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.     

The Azospirillum brasilense Che1 chemotaxis pathway controls swimming velocity, which affects transient cell-to-cell clumping

The Che1 chemotaxis-like pathway of Azospirillum brasilense contributes to chemotaxis and aerotaxis, and it has also been found to contribute to regulating changes in cell surface adhesive properties that affect the propensity of cells to clump and to flocculate. The exact contribution of Che1 to the control of chemotaxis and flocculation in A. brasilense remains poorly understood. Here, we show that Che1 affects reversible cell-to-cell clumping, a cellular behavior in which motile cells transiently interact by adhering to one another at their nonflagellated poles before swimming apart. Clumping precedes and is required for flocculation, and both processes appear to be independently regulated. The phenotypes of a ΔaerC receptor mutant and of mutant strains lacking cheA1, cheY1, cheB1, or cheR1 (alone or in combination) or with che1 deleted show that Che1 directly mediates changes in the flagellar swimming velocity and that this behavior directly modulates the transient nature of clumping. Our results also suggest that an additional receptor(s) and signaling pathway(s) are implicated in mediating other Che1-independent changes in clumping identified in the present study. Transient clumping precedes the transition to stable clump formation, which involves the production of specific extracellular polysaccharides (EPS); however, production of these clumping-specific EPS is not directly controlled by Che1 activity. Che1-dependent clumping may antagonize motility and prevent chemotaxis, thereby maintaining cells in a metabolically favorable niche.     

Calcofluor- and lectin-binding exocellular polysaccharides of Azospirillum brasilense and Azospirillum lipoferum.

Extracellular polysaccharides synthesized by Azospirillum brasilense and A. lipoferum were shown on agar plates and liquid flocculating cultures. The six strains used in this work expressed a mucoid phenotype, yielding positive calcofluor fluorescence under UV light. The calcofluor-binding polysaccharides were distributed between the capsular and exopolysaccharide fractions, suggesting exocellular localization. No calcofluor fluorescence was observed in residual cells after separation of the capsular and exopolysaccharide fractions. Cellulose content was significantly higher in flocculating than in nonflocculating cultures. Failure to induce flocculation by addition of cellulose (100 mg/ml) to nonflocculating cultures, together with the sensitivity of flocs to cellulase digestion, suggested that cellulose is involved in maintenance of floc stability. Different A. brasilense and A. lipoferum strains bound to a wheat lectin (fluorescein isothiocyanate-wheat germ agglutinin), indicating the occurrence of specific sugar-bearing receptors for wheat germ agglutinin on the cell surface. The biochemical specificity of the reaction was shown by hapten inhibition with N-acetyl-D-glucosamine. All six strains failed to recognize fluorescein isothiocyanate-soybean seed lectin under our experimental conditions. We conclude that azospirilla produce exocellular polysaccharides with calcofluor- and lectin-binding properties.     

Azospirillum lipoferum and Azospirillum brasilense surface polysaccharide mutants that are affected in flocculation

M ichiels , K., V erreth , C. & V anderleyden , J. 1990. Azospirillum lipoferum and Azospirillum brasilense surface polysaccharide mutants that are affected in flocculation. Journal of Applied Bacteriology 69 , 705–711.
Surface polysaccharide production by Azospirillum is demonstrated by fluorescence of colonies grown on media containing the fluorescent dye Calcofluor, which binds to β-linked polysaccharides. Mutants showing decreased and increased levels of fluorescence are obtained from Azospirillum lipoferum strain Sp59b by chemical mutagenesis, and from A. brasilense strain 7030 by Tn5 mutagenesis.
The A. brasilense 7030 fluorescence mutants produce wild-type levels of exo-polysaccharide in their culture supernatant fluids, but are affected in flocculation in liquid culture. On the basis of these observations, we postulate that an A. brasilense surface polysaccharide, different from the exopolysaccharide, is involved in both Calcofluor staining and flocculation.
It is shown by DNA hybridization that the genetic loci affected in the A. brasilense 7030 fluorescence mutants are different from the A. brasilense exoB and exoC loci, which are involved in exopolysaccharide production.     

