Function of a chemotaxis-like signal transduction pathway in modulating motility, cell clumping, and cell length in the alphaproteobacterium Azospirillum brasilense

A chemotaxis signal transduction pathway (hereafter called Che1) has been previously identified in the alphaproteobacterium Azospirillum brasilense. Previous experiments have demonstrated that although mutants lacking CheB and/or CheR homologs from this pathway are defective in chemotaxis, a mutant in which the entire chemotaxis pathway has been mutated displayed a chemotaxis phenotype mostly similar to that of the parent strain, suggesting that the primary function of this Che1 pathway is not the control of motility behavior. Here, we report that mutants carrying defined mutations in the cheA1 (strain AB101) and the cheY1 (strain AB102) genes and a newly constructed mutant lacking the entire operon [Δ(cheA1-cheR1)::Cm] (strain AB103) were defective, but not null, for chemotaxis and aerotaxis and had a minor defect in swimming pattern. We found that mutations in genes of the Che1 pathway affected the cell length of actively growing cells but not their growth rate. Cells of a mutant lacking functional cheB1 and cheR1 genes (strain BS104) were significantly longer than wild-type cells, whereas cells of mutants impaired in the cheA1 or cheY1 genes, as well as a mutant lacking a functional Che1 pathway, were significantly shorter than wild-type cells. Both the modest chemotaxis defects and the observed differences in cell length could be complemented by expressing the wild-type genes from a plasmid. In addition, under conditions of high aeration, cells of mutants lacking functional cheA1 or cheY1 genes or the Che1 operon formed clumps due to cell-to-cell aggregation, whereas the mutant lacking functional CheB1 and CheR1 (BS104) clumped poorly, if at all. Further analysis suggested that the nature of the exopolysaccharide produced, rather than the amount, may be involved in this behavior. Interestingly, mutants that displayed clumping behavior (lacking cheA1 or cheY1 genes or the Che1 operon) also flocculated earlier and quantitatively more than the wild-type cells, whereas the mutant lacking both CheB1 and CheR1 was delayed in flocculation. We propose that the Che1 chemotaxis-like pathway modulates the cell length as well as clumping behavior, suggesting a link between these two processes. Our data are consistent with a model in which the function of the Che1 pathway in regulating these cellular functions directly affects flocculation, a cellular differentiation process initiated under conditions of nutritional imbalance.     

The chemotaxis-like Che1 pathway has an indirect role in adhesive cell properties of Azospirillum brasilense

The Azospirillum brasilense chemotaxis-like Che1 signal transduction pathway was recently shown to modulate changes in adhesive cell surface properties that, in turn, affect cell-to-cell aggregation and flocculation behaviors rather than flagellar-mediated chemotaxis. Attachment to surfaces and root colonization may be functions related to flocculation. Here, the conditions under which A. brasilense wild-type Sp7 and che1 mutant strains attach to abiotic and biotic surfaces were examined using in vitro attachment and biofilm assays combined with atomic force microscopy and confocal microscopy. The nitrogen source available for growth is found to be a major modulator of surface attachment by A. brasilense and could be promoted in vitro by lectins, suggesting that it depends on interaction with surface-exposed residues within the extracellular matrix of cells. However, Che1-dependent signaling is shown to contribute indirectly to surface attachment, indicating that distinct mechanisms are likely underlying flocculation and attachment to surfaces in A. brasilense.     

Tapping-mode atomic force microscopy in fluid of hydrated extracellular matrix

Fragments of native, hydrated rat tail tendon were imaged by tapping-mode atomic force microscopy while immersed in fluid. The specimens were soft and sensitive to the operating parameters, and with minimal imaging pressure the collagen fibrils appeared covered by irregular blobs or by filamentous material. A slight increase in pressure caused the underlying fibril surface to appear, with an evident D-period, gap- and overlap-zones and three intraperiod ridges. Fibrils often ran parallel and in phase, implying some coupling mechanism. Longitudinal subfibrils, 8-9 nm thick, occasionally appeared. The simultaneous acquisition of the "tapping amplitude" along with the usual "height" channel clearly confirmed the presence of longitudinal subfibrils, indicative of the inner architecture of the fibril.     

Posttranslational regulation of nitrogenase activity in Azospirillum brasilense ntrBC mutants: ammonium and anaerobic switch-off occurs through independent signal transduction pathways.

