Development of "Intelligent particles" for the treatment of dental caries.

Dental caries is one of the most prevalent non-communicable diseases worldwide, mediated by a multispecies biofilm that consists of high levels of acidogenic bacteria which ferment sugar to acid and cause teeth demineralization. Current treatment practice remains insufficient in addressing 1) rapid clearance of therapeutic agents from the oral environment 2) destroying bacteria that contribute to the healthy oral microbiome. In addition, increasing concerns over antibiotic resistance calls for innovative alternatives. In this study, we developed a pH responsive nano-carrier for delivery of polycationic silver nanoparticles. Branched-PEI capped silver nanoparticles (BPEI-AgNPs) were encapsulated in a tannic acid - Fe (III) complex-modified poly(D,L-lactic-co-glycolic acid) (PLGA) particle (Fe(III)-TA/PLGA@BPEI-AgNPs) to enhance binding to the plaque biofilm and demonstrate "intelligence" by releasing BPEI-AgNPs under acidic conditions that promote dental caries The constructed Fe(III)-TA/PLGA@BPEI-AgNPs (intelligent particles - IPs) exhibited significant binding to an axenic S. mutans biofilm grown on hydroxyapatite. Ag+ ions were released faster from the IPs at pH 4.0 (cariogenic pH) compared to pH 7.4. The antibiofilm results indicated that IPs can significantly reduce S. mutans biofilm volume and viability under acidic conditions. Cytotoxicity on differentiated Caco-2 cells and human gingival fibroblasts indicated that IPs were not cytotoxic. These findings demonstrate great potential of IPs in the treatment of dental caries.

Iron(III) cross-linked hydrogels based on Alteromonas macleodii Mo 169 exopolysaccharide.

Recently, polysaccharide-based hydrogels crosslinked with the trivalent iron cation have attracted interest due to their remarkable properties that include high mechanical stability, stimuli-responsiveness, and enhanced absorptivity. In this study, a Fe3+ crosslinked hydrogel was prepared using the biocompatible extracellular polysaccharide (EPS) secreted by the marine bacterium Alteromonas macleodii Mo169. Hydrogels with mechanical strengths (G') ranging from 0.3 kPa to 44.5 kPa were obtained as a result of the combination of different Fe3+ (0.05-9.95 g L-1) and EPS (0.3-1.7 %) concentrations. All the hydrogels had a water content above 98 %. Three different hydrogels, named HA, HB, and HC, were chosen for further characterization. With strength values (G') of 3.2, 28.9, and 44.5 kPa, respectively, these hydrogels might meet the strength requirements for several specific applications. Their mechanical resistance increased as higher Fe3+ and polymer concentrations were used in their preparation (the compressive hardness increased from 8.7 to 192.1 kPa for hydrogel HA and HC, respectively). In addition, a tighter mesh was noticed for HC, which was correlated to its lower swelling ratio value compared to HA and HB. Overall, this preliminary study highlighted the potential of these hydrogels for tissue engineering, drug delivery, or wound healing applications.

Influence of Biofilm Maturity on the Antibacterial Efficacy of Cold Atmospheric Plasma in Oral Microcosm Biofilms.

As biofilms mature, biomass and extracellular polysaccharide (EPS) content increases, enhancing pathogenicity. Therefore, this study aimed to evaluate the antibacterial efficacy of cold atmospheric plasma (CAP) against oral microcosm biofilms and the influence of biofilm maturity on treatment. Oral microcosm biofilms were cultured on hydroxyapatite disks for 2 and 6 days. Based on the treatment and biofilm maturity, these were subsequently allocated into six groups (N = 19 each): Groups 1 and 2 were incubated with distilled water for 1 min; Groups 3 and 4 were treated with CAP for 2 min, and Groups 5 and 6 were treated with 0.12% chlorhexidine gluconate for 1 min. Groups 1, 3, and 5 represent 2-day biofilms, and Groups 2, 4, and 6 represent 6-day biofilms. Treatments were repeated daily for 5 days. Antibacterial efficacy was analyzed by measuring oral biofilms' red fluorescence intensity (RatioR/G) and quantifying EPS content and bacterial viability. The RatioR/G was 1.089-fold and 1.104-fold higher in Groups 4 and 6 than in Groups 3 and 5 following antibacterial treatment, respectively (p < 0.001). EPS content increased by 1.71-fold in Group 6 than in Group 5 (p < 0.001). Bacterial survival rate was the lowest in Group 3 (p = 0.005). These findings underscore the relevance of CAP treatment in maintaining antibacterial efficacy regardless of the biofilm development stage, highlighting its potential utility in oral care.

Enhanced polysaccharide production through quorum sensing system in Cordyceps militaris.

