed real-time monitoring of the infection and of treatment efficacy in the same animal; however, only short-term monitoring (12 h) could be performed.Because luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. After oral administration of bioluminescent Salmonella to susceptible mice, the bioluminescent signal recorded in the abdominal region was greatly enhanced after air exposure (9). It was therefore assumed that direct bioluminescence imaging of intestine-colonizing microorganisms would not be optimal unless oxygen was provided exogenously or as the result of the close interaction between cells and the bacteria (9). However, the bacterial luciferase was used to trace in real time the colonization dynamics by Citrobacter rodentium of the gastrointestinal tracts of living animals, demonstrating that the gut represents a semianaerobic environment that allows the study of bacterial colonization by BLI (33).Factors essential for colonization are best studied in cocolonization experiments (7, 17). There are several luciferases with distinct emission spectra (34) that could be used in competition experiments to trace simultaneously two bacterial populations in the same living animal. However, in order not to impose additional and different metabolic burdens on the bacteria under study, the exogenous luciferases ideally have to be similar to allow comparison between strains. The thermostable luciferase variants PpyRE-TS and PpyGR-TS, derived from wild-type luciferase from the North American firefly Photinus pyralis, emit red (612 nm) and green (552 nm) light, respectively, at 37°C and are encoded by single genes of 1,650 bp, differing by only 9 bp (4). Bioluminescence color is determined by the Ser₂₈₄Thr (PpyRE-TS) and Val₂₄₁Ile, Gly₂₄₆Ala, and Phe₂₅₀Ser (PpyGR-TS) amino acid changes (5, 34). By use of optical filters, the emission spectra are readily distinguishable (4, 5). Five additional mutations provide enhanced thermostability at 37°C (4), improving the compatibility of the enzymes with bacterial culture conditions and BLI in animal models.While the luciferase mutants and all firefly luciferases use as substrates firefly luciferin and ATP to produce light, in vivo imaging is commonly performed with endogenous ATP and requires only exogenous administration of the luciferase substrate.The aim of this study was to develop a dynamic mouse model using in vivo bioluminescence imaging systems to monitor bacterial colonization in situ and in real time in whole living animals. Various strains of E. coli harboring the genes for the bacterial luciferase from P. luminescens or the firefly luciferase mutants (PpyRE-TS and PpyGR-TS) from P. pyralis have been engineered and used to follow bacterial intestinal colonization in mice. BLI was found to be well adapted to compare the intestine-colonizing capacities of various E. coli strains and to monitor cocolonization in vivo by use of dual bioluminescence emission.     

Characterization of two bioluminescentRhizobium meliloti strains constructed for field releases

The deliberate release of genetically engineered microorganisms requires a thorough characterization of the microbes in question. For the two bioluminescentRhizobium meliloti strains, L1 and L33 [Selbitschka et al. (1992) Mol Ecol 1: 9–19; Selbitschka et al. (1995) FEMS Microbiol Ecol 16:223–232], designated for field release, the sites of genetic modifications in the chromosomes were sequenced from amplified genomic DNA. This indicated no unexpected alterations in the nucleotide sequence. The bioluminescent phenotype was stably inherited over more than 100 generations in liquid cultures. The presence of the luciferase gene in both strains did not have secondary effects on a variety of metabolic pathways as assessed by the Biolog GN system. A specific polymerase chain reaction amplification, based on the chromosomal insertion site of theluc cassette, allowed the discrimination between the two strains and thus simplifies monitoring. The RecA-deficient strain L1 showed a strongly (more than 90%) reduced ability to nodulate alfalfa in competition with its parent strainR. meliloti 2011 and its RecA⁺ counterpart L33.     

Alternative Luciferase for Monitoring Bacterial Cells under Adverse Conditions

The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.     

Effect of heterologous bacteria and combined nitrogen on the adsorption ofRhizobium meliloti to lucerne seedling roots

Summary The binding ofRhizobium meliloti strains A₂ (effective) and V₆ (ineffective),Agrobacterium tumefaciens strain B₆S₃ andR. trifolii strain TL₅ to lucerne seedling roots was studied by using¹⁴C or³H-labelled bacteria. When added singly or in combination with the heterologous bacteria, the number of A₂ cells attached to the roots was significantly less than the number of B₆S₃ or TL₅ cells. However, the presence of the heterologous bacteria did not decrease the proportion of A₂ cells added in the inoculum that bind to the roots, suggesting thatR. meliloti is attached to specific sites. In fact, the same number of A₂ or V₆ cells bind to the roots and in mixed inoculation the 2 strains share equally the binding sites. When added to the seedlings growth medium NO ₃ ^– at 5 or 16 mM significantly decreased the number of A₂ cells adhering to lucerne seedling roots. The results suggest that the lectin-recognition hypothesis is probably involved in the attachment ofR. meliloti to lucerne seedling roots.Contribution No. 252 Station de recherches, Agriculture Canada     

Characterization and isolation of a T-DNA tagged banana promoter active during in vitro culture and low temperature stress

Background  

Next-generation transgenic plants will require a more precise regulation of transgene expression, preferably under the control of native promoters. A genome-wide T-DNA tagging strategy was therefore performed for the identification and characterization of novel banana promoters. Embryogenic cell suspensions of a plantain-type banana were transformed with a promoterless, codon-optimized luciferase (luc ⁺) gene and low temperature-responsive luciferase activation was monitored in real time.     

