Quorum sensing activity of the plant growth-promoting rhizobacterium <Emphasis Type="Italic">Serratia glossinae</Emphasis> GS2 isolated from the sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) rhizosphere

Plant growth-promoting rhizobacteria (PGPR) affect plant growth through various mechanisms, such as indole-3-acetic acid (IAA) production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and biofilm formation. The aim of the study reported here was to isolate and characterize rhizobacteria that produce quorum-sensing signal molecules and other PGPR-related molecules. A biofilm-forming bacterium, GS2, was isolated from the rhizosphere of a sesame plant and subsequently found to produce two quorum-sensing signal molecules that were identified as N-hexanoyl-L-homoserine lactone (m/z 200) and N-octanoyl-L-homoserine lactone (m/z 228) by liquid chromatography–tandem mass spectrometry analysis. The strain was also found to produce IAA (17.2 μg mL^?1), gibberellins (113.7 μg mL^?1), and ACC deaminase (9.7 μM α-ketobutyrate mg^?1 protein h^?1). The strain was identified as Serratia glossinae based on a comparison of 16S rRNA gene sequences. Inoculation of the strain promoted growth of a gibberellin-deficient rice dwarf mutant (Waito-C). Different growth attributes, including shoot and root elongation, chlorophyll content, and plant weight could be attributed to the PGPR characteristics of strain GS2. These results suggest that S. glossinae strain GS2 can serve as a microbial agent that improves plant growth.     

Culturable bacterial diversity from a feed water of a reverse osmosis system,evaluation of biofilm formation and biocontrol using phages

Biofilm formation on reverse osmosis (RO) systems represents a drawback in the application of this technology by different industries, including oil refineries. In RO systems the feed water maybe a source of microbial contamination and thus contributes for the formation of biofilm and consequent biofouling. In this study the planktonic culturable bacterial community was characterized from a feed water of a RO system and their capacities were evaluated to form biofilm in vitro. Bacterial motility and biofilm control were also analysed using phages. As results, diverse Protobacteria, Actinobacteria and Bacteroidetes were identified. Alphaproteobacteria was the predominant group and Brevundimonas, Pseudomonas and Mycobacterium the most abundant genera. Among the 30 isolates, 11 showed at least one type of motility and 11 were classified as good biofilm formers. Additionally, the influence of non-specific bacteriophage in the bacterial biofilms formed in vitro was investigated by action of phages enzymes or phage infection. The vB_AspP-UFV1 (Podoviridae) interfered in biofilm formation of most tested bacteria and may represent a good alternative in biofilm control. These findings provide important information about the bacterial community from the feed water of a RO system that may be used for the development of strategies for biofilm prevention and control in such systems.     

Dependence of Continuous-Flow Biofilm Formation by Listeria monocytogenes EGD-e on SOS Response Factor YneA