Nitrogenase activity is regulated by reversible ADP-ribosylation in response to NH4+ and anaerobic conditions in Azospirillum brasilense. The effect of mutations in ntrBC on this regulation was examined. While NH4+ addition to ntrBC mutants caused a partial loss of nitrogenase activity, the effect was substantially smaller than that seen in ntr+ strains. In contrast, nitrogenase activity in these mutants was normally regulated in response to anaerobic conditions. The analysis of mutants lacking both the ntrBC gene products and dinitrogenase reductase activating glycohydrolase (DRAG) suggested that the primary effect of the ntrBC mutations was to alter the regulation of DRAG activity. Although nif expression in the ntr mutants appeared normal, as judged by activity, glutamine synthetase activity was significantly lower in ntrBC mutants than in the wild type. We hypothesize that this lower glutamine synthetase activity may delay the transduction of the NH4+ signal necessary for the inactivation of DRAG, resulting in a reduced response of nitrogenase activity to NH4+. Finally, data presented here suggest that different environmental stimuli use independent signal pathways to affect this reversible ADP-ribosylation system.     

Imaging surface of gold-immunolabeled thin sections by atomic force microscopy

Atomic force microscopy (AFM) has been used to image the surface of thin sections of fungal infected plant tissue, with or without post-embedding immunocytochemical labeling with gold conjugates. Plant and fungal cells are easily identified from their size, shape and roughness. The cellular shape is similar to that observed by light or electron microscopy (LM or EM) and some internal organelles can even be individualized. The gold beads are easily observed and counted. Their dimensions varied according to the roughness of the surface, but fit with the expected sizes.     

Topography of cell traces studied by atomic force microscopy

Migrating adherent cells release material onto artificial substrates like glass and silicon while moving. Traces of mouse fibroblasts (L929) have been visualised by atomic force microscopy (AFM). “Non-contact” mode AFM in a liquid environment can extract topographic information from these traces. This dynamic mode allows the study of these soft structures without damage or compression. The AFM images show crossing and branching networks (with specific angles of branching), structured patches, nodular elements, linear elements with irregular height and other features. Fourier analysis of segment spacing in the strands is presented. These spatial features of fibroblast traces are strong indications that actin linked to structural proteins is involved in the formation of cell traces. We also give methods for trace preparation and undistorted imaging and discuss further perspectives. Received: 11 January 1999 / Revised version: 1 April 1999 / Accepted: 8 April 1999     

Visualization of plant cell walls by atomic force microscopy.

Atomic force microscopy has been used to visualize the ultrastructure of hydrated plant cell wall material from prepared apple (Malus pumila MILL; Cox orange pippin), water chestnut (Eleocharis dulcis L.), potato (Solanum tuberosum L.; Bintje), and carrot (Daucus carota L.; Amsterdamse bak) parenchyma. Samples of cell wall material in aqueous suspension were deposited onto freshly cleaved mica. Excess water was blotted away and the moist samples were imaged in air at ambient temperature and humidity. The three-dimensional images obtained highlighted the layered structure of the plant cell walls and revealed features interpreted as individual cellulose microfibrils and plasmodesmata.     

LRP1 regulates remodeling of the extracellular matrix by fibroblasts

Low density lipoprotein receptor-related protein (LRP1) is an endocytic receptor for diverse proteases, protease inhibitors, and other plasma membrane proteins, including the urokinase receptor (uPAR). LRP1 also functions in cell-signaling and regulates gene expression. The goal of this study was to determine whether LRP1 regulates remodeling of provisional extracellular matrix (ECM) by fibroblasts. To address this problem, we utilized an in vitro model in which type I collagen was reconstituted and overlaid with fibronectin. Either the collagen or fibronectin was fluorescently-labeled. ECM remodeling by fibroblasts deficient in LRP1, uPAR, or MT1-MMP was studied. MT1-MMP was required for efficient remodeling of the deep collagen layer but not involved in fibronectin remodeling. Instead, fibronectin was remodeled by a system that required urokinase-type plasminogen activator (uPA), uPAR, and exogenously-added plasminogen. LRP1 markedly inhibited fibronectin remodeling by regulating cell-surface uPAR and plasminogen activation. LRP1 also regulated remodeling of the deep collagen layer but not by controlling MT1-MMP. Instead, LRP1 deficiency or inhibition de-repressed a secondary pathway for collagen remodeling, which was active in MT1-MMP-deficient cells but not in uPAR-deficient cells. These results demonstrate that LRP1 regulates ECM remodeling principally by repressing pathways that require plasminogen activation by uPA in association with uPAR.     

Optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy

Fixation ability of five common fixation solutions, including 2.5% glutaraldehyde, 10% formalin, 4% paraformaldehyde, methanol/acetone (1:1), and ethanol/acetic acid (3:1) were evaluated by using atomic force microscopy in the present study. Three model bacteria, i.e., Escherichia coli, Pseudomonas putida, and Bacillus subtilis were applied to observe the above fixation methods for the morphology preservation of bacterial cells and surface ultrastructures. All the fixation methods could effectively preserve cell morphology. However, for preserving bacterial surface ultrastructures, the methods applying aldehyde fixations performed much better than those using alcohols, since the alcohols could detach the surface filaments (i.e., flagella and pili) significantly. Based on the quantitative and qualitative assessments, the 2.5% glutaraldehyde was proposed as a promising fixation solution both for observing morphology of both bacterial cell and surface ultrastructures, while the methonal/acetone mixture was the worst fixation solution which may obtain unreliable results.     

