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.     

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.     

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 and form microcolonies. Spontaneous variants of A. brasilense with rapid swarming 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 (a) necessary for swarming and/or spreading with the formation of microcolonies and (b) capable of interacting with Congo Red.     

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.     

A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov.

Sixty-one strains of the root-associated nitrogen fixer Spirillum lipoferum exhibited a similar morphology in peptone--succinate salts medium: vibrioid cells having a diameter of 1.0 micrometer. When grown in broth the cells had a single polar flagellum, but when grown on agar at 30 degrees C lateral flagella of shorter wavelength were also formed. The DNA base composition was 69--71 mol% guanine + cytosine when determined by thermal denaturation. DNA homology experiments indicated the occurrence of two distinct but related homology groups: 46 strains were in group I and 15 strains were in group II. Group II strains were distinguished by their ability to use glucose as a sole carbon source for growth in nitrogen-free medium, by their production of an acidic reaction in a peptone-based glucose medium, by their requirement for biotin, and by their formation of wider, longer, S-shaped or helical cells in semisolid nitrogen-free malate medium. The results indicate that two species exist, and on the basis of their characteristics it is proposed that they be assigned to a new genus, Azospirillum. Strians belonging to group II are named A. lipoferum (Beijerinck) comb. nov., while those belonging to group I are named A. brasilense sp. nov. Strain Sp 59b (ATCC29707) is proposed as the neotype strain for A. lipoferum, and strain Sp 7 (ATCC 29145) is proposed as the type strain for A. brasilense.     

Chemotactic control of the two flagellar systems of Vibrio parahaemolyticus.

Vibrio parahaemolyticus synthesizes two distinct flagellar organelles, the polar flagellum (Fla), which propels the bacterium in a liquid environment (swimming), and the lateral flagella (Laf), which are responsible for movement over surfaces (swarming). Chemotactic control of each of these flagellar systems was evaluated separately by analyzing the behavioral responses of strains defective in either motility system, i.e., Fla+ Laf- (swimming only) or Fla- Laf+ (swarming only) mutants. Capillary assays, modified by using viscous solutions to measure swarming motility, were used to quantitate chemotaxis by the Fla+ Laf- or Fla- Laf+ mutants. The behavior of the mutants was very similar with respect to the attractant compounds and the concentrations which elicited responses. The effect of chemotaxis gene defects on the operation of the two flagellar systems was also examined. A locus previously shown to encode functions required for chemotactic control of the polar flagellum was cloned and mutated by transposon Tn5 insertion in Escherichia coli, and the defects in this locus, che-4 and che-5, were then transferred to the Fla+ Laf- or Fla- Laf+ strains of V. parahaemolyticus. Introduction of the che mutations into these strains prevented chemotaxis into capillary tubes and greatly diminished movement of bacteria over the surface of agar media or through semisolid media. We conclude that the two flagellar organelles, which consist of independent motor-propeller structures, are directed by a common chemosensory control system.     

Effect of the Integration of Vector pJFF350 into Plasmid 85-MDa of Azospirillum brasilense Sp245 on Bacterial Flagellation and Motility

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.     

Motility, chemokinesis, and methylation-independent chemotaxis in Azospirillum brasilense.

Observations of free-swimming and antibody-tethered Azospirillum brasilense cells showed that their polar flagella could rotate in both clockwise and counterclockwise directions. Rotation in a counterclockwise direction caused forward movement of free-swimming cells, whereas the occasional change in the direction of rotation to clockwise caused a brief reversal in swimming direction. The addition of a metabolizable chemoattractant, e.g., malate or proline, had two distinct effects on the swimming behavior of the bacteria: (i) a short-term decrease in reversal frequency from 0.33 to 0.17 s-1 and (ii) a long-term increase in the mean population swimming speed from 13 to 23 microns s-1. A. brasilense therefore shows both chemotaxis and chemokinesis in response to temporal gradients of some chemoeffectors. Chemokinesis was dependent on the growth state of the cells and may depend on an increase in the electrochemical proton gradient above a saturation threshold. Analysis of behavior of a methionine auxotroph, assays of in vivo methylation, and the use of specific antibodies raised against the sensory transducer protein Tar of Escherichia coli all failed to demonstrate the methylation-dependent pathway for chemotaxis in A. brasilense. The range of chemicals to which A. brasilense shows chemotaxis and the lack of true repellents indicate an alternative chemosensory pathway probably based on metabolism of chemoeffectors.     