Listeria monocytogenes was previously shown to form biofilms composed of a network of knitted chains under continuous-flow conditions. Here we show that the SOS response is activated under these conditions and that deletion of its regulon member yneA results in diminished biofilm formation under continuous-flow conditions.The food-borne pathogen Listeria monocytogenes is widely distributed in the environment and is able to grow in soil and on plant materials, thereby facilitating environmental transmission of this pathogen. L. monocytogenes is therefore frequently encountered in food processing facilities, on food contact surfaces, in pipelines, on floors, and in drains, which in turn may result in contamination of food products. It is expected that the formation of biofilms and subsequent dispersal plays an important role in recontamination processes. Biofilms are structured communities of microorganisms adhering to a surface that may be encapsulated within a self-produced protective and adhesive matrix of extracellular polymeric substances (EPS) (9). Most studies of L. monocytogenes biofilm formation focus on biofilm formation under static conditions on polystyrene, glass, or stainless steel surfaces. L. monocytogenes biofilms on polystyrene and glass consist of a homogeneous layer, while on stainless steel L. monocytogenes biofilms consist of single attached cells or microcolonies (2, 6, 11). The small, rod-shaped morphology of these static biofilm cells is very similar to the morphology of planktonic cells. However, L. monocytogenes biofilms formed under continuous-flow conditions, conceivably encountered in industrial pipelines, consist of a dense network of knitted chains composed of elongated cells and surrounding ball-shaped microcolonies (10). Recently, it has been shown that activation of the L. monocytogenes SOS response factor YneA resulted in cell elongation (14). The SOS response is involved in DNA repair, restart of stalled replication forks (3, 8), and mutagenesis (12). It is regulated by RecA (activator) and LexA (repressor) and furthermore contains DNA repair systems and translesion DNA polymerases such as DinB (15). To prevent transaction of the genome during replication fork stalling, septum formation at the midcell is inhibited by YneA, which results in cell elongation (7, 14). Recently, RecA-dependent genetic recombination was described for Pseudomonas aeruginosa biofilm cells harvested from a drip flow reactor, pointing to activation of the SOS response under these conditions (1). In this study, we investigated whether the SOS response is activated during L. monocytogenes EGD-e biofilm formation and whether there is a role for YneA in knitted chain biofilm formation.L. monocytogenes EGD-e (5), its isogenic in-frame ΔrecA and ΔyneA deletion mutants, its yneA complementation mutant, and its recA and yneA promoter reporter mutants (14) were grown in brain heart infusion (BHI; Difco) broth. No significant difference in planktonic growth between wild-type and mutant cultures was observed (results not shown). Continuous-flow biofilm formation experiments were performed as described previously (10) with small modifications. Biofilms were grown in a flow cell (BST FC 281; Biosurface Technologies Corporation) at 20°C, using BHI with a flow rate of 10 ml/h. Static biofilm experiments were performed as described previously (4) with small modifications. Biofilms were grown in BHI in 12-well polystyrene microtiter plates (Greiner) using a 1% inoculum of an overnight-grown culture. For quantification, the biofilm cells were harvested in phosphate-buffered saline (PBS), serially diluted in PBS, and plated on BHI agar. Colonies were enumerated after 2 days of incubation at 30°C. Quantitative real-time PCR analysis was performed as described previously (13) using primers shown in appendix S1 in the supplemental material. Shortly, biofilms were quenched in RNAprotect (Qiagen) following the manufacturer''s protocol and harvested. Expression levels were normalized using the housekeeping genes tpi, rpoB, and 16S rRNA. Biofilm formation experiments and quantitative PCR (Q-PCR) analysis were performed in two independent biological experiments using two replicates each. Statistically significant differences were identified using a two-tailed Student t test (P < 0.05).To investigate activation of L. monocytogenes EGD-e SOS response during continuous-flow biofilm formation, Q-PCR analysis of the SOS response genes recA, lexA, yneA, and dinB and promoter reporter studies using the promoters for recA and yneA were performed (Fig. ​(Fig.1).1). Compared with the reference (planktonic cells from a 48-h liquid culture), all four tested SOS response genes were upregulated in wild-type strain cells isolated from a 48-h continuous-flow biofilm (P < 0.05, t test), but not in cells isolated from a 48-h static biofilm (Fig. ​(Fig.1A).1A). Furthermore, the ΔrecA mutant strain did not show upregulation of yneA and the other SOS response genes during continuous-flow biofilm formation, indicating that RecA is required for activation of the SOS response during continuous-flow biofilm formation. Furthermore, continuous-flow biofilm formation also resulted in visible expression of enhanced green fluorescent protein (EGFP) for both yneA and recA promoter reporters (Fig. 1B and C). Expression of EGFP was not observed for these promoter reporters in planktonic cells grown in liquid culture or during static biofilm formation (results not shown). These results indicate that the SOS response is specifically activated during continuous-flow biofilm formation.Open in a separate windowFIG. 1.Activation of the SOS response during biofilm formation. (A) The graph shows differential expression of four SOS response genes in the wild-type and ΔrecA mutant strain between 48-h stationary-phase cultures (black), 48-h static biofilms (light gray), and 48-h continuous-flow biofilms (dark gray). Expression for each SOS response gene in the wild-type 48-h stationary-phase cultures is set at 1. (B and C) Micrographs show fluorescence (1) and phase-contrast (2) pictures of cells expressing EGFP from the recA (B) and yneA (C) promoters after 48 h of biofilm formation in BHI at 20°C under continuous-flow conditions.The impact of RecA and YneA on biofilm formation was assessed using the wild-type strain and in-frame ΔyneA and ΔrecA strains (Fig. ​(Fig.2).2). Both ΔrecA and ΔyneA mutants showed a significant deficiency in total biofilm produced under continuous-flow conditions (P < 0.05, t test), which was approximately 100-fold lower than that of the wild-type strain. No significant difference in static biofilm formation between wild-type and mutant strains was observed. Apparently, YneA and RecA are not required for static biofilm formation, which is in line with the lack of activation of the SOS response under these conditions. The wild-type, ΔyneA, and ΔrecA strains were microscopically examined during continuous-flow biofilm formation (Fig. ​(Fig.3).3). Analysis of the number of adherent cells 1 h after the start of the experiment did not reveal differences between the wild-type strain and the two mutants, which indicates that initial attachment is similar. After 24 h, the wild-type strain biofilm appeared to be composed of a complex structure of elongated cells forming a network of knitted chains, which after 48 h had developed into a denser network containing ball-shaped microcolonies. These results are in concordance with the study by Rieu et al. (10). However, both ΔyneA and ΔrecA mutant strains showed only some patches of adherent cells after 24 h, which developed into very small microcolonies after 48 h. Thus, formation of elongated cells in a network of knitted chains was not observed for these mutants. These results indicate that RecA and YneA are required to form this type of biofilm. To verify the specific role of YneA in continuous-flow biofilm formation, a yneA complementation mutant was constructed, which indeed showed biofilm formation capacity similar to that of the wild type, under both continuous-flow and static conditions (results not shown).Open in a separate windowFIG. 2.Comparative analysis of biofilm formation between wild-type strain and ΔrecA and ΔyneA mutants under continuous-flow and static conditions. The graph shows the amount of biofilm produced by wild-type and mutant strains after 48 h of biofilm formation at 20°C under continuous-flow (dark gray) and static (light gray) conditions. *, significantly different from wild-type strain (P < 0.05, t test).Open in a separate windowFIG. 3.Knitted chain biofilm formation under continuous-flow conditions is dependent on RecA and YneA. The micrographs show biofilms formed after 1, 24, and 48 h in BHI at 20°C for the wild-type strain (A), the ΔrecA mutant strain (B), and the ΔyneA mutant strain (C).This study established a clear link between the SOS response and knitted chain biofilm formation under continuous-flow conditions. RecA-dependent activation of the SOS response and in particular of yneA under continuous-flow conditions resulted in cell elongation and the formation of knitted chain biofilms. The signals that activate the L. monocytogenes SOS response are currently being studied and may provide tools for control of biofilm formation under continuous-flow conditions.      