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.     

Applications of yeast flocculation in biotechnological processes

A review on the main aspects associated with yeast flocculation and its application in biotechnological processes is presented. This subject is addressed following three main aspects—the basics of yeast flocculation, the development of “new” flocculating yeast strains and bioreactor development. In what concerns the basics of yeast flocculation, the state of the art on the most relevant aspects of mechanism, physiology and genetics of yeast flocculation is reported. The construction of flocculating yeast strains includes not only the recombinant constitutive flocculent brewer's yeast, but also recombinant flocculent yeast for lactose metabolisation and ethanol production. Furthermore, recent work on the heterologous β-galactosidase production using a recombinant flocculentSaccharomyces cerevisiae is considered. As bioreactors using flocculating yeast cells have particular properties, mainly associated with a high solid phase hold-up, a section dedicated to its operation is presented. Aspects such as bioreactor productivity and culture stability as well as bioreactor hydrodynamics and mass transfer properties of flocculating cell cultures are considered. Finally, the paper concludes describing some of the applications of high cell density flocculation bioreactors and discussing potential new uses of these systems.     

Antigenic identity of the capsule lipopolysaccharides,exopolysaccharides, and O-specific polysaccharides in Azospirillum brasilense

The antigenic identity (and close values of electrophoretic mobility) of capsular polysaccharides, exopolysaccharides, and O-specific polysaccharides was revealed in the Azospirillum brasilense strains Sp7 and Sp245 by the immunodiffusion and immunoelectrophoretic methods. Together with the literature data on the identity of the monosaccharides composition of these polymers, this gives evidence of the absence of a specific capsular antigen in the bacteria studied. Thus, extracellular Azospirillum brasilense polysaccharides are likely to represent O-antigenic lipopolysaccharide fragments excreted by the bacteria into the culture medium, and their identification as a capsule or as an exopolysaccharide depends on the strength of the attachment of these polysaccharides to the cell surface.     

The role of cell surface bacterial lectins in the aggregation of Azospirilla

The mutant strain Azospirillum brasilense Sp7.2.3 with impaired lectin activity exhibited poorer cell aggregation than its parent strain A. brasilense Sp7(S) both in the exponential and stationary growth phases. The pretreatment of bacterial cells with the specific haptens (L-fucose and D-galactose) of a lectin located at the cell surface of the mutant strain was found to inhibit the aggregation of azospirilla. The specific binding of the A. brasilense Sp7(S) lectin to the extracellular polysaccharide-containing complexes of this strain was revealed by dot immunoblotting on nitrocellulose membrane filters. The interaction of the lectins of A. brasilense 75, A. brasilense Sp7, and A. lipoferum 59b with the polysaccharide-containing complexes that were isolated from these strains was not specific. No interstrain cross-interaction between the exopolysaccharides and lectins of azospirilla was found. A coflocculation of A. brasilense Sp7 cells with Bacillus polymyxa 1460 cells was shown. The involvement of autogenous lectins in the aggregation of bacterial cells is discussed.     

Identification and isolation of genes involved in poly(beta-hydroxybutyrate) biosynthesis in Azospirillum brasilense and characterization of a phbC mutant

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly(beta-hydroxybutyrate) (PHB) under suboptimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments, thus showing great potential for the improvement of bacterial inoculants for plants and soils. The three genes that are considered to be essential in the PHB biosynthetic pathway, phbA (beta-ketothiolase), phbB (acetoacetyl coenzyme A reductase), and phbC (PHB synthase), were identified in Azospirillum brasilense strain Sp7, cloned, and sequenced. The phbA, -B, and -C genes were found to be linked together and located on the chromosome. An A. brasilense phbC mutant was obtained by insertion of a kanamycin resistance cassette within the phbC gene. No PHB production was detected in this mutant. The capability of the wild-type strain to endure starvation conditions was higher than that of the mutant strain. However, motility, cell aggregation, root adhesion, and exopolysaccharide (EPS) and capsular polysaccharide (CPS) production were higher in the phbC mutant strain than in the wild type.     