This study aimed to enhance extracellular polysaccharide (EPS) production in Cordyceps militaris by constructing a quorum sensing (QS) system to regulate the expression of biosynthetic enzyme genes, including phosphoglucomutase, hexokinase, phosphomannomutase, polysaccharide synthase, and UDP-glucose 4-epimerase genes. The study found higher EPS concentrations in seven recombinant strains compared to the wild-type C. militaris, indicating that the overexpression of key enzyme genes increased EPS production. Among them, the CM-pgm-2 strain exhibited the highest EPS production, reaching a concentration of 3.82 ± 0.26 g/L, which was 1.52 times higher than the amount produced by the wild C. militaris strain. Additionally, the regulatory effects of aromatic amino acids on the QS system of the CM-pgm-2 strain were investigated. Under the influence of 45 mg/L tryptophan, the EPS production in CM-pgm-2 reached 4.75 ± 0.20 g/L, representing a 1.90-fold increase compared to wild C. militaris strains. This study provided an effective method for the large-scale production of EPSs in C. militaris, and opened up new avenues for research into fungal QS mechanisms.

Isolation, structural characterization and immunomodulatory activity on RAW264.7 cells of a novel exopolysaccharide of Dictyophora rubrovalvata.

Fungal polysaccharides have been explored by many for both structural studies and biological activities, but few studies have been done on the extracellular polysaccharides of Dictyophora rubrovalvata, so a new exopolysaccharide was isolated from Dictyophora rubrovalvata and its structure and its immunological activity were investigated. The crude exopolysaccharide (EPS) was purified by DEAE52 cellulose and Sephadex G-200 to obtain a new acidic polysaccharide (DR-EPS). DR-EPS (2.66 × 103 kDa) was consisted mainly of mannose, glucose, galactose and glucuronic acid with a molar ratio of 1: 0.86: 0.20: 0.01. In addition, DR-EPS increased the phagocytic activity of RAW264.7 cells up to 2.67 times of the blank control group. DR-EPS improved intracellular nucleic acid and glycogen metabolism as observed by AO and PAS staining. DR-EPS(40 µg/mL) promoted NO production up to 30.66 µmol, enhanced acid phosphatase (ACP) and superoxide dismutase (SOD) activities, with activity maxima of 660 U/gprot and 96.27 U/mgprot, respectively, and DR-EPS (160 µg / mL) significantly increased the lysozyme content as 2.73 times of the control group. The good immunological activity of extracellular polysaccharides of Dictyophora rubrovalvata provides directions for the use of fermentation broths.

The Anti-Microbial Peptide Citrocin Controls Pseudomonas aeruginosa Biofilms by Breaking Down Extracellular Polysaccharide.

Citrocin is an anti-microbial peptide that holds great potential in animal feed. This study evaluates the anti-microbial and anti-biofilm properties of Citrocin and explores the mechanism of action of Citrocin on the biofilm of P. aeruginosa. The results showed that Citrocin had a significant inhibitory effect on the growth of P. aeruginosa with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 0.3 mg/mL. All five concentrations (1/4MIC, 1/2MIC, MIC, 2MIC, and 4MIC) of Citrocin inhibited P. aeruginosa biofilm formation. Citrocin at the MIC, 2MIC and 4MIC removed 42.7%, 76.0% and 83.2% of mature biofilms, respectively, and suppressed the swarming motility, biofilm metabolic activity and extracellular polysaccharide production of P. aeruginosa. Metabolomics analysis indicated that 0.3 mg/mL of Citrocin up- regulated 26 and down-regulated 83 metabolites, mainly comprising amino acids, fatty acids, organic acids and sugars. Glucose and amino acid metabolic pathways, including starch and sucrose metabolism as well as arginine and proline metabolism, were highly enriched by Citrocin. In summary, our research reveals the anti-biofilm mechanism of Citrocin at the metabolic level, which provides theoretical support for the development of novel anti-biofilm strategies for combatting P. aeruginosa.

[Effect of Tween 20 on enhancing extracellular polysaccharide synthesis by Pantoea alhagi NX-11].

Pantoea alhagi NX-11 exopolysaccharide (PAPS) is a novel microbial biostimulant that enhances crop resistance to salt and drought stress. It is biodegradable and holds promising applications in improving agricultural yield and efficiency. However, the fermentation process of PAPS exhibits a high viscosity due to low oxygen transfer efficiency, which hinders yield improvement and downstream processing. This study aimed to investigate the effects of seven oxygen carriers (Span 80, Span 20, Tween 80, Tween 20, glycerin, olive oil, and soybean oil) on fermentation yield. The results showed that the addition of 0.5% (V/V) Tween 20 significantly enhanced the production of PAPS. Moreover, the introduction of 0.5% (V/V) Tween 20 in a 7.5 L fermenter resulted in a PAPS titer of (16.85±0.50) g/L, which was 17.70% higher than that of the control group. Furthermore, the rheological characterization and the microstructure analysis of the polysaccharide products revealed that the characteristic structure of polysaccharides remained unchanged in the oxygen carrier treated group, but their viscosity increased. These findings may facilitate enhancing the biosynthesis efficiency of other polymer products.