Molecular diversity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing PGPR from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) rhizosphere

Aims

The present study was planned to investigate the diversity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing bacteria from the rhizosphere of wheat plants and subsequent evaluation of selected PGPR on growth enhancement of wheat seedlings under drought and saline conditions.

Methods

ACC deaminase producing plant growth promoting rhizobacteria (PGPR) were isolated from the rhizosphere of wheat and identified using 16S rRNA gene sequence analysis. Isolates were evaluated for various direct and indirect plant growth promoting (PGP) traits. Plant inoculation experiment was conducted using isolates IG 19 and IG 22 in wheat to assess their plant growth promotion potential under salinity and drought stress.

Results

Thirty-eight ACC deaminase producing PGPR were isolated which belonged to 12 distinct genera and falling into four phyla γ-proteobacteria, β-proteobacteria, Flavobacteria and Firmicutes. Klebsiella sp. was the most abundant genera and followed by Enterobacter sp. The isolates exhibited ACC deaminase activities ranging from 0.106–0.980 μM α- ketobutyrate μg protein^?1 h^?1. The isolates showed multiple PGP traits such as IAA production, phosphate, zinc, potassium solubilization and siderophore production. Enterobacter cloacae (IG 19) and Citrobacter sp. (IG 22) inoculated wheat seedlings showed notable increases in fresh and dry biomass under non-stress as well as under stressed condition.

Conclusion

To the best of our knowledge this is the first report of presence of ACC deaminase activity and other PGP traits from the genus Citrobacter and Empedobacter. Our finding revealed that the γ-proteobacteria group dominated the wheat rhizosphere. Plant inoculation with PGPR could be a sustainable approach to alleviate abiotic stresses in wheat plants. These native PGPR isolates could be used as potential biofertilizers for sustainable agriculture.

     

Potent in vitro synergism of fusidic acid (FA) and berberine chloride (BBR) against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA)

It was found in the present study that combined use of fusidic acid (FA) and berberine chloride (BBR) offered an in vitro synergistic action against 7 of the 30 clinical methicillin-resistant Staphylococcus aureus (MRSA) strains, with a fractional inhibitory concentration (FIC) index ranging from 0.5 to 0.19. This synergistic effect was most pronounced on MRSA 4806, an FA-resistant isolate, with a minimum inhibitory concentration (MIC) value of 1,024 μg/ml. The time-kill curve experiment showed that FA plus BBR yielded a 4.2 log₁₀ c.f.u./ml reduction in the number of MRSA 4806 bacteria after 24-h incubation as compared with BBR alone. Viable count analysis showed that FA plus BBR produced a 3.0 log₁₀ c.f.u./ml decrease in biofilm formation and a 1.5 log₁₀ c.f.u./ml decrease in mature biofilm in viable cell density as compared with BBR alone. In addition, phase contrast micrographs confirmed that biofilm formation was significantly inhibited and mature biofilm was obviously destructed when FA was used in combination with BBR. These results provide evidence that combined use of FA and BBR may prove to be a promising clinical therapeutic strategy against MRSA.     

