Effect of congo red on the motility of the bacterium Azospirillum brasilense

In semiliquid laboratory media, the bacterium Azospirillum brasilense migrates with the formation of swarming rings. It is demonstrated that adsorption of the sulfonated azodye Congo Red confers on A. brasilense the ability to consistently spread in a semiliquid agar with formation of microcolonies. Spontaneous variants of A. brasilense with increased swarming rate are described, as well as variants that swarm in the presence of Congo Red. It is assumed that at least two types of compounds are formed, which are necessary for swarming and/or spreading with the formation of microcolonies and are capable of interacting with Congo Red.     

Analysis of Congo red-induced changes in the cell surface and macrocolony structure of the bacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

Adsorption of the vital dye Congo red suppresses swarming of Azospirillum brasilense in a semiliquid medium, and the bacteria become able to spread with the formation of microcolonies. By using direct and stereoscopic light microscopy, the patterns of the front of Azospirillum spreading in a semiliquid medium containing the dye were analyzed. It was found that in a medium with Congo red, small motile colonies were formed among the individual cells, and once formed, they left the boundaries of the swarming front. The microcolonies produced by azospirilla in the presence of the dye were ordered bacterial structures, rather than random cell aggregates. Transmission electron microscopy revealed that the cells grown without the dye had polar flagella, whereas the cells from the medium with Congo red had no flagella and were covered with a layer of fibrillike material. Immunochemical data for the cell surface changes resulting from interaction with the dye make it possible to consider Azospirillum lipopolysaccharide as a probable Congo red receptor.     

Formation of polar bundles of pili on a cell and the behavior of Azospirillum brasilense in semiliquid agar]

This paper describes the formation of single polar bundles of pili on Azospirillum brasilense cells, the twitching motility of cell aggregates, and a new type of social behavior--the dispersal of bacterial cells in semiliquid agar associated with the formation of granular inclusions (the so-called Gri+ phenotype)--which is an alternative to swarming (the Swa+ phenotype). The wild-type A. brasilense cells occurring in a semiliquid agar may show either the Swa+Gri-, or Swa-Gri-, or Swa-Gri+ phenotype. The formation of single polar flagella (Fla) or polar bundles of pili may reflect two alternative states of A. brasilense cells. The components of the Fla system may be involved in the regulation of the phenotypic variation of azospirilla.     

Changes in motility of the rhizobacterium Azospirillum brasilense in the presence of plant lectins

The plant-beneficial bacterium Azospirillum brasilense can swim in liquids and swarm or migrate with the formation of microcolonies in soft media. To get closer to understanding the influence of natural environments on A. brasilense motility, we studied the individual and social movement of the bacterium in the presence of various plant lectins. The lectins with specificity for N-acetyl-beta-d-glucosamine oligomers (wheat germ, Solanum tuberosum and Ulex europeus agglutinins) decreased A. brasilense swimming speed and induced the formation of branched-granular colonies instead of the swarming rings. These effects seemed to be a consequence of specific interactions between the agglutinins and the lectin-binding polymers present in the A. brasilense cell envelope. Concanavalin A (with an affinity for terminal alpha-d-mannosyl and alpha-d-glucosyl residues) and Phaseolus vulgaris phytohemagglutinin P (with unknown specificity) almost did not affect the motility of A. brasilense.     

Complementation analysis of mutants of the associative bacteria Azospirillum brasilense Sp245 and S27, defective in mobility and flagellation

Three mutants of Azospirillum brasilense Sp245 incapable of both formation of the polar flagellum (Fla-phenotype) and swarming in semisolid media (Swa-phenotype) were characterized. These mutants were shown to have lost the 85-MDa plasmid and to carry the Tn5-Mob transposon and pSUP5011 vector in different regions of their genomes. With the use of A. brasilense Sp245 gene bank, the capacity for both polar flagellum formation and swarming was restored in the above mutants and in the previously generated transposon mutants A. brasilense Sp245 and S27. The transconjugants obtained were only slightly motile in the liquid culture. In the gene bank of Sp245, the recombinant plasmids carrying wild-type fla/swa loci were identified.     