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.     

Swa2p-dependent clathrin dynamics is critical for Flo11p processing and ‘Mat’ formation in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is able to form complex multicellular structures called mats on low-density agar Petri plates. Mat formation strictly depends on Flo11p, a cell surface mannoprotein that mediates the adhesion of yeast cells to the agar surface. Here, we show that Swa2p, an auxilin ortholog required for clathrin-coated vesicle uncoating, is strictly required for biofilm formation. We show that the maturation and cellular levels of Flo11p are affected in Δswa2 cells, yet without compromising invasive growth. Both the TPR and J-domains of Swa2p, but not its clathrin-binding and ubiquitin-association motifs, are required for its function in Flo11p processing.     

Stalkless mutants of Caulobacter crescentus.

A stalk, a single falgellum, several pili, and deoxyribonucleic acid (DNA) phage receptors are polar surface structures expressed at a defined time in the Caulobacter crescentus cell cycle. When mutants were isolated as DNA phage phiCbK-resistant or ribonucleic acid (RNA) phage phiCp2-resistant, as well as nonmotile, strains, 5 out of 30 such mutant isolates were found not to possess stalks, but did possess inactive flagella. These stalkless mutants were resistant simultaneously to both DNA and RNA phages and did not possess pili and DNA pendent stalkless mutants. All motile revertants simultaneously regained the capacity to form stalks and susceptibility to DNA and RNA phages. It is suggested that a single mutation pleiotropically affects stalk formation, flagella motility, and coordinate polar morphogenesis of pili and DNA phage receptors. The stalkless mutants grew at a generation time similar to that of the wild-type strain at 30 degrees C. Cell size and morphology of a stalkless mutant, C. crescentus CB13 pdr-819, were also similar to those of the wild-type strain, except for the absence of a stalk. In addition, the CB13 pdr-819 predivisional cells were partitioned into smaller and larger portions, indicating asymmetrical cell division, as in the wild-type strain. From these results, it is suggested that swarmer cells undergo transition to cells of a stalked-cell nature without stalk formation and that the cell cycle of the stalkless mutant proceeds in an ordered sequence similar to that defining the wild-type cell cycle.     

Immortalization of human fibroblasts using tsA mutant of SV40 and pSV3neo plasmid]

Clones of immortalized human fibroblasts with an extended life span in culture and a capability of subloning were obtained after the infection with a temperature sensitive mutant (tsA 239) of SV40 virus and pSV3neo plasmid. As compared with the parental cells, the obtained clones exhibited increased plating efficiency, decreased doubling time, and serum dependence. We did not obtained the colony formation during cultivation of immortalized cells in semiliquid agar. This means that our cells were not completely malignant. The PCR (polymerase chain reaction)-analysis has revealed the presence of viral DNA at early passages (25th passage) after the infection by tsA SV40, and its absence after a prolonged cultivation (46th passage). PCR-analysis of the clones obtained after pSV3neo transfection has revealed the presence of gene A sequences either at early (9-15), or later (62) passages. The expression of the gene A product in cells of these clones was revealed only early passages (11 and 35). Possible mechanisms of immortal phenotype origin in human diploid cells after the action of ts-mutant and other constructions of SV40 are discussed.     