Molecular charcterization of tatD DNAse gene from Ralstonia paucula RA4T soil bacterium

Ralstonia paucula strain RA4^T, a gram negative, non-spore forming, motile bacterium having positive catalase and oxidase test, was isolated from surface soil. Twin arginine translocation protein type D (TatD) is shown to be located in cytoplasm and exhibits magnesium-dependent DNase. A tatD DNase gene was isolated and cloned from Ralstonia paucula RA4^T genome. Nucleotide sequence analysis of the gene revealed 813 nucleotides encoding a protein of 270 amino acid residues. The tatD gene showed a high similarity to homolog gene from Ralstonia pickettii strain 12D. The deduced polypeptide sequence of TatD DNase from R. paucula RA4^T had a typical catalytic site, HHPLDEHRHDP, and its calculated molecular mass and predicted isoelectric point were 29616 Da and 5.33, respectively. The deduced amino acid sequence showed a high degree of similarity to TatD DNase isoforms from Ralstonia genus and other sources. Predicted three-dimensional structure of TatD confirmed the presence of active site and theoretical function as DNase.     

Effects of different plant root exudates and their organic acid components on chemotaxis, biofilm formation and colonization by beneficial rhizosphere-associated bacterial strains

Aim

It is necessary to understand the roles of root exudates involved in plant-microbe interactions to inform practical application of beneficial rhizosphere microbial strains.

Methods

Colonization of Bacillus amyloliquefaciens SQR9 (isolated from cucumber rhizosphere) and Bacillus subtilis N11 (isolated from banana rhizosphere) of their original host was found to be more effective as compared to the colonization of the non-host plant. Organic acids in the root exudates of the two plants were identified by High performance liquid chromatography (HPLC). The chemotactic response and effects on biofilm formation were assessed for SQR9 and N11 in response to cucumber and banana root exudates, as well as their organic acids components.

Results

Citric acid detected exclusively in cucumber exudates could both attract SQR9 and induce its biofilm formation, whereas only chemotactic response but not biofilm formation was induced in N11. Fumaric acid that was only detected in banana root exudates revealed both significant roles on chemotaxis and biofilm formation of N11, while showing only effects on biofilm formation but not chemotaxis of SQR9.

Conclusion

The relationship between PGPR strain and root exudates components of its original host might contribute to preferential colonization. This study advances a clearer understanding of the mechanisms relevant to application of PGPR strains in agricultural production.     

Pronounced Effect of the Nature of the Inoculum on Biofilm Development in Flow Systems