Self-assembly of Congo Red—A theoretical and experimental approach to identify its supramolecular organization in water and salt solutions

The supramolecular organization of Congo Red molecules was studied to approach an understanding of the unusual complexation characteristics associated with the liquid crystalline nature of this dye. Differential scanning calorimetry (DSC) and nmr data indicate that Congo Red assembly arrangements differ in water and salt solutions. Compact, highly ordered material with a distinct melting transition is created, but not below 0.3% sodium chloride concentration. The twist in the assembly arrangement of Congo Red molecules, caused in water by repulsion, decreases when the charges are shielded, allowing for more overlapping of the naphthalene rings and their engagement in stacking interaction. The crystallization transition observed in DSC analysis of Congo Red fast-assembled by cooling in salt solutions indicates that the formation of compact crystalline mesophase material is a time-consuming process in which coplanarity and a highly ordered organization must be achieved. Two different superposition variants, called “direct” and “reversed” here, were considered fundamental to compact Congo Red organization. They correspond to optimal face-to-face ring stackings, and are formed by simple direct translation or alternative imposition of reversed (180° rotated) molecules, respectively. In NaCl solution (2.8%) there is a significant downfield chemical shift alteration of the nmr signal related to proton 8, which is in the naphthalene ring on the side opposite to the charged sulfonic group. It was associated selectively with the transition of Congo Red to compact form. This effect confirms the close approach of the sulfonic groups and proton 8, and indicates that formation of the reversed arrangement is favored in the Congo Red supramolecular organization. Molecular dynamics simulation based on AMBER 4.1 force field and analysis of electrostatic field densities around the molecule were used for comparative modeling. Molecular dynamics (150 ps) were simulated for two eight-molecule micelle models constructed to reflect direct and reversed arrangements of Congo Red molecules. Although both versions generally preserved their initial assembly structure in the simulations, the reversed version proved more stable. The proximity of the sulfonic group and proton 8, confirmed by computer analysis, explains the correlation between the formation of Congo Red micellar organization and the distinct shift alteration related to this proton, as found by nmr. © 1998 John Wiley & Sons, Inc. Biopoly 46: 267–281, 1998     

Effect of integrating the pJFF350 vector into the 85-MDa plasmid of Azospirillum brasilense Sp245 on bacterial flagellation and mobility

Results of genetic analysis of three derivatives of Azospirillum brasilense Sp245 (strains BK570, SK051, and SK248) carrying cointegrates of plasmids 85-MDa and pJFF350 (the vector for omegon mutagenesis), which manifest abnormalities in flagellation and motility, are presented. It was shown for the first time that the integration of the suicide vector into one of Azospirillum resident plasmids is accompanied by the formation of various fusion products and changes in flagellation and motility of these bacteria, such as the loss of the polar (Fla) and lateral (Laf) flagella in SK051; inactivation of Fla and Laf in SK248; and Fla-dependent acceleration of expansion in semiliquid media in BK570.     

Transposon tnPhoA mutagenesis as a method of obtaining mutants of Azospirillum brasilense by motility and flagellation

Three A. brasilense strains (S27, SpBr14, and KR77) did not hydrolyze the chromogenic substrate of alkaline phosphatase (PhoA), X-phosphate, in situ, and were used as recipients in experiments on TnphoA mutagenesis. KMR transconjugates were obtained only for A. brasilense S27, 85% of them were also PhoA+. About 12% TnphoA mutants of A. brasilense S27 had reduces capacity to swarming and 3% of mutants neither swam nor swarmed. These totally immotile clones were examined under transmission electron microscope and were classified as Fla-Laf-, Fla-leakyLaf-, and Fla-Laf+ mutants. In Fla-Laf+ TnphoA mutants of S27, the expression of their lateral flagella (Laf) retained the wild-type inducibility. The presence of intact polar flagellum (Fla) did not seem to be obligatory for controllable expression of Laf in A. brasilense S27. The data suggest that A. brasilense S27 Fla and Laf systems have common structural and/or regulatory components. The PhoA+ phenotype of S27 Fla- mutants suggested a periplasmic and/or membrane localization of the hybrid proteins, the formation of which blocks the flagellar assembly or functioning. Immunochemical analysis with antibodies to alkaline phosphatase will identify these proteins.     