Terrestrial algae: pioneer organisms of carbonate rock solutional weathering in South China karst.

The formation of soil in karst ecosystem has always been a scientific problem of great concern to human beings. Algae can grow on the exposed and non-nutrition carbonate surface, inducing and accelerating weathering of rock substrates, thus promoting soil formation. Yet the actual contribution of algae to solutional weathering intensity remains unclear. In this study, we performed weathering simulation experiment on two algae species (Klebsormidium dissectum (F.Gay) H.Ettl & G.Gärtner and Chlorella vulgaris Beijerinck), which were screened from carbonated rock surfaces from a typical karst region in South China. The results showed: (1) both algae have solutional weathering effect on carbonate rock, (2) there is no difference of solutional intensity observed, yet the solutional modes are different, suggesting different ecological adaptative strategies, (3) algae on carbonate rocks have higher carbonic anhydrase activity (CAA) and secrete more extracellular polysaccharide (EPS), accelerating rock weathering. (4) The absolute dissolution amount of carbonate rock with algae participation is 3 times of that of without algae. These results indicate the significant impact of terrestrial algae on carbonate rock solutional weathering and provides quantitative evidence that terrestrial algae are pioneer species. It also contributes to our further understanding of soil formation in karst ecosystems in South China.

Exopolysaccharides of lactic acid bacteria: Structure, biological activity, structure-activity relationship, and application in the food industry: A review.

Over the past few decades, researchers have discovered that probiotics play an important role in our daily lives. With the further deepening of research, more and more evidence show that bacterial metabolites have an important role in food and human health, which opens up a new direction for the research of lactic acid bacteria (LAB) in the food and pharmaceutical industry. Many LAB have been widely studied because of the ability of exopolysaccharides (EPS). Lactic acid bacteria exopolysaccharides (LAB EPS) not only have great potential in the treatment of human diseases but also can become natural ingredients in the food industry to provide special qualitative structure and flavor. This paper has organized and summarized the biosynthesis, strain selection, production process parameters, structure, and biological activity of LAB EPS, filling in the monotony and incompleteness of previous articles' descriptions of LAB EPS. Therefore, this paper focuses on the general biosynthetic pathway, structural characterization, structure-activity relationship, biological activity of LAB EPS, and their application in the food industry, which will help to deepen people's understanding of LAB EPS and develop new active drugs from LAB EPS. Although the research results are relatively affluent, the low yield, complex structure, and few clinical trials of EPS are still the reasons that hinder its development. Therefore, future knowledge expansion should focus on the regulation of structure, physicochemical properties, function, higher production of EPS, and clinical trial applications, which can further increase the commercial significance and value of EPS. Furthermore, better understanding the structure-function relationship of EPS in food remains a challenge to date.

The extracellular polysaccharide determine the physico-chemical surface properties of Microcystis.

Microcystis possesses the capacity to form colonies and blooms in lakes and reservoirs worldwide, causing significant ecological challenges in aquatic ecosystems. However, little is known about the determining factors of physico-chemical surface properties that govern the competitive advantage of Microcystis. Here, The physico-chemical surface properties of Microcystis wesenbergii and Microcystis aeruginosa, including specific surface area (SSA), hydrophobicity, zeta potential, and functional groups were investigated. Additionally, the extracellular polysaccharide (EPS) were analyzed. Laboratory-cultured Microcystis exhibited hydrophilic, a negative zeta potential and negatively charged. Furthermore, no significant relationship was shown between these properties and the cultivation stage. Microcystis wesenbergii exhibited low free energy of cohesion, high surface free energy, high growth rate, and high EPS content during the logarithmic phase. On the other hand, M. aeruginosa displayed lower free energy of cohesion, high surface free energy, high EPS content, and high growth rate during the stationary phase. These characteristics contribute to their respective competitive advantage. Furthermore, the relationship between EPS and surface properties was investigated. The polysaccharide component of EPS primarily influenced the SSA and total surface energy of Microcystis. Likewise, the protein component of EPS influenced hydrophobicity and surface tension. The polysaccharide composition, including glucuronic acid, xylose, and fructose, mainly influenced surface properties. Additionally, hydrophilic groups such as O-H and P-O-P played a crucial role in determining hydrophobicity in Microcystis. This study elucidates that EPS influenced the SSA, hydrophobicity, and surface free energy of Microcystis cells, which in turn impact the formation of Microcystis blooms and the collection.

The extracellular polysaccharide inhibit porcine epidemic diarrhea virus with extract and gene editing Lacticaseibacillus.