Biofilm formation renders sessile microbial populations growing in continuous-flow systems less susceptible to variation in dilution rate than planktonic cells, where dilution rates exceeding an organism''s maximum growth rate (μ_max) results in planktonic cell washout. In biofilm-dominated systems, the biofilm''s overall μ_max may therefore be more relevant than the organism''s μ_max, where the biofilm μ_max is considered as a net process dependent on the adsorption rate, growth rate, and removal rate of cells within the biofilm. Together with lag (acclimation) time, the biofilm''s overall μ_max is important wherever biofilm growth is a dominant form, from clinical settings, where the aim is to prevent transition from lag to exponential growth, to industrial bioreactors, where the aim is to shorten the lag and rapidly reach maximum activity. The purpose of this study was to measure CO₂ production as an indicator of biofilm activity to determine the effect of nutrient type and concentration and of the origin of the inoculum on the length of the lag phase, biofilm μ_max, and steady-state metabolic activity of Pseudomonas aeruginosa PA01 (containing gfp), Pseudomonas fluorescens CT07 (containing gfp), and a mixed community. As expected, for different microorganisms the lengths of the lag phase in biofilm development and the biofilm μ_max values differ, whereas different nutrient concentrations result in differences in the lengths of lag phase and steady-state values but not in biofilm μ_max rates. The data further showed that inocula from different phenotypic origins give rise to lag time of different lengths and that this influence persists for a number of generations after inoculation.Microbial growth in batch cultures has been studied for a long time, and the observed phases have been designated the lag phase, the acceleration phase, the exponential phase, the retardation phase, the stationary phase, and the phase of decline although not each culture displays all of the mentioned phases (16). In contrast to batch cultures and static (no flow) biofilms (e.g., those that form in 96-well plates), the increase in biofilm cells in a flowing environment is a net process that is dependent on the irreversible adsorption rate of cells to the surface, the growth rate of the microorganisms, and the removal rate of cells lost to the bulk flow (18). There are numerous benefits for the cells in biofilms, e.g., protection against antimicrobials and the opportunity for and proliferation by continuous cell dispersion. There is also a possible competitive advantage if cells colonize surfaces at multiple sites and grow in such a manner that the resulting three-dimensional architecture exposes the maximum biofilm surface area to surrounding nutrients. The most successful colonizers would therefore be the cells with the ability to adhere to the surface (and stay adhered) and to start multiplying at maximum rate. The process of events from being free-floating cells to the so-called permanently surface-attached phase involves early steps including reversible attachment and a phenotypic change in the cells from a planktonic state to a sessile state, with concomitant changes in gene expression; these steps contribute to a lag phase that will occur before maximal growth/biofilm development can take place (23). Clearly, the ability to progress from the lag phase to a fast-growing phase, as well as the duration of the lag phase, is an important determinant of biofilm function and has an impact in a diverse range of environments, often with implications for infection or contamination control, as well as in industrial processes.At the cell level, an extended lag phase and slower growth create the risk that the cells will be displaced by faster-growing microcolonies, as was demonstrated by Klayman et al. (13) in dual species biofilms. A microorganism''s competence in dominating a surface area can therefore be evaluated by comparing the lag phases and maximal growth rates (μ_max) of a biofilm growth curve. Knowing a bacterial population''s specific growth rate is a requirement for its cultivation at optimum rates in a chemostat or other continuously fed bioreactor. A key assumption for this type of cultivation is that wall growth has a negligible effect, which is in stark contrast to systems where surface-associated growth dominates. Indeed, while dilution rates exceeding an organism''s μ_max results in cell washout in a conventional chemostat setting, biofilm formation enables microbial populations to persist at dilution rates much higher than the organism''s μ_max.Biofilm growth rates have been determined by various techniques, such as fluorescence in situ hybridization (FISH) (12, 31), measuring the incorporation of radioactive substances like [³H]thymidine and ³²P (8, 9), microscopy (3, 13, 17), measuring the total increase in biofilm mass (both cells and extracellular polymeric substances) (24, 28), colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-amino)carbonyl]-2H- tetrazolium hydroxide (XTT) assays (26), or measurement of amide II bands, as determined by attenuated total reflectance-Fourier transform infrared spectroscopy (6). Some of the above-mentioned techniques suffer the drawback that the biofilms have to be sacrificed with sampling or that the measured increases do not distinguish between live and dead matter in the biofilm (i.e., increases measured might not represent an accurate increase in viable cell numbers).In this study a carbon dioxide evolution measurement system (CEMS) (15) was used to track the biofilm development rate in real time. The advantage of using this system is that the measured rates represent the metabolic activity of the active cell mass and can be done nondestructively for any biofilm-forming microorganism. In the past, measurement of oxygen uptake rates has been used for determination of growth rates in batch cultures (19) and of the localized growth rate in biofilms (32). CO₂ measurements by a gas chromatograph have been used to determine growth rates in batch systems (4), but to our knowledge this is the first time that CO₂ measurements have been used to determine whole-biofilm specific growth rates. We applied this technique to compare the biofilm μ_max values for two well-described pseudomonads and a mixed microbial community when the organisms are grown on different nutrients and to test the premise that the origin of the inoculum has an impact on early biofilm development.     

Influence of the shear stress and salinity on Anammox biofilms formation: modelling results

Anammox biomass has a long duplication time and low yield, thus the process must be operated in reactors with good sludge retention, such as biofilm systems. Therefore, it would be important to research the ability of Anammox biomass to form biofilms under different conditions. The effects of shear stress and salinity (NaCl and CaCl₂) on Anammox biofilm formation were studied. Anammox bacteria showed good attachment capacity, with an initial adhesion phase lasting for 5–7 days at the different flow rates tested (Reynolds numbers 54, 63, 188 and 400). A four-parameter model was developed and the experimental data fitted well into the model. The presence of 5 g/L of each of the two salts favoured the formation of Anammox biofilm. The effects of CaCl₂ were stronger than those caused by NaCl. 15 g/L of NaCl was detrimental for the biofilm, probably due to an inhibitory effect.     