茎瘤固氮根瘤菌环二鸟苷酸代谢相关基因的功能鉴定

【目的】考察茎瘤固氮根瘤菌ORS571中c-di-GMP合成酶AZC-2412的编码基因缺失的突变表型,初步探究其功能机理。【方法】本实验构建基于cre-loxp重组酶系统的根瘤菌基因敲除系统,以及采用三亲接合技术构建突变株。测定野生型和突变株的生长速率、趋化能力、胞外多糖产量、生物膜形成等表型。【结果】突变株与野生型生长速率几乎相同。与野生型相比突变株由于细胞内c-di-GMP水平降低,胞外多糖、生物膜产量等均有所下降。【结论】实验表明,环二鸟苷酸合成酶AZC-2412缺失,使得c-di-GMP水平降低,对胞外多糖生成、细菌的运动能力、生物膜的形成、细胞絮凝、与植物的互作等均有调控作用。     

Flocculation behavior and mechanism of an exopolysaccharide from the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913

Flocculation behavior and mechanism of the exopolysaccharide secreted by Pseudoalteromonas sp. SM9913 (EPS SM9913), a psychrophilic bacterium isolated from 1855m deep-sea sediment, has been studied in this paper. EPS SM9913 showed a peak flocculating activity of 49.3 in 1g/L kaolin suspension with 4.55mmol/L CaCl2 and the optimum pH range of 5-8. It appears that the flocculating activity of EPS SM9913 was stimulated by Ca2+ and Fe2+. This study found that EPS SM9913 showed a better flocculation performance than Al2(SO4)3 at salinity of 5-100 per thousand or temperatures of 5-15 degrees C. In addition, this EPS was effective to flocculate several other suspended solids. The measured zeta-potentials, the size of flocs formed during the flocculation process and the surface profile of flocs revealed by scan electron micrograph suggest that bridging is the main flocculation mechanism of the studied EPS. Deacetylation of EPS SM9913 resulted in a significant decrease in its flocculating activity indicating that the large number of acetyl groups in EPS SM9913 played an important role in its flocculation performance.     

Influence of O Polysaccharides on Biofilm Development and Outer Membrane Vesicle Biogenesis in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is a common opportunistic human pathogen known for its ability to adapt to changes in its environment during the course of infection. These adaptations include changes in the expression of cell surface lipopolysaccharide (LPS), biofilm development, and the production of a protective extracellular exopolysaccharide matrix. Outer membrane vesicles (OMVs) have been identified as an important component of the extracellular matrix of P. aeruginosa biofilms and are thought to contribute to the development and fitness of these bacterial communities. The goal of this study was to examine the relationships between changes in the cell surface expression of LPS O polysaccharides, biofilm development, and OMV biogenesis in P. aeruginosa. We compared wild-type P. aeruginosa PAO1 with three chromosomal knockouts. These knockouts have deletions in the rmd, wbpM, and wbpL genes that produce changes in the expression of common polysaccharide antigen (CPA), O-specific antigen (OSA), or both. Our results demonstrate that changes in O polysaccharide expression do not significantly influence OMV production but do affect the size and protein content of OMVs derived from both CPA⁻ and OSA⁻ cells; these mutant cells also exhibited different physical properties from wild-type cells. We further examined biofilm growth of the mutants and determined that CPA⁻ cells could not develop into robust biofilms and exhibit changes in cell morphology and biofilm matrix production. Together these results demonstrate the importance of O polysaccharide expression on P. aeruginosa OMV composition and highlight the significance of CPA expression in biofilm development.     