Influence of cell surface structures on crenarchaeal biofilm formation using a thermostable green fluorescent protein

The thermoacidophilic crenarchaeote Sulfolobus acidocaldarius displays three distinct type IV pili-like structures on its surface: (i) the flagellum, (ii) the UV-induced pili and (iii) the adhesive pili. In bacteria, surface appendages play an important role in the spatial organization of cells from initial surface attachment to the development of mature community structures. To investigate the influence of the diverse set of type IV pili-like structures in S. acidocaldarius, single, double and triple mutants lacking the cell surface appendages were constructed and analysed for their behaviour in attachment assays and during biofilm formation. A heat stable green fluorescent protein was employed the first time in a hyperthermophilic archaeon. A codon adjusted eCGP123 was expressed to study mixed biofilms of different deletion mutants to understand the interplay of the surface structures during biofilm formation. During this process the deletion of the adhesive pili and UV-induced pili led to the most pronounced effects, either an increase in cell density or increased cluster formation respectively. However, all three cell surface appendages played a role in the colonization of surfaces and only the interplay of all three appendages leads to the observed wild-type biofilm phenotype.     

Role of membrane potential and ATP in complex formation between Escherichia coli male cells and filamentous phage fd

Mutant strains of Escherichia coli male cells defective in Ca2+,Mg2+-dependent ATPase (unc) were constructed and tested for their ability to form a complex between sex pili and the filamentous phage fd under conditions where either the membrane potential or the cellular concentration of ATP was lowered. The uncoupler carbonyl cyanide m-chlorophenylhydrazone and the respiratory inhibitor cyanide, as well as valinomycin-K+ and colicin E1, all markedly diminished complex formation, indicating that the maintenance of a membrane potential, but probably not the pH gradient, is essential for the formation of the complex. Since complex formation with freshly centrifuged cells (which initially lacked sex pili) as well as with preincubated cells (in which pre-existing pili were available for complex formation) was inhibited by exposure to the inhibitors, energy seems to be required for both the reappearance (probably assembly) and the maintenance of sex pili on the cell surface. Brief exposure of freshly centrifuged cells to arsenate resulted in only partial inhibition of complex formation. However, marked inhibition of complex formation was observed following exposure to arsenate of preincubated cells possessing sex pili. This indicates that compounds such as ATP may also be required for maintenance of sex pili on the cell surface.     

Variations in the expression of pili: the effect on adherence of Neisseria meningitidis to human epithelial and endothelial cells

The effect of variations in Neisseria meningitidis pili on bacterial interactions with three epithelial cell lines as well as human umbilical vein endothelial cells was studied using a panel of seven strains expressing Class I or Class II pili. Comparison of adherence of piliated and pilus-deficient variants of each strain to epithelial cells suggested that Class I pili may mediate bacterial adherence with all three epithelial cell lines. In contrast, Class II pili of the strains used did not increase bacterial adherence to Hep-2 larynx carcinoma cells, although an increase in adherence to Chang conjunctival and A549 lung carcinoma epithelial cells was observed in the Class II pili-expressing strains. In addition to these interclass functional variations, differences in adherence to epithelial cells were also observed among Class I and Class II strains. Functionally different pilin variants of one Class I strain, MC58, were obtained by single colony isolation. One piliated variant was identified which had concurrently lost the ability to adhere to both Chang and Hep-2 cells ('non-adherent' phenotype; adherence of less than 2 bacteria per cell). In addition, several adherent pilin variants were isolated from non-adherent Pil- and Pil+ bacteria by selection on Chang cells (adherence of 10-25 bacteria per cell). In contrast to epithelial cells, all variant pili, whether of Class I or Class II, adhered to endothelial cells in substantially larger numbers (greater than 50 bacteria per cell) and therefore implied the existence of distinct mechanisms in pilus-facilitated interactions of N. meningitidis with endothelial and epithelial cells.     