Characterization of cellulose whiskers and their nanocomposites by atomic force and electron microscopy

The aim of this work was to compare and explore electron microscopy and atomic force microscopy (AFM) for structure determination of cellulose whiskers and their nanocomposite with poly(lactic acid). From conventional bright-field transmission electron microscopy (TEM) it was possible to identify individual whiskers, which enabled determination of their sizes and shape. AFM overestimated the width of the whiskers due to the tip-broadening effect. Field emission scanning electron microscopy (FESEM) allowed for a quick examination giving an overview of the sample; however, the resolution was considered insufficient for detailed information. Ultramicrotomy of nanocomposite films at cryogenic temperatures enabled detailed inspection of the cellulose whiskers in the poly(lactic acid) matrix by AFM. FESEM applied on fractured surfaces allowed insight into the morphology of the nanocomposite, although rather restricted due to the metal coating and limited resolution. Detailed information was obtained from TEM; however, this technique required staining and suffered in general from limited contrast and beam sensitivity of the material.     

Analysis of micro- and nano-structures of the corneal surface of Drosophila and its mutants by atomic force microscopy and optical diffraction

Drosophila melanogaster is a model organism instrumental for numerous biological studies. The compound eye of this insect consists of some eight hundred individual ommatidia or facets, ca. 15 μm in cross-section. Each ommatidium contains eighteen cells including four cone cells secreting the lens material (cornea). High-resolution imaging of the cornea of different insects has demonstrated that each lens is covered by the nipple arrays--small outgrowths of ca. 200 nm in diameter. Here we for the first time utilize atomic force microscopy (AFM) to investigate nipple arrays of the Drosophila lens, achieving an unprecedented visualization of the architecture of these nanostructures. We find by Fourier analysis that the nipple arrays of Drosophila are disordered, and that the seemingly ordered appearance is a consequence of dense packing of the nipples. In contrast, Fourier analysis confirms the visibly ordered nature of the eye microstructures--the individual lenses. This is different in the frizzled mutants of Drosophila, where both Fourier analysis and optical imaging detect disorder in lens packing. AFM reveals intercalations of the lens material between individual lenses in frizzled mutants, providing explanation for this disorder. In contrast, nanostructures of the mutant lens show the same organization as in wild-type flies. Thus, frizzled mutants display abnormal organization of the corneal micro-, but not nano-structures. At the same time, nipples of the mutant flies are shorter than those of the wild-type. We also analyze corneal surface of glossy-appearing eyes overexpressing Wingless--the lipoprotein ligand of Frizzled receptors, and find the catastrophic aberration in nipple arrays, providing experimental evidence in favor of the major anti-reflective function of these insect eye nanostructures. The combination of the easily tractable genetic model organism and robust AFM analysis represents a novel methodology to analyze development and architecture of these surface formations.     

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.     

Imaging of the membrane surface of MDCK cells by atomic force microscopy.

The membrane surface of polarized renal epithelial cells (MDCK cells) grown as a monolayer was imaged with the atomic force microscope. The surface topography of dried cells determined by this approach was consistent with electron microscopy images previously reported. Fixed and living cells in aqueous medium gave more fuzzy images, likely because of the presence of the cell glycocalix. Treatment of living cells with neuraminidase, an enzyme that partly degrades the glycocalix, allowed sub-micrometer imaging. Protruding particles, 10 to 60 nm xy size, occupy most of the membrane surface. Protease treatment markedly reduced the size of these particles, indicating that they corresponded to proteins. Tip structure effects were probably involved in the exaggerated size of imaged membrane proteins. Although further improvements in the imaging conditions, including tip sharpness, are required, atomic force microscope already offers the unique possibility to image proteins at the membrane surface of living cells.     