Crystal structure of native and a mutant of Lampyris turkestanicus luciferase implicate in bioluminescence color shift

Firefly bioluminescence reaction in the presence of Mg^2 +, ATP and molecular oxygen is carried out by luciferase. The luciferase structure alterations or modifications of assay conditions determine the bioluminescence color of firefly luciferase. Among different beetle luciferases, Phrixothrix hirtus railroad worm emits either yellow or red bioluminescence color. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional arginine residue at 353 that is absent in other firefly luciferases. It was reported that insertion of Arg in an important flexible loop350–359 showed changes in bioluminescence color from green to red and the optimum temperature activity was also increased. To explain the color tuning mechanism of firefly luciferase, the structure of native and a mutant (E354R/356R/H431Y) of Lampyris turkestanicus luciferase is determined at 2.7 Å and 2.2 Å resolutions, respectively. The comparison of structure of both types of Lampyris turkestanicus luciferases reveals that the conformation of this flexible loop is significantly changed by addition of two Arg in this region. Moreover, its surface accessibility is affected considerably and some ionic bonds are made by addition of two positive charge residues. Furthermore, we noticed that the hydrogen bonding pattern of His431 with the flexible loop is changed by replacing this residue with Tyr at this position. Juxtaposition of a flexible loop (residues 351–359) in firefly luciferase and corresponding ionic and hydrogen bonds are essential for color emission.     

Congo Red Absorption by Rhizobium leguminosarum

Congo red absorption is generally considered a contraindication of Rhizobium. However, R. leguminosarum takes up the dye on yeast extract-mannitol agar. The uptake of congo red varies among strains of R. leguminosarum, as shown elsewhere with strains of R. trifolii and R. meliloti. Congo red absorption does not distinguish rhizobia from other bacteria, but may be useful as a strain marker.     

Comparison of DNA Polymerase of Rhizobium meliloti and Alfalfa Bacteroids

DNA dependent-DNA polymerase activity was established and partially purified from extracts of cultured Rhizobium meliloti, F-28, and nodule bacteroids (R. meliloti, F-28) of alfalfa plants (Medicago sativa). Polymerase activity in the partially purified fractions showed characteristic dependence on Mg²⁺, DNA, and a full complement of deoxyribonucleoside triphosphates. DNase activity, preference of “activated” double strand DNA, and inhibition by p-chloromercuribenzoate and MnCl₂ were responses common to both systems. The two systems however did exhibit some differences in pH, Mg²⁺, and primer optima. Polymerase activity in crude extracts of the cultured bacteria was more stable and had 10- to 18-fold greater specific activity than the bacteroid extracts. Preliminary measurements of specific DNA polymerase activity in crude extracts of cultured Rhizobium japonicum were not significantly higher than that in the crude extracts of soybean nodule bacteroids. A possible correlation between DNA synthesis and the successful establishment of rhizobia-legume symbiosis is discussed.     

Quantitation of Adsorption of Rhizobia in Low Numbers to Small Legume Roots

Bacteria adsorbed in low numbers to alfalfa or clover root surfaces were counted after incubation of seedlings in mineral solution with very dilute inocula (less than 10⁵ bacteria per ml) of an antibiotic-resistant strain under defined conditions. After specified washing, bacteria which remained adsorbed to roots were selectively quantitated by culturing the roots embedded in yeast extract-mannitol-antibiotic agar and counting the microcolonies along the root surface; the range was from about 1 bacterium per root (estimated as the most probable number) to 50 bacteria per cm of root length (by direct counting). This simple procedure can be used with any pair of small-rooted plant and antibiotic-resistant bacterium, requires bacterial concentrations comparable to those frequently found in soils, and yields macroscopic localization and distribution data for adsorbed bacteria over the root surface. The number of adsorbed bacteria was proportional to the size of the inoculum. One of every four Rhizobium meliloti cells adsorbed in very low numbers to alfalfa roots resulted in the formation of a nodule. Overall adsorption of various symbiotic and nonsymbiotic bacterial strains to alfalfa and clover roots did not reflect the specificities of these legumes for their respective microsymbionts, R. meliloti and R. trifolii.     

Quantitative and Qualitative Analysis of Bacteria in Er(III) Solution by Thin-Film Magnetopheresis

Magnetic deposition, quantitation, and identification of bacteria reacting with the paramagnetic trivalent lanthanide ion, Er³⁺, was evaluated. The magnetic deposition method was dubbed thin-film magnetopheresis. The optimization of the magnetic deposition protocol was accomplished with Escherichia coli as a model organism in 150 mM NaCl and 5 mM ErCl₃ solution. Three gram-positive bacteria, Staphylococcus epidermidis, Staphylococcus saprophyticus, and Enterococcus faecalis, and four gram-negative bacteria, E. coli, Pseudomonas aeruginosa, Proteus mirabilis, and Klebsiella pneumoniae, were subsequently investigated. Quantitative analysis consisted of the microscopic cell count and a scattered-light scanning of the magnetically deposited material aided by the computer data acquisition system. Qualitative analysis consisted of Gram stain differentiation and fluorescein isothiocyanate staining in combination with selected antisera against specific types of bacteria on the solid substrate. The magnetic deposition protocol allowed quantitative detection of E. coli down to the concentration of 10⁵ CFU ml^-1, significant in clinical diagnosis applications such as urinary tract infections. Er³⁺ did not interfere with the typical appearance of the Gram-stained bacteria nor with the antigen recognition by the antibody in the immunohistological evaluations. Indirect antiserum-fluorescein isothiocyanate labelling correctly revealed the presence of E. faecalis and P. aeruginosa in the magnetically deposited material obtained from the mixture of these two bacterial species. On average, the reaction of gram-positive organisms was significantly stronger to the magnetic field in the presence of Er³⁺ than the reaction of gram-negative organisms. The thin-film magnetophoresis offers promise as a rapid method for quantitative and qualitative analysis of bacteria in solutions such as urine or environmental water.     

Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and “Sarcina lutea” did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and “S. lutea” were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.