Formation of Polar Bundles of Pili and the Behavior of Azospirillum brasilenseCells in a Semiliquid Agar

This paper describes the formation of single polar bundles of pili on Azospirillum brasilensecells, the twitching motility of cell aggregates, and a new type of social behavior—the dispersal of bacterial cells in semiliquid agar associated with the formation of granular inclusions (the so-called Gri⁺phenotype)—which is an alternative to swarming (the Swa phenotype). The wild-type A. brasilensecells occurring in a semiliquid agar may show either the Swa⁺Gri^–, or Swa^–Gri^–, or Swa^–Gri⁺phenotype. The formation of single polar flagella (Fla) or polar bundles of pili may reflect two alternative states of A. brasilensecells. The components of the Fla system may be involved in the regulation of the phenotypic variation of azospirilla.     

Enhanced swarming of bacteria on agar plates containing the surfactant Tween 80

A widely used method for quantifying swarming motility is the swarm plate assay. A significant increase in the motility halo size formed by Escherichia coli or Azospirillum brasilense was measured on Tween 80-containing agar relative to untreated agar. This improvement could benefit the identification of mutants in swarming motility.     

The use of fragments of the 85- and 120-MDa plasmids of Azospirillum brasilense Sp245 to study the plasmid rearrangement in this bacterium and to search for homologous sequences in plasmids of Azospirillum brasilense Sp7

A spontaneous loss of the 85- (p85) and 120-MDa (p120) replicons and simultaneous generation of a plasmid of more than 300 MDa were associated with defects in synthesis of O-specific and Calcofluor-binding polysaccharides and had no effect on flagellation and motility of the Azospirillum brasilense Sp245.5 mutant. The plasmid rearrangement was studied by hybridization of DNAs from the wild-type Sp245 strain and the Sp245.5 mutant with p85 and p120 fragments that contained loci involved in formation of the polar (fla) and lateral (laf) flagella, synthesis of O-specific and Calcofluor-binding polysaccharides (lps/cal), swimming (mot), and swarming (swa) of bacteria. Hybridization with the p120 fragments revealed incorporation of the intact fla/swa loci and the altered lps/cal loci into a new megaplasmid. Two EcoRI fragments homologous to the fla/laf/mot/swa loci of p85 were found in A. brasilense Sp245 DNA, whereas only one copy was preserved in the Sp245.5 mutant. Hybridization of the p120 and p85 fragments of Sp245 to the A. brasilense Sp7 DNA for the first time revealed regions of substantial homology to these fragments in the 90- and 115-MDa Sp7 plasmids, respectively.     

Effects of sequential proline substitutions on amyloid formation by human amylin20-29

Amylin, also known as islet amyloid polypeptide (IAPP), is the major protein component of the fibril deposits found in the pancreas of individuals with type II diabetes. The central region of amylin, residues 20-29, has been implicated as a key determinate of amyloid formation. To establish which positions are most important for amyloid formation, the wild-type sequence of the 20-29 fragment and a set of 10 variants have been synthesized in which a proline was placed at each position. Proline is energetically unfavorable in the extended cross-beta structure found in amyloid. If a particular position is critical for amyloid formation, then substitution with a proline should inhibit amyloid formation. A proline substitution at any position inhibited aggregation and amyloid formation. Substitution of Asn22, Gly24, and residues 26-28 had the largest effect. Fourier transform infrared (FTIR) spectroscopy showed little secondary structure in these peptides, and transmission electron microscopy (TEM) showed mostly amorphous material. The peptides were much more soluble than the wild-type sequence, and no birefringence was observed with Congo Red staining. Proline substitutions at the N (residues 20 and 21) and C termini showed the least effect. These peptides showed the classic fibril morphology, a significant amount of beta-sheet structure, and exhibited green birefringence when stained with Congo Red. The results indicate that residues 22, 24, and 26-28 play a key role in formation of amyloid by amylin. Positions 23 and 25 also appear to be important, but may be less critical than positions 22, 24, and 26-28.     