Lacticaseibacillus is one of the predominant microorganisms in gut from human and animal, and the lacticaseibacillus have effective applications against the viral diarrhea of piglets in the farm. However, the function and the concrete cell single pathways of the active ingredient from lacticaseibacillus was not clear within anti-infection in the postbiotics research. Here, we compared the biological function of extracellular polysaccharides (EPS) purified from lacticaseibacillus casei (L. casei) and gene editing lacticaseibacillus casei with the CRISPER-Cas9 technology, which were with the ability of antioxidation and anti-inflammation, and the EPS could also inhibit the ROS production within the Porcine Small Intestinal Epithelial Cells-J2 (IPEC-J2). Interestingly, we found that both of EPS and genome editing lacticaseibacillus casei could specifically target the IFN-λ expression in the IPEC-J2, which was beneficial against the PEDV infection in the virus replication and production with the qRT-PCR and indirect immunofluorescence methods. Finally, the STAT3 cell single pathway was stimulated to transcribe IFN-λ with the EPS to elucidate the detailed mechanism of activating type III IFN signals receptor of IL-10R2, which play the function between anti-inflammation and anti-virus in the PEDV infection. Taken together, our research linked a postbiotics of EPS with the antiviral infection of PEDV, which suggest that the lacticaseibacillus itself still have displayed the potential immunomodulatory activities, and highlight the immunomodulatory potential of EPS-producing microbes.

Characterization of Klebsiella pneumoniae Extracellular Polysaccharides.

Klebsiella pneumoniae is a clinically significant, Gram-negative pathogen in which the production of extracellular polysaccharides is a key virulence factor. Extracellular polysaccharides such as the capsule and its mucoviscosity play a significant role in K. pneumoniae infection. In this article, we explain several standard protocols used to characterize the extracellular polysaccharides of K. pneumoniae. Several of these protocols are adapted specifically for K. pneumoniae and describe methods to purify and quantify the extracellular polysaccharide of K. pneumoniae. We also present a standardized protocol to quantify K. pneumoniae mucoviscosity, a unique feature of K. pneumoniae extracellular polysaccharide. These protocols will help create uniformity in standard protocols used in K. pneumoniae extracellular polysaccharide studies. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extracellular polysaccharide isolation and purification Basic Protocol 2: Large-scale isolation and purification of extracellular polysaccharide Basic Protocol 3: Uronic acid quantification of extracellular polysaccharide Basic Protocol 4: Extracellular polysaccharide visualization by SDS-PAGE Basic Protocol 5: Klebsiella pneumoniae mucoviscosity measurement by sedimentation resistance assay Alternate Protocol 5: 96-well plate-based Klebsiella pneumoniae sedimentation resistance assay Support Protocol 5: Determination of plate to cuvette conversion factor.

More than just a gel: the extracellular matrixome of Pseudomonas aeruginosa.

Armed with an arsenal of protein secretion systems, antibiotic efflux pumps, and the occasional proclivity for explosive self-destruction, Pseudomonas aeruginosa has become a model for the study of bacterial pathogenesis and biofilm formation. There is accruing evidence to suggest that the biofilm matrix-the bioglue that holds the structure together-acts not only in a structural capacity, but is also a molecular "net" whose function is to capture and retain certain secreted products (including proteins and small molecules). In this perspective, we argue that the biofilm matrixome is a distinct extracellular compartment, and one that is differentiated from the bulk secretome. Some of the points we raise are deliberately speculative, but are becoming increasingly accessible to experimental investigation.

Bioinformatic and functional characterization of cyclic-di-GMP metabolic proteins in Vibrio alginolyticus unveils key diguanylate cyclases controlling multiple biofilm-associated phenotypes.

The biofilm lifestyle is critical for bacterial survival and proliferation in the fluctuating marine environment. Cyclic diguanylate (c-di-GMP) is a key second messenger during bacterial adaptation to various environmental signals, which has been identified as a master regulator of biofilm formation. However, little is known about whether and how c-di-GMP signaling regulates biofilm formation in Vibrio alginolyticus, a globally dominant marine pathogen. Here, a large set of 63 proteins were predicted to participate in c-di-GMP metabolism (biosynthesis or degradation) in a pathogenic V. alginolyticus strain HN08155. Guided by protein homology, conserved domains and gene context information, a representative subset of 22 c-di-GMP metabolic proteins were selected to determine which ones affect biofilm-associated phenotypes. By comparing phenotypic differences between the wild-type and mutants or overexpression strains, we found that 22 c-di-GMP metabolic proteins can separately regulate different phenotypic outputs in V. alginolyticus. The results indicated that overexpression of four c-di-GMP metabolic proteins, including VA0356, VA1591 (CdgM), VA4033 (DgcB) and VA