Characterization of cell elasticity correlated with cell morphology by atomic force microscope

Biomechanical properties of cells have been identified as an important factor in a broad range of biological processes. Based on measurements of mechanical properties by atomic force microscopy (AFM) particularly cell elasticity has been linked with human diseases, such as cancer. AFM has been widely used as a nanomechanical tool to probe the elasticity of living cells, however, standard methods for characterizing cell elasticity are still lacking. The local elasticity of a cell is conventionally used to represent the mechanical property of the cell. However, since cells have highly heterogeneous regions, elasticity mapping over the entire cell, rather than at a few points of measurement, is required. Using human aortic endothelial cells (HAECs) as a model, we have developed in this study a new method to evaluate cell elasticity more quantitatively. Based on the height information of the cell, a new characterization method was proposed to evaluate the elasticity of a cell. Using this method, elasticities of cells on different substrates were compared. Results showed that the elasticity of HAECs on softer substrate also has higher value compared to those on harder substrate given a certain height where the statistical distribution analysis confirmed that higher actin filaments density was located. Thus, the elasticity of small portions of a cell could not represent the entire cell property and may lead to invalid characterization. In order to gain a more comprehensive and detailed understanding of biomechanical properties for future clinical use, elasticity and cell morphology should therefore be correlated with discussion.     

Reproducible acquisition of Escherichia coli porin surface topographs by atomic force microscopy.

Crystalline membranes reconstituted from Escherichia coli OmpF porin and phospholipids were adsorbed to freshly cleaved mica and imaged in solution by the atomic force microscope. The extracellular as well as the periplasmic side of the porin trimers could be identified and the conditions to record topographs at 1-nm lateral and 0.1-nm vertical resolution were established.     

Characterization of changes to the cell surface during the life cycle of Streptomyces coelicolor: atomic force microscopy of living cells

Cell surface changes that accompany the complex life cycle of Streptomyces coelicolor were monitored by atomic force microscopy (AFM) of living cells. Images were obtained using tapping mode to reveal that young, branching vegetative hyphae have a relatively smooth surface and are attached to an inert silica surface by means of a secreted extracellular matrix. Older hyphae, representing a transition between substrate and aerial growth, are sparsely decorated with fibers. Previously, a well-organized stable mosaic of fibers, called the rodlet layer, coating the surface of spores has been observed using electron microscopy. AFM revealed that aerial hyphae, prior to sporulation, possess a relatively unstable dense heterogeneous fibrous layer. Material from this layer is shed as the hyphae mature, revealing a more tightly organized fibrous mosaic layer typical of spores. The aerial hyphae are also characterized by the absence of the secreted extracellular matrix. The formation of sporulation septa is accompanied by modification to the surface layer, which undergoes localized temporary disruption at the sites of cell division. The characteristics of the hyphal surfaces of mutants show how various chaplin and rodlin proteins contribute to the formation of fibrous layers of differing stabilities. Finally, older spores with a compact rodlet layer develop surface concavities that are attributed to a reduction of intracellular turgor pressure as metabolic activity slows.     

Direct probing by atomic force microscopy of the cell surface softness of a fibrillated and nonfibrillated oral streptococcal strain

In this paper, direct measurement by atomic force microscopy (AFM) of the cell surface softness of a fibrillated oral streptococcal strain Streptococcus salivarius HB and of a nonfibrillated strain S. salivarius HBC12 is presented, and the data interpretation is validated by comparison with results from independent techniques. Upon approach of the fibrillated strain in water, the AFM tip experienced a long-range repulsion force, starting at approximately 100 nm, attributed to the compression of the soft layer of fibrils present at the cell surface. In 0.1 M KCl, repulsion was only experienced when the tip was closer than approximately 10 nm, reflecting a stiffer cell surface due to collapse of the fibrillar mass. Force-distance curves indicated that the nonfibrillated strain, probed both in water and in 0.1 M KCl, was much stiffer than the fibrillated strain in water, and a repulsion force was experienced by the tip at close approach only (20 nm in water and 10 nm in 0.1 M KCl). Differences in cell surface softness were further supported by differences in cell surface morphology, the fibrillated strain imaged in water being the only specimen that showed characteristic topographical features attributable to fibrils. These results are in excellent agreement with previous indirect measurements of cell surface softness by dynamic light scattering and particulate microelectrophoresis and demonstrate the potential of AFM to directly probe the softness of microbial cell surfaces.     

Production of bacteriocins and siderophore-like activity by Azospirillum brasilense

Sixty Azospirillum strains were tested for their bacteriocin production ability; twenty-seven (45%) were able to produce bacteriocins and inhibited the growth of one or more indicator strains in solid medium. Mitomycin C treatment enhanced the proportion to 80%. Sometimes large growth inhibition zones were formed, but not when FeCl3 was added in the medium. These inhibition zones probably result from the activity of siderophores. Partially purified bacteriocins produced by four strains were inactivated at pH 4, but were very stable between pH 5 to 10; bacteriocins produced by three strains lost their activity between 55 and 80 degrees C. Loss or decrease in the bacteriocin activity was observed with pronase E treatment; trypsin, lysozyme and alpha-amylase did not have an effect on bacteriocin activity. These findings show that the antagonism among azospirilla was due principally to the bacteriocins and sometimes probably due to siderophores, but not to bacteriophages or other substances.