Yeast flocculation: New story in fuel ethanol production

Yeast flocculation has been used in the brewing industry to facilitate biomass recovery for a long time, and thus its mechanism of yeast flocculation has been intensively studied. However, the application of flocculating yeast in ethanol production garnered attention mainly in the 1980s and 1990s. In this article, updated research progress in the molecular mechanism of yeast flocculation and the impact of environmental conditions on yeast flocculation are reviewed. Construction of flocculating yeast strains by genetic approach and utilization of yeast flocculation for ethanol production from various feedstocks were presented. The concept of self-immobilized yeast cells through their flocculation is revisited through a case study of continuous ethanol fermentation with the flocculating yeast SPSC01, and their technical and economic advantages are highlighted by comparing with yeast cells immobilized with supporting materials and regular free yeast cells as well. Taking the flocculating yeast SPSC01 as an example, the ethanol tolerance of the flocculating yeast was also discussed.     

Arabinose content of extracellular polysaccharide plays a role in cell aggregation of Azospirillum brasilense

Extracellular polysaccharides play an important role in aggregation and surface colonization of plant-associated bacteria. In this work, we report the time course production and monomer composition of the exopolysaccharide (EPS) produced by wild type strain and several mutants of the plant growth promoting rhizobacterium (PGPR) Azospirillum brasilense. In a fructose synthetic medium, wild type strain Sp7 produced a glucose-rich EPS during exponential phase growth and an arabinose-rich EPS during stationary and death phase growth. D-glucose or L-arabinose did not support cell growth as sole carbon sources. However, glucose and arabinose-rich EPSs, when used as carbon source, supported bacterial growth. Cell aggregation of Sp7 correlated with the synthesis of arabinose-rich EPS. exoB (UDP-glucose 4'-epimerase), exoC (phosphomannomutase) and phbC (poly-beta-hydroxyburyrate synthase) mutant strains, under tested conditions, produced arabinose-rich EPS and exhibited highly cell aggregation capability. A mutant defective in LPS production (dTDP 4-rhamnose reductase; rmlD) produced glucose-rich EPS and did not aggregate. These results support that arabinose content of EPS plays an important role in cell aggregation. Cell aggregation appears to be a time course phenomenon that takes place during reduced metabolic cell activity. Thus, aggregation could constitute a protected model of growth that allows survival in a hostile environment. The occurrence of exoC and rmlD was detected in several species of Azospirillum.     

Production of an exopolysaccharide bioflocculant by Sorangium cellulosum

AIMS: To isolate a new exopolysaccharide bioflocculant produced by the myxobacterium Sorangium cellulosum NUST06, and to characterize its chemical composition and expolysaccharide production relative to carbon source. METHODS AND RESULTS: Exopolysaccharide levels and biomass production by S. cellulosum NUST06 were analysed relative to carbon source. Glucose in the medium at a level of 3 g l(-1) completely inhibited cell growth and exopolysaccharide production, but low concentrations of glucose (1-2 g l(-1)) could stimulate cell utilization of starch. The chemical composition and flocculating activity of the NUST06 exopolysaccharide was investigated. The flocculant comprised 38.3% proteins and 58.5% carbohydrates, of which glucose, mannose and glucuronic acid were present at 51.3%, 39.2% and 10.5%, respectively. The flocculating activity of the NUST06 flocculant depended strongly on cations. CONCLUSIONS: It is feasible to produce an exopolysaccharide bioflocculant by the strain NUST06 in a mineral salts medium using starch as a carbon source. SIGNIFICANCE AND IMPACT OF THE STUDY: This strain may be advantageous for commercial bioflocculant production and may enrich existing knowledge of myxobacteria.     