Alleviation of Mercury Toxicity in Wheat by the Interaction of Mercury-Tolerant Plant Growth-Promoting Rhizobacteria

Heavy metal contamination of agricultural soils has increased along with industrialization. Mercury is a toxic heavy metal and a widespread pollutant in the ecosystem. Mercury-tolerant and plant growth-promoting rhizobacteria (PGPR) HG 1, HG 2, and HG 3 were isolated from the rhizosphere of plants growing in a mercury-contaminated site. These isolates were able to grow in the presence of mercury ranging from 10 to 200 µM in minimal medium and 25 to 500 µM in LB medium. The strains were characterized by morphological, biochemical, and plant growth-promoting traits. In the present study, these PGPR strains were analyzed for their involvement in metal stress tolerance in Triticum aestivum (wheat). Two bacterial strains, namely, Enterobacter ludwigii (HG 2) and Klebsiella pneumoniae (HG 3), showed better growth promotion of T. aestivum seedlings under metal stress. Different growth parameters like, water content and biochemical properties were analyzed in the PGPR-inoculated wheat plants under 75 µM HgCl₂. Shoot length, root length, shoot dry weight, root dry weight and relative water content (RWC) were significantly higher in inoculated plants compared to uninoculated plants under stress condition. Proline content, electrolyte leakage, and malondialdehyde content (shoots and roots) were significantly lower in inoculated plants with respect to uninoculated plants under mercury stress. Therefore, it could be assumed that all these parameters collectively improve plant growth under mercury stress conditions in the presence of PGPR. Hence, these PGPRs can serve as promising candidates for increasing plant growth and also have immense potential for bioremediation of mercury-contaminated soils.     

Kinetics of nitrification in a fixed biofilm reactor using dewatered sludge-fly ash composite ceramic particle as a supporting medium

A mathematical model system was derived to describe the kinetics of ammonium nitrification in a fixed biofilm reactor using dewatered sludge-fly ash composite ceramic particle as a supporting medium. The model incorporates diffusive mass transport and Monod kinetics. The model was solved using a combination of the orthogonal collocation method and Gear’s method. A batch test was conducted to observe the nitrification of ammonium-nitrogen ( \({\text{NH}}_{4}^{ + }\) -N) and the growth of nitrifying biomass. The compositions of nitrifying bacterial community in the batch kinetic test were analyzed using PCR–DGGE method. The experimental results show that the most staining intensity abundance of bands occurred on day 2.75 with the highest biomass concentration of 46.5 mg/L. Chemostat kinetic tests were performed independently to evaluate the biokinetic parameters used in the model prediction. In the column test, the removal efficiency of \({\text{NH}}_{4}^{ + }\) -N was approximately 96 % while the concentration of suspended nitrifying biomass was approximately 16 mg VSS/L and model-predicted biofilm thickness reached up to 0.21 cm in the steady state. The profiles of denaturing gradient gel electrophoresis (DGGE) of different microbial communities demonstrated that indigenous nitrifying bacteria (Nitrospira and Nitrobacter) existed and were the dominant species in the fixed biofilm process.     

Synergistic effect of Pseudomonas putida and Bacillus amyloliquefaciens ameliorates drought stress in chickpea (Cicer arietinum L.)

Two plant growth promoting rhizobacteria (PGPR) Pseudomonas putida NBRIRA and Bacillus amyloliquefaciens NBRISN13 with ability to tolerate abiotic stress along with multiple PGP traits like ACC deaminase activity, minerals solubilisation, hormones production, biofilm formation, siderophore activity were evaluated for their synergistic effect to ameliorate drought stress in chickpea. Earlier we have reported both the strains individually for their PGP attributes and stress amelioration in host plants. The present study explains in detail the possibilities and benefits of utilizing these 2 PGPR in consortium for improving the chickpea growth under control and drought stressed condition. In vitro results clearly demonstrate that both the PGPR strains are compatible to each other and their synergistic growth enhances the PGP attributes. Greenhouse experiments were conducted to evaluate the effect of inoculation of both strains individually and consortia in drought tolerant and sensitive cultivars (BG362 and P1003). The growth parameters were observed significantly higher in consortium as compared to individual PGPR. Colonization of both PGPR in chickpea rhizosphere has been visualized by using gfp labeling. Apart from growth parameters, defense enzymes, soil enzymes and microbial diversity were significantly modulated in individually PGPR and in consortia inoculated plants. Negative effects of drought stress has been ameliorated and apparently seen by higher biomass and reversal of stress indicators in chickpea cultivars treated with PGPR individually or in consortia. Findings from the present study demonstrate that synergistic application has better potential to improve plant growth promotion under drought stress conditions.     