In Rhodobacter sphaeroides, chemotactic operon 1 regulates rotation of the flagellar system 2

Rhodobacter sphaeroides is able to assemble two different flagella, the subpolar flagellum (Fla1) and the polar flagella (Fla2). In this work, we report the swimming behavior of R. sphaeroides Fla2(+) cells lacking each of the proteins encoded by chemotactic operon 1. A model proposing how these proteins control Fla2 rotation is presented.     

Cloning, sequencing, and phenotypic analysis of laf1, encoding the flagellin of the lateral flagella of Azospirillum brasilense Sp7.

Azospirillum brasilense can display a single polar flagellum and several lateral flagella. The A. brasilense Sp7 gene laf1, encoding the flagellin of the lateral flagella, was isolated and sequenced. The derived protein sequence is extensively similar to those of the flagellins of Rhizobium meliloti, Agrobacterium tumefaciens, Bartonella bacilliformis, and Caulobacter crescentus. An amino acid alignment shows that the flagellins of these bacteria are clustered and are clearly different from other known flagellins. A laf1 mutant, FAJ0201, was constructed by replacing an internal part of the laf1 gene by a kanamycin resistance-encoding gene cassette. The mutant is devoid of lateral flagella but still forms the polar flagellum. This phenotype is further characterized by the abolishment of the capacities to swarm on a semisolid surface and to spread from a stab inoculation in a semisolid medium. FAJ0201 shows a normal wheat root colonization pattern in the initial stage of plant root interaction.     

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.     

Regulation of phase variation in type I pili formation in <Emphasis Type="Italic">Escherichia coli</Emphasis>: Role of alkylresorcinols,microbial autoregulators

Formation of virulence-associated type I pili in Escherichia coli should be considered as one of the most efficient models for investigating the mechanisms of regulating the heterogeneity of populations of genetically identical microbial cells. The present work focused on the role of alkylhydroxybenzenes (AHBs), density-dependent intercellular regulators, in controlling phase variations in type I pili formation (fimbriogenesis). The tested AHB homologue was C12-AHB; a genetically constructed strain E. сoli dsp250 containing the fimA-lacZ hybrid operon was used. In this operon, the fimA gene encodes the main subunit of the pili protein, and its expression results in β-galactosidase synthesis; pili-forming cells, therefore, become blue on the medium with the Х-gal substrate. Expression of fimA depends on the inversion of the fimS region that is located upstream of it. If the inversion is on, pili formation takes place, if it is off, no pili are formed. An increase in C12-AHB concentration (within the 5 × 10^–5–2 × 10^–4 M range) in the exponential-phase culture of strain dsp250 causes a dose-dependent change in the dominant phenotype that is displayed by up to 98–99% of the cells. Cells with this phenotype form colonies with a blue center and white edges. Up to 60% of the cells with this phenotype assume a metastable state and up to 11% and 44% of them transition to the alternative phenotypes of pili-forming and pili-less cells, respectively. The influence of C12-AHB on off-switching, i.e. the formation of the avirulent phenotype, was observed irrespective of the growth conditions of strain dsp250. Addition of glucose to the LB medium (5 or 10 mg/mL) resulted in catabolic repression via regulation by the cAMP-CNR complex and predictably induced pili formation in 49 and 75% of the cells, respectively. Against this background, C12-AHB caused a dose-dependent decrease in the share of pili-forming cells to 33–61% and an increase in the share of pili-less cells to 32–61%. If glucose was added in excess (2.5, 5 or 10 mg/mL) to the diluted LB/2 medium, pili formation was completely repressed, while C12-AHB still induced the off inversion to the pili-less phenotype in up to 30% of the cells. The conclusion can be drawn that C12-AHB is not involved in the pathway of fimbriogenesis regulation via cAMP. Since C12-AHB functions as an extracellular alarmon (activating the rpoS regulon and the SOS response as shown earlier, see Golod et al., 2009), its mechanism of action apparently involves stress signal transduction. It induces the synthesis of global regulators RpoS and H-NS and of intracellular alarmon (p) ppGpp; these factors are responsible for the on → off inversion and the proliferation of pili-less cells.