Ssg, a putative glycosyltransferase, functions in lipo- and exopolysaccharide biosynthesis and cell surface-related properties in Pseudomonas alkylphenolia

In the presence of vaporized p-cresol, Pseudomonas alkylphenolia KL28 forms specialized aerial structures (SAS). A transposon mutant of strain KL28 (C23) incapable of forming mature SAS was isolated. Genetic analysis of the C23 mutant revealed the transposon insertion in a gene (ssg) encoding a putative glycosyltransferase, which is homologous to the Pseudomonas aeruginosa PAO1 PA5001 gene. Deletion of ssg in KL28 caused the loss of lipopolysaccharide O antigen and altered the composition of the exopolysaccharide. Wild-type KL28 produced a fucose-, glucose- and mannose-rich exopolysaccharide, while the mutant exopolysaccharide completely lacked fucose and mannose, resulting in an exopolysaccharide with glucose as the major component. The mutant strain showed reduced surface spreading, pellicle and biofilm formation, probably due to the cumulative effect of lipopolysaccharide truncation and altered exopolysaccharide composition. Our results show that the ssg gene of KL28 is involved in both lipopolysaccharide and exopolysaccharide biosynthesis and thus plays an important role in cell surface properties and cell-cell interactions of P. alkylphenolia.     

Characterization of a novel bioflocculant, p-KG03, from a marine dinoflagellate, Gyrodinium impudicum KG03

The flocculating activity of an exopolysaccharide, p-KG03, produced by a marine dinoflagellate Gyrodinium impudicum KG03 was investigated. The p-KG03 was a highly sulfated exopolysaccharide that showed strong antiviral activity against encephalomyocarditis virus (EMCV) and immunostimulating activity by NK cell activation. For the industrial applications of p-KG03, as the bioflocculant agent, p-KG03 showed that more than 90% of the flocculating activity in kaolin suspension occurred at concentrations of 0.5 mg/l with the maximum at 1.0 mg/l. However, flocculation decreased from 2.5 mg/l. The flocculation rate increased linearly with concentration and was higher than that observed in commercial products such as polyacrylamide (approximately 1.0 mg/l) or zooglan (approximately 3.0 mg/l). The p-KG03 was an effective flocculant under acidic conditions (pH 3-6) and over a wide temperature range (4-90 degrees C). The presence of cations did not enhance flocculating activity. The average molecular mass, as determined by gel filtration chromatography, was about 1.87 x 10(3) KDa. Galactose was the main sugar in p-KG03, which also contained uronic acid (2.9%, w/w) and sulfate groups (10.3%, w/w). The infrared spectrum of p-KG03 showed absorption bands of carboxylate groups. Thermogravimetric analysis indicated a degradation temperature (T(d)) of 250 degrees C. Several other properties of p-KG03 such as intrinsic viscosity, the rheological behavior, consistency index (k) and flow behavior index (eta) were also studied.     

Formation of TRA-dependent surface structures in Agrobacterium tumefaciens and their absence in R1 (deltatraR) mutant

Agrobacteria have Ti plasmid DNA delivering systems for the transfer to recipient cells by the conjugation mechanism. This transfer is absolutely dependent on induction tra genes. It is not clear which tra-dependent surface (extracellular) proteins (structures) are involved in the transport mechanism and whether these proteins also play a role in the contact formation. SDS-PAGE electrophoresis of proteins released from the cell showed disappearance of 63 and 67 kD proteins in R1(delta traR) strain, which were found in the growth medium and triton extract from the outer membrane of Ti plasmid-harboring A. tumefaciens R10 strains. The traR defective mutant did not express these proteins and had a higher hemagglutination and flocculation capacity than the wild strain. On the other hand, the wild strain showed D-galactose and N-acetyl-galactosamine specific hemagglutination which was not shown by traR mutant. Motility and chemotactic behavior of traR mutant in semisolid medium were defective. As a rule, one (or rarely two) thread-like connections in vir(-) and tra(+) conditions were observed on the agrobacterial cell surface. SDS pretreatment of agrobacterial cells had a significant effect on the expression of tra-dependent surface structures.