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

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

The Hofmeister effect on amyloid formation using yeast prion protein

A variety of proteins are capable of converting from their soluble forms into highly ordered fibrous cross‐β aggregates (amyloids). This conversion is associated with certain pathological conditions in mammals, such as Alzheimer disease, and provides a basis for the infectious or hereditary protein isoforms (prions), causing neurodegenerative disorders in mammals and controlling heritable phenotypes in yeast. The N‐proximal region of the yeast prion protein Sup35 (Sup35NM) is frequently used as a model system for amyloid conversion studies in vitro. Traditionally, amyloids are recognized by their ability to bind Congo Red dye specific to β‐sheet rich structures. However, methods for quantifying amyloid fibril formation thus far were based on measurements linking Congo Red absorbance to concentration of insulin fibrils and may not be directly applicable to other amyloid‐forming proteins. Here, we present a corrected formula for measuring amyloid formation of Sup35NM by Congo Red assay. By utilizing this corrected procedure, we explore the effect of different sodium salts on the lag time and maximum rate of amyloid formation by Sup35NM. We find that increased kosmotropicity promotes amyloid polymerization in accordance with the Hofmeister series. In contrast, chaotropes inhibit polymerization, with the strength of inhibition correlating with the B‐viscosity coefficient of the Jones‐Dole equation, an increasingly accepted measure for the quantification of the Hofmeister series.     

Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities

Pseudomonas aeruginosa is a ubiquitous environmental bacterium capable of forming biofilms on surfaces as a survival strategy. It exhibits a large variety of competition/virulence factors, such as three types of motilities: flagellum-mediated swimming, flagellum-mediated swarming, and type IV pilus-mediated twitching. A strategy frequently used by bacteria to survive changing environmental conditions is to create a phenotypically heterogeneous population by a mechanism called phase variation. In this report, we describe the characterization of phenotypic variants forming small, rough colonies that spontaneously emerged when P. aeruginosa 57RP was cultivated as a biofilm or in static liquid cultures. These small-colony (S) variants produced abundant type IV fimbriae, displayed defective swimming, swarming, and twitching motilities, and were impaired in chemotaxis. They also autoaggregated in liquid cultures and rapidly initiated the formation of strongly adherent biofilms. In contrast, the large-colony variant (parent form) was poorly adherent, homogeneously dispersed in liquid cultures, and produced scant polar fimbriae. Further analysis of the S variants demonstrated differences in a variety of other phenotypic traits, including increased production of pyocyanin and pyoverdine and reduced elastase activity. Under appropriate growth conditions, cells of each phenotype switched to the other phenotype at a fairly high frequency. We conclude that these S variants resulted from phase variation and were selectively enriched when P. aeruginosa 57RP was grown as a biofilm or in static liquid cultures. We propose that phase variation ensures the prior presence of phenotypic forms well adapted to initiate the formation of a biofilm as soon as environmental conditions are favorable.     

Bacterial lateral flagella: an inducible flagella system

Flagella are complex surface organelles that allow bacteria to move towards favourable environments and that contribute to the virulence of pathogenic bacteria through adhesion and biofilm formation on host surfaces. There are a few bacteria that possess functional dual flagella systems, such as Vibrio parahaemolyticus, some mesophilic Aeromonas spp., Rhodospirillum centenum and Azospirillum brasilense. These bacteria are able to express both a constitutive polar flagellum required for swimming motility and a separate lateral flagella system that is induced in viscous media or on surfaces and is essential for swarming motility. As flagella synthesis and motility have a high metabolic cost for the bacterium, the expression of the inducible lateral flagella system is highly regulated by a number of environmental factors and regulators.     