Cytophysiological characteristics of Arabidopsis thaliana cultivated cells with disable perception of ethylene signal by the ETR1 receptor

Contradictory data about ethylene influence on cell growth and division prompted us to investigate cytophysiological characteristics of suspension cultures of Arabidopsis thaliana of wild type Col-0 and ert1-1 mutant carrying a point mutation in the site of ethylene binding by the ETR1 receptor. Some cytophysiological characteristics of the etr1-1 cultivated cells differed from those of Col-0: the growth rate of mutant cells was less and cell sizes were smaller, the culture was committed to the formation of tracheary elements (TE), had a pronounced modal class of nuclei (54%) with the amount of DNA 8C and a tendency to expand the ploidy toward 32C. Despite the absence of ethylene perception by the ETR1 receptor, the cell culture of mutant responded to treatment with ethylene by growth acceleration, an increase in cell viability and in the number of cells in the S-phase of the cell cycle. The inhibitor of ethylene binding to receptors, 1-methylcyclopropene, suppressed growth and viability of the cells of both genotypes. In the etr1-1 cell culture, the inhibitor reduced the number of S-phase nuclei and activated TE formation. All data obtained indicate that ethylene perception and transduction of ethylene signal are required for the maintenance of cell viability and active in vitro growth. It is supposed that the functional activity of the ETR1 receptor is necessary for optimal cell expansion, whereas other receptors are responsible for cell proliferation.     

In vitro culture of sweet basil: gas exchanges,growth, and rosmarinic acid production

Five in vitro culture systems with different ventilation rates were used to investigate the influence of vessel environment on photosynthesis, dark respiration, ethylene evolution, and rosmarinic acid (RA) production in sweet basil (Ocimum basilicum L.) micropropagated shoots. The systems under comparison were two bioreactors with either temporary (RITA?) or stationary (Growtek?) immersion, and three types of vessels (Magenta?, Microbox ECO ₂ ?, and PCCV25?) that are largely used for plant micropropagation. Shoots of green-leaved cv. Genovese and purple-leaved cv. Dark Opal were cultured on a modified Murashige and Skoog medium containing 0.25 mg dm^?3 6-benzylaminopurine. The instantaneous rates of photosynthesis, dark respiration, and ethylene production were determined by gas chromatography measuring CO₂ and ethylene concentrations in vessel headspaces. The tissue RA content was determined by HPLC in HCl-methanol extracts. The explant growth and morphology were significantly affected by culture conditions and cultivars. The largest biomass production was observed under the photomixotrophic culture conditions provided by Growtek?, whereas the highest RA content in shoot tissues was found in the RITA? photomixotrophic system, where ethylene accumulated to the greatest extent.     

The Cyclic-di-GMP Phosphodiesterase BinA Negatively Regulates Cellulose-Containing Biofilms in Vibrio fischeri

Bacteria produce different types of biofilms under distinct environmental conditions. Vibrio fischeri has the capacity to produce at least two distinct types of biofilms, one that relies on the symbiosis polysaccharide Syp and another that depends upon cellulose. A key regulator of biofilm formation in bacteria is the intracellular signaling molecule cyclic diguanylate (c-di-GMP). In this study, we focused on a predicted c-di-GMP phosphodiesterase encoded by the gene binA, located directly downstream of syp, a cluster of 18 genes critical for biofilm formation and the initiation of symbiotic colonization of the squid Euprymna scolopes. Disruption or deletion of binA increased biofilm formation in culture and led to increased binding of Congo red and calcofluor, which are indicators of cellulose production. Using random transposon mutagenesis, we determined that the phenotypes of the ΔbinA mutant strain could be disrupted by insertions in genes in the bacterial cellulose biosynthesis cluster (bcs), suggesting that cellulose production is negatively regulated by BinA. Replacement of critical amino acids within the conserved EAL residues of the EAL domain disrupted BinA activity, and deletion of binA increased c-di-GMP levels in the cell. Together, these data support the hypotheses that BinA functions as a phosphodiesterase and that c-di-GMP activates cellulose biosynthesis. Finally, overexpression of the syp regulator sypG induced binA expression. Thus, this work reveals a mechanism by which V. fischeri inhibits cellulose-dependent biofilm formation and suggests that the production of two different polysaccharides may be coordinated through the action of the cellulose inhibitor BinA.Bacterial biofilms play important roles in the environment and in interactions with eukaryotic hosts (for reviews, see references 17 and 32). Exopolysaccharides are a major component of biofilms (23), and many bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Salmonella spp., have the ability to produce multiple different exopolysaccharides (23). For some of these bacteria, it has been demonstrated that the different polysaccharides contribute to biofilm formation in different settings. For example, several strains of Salmonella require an exopolysaccharide called O-antigen capsule to form biofilms on human gallstones but not to form biofilms on glass or plastic (11). Conversely, the exopolysaccharides cellulose and colanic acid are required for optimal biofilm formation by Salmonella spp. on glass and plastic but are not required for biofilm formation on human gallstones (11, 35). For some bacteria, a particular exopolysaccharide promotes attachment to one surface but seems to interfere with attachment to other surfaces. One example of this is E. coli O157:H7, which requires the exopolysaccharides poly-β-1,6-N-acetylglucosamine (PGA), colanic acid, and cellulose for optimal binding to alfalfa sprouts and plastic (27). In contrast, these polysaccharides are not required for binding by E. coli O157:H7 cells to human intestinal epithelial (Caco-2) cells and binding was actually enhanced in cellulose and PGA mutants, suggesting that while these polysaccharides are important for attachment to sprouts and plastic, they interfere with attachment to Caco-2 cells (27).The marine bacterium Vibrio fischeri is known to produce at least two different exopolysaccharides that play roles in biofilm formation, the symbiosis polysaccharide (Syp) and cellulose (12, 59, 60). The Syp polysaccharide is critical for the formation of a biofilm-like aggregate at the initiation of symbiosis with the Hawaiian bobtail squid Euprymna scolopes (59). The natural condition(s) under which V. fischeri cells use cellulose in biofilm formation is not yet known. However, in other bacteria, cellulose contributes to the ability to attach to a variety of surfaces, including plant roots, other plant cells, mammalian epithelial cells, glass, and plastic (27, 28, 31, 35, 37, 45).Although V. fischeri biofilm formation appears to be important for interaction with its symbiotic host and is likely also to be important in the marine environment outside the host, wild-type V. fischeri cells do not produce substantial biofilms under a variety of standard laboratory conditions. V. fischeri''s ability to form biofilms in culture, however, is greatly enhanced when the syp biosynthetic gene cluster (Fig. ​(Fig.1)1) is induced by overexpression of the response regulator SypG, the sensor kinase RscS, or the sensor kinase SypF (12, 59, 60).Open in a separate windowFIG. 1.The binA gene and its predicted protein. (A) The binA gene (VFA1038) is located downstream from and oriented in the same direction as the syp gene locus. Individual genes are indicated by block arrows, and the four known or putative promoters within the syp locus are indicated by line arrows. (B) The BinA protein (630 amino acids [aa]) is predicted to have three domains, GAF (∼Q20 to L151), GGDEF (∼H205 to A338), and EAL (∼L374 to D611), as indicated. Only the EAL domain is well conserved.In culture, V. fischeri also forms biofilms when cellulose production is induced. Cellulose contributes to the biofilms formed when either SypF or the putative response regulator VpsR is overexpressed (12). Additionally, overexpression of the diguanylate cyclase MifA induces biofilm formation and increases binding to two dyes that are cellulose indicators, Congo red and calcofluor (34, 37, 54).The product of diguanylate cyclase activity, cyclic diguanylate (c-di-GMP), is an intracellular signaling molecule that plays an important role in regulating biofilm formation and motility (reviewed in references 10, 22, 38, 39, 48, and 56). C-di-GMP is produced from two GTP molecules by diguanylate cyclases with conserved GGDEF domains and is depleted by hydrolysis to linear pGpG by phosphodiesterases (PDEs) with EAL or HD-GYP domains. There is evidence of increased cellulose production in response to increased c-di-GMP levels in many bacteria (36, 41). In general, high levels of c-di-GMP enhance biofilm formation and low levels of c-di-GMP enhance motility (39).Here we report that V. fischeri biofilm formation also increases in the absence of BinA, a GGDEF/EAL domain protein encoded by a gene that is adjacent to the syp cluster. We also report the discovery of genes involved in biofilm formation by binA mutants and the identification of amino acids that are critical to BinA activity. Finally, we suggest a mechanism by which the production of two different polysaccharides may be coordinated by BinA.