Studies on the Physiology of Nodule Formation: VIII. The Influence of the Size of the Rhizosphere Population of Nodule Bacteria on Root Hair Infection in Clover

With an adequate inoculum the number of infected root hairsin three species of clover (Trifolium parviflorum, T. patensand T. glomeratum) increased exponentially with time in twophases; the increase was rapid during the first 8-10 days beforenodulation begins, but slower afterwards. T. parviflorum hadmost infections and T. glomeratum the fewest. Experiments on varying inoculum size, using an avirulent mutanstrain of Rhizobhtm trifolii as diluent, showed that root-hairinfection was differentially limited by inoculum size duringthe two phases. Infection in all three species was about doubledby doubling the density of the virulent bacteria in the rhizospherebefore nodulation begins. After nodulation bacterial densityhad to be increased much more than twice to double the numberof infections. This increase in the infecting population wasinversely related to the numbers of infections formed on thethree host species. Early infection and nodulation were promoted by high bacterialdensity in the rhizosphere.     

Measurement of virulence of aeromonads using a suckling mouse model of infection

Abstract Virulent and avirulent strains of Aeromonas spp. were identified and virulence quantified using an animal model. Virulence was measured by determining a 50% lethal dose (LD₅₀) 43 h after oral administration of live bacteria. The LD₅₀ of virulent Aeromonas isolates ranged from log₁₀ 7.53 (mean) organisms to log₁₀ 8.88 (mean). Some isolates were avirulent in this model. Detection of cytotoxic activity in culture supernatants correlated with virulence (Fisher exact test, P = 0.0029). There was no correlation between LD₅₀ and the source of the isolate, β-haemolysis or lipopolysaccharide (LPS) banding profile on SDS-PAGE. In this animal model, virulence was multifactorial in that: (i) bacterial multiplication in the gut was associated with fatal infection; (ii) the increase in bacterial numbers in the gut of mice administered a lethal dose of bacteria was accompanied by accumulation of fluid; and (iii) there was evidence of extraintestinal spread of infection. Protection of suckling mice by rabbit antiserum to Aeromonas cell envelopes was observed.     

Effect of the influenza A virus on the sensitivity of mice to infection caused by Streptococcus group B

Study of the capacity of group B streptococci for causing the development of infection in mice has revealed the virulence of the cultures for mice to be determined by the serovar of the streptococcus, the infective dose, and the amount of type-specific polysaccharide. Under the conditions of mixed viral-bacterial infection, influenza A virus was shown to influence the development of bacterial infection in the animals in two ways: to increase the virulence of an avirulent strain and to decrease the pathogenicity of a virulent one in streptococcal monoinfections. Simultaneously with viral infection, the stimulation of the multiplication of an avirulent strain in the lungs of mice was observed, while in the control groups of the animals the elimination of bacteria from the lungs was registered. No additional accumulation of the infective virus in the lungs of mice in the presence of streptococci was found.     

Competitiveness of Rhizobium leguminosarum bv. trifolii strains in mixed inoculation of clover (Trifolium pratense)

Rhizobium leguminosarum by. trifolii (Rlt) establishes beneficial root nodule symbiosis with clover. Twenty Rlt strains differentially marked with antibiotic-resistance markers were investigated in terms of their competitiveness and plant growth promotion in mixed inoculation of clover in laboratory experiments. The results showed that the studied strains essentially differed in competition ability. These differences seem not to be dependent on bacterial multiplication in the vicinity of roots, but rather on complex physiological traits that affect competitiveness. The most remarkable result of this study is that almost half of the total number of the sampled nodules was colonized by more than one strain. The data suggest that multi-strain model of nodule colonization is common in Rhizobium-legume symbiosis and reflects the diversity ofrhizobial population living in the rhizosphere.     

Comparative characteristics of cell-free filtrates of Shigella flexneri of different virulence]

It was shown for the first time that the virulent Sh. flexneri strain grown on Luria broth differed from the avirulent one by the yield of readily released surface-located complexes--lipopolysaccharide (determined by rhamnose) and protein into the filtrate. There was no distinct correlation between the strain virulence and the content of rhamnose-determined lipopolysaccharide in the filtrate; growing bacteria in the presence of Ca and Mg ions had no significant influence on the lipopolysaccharide release into the filtrate. Protein release into the cell-free filtrate was thrice that in the virulent shigella strain than in the avirulent one. When bacteria were grown in the presence of Ca ions protein release from the virulent strain increased 1.5-fold and changed but little in the avirulent culture. Cell-free filtrates of the virulent strain produced toxic action on L tissue culture cells; in conjunctival infection of guinea pigs they caused some reduction of the LD50 of the virulent strain and sharply aggravated the course of the infectious process. Heating of the filtrate at 100 degrees C for 15 min decreased their toxic action on L cells. The data obtained indicated that the active biological factor revealed in the virulent strain of Sh. flexneri was protein or its derivative.     

Effects of Gypsophila saponins on bacterial growth kinetics and on selection of subterranean clover rhizosphere bacteria

Plant secondary metabolites, such as saponins, have a considerable impact in agriculture because of their allelopathic effects. They also affect the growth of soil microorganisms, especially fungi. We investigated the influence of saponins on rhizosphere bacteria in vitro and in soil conditions. The effects of gypsophila saponins on the growth kinetics of rhizosphere bacteria were studied by monitoring the absorbance of the cultures in microtiter plates. Gypsophila saponins (1%) increased the lag phase of bacterial growth. The impact of gypsophila saponins on subterranean clover rhizosphere was also investigated in a pot experiment. The addition of gypsophila saponins did not modify clover biomass but significantly increased (twofold with 1% saponins) the weight of adhering soil. The number of culturable heterotrophic bacteria of the clover rhizosphere was not affected by the addition of gypsophila saponins. Nevertheless, the phenotypical characterization of the dominant Gram-negative strains of the clover rhizosphere, using the Biolog system, showed qualitative and quantitative differences induced by 1% saponins. With the addition of saponins, the populations of Chryseomonas spp. and Acinetobacter spp., the two dominant culturable genera of control clover, were no longer detectable or were significantly decreased, while that of Aquaspirillum dispar increased and Aquaspirillum spp. became the major genus. Aquaspirillum dispar and Aquaspirillum spp. were also the dominant rhizosphere bacteria of Gypsophila paniculata, which greatly accumulates these saponins in its roots. These results suggest that saponins may control rhizosphere bacteria in soil through rhizodeposition mechanisms.     

Population genetics of rhizobia: construction and analysis of an "Infection and Release" model

A mathematical model is created to assess the inputs of sym gene transfer of in planta multiplication and of interstrain competition into dynamics of the rhizobia populations. Their microevolution is presented as a series of the "infection and release" cycles; each cycle includes transfer of sym genes from virulent initial symbionts to avirulent local bacteria yielding the virulent novel symbionts; competition between initial symbionts and novel symbionts for the host nodulation; multiplication of initial symbionts and novel symbionts in planta and their release into soil; competition between the released novel symbionts and resident local bacteria for ex planta survival. A recurrent equation is created to determine the number of novel symbionts at each cycle of evolution of the closed bacteria-plant system. Its analysis demonstrates that under certain, really allowable values of the introduced parameters two major effects may occur: (a) rapid multiplication of novel symbionts arisen from sym gene transfer; and (b) increase of frequency of rare local bacteria genotypes after acquisition of virulence. Multiplication of very rare strains (p<10(-19)) in the plant-associated bacteria population is possible at certain parameters of the system. Variation of the sizes of bacteria populations and of the parameters for interstrain competition may influence the evolutionary rate of the bacteria population. The "infection and release" model represents a selective mechanism which may be responsible for a high taxonomic diversity of rhizobia and for a panmictic structure of their populations.     

Ligionella pneumophila: Avirulent to virulent conversion through passage in cultured human embryonic lung fibroblasts

Legionella pneumophila isolated in guinea pigs from human lung tissues was highly virulent as determined by its infectivity and lethality in guinea pigs. Repeated passages of the bacteria on agar media resulted in the loss of virulence in guinea pigs. Virulence, however, was restored by cultivating the avirulent bacteria in cell cultures of human embryonic lung fibroblasts. Death of the host animals was the result of infection; no lethal toxin was detected in the cultural filtrate. These findings indicate that the virulent form ofL. pneumophilia is capable of surviving inside the host cells either through its endogenous resistance to environmental factors within the host cells or by host cell selection. Intracellular multiplication of the virulent bacteria followed by destruction of host cells appears to be an important pathogenic mechanism of Legionnaires' disease.     

Hsp70 protects macrophages infected with Salmonella choleraesuis against TNF-α-induced cell death

Hsp70 plays an important role in cytoprotection against tumor necrosis factor (TNF) α-mediated cytotoxicity. To investigate the role of Hsp70 in cytoprotein during Salmonella infection, we examined endogenous Hsp70 induction and TNF-α production in a monocyte/macrophage line, J774A.1, after infection with a virulent strain of Salm.choleraesuis RF-1 carrying a 50 kb virulent plasmid or the plasmid-cured avirulent strain 31N-1. Intracellular bacteria progressively increased in J774A.1 cells phagocytosing avirulent 31N-1 bacteria, whereas such progressive growth was not evident in J774A.1 cells phagocytosing avirulent 31N-1 bacteria. On the contrary, J774A.1 cells infected with virulent RF-1 bacteria expressed less Hsp70 than those infected with avirulent 31N-1 bacteria. The level of TNF-α production by J774A.1 infected with virulent RF-1 was much the same as that by J774A.1 infected with avirulent 31N-1. J774A.1 infected with virulent RF-1 died spontaneously; death was inhibited by the addition of anti-TNF-α mAb. Although the frequency of dead J774A.1 with hypodiploid DNA content increased only marginally after infection with avirulent 31N-1, treatment with Hsp70 anti-sense oligonucleotide resulted in a dramatic increase of dead cells in the infected macrophages. Taken together, these results suggest that Hsp70 induced macrophages plays an important role in host defense against Salmonella infection by protecting the macrophages against TNF α-induced cell death. Furthermore, cell death due to impaired endogenous Hsp synthesis in the phagocytes implies a novel pathogenic mechanism for virulence of Salm. choleraesuis RF-1.     

Characteristics of the virulence of a strain of S. typhimurium and its glycerin mutant in a HEp-2 cell line model]

Sensitivity of the HEp-2 cell culture to the infection with the virulent salmonella strains was shown; active penetration and multiplication of bacteria in the monolayer cells indicated this. Gly 90, a glycerine mutant defective by glycerokinase, obtained under the action of ethylmethanesulphonate, displayed a distinct difference from the initial virulent strains of salmonella by decreased invasive properties and the absence of any capacity to multiplication in the epithelial cells. The data on the avirulence of the glycerine mutant obtained on a model in vitro confirmed the observations carried out in vivo in intraperitoneal infection of albino mice (LD50 = 1-10(7) cells) and in keratoconjunctival infection of guinea pigs.     

Genetical changes in rhizobium bacteria and in their bacteriophages during coexistence

Summary Three systems, each consisting of a virulent phage and a susceptible bacterium, were incubated for 24 weeks in liquid culture and the processes of interaction and genetical change in both partners investigated. The initial stage of interaction in all systems was identical; all susceptible bacterial cells were lysed and only cells that originally were resistant or mutated to resistance, remained alive. In two systems the multiplication of resistant cells was not restricted by large concentration of phage; in the third there was no bacterial multiplication. Phage concentration was maintained at a high titre for the duration of the experiment by the presence of the resistant mutant bacteria. Phage resistant mutants were genetically less stable than the parent forms; some also had changed colony morphology and symbiotic properties. Mutations in these different characteristics were independent. In two systems virulent phage mutated into temperate form; hence some of the phage-resistant mutant cells were genetically suitable for establishment of lysogeny.     

Interactions Between Macrophages of Guinea Pigs and Salmonellae I. Fate of Salmonella typhimurium Within Macrophages of Normal Guinea Pigs

A suspended cell culture procedure was described for the cultivation of guinea pig macrophages infected with Salmonella typhimurium. The fate of the intracellular bacteria was assessed by quantitative recovery of viable bacteria with 0.5% solution of sodium desoxycholate. Two strains of S. typhimurium with different degrees of virulence for mice were compared. There was an initial destruction of intracellular bacteria of both strains; however, the extent of this destruction differed. Approximately 1% of the avirulent bacteria initially phagocytized survived at the end of 4 hr, whereas approximately 8% of the virulent bacteria survived at the end of 3 hr. After this initial killing, the intracellular bacteria began to multiply at a logarithmic rate between 3 and 21 hr after phagocytosis, and then a stationary phase was attained. The rate of this multiplication was comparable for both strains.     

Confocal microscopy reveals in planta dynamic interactions between pathogenic,avirulent and non‐pathogenic Pseudomonas syringae strains

Recent advances in genomics and single‐cell analysis have demonstrated the extraordinary complexity reached by microbial populations within their hosts. Communities range from complex multispecies groups to homogeneous populations differentiating into lineages through genetic or non‐genetic mechanisms. Diversity within bacterial populations is recognized as a key driver of the evolution of animal pathogens. In plants, however, little is known about how interactions between different pathogenic and non‐pathogenic variants within the host impact on defence responses, or how the presence within a mixture may affect the development or the fate of each variant. Using confocal fluorescence microscopy, we analysed the colonization of the plant apoplast by individual virulence variants of Pseudomonas syringae within mixed populations. We found that non‐pathogenic variants can proliferate and even spread beyond the inoculated area to neighbouring tissues when in close proximity to pathogenic bacteria. The high bacterial concentrations reached at natural entry points promote such interactions during the infection process. We also found that a diversity of interactions take place at a cellular level between virulent and avirulent variants, ranging from dominant negative effects on proliferation of virulent bacteria to in trans suppression of defences triggered by avirulent bacteria. Our results illustrate the spatial dynamics and complexity of the interactions found within mixed infections, and their potential impact on pathogen evolution.     

Stuides on the Physiology of Nodle Formation: V. Further Experiments on the Stimulating and Inhibitory Effects of Roots Secretions

The influence of root secretions on nodulation of clover andlucerne in agar culture is examined using a technique of preplanting.Root secretions are shown to have two effects: nodulation ofthe seedling is induced at an earlier stage (stimulation), buttakes place at a reduced rate (inhibition). These effects donot depend upon the presence of the bacteria in the preplantingphase of an experiment. The species used differed both in productionof and reaction to secretions giving these effects. As a donatingplant lucerne is more active than clover, and selected earlynodulating lines of clover are more active than late lines.Stimulation of lucerne is promoted by lower levels of secretionthan clover and early nodulating lines are less affected thanlate lines. The influence of secretions on nodulation rate isgreater for lucerne than for clover at lower levels and doesnot differ between early and late nodulating lines. In contrastto lucerne, inhibition of nodulation on clover is proportionalto duration of the preplanting suggesting that secretions accumulateduring growth. The hypothesis is put forward that the stimulating reactionrepresents an increase in the availability of the infectivefoci already present on the root and that the inhibiting reactionrepresents a reduction in the number of these foci.     

Type IV Secretion-Dependent Activation of Host MAP Kinases Induces an Increased Proinflammatory Cytokine Response to Legionella pneumophila

The immune system must discriminate between pathogenic and nonpathogenic microbes in order to initiate an appropriate response. Toll-like receptors (TLRs) detect microbial components common to both pathogenic and nonpathogenic bacteria, whereas Nod-like receptors (NLRs) sense microbial components introduced into the host cytosol by the specialized secretion systems or pore-forming toxins of bacterial pathogens. The host signaling pathways that respond to bacterial secretion systems remain poorly understood. Infection with the pathogen Legionella pneumophila, which utilizes a type IV secretion system (T4SS), induced an increased proinflammatory cytokine response compared to avirulent bacteria in which the T4SS was inactivated. This enhanced response involved NF-κB activation by TLR signaling as well as Nod1 and Nod2 detection of type IV secretion. Furthermore, a TLR- and RIP2-independent pathway leading to p38 and SAPK/JNK MAPK activation was found to play an equally important role in the host response to virulent L. pneumophila. Activation of this MAPK pathway was T4SS-dependent and coordinated with TLR signaling to mount a robust proinflammatory cytokine response to virulent L. pneumophila. These findings define a previously uncharacterized host response to bacterial type IV secretion that activates MAPK signaling and demonstrate that coincident detection of multiple bacterial components enables immune discrimination between virulent and avirulent bacteria.     

Virulent and Avirulent Strains of Babesia bovis: The Relationship Between Parasite Protease Content and Pathophysiological Effect of the Strain

A virulent strain of Babesia bovis (“L” strain) was rendered avirulent by irradiation with 35 krads with a γ source. Another virulent strain of B. bovis (“C” strain) was made avirulent by rapid blood passage through 12 splenectomised calves. Both the parent virulent and their respective avirulent strains were injected into susceptible cattle. A nonfatal disease was observed in those intact cattle that had avirulent parasites; however, a fatal disease was produced in those animals that had received virulent parasites and in splenectomised calves that had received avirulent parasites. Blood kinin levels rose and plasma kininogen levels fell significantly in those animals infected with both virulent strains. Nonsignificant changes occurred with these parameters in animals infected with avirulent parasites. Preparations of disrupted parasites were obtained from the four parasite populations. Both virulent strains contained high levels of protease. The avirulent forms contained insignificant amounts. As parasite doubling times and maximum parasitaemias were the same for all four parasite populations, we conclude that these enzymes are not obligatory for parasite multiplication in the vertebrate host. Their role in producing pathological changes in the host is discussed.     

A pathogen-inducible patatin-like lipid acyl hydrolase facilitates fungal and bacterial host colonization in Arabidopsis

Genes and proteins related to patatin, the major storage protein of potato tubers, have been identified in many plant species and shown to be induced by a variety of environmental stresses. The Arabidopsis patatin-like gene family (PLPs) comprises nine members, two of which (PLP2 and PLP7) are strongly induced in leaves challenged with fungal and bacterial pathogens. Here we show that accumulation of PLP2 protein in response to Botrytis cinerea or Pseudomonas syringae pv. tomato (avrRpt2) is dependent on jasmonic acid and ethylene signaling, but is not dependent on salicylic acid. Expression of a PLP2-green fluorescent protein (GFP) fusion protein and analysis of recombinant PLP2 indicates that PLP2 encodes a cytoplasmic lipid acyl hydrolase with wide substrate specificity. Transgenic plants with altered levels of PLP2 protein were generated and assayed for pathogen resistance. Plants silenced for PLP2 expression displayed enhanced resistance to B. cinerea, whereas plants overexpressing PLP2 were much more sensitive to this necrotrophic fungus. We also established a positive correlation between the level of PLP2 expression in transgenic plants and cell death or damage in response to paraquat treatment or infection by avirulent P. syringae. Interestingly, repression of PLP2 expression increased resistance to avirulent bacteria, while PLP2-overexpressing plants multiplied avirulent bacteria close to the titers reached by virulent bacteria. Collectively, the data indicate that PLP2-encoded lipolytic activity can be exploited by pathogens with different lifestyles to facilitate host colonization. In particular PLP2 potentiates plant cell death inflicted by Botrytis and reduces the efficiency of the hypersensitive response in restricting the multiplication of avirulent bacteria. Both effects are possibly mediated by providing fatty acid precursors of bioactive oxylipins.     

Bacterium release into host cells of nitrogen-fixing soybean nodules: the symbiosome membrane comes from three sources

The release process of bacteria into the cytoplasm of soybean nodule cells has been studied, and three functional zones of the infection thread are delineated. Zone 1 is found over the greatest length of very long infection threads. Zone 2 is a short region where membrane mobilization by exocytosis of endoplasmic reticulum (ER) into the infection-thread membrane takes place; the result is that much new membrane and wall degradation enzymes can be provided. In addition, de novo membrane formation takes place inside the infection thread in apposition to the bacterial outer membrane. Zone 3 is the endocytic region where both bacteria and infection-thread wall degradation vesicles are released into the host cytoplasm and constitute a second product of endocytosis at the infection thread tip. Evidence is presented indicating that the symbiosome membrane, even at its time of origin, is composed of membrane from three sources: the host infection-thread membrane, ER, and de novo synthesis; the membrane formation that is so large for these purposes is probably carried out both from the ER directly and also through the Golgi-apparatus synthesis. Evidence is also given that the bacteria have lost their exopolysaccharide coatings before release into symbiosomes.     

Soil acidity factors and nodulation ofTrifolium repens

Summary Effects of factors associated with soil acidity (low pH, low calcium, high aluminium and high manganese) on theTrifolium repens-Rhizobium trifolii symbiosis were investigted under laboratory conditions using an axenic solution-culture technique. 200 μM manganese increased root elongation in the range pH 4.3–5.5, but had no effect on root hair formation, the number of Rhizobium in the rhizosphere, or nodule formation. Root elongation and root hair formation were unaffected at pH 4.3 when 500 or 1000μM calcium was supplied, whereas multiplication of Rhizobium in the rhizosphere and nodulation were inhibited at pH 4.3 and 4.7.50–1000μM calcium had no effect either on the multiplication of Rhizobium in the range pH 4.3–5.5, or on nodule formation in the absence of aluminium. 50 μM aluminium inhibited, root elongation and root hair formation at pH 4.3 and 4.7; the effect on root elongation was reduced by increasing the calcium concentration from 50 to 1000μM. 50μM aluminium also inhibited Rhizobium multiplication in the rhizosphere and reduced nodule formation at pH 5.5 (at which aluminium precipitated out of solution), but root elongation and root hair formation were unaffected. These, effects of aluminium at pH 5.5 may explain the poor response to inoculation by white clover in acid mineral soils after liming.     

Modulation of the mitogenic PHA response of carp, Cyprinus carpio L., by extracellular products of Aeromonas salmonicida

The influence of bacteria-free supernatants from cultures of atypical virulent (V234/81, auto-agglutinating. A-layer positive) and avirulent (126/68, non-agglutinating, A-layer negative) strains of A. salmonicida , obtained after different culture times in yeast-tryptone broth at 20°C, was tested on the PHA response of carp pronephric leucocytes in vitro . Supernatants from virulent cultures modulated the response, whereas avirulent supernatants had no effect. The response was enhanced (400%) by supernatant from early virulent cultures (20 h), but severely depressed (<3%) by later ones (96 h). The effects were dose-dependent. Inhibitory activity of 96-h supernatant was lost by heating (70°C, 30 min), suggesting that the inhibiting factors are all proteinaceous.
Heated 96-h supernatant was as stimulatory as early supernatant. Stimulation of leucocytes also occurred in the absence of PHA with early and heat-treated 96-h supernatants, but at a tenth of the level, suggesting that only stimulated cells (blasts) might respond to the substance(s) present in supernatants. Membrane fragments from virulent and avirulent bacteria, and purified LPS from virulent bacteria, were stimulatory with or without PHA. Endotoxin-free, heat-treated, 96-h culture supernatants were also stimulatory, suggesting that an additional mitogenic factor(s), other than LPS, is present in the supernatants. The modulating in vitro effects of extracellular products from A. salmonicida might explain the immunosuppression seen during later stages of erythrodermatitis in vivo.