Phenotypic variability in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains Sp7 and Sp245: Association with dormancy and characteristics of the variants

The state of metabolic dormancy in diazotrophic bacteria Azospirillum brasilense Sp7 (non-endophytic strain) and Sp245 (endophytic strain) was found to be associated with phenotypic variability. The latter manifested itself in the extension of the spectrum of A. brasilense phenotypic variants resulting from plating of cyst-like resting cells (CRC) on solid media and was more pronounced in strain Sp7. The major colony’s morphological variants of strain Sp7 were (1) the dominant S type; (2) the highly pigmented Pg type; (3) the R type; (4) the Sm type, forming small colonies; and (5) the Sg type, forming segmented colonies. In addition to their colony morphology, the variants differed in the phenotype stability during transfers on the standard solid medium and in their motility in semisolid agar. The occurrence frequency of the phenotypic variants depended on the conditions and duration of incubation (storage) of the CRC of strain Sp7, as well as on heat treatment (at 55 and 60°C for 10 min) of the cells prior to inoculation. The maximum frequency of S → Pg transitions (up to 74%) was observed during the germination of CRC stored in a spent culture medium at −20°C for 4 months; the maximum frequency (up to 100%) of S → Sm transitions was observed after inoculation of the CRC subjected to heat treatment. The Pg variants were the most stable, whereas other types reverted rapidly to the S or Pg variant. The S variant grown in semisolid agar exhibited the mixed type of motility (Swa⁺Gri⁺, swarming and migration in the form of microcolonies); the Pg and Sg variants showed the Swa⁺Gri⁻ (swarming) phenotype and the Sm variant was nonmotile (Swa⁻Gri⁻ phenotype). The spectrum of phenotypic variants of the endophytic strain Sp245 was narrower than that of strain Sp7 and was represented by S, Sm, and M (mucoid) variants that differed in the patterns of cell motility: the dominant S type displayed the swarming pattern (Swa⁺Gri⁻), the mucoid M type showed the mixed type (Swa⁺Gri⁺) of motility, and the Sm variant was nonmotile. The differences between the nonendophytic strain Sp7 and the endophytic strain Sp245 in their capacity for phenotypic dissociation and cell motility in semisolid media may reflect their ability to adapt to changing ambient conditions and specificity of plant-microbial interactions.     

Demonstration of amyloid with Mesitol WLS-Congo Red: Application of a textile auxiliary to histochemistry

Summary Previous histochemical investigations demonstrated similarities in the binding of Congo Red and other direct cotton dyes by amyloid and cellulose. It seemed threfore of interest to determine whether or not the cellulose-like reactivity of amyloid extends also to dye solutions containing an anionic reserving agent. These reagents are used in the dyeing of wool-cellulose (Halbwolle) fabrics to prevent binding of direct cotton dyes by proteins. Mesitol WLS-Congo Red solutions stained amyloid selectively; other tissue structures, except some hyaline deposits in arterioles, remained unstained. The cause of this non-specific reaction could not be determined with certainty. Therefore, the alkaline Congo Red method is recommended for histochemical identification of amyloid. However, the Mesitol WLS-Congo Red technic was very useful for demonstration of amyloid after prolonged storage of tissues in formalin; amyloid in such material showed little or no reactivity with the alkaline Congo Red or the Sirius dye methods. This pilot study indicates that anionic reserving agents can be effectively employed under conditions of histochemical technics.     

Self-produced extracellular stimuli modulate the Pseudomonas aeruginosa swarming motility behaviour

Pseudomonas aeruginosa presents three types of motilities: swimming, twitching and swarming. The latter is characterized by rapid and coordinated group movement over a semisolid surface resulting from morphological differentiation and intercellular interactions. A striking feature of P. aeruginosa swarming motility is the formation of migrating tendrils producing colonies with complex fractal-like patterns. Previous studies have shown that normal swarming motility is intimately related to the production of extracellular surface-active molecules: rhamnolipids (RLs), composed of monorhamnolipids (mono-RLs) and dirhamnolipids (di-RLs), and 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs). Here, we report that (i) di-RLs attract active swarming cells while HAAs behave as strong repellents, (ii) di-RLs promote and HAAs inhibit tendril formation and migration, (iii) di-RLs and HAAs display different diffusion kinetics on a surface as di-RLs spread faster than HAAs in agar, (iv) di-RLs and HAAs have no effect on swimming cells, suggesting that swarming cells are different from swimming cells not only in morphology but also at the regulatory level and (v) mono-RLs act as wetting agents. We propose a model explaining how HAAs and di-RLs together modulate the behaviour of swarming migrating cells by acting as self-produced negative and positive chemotactic-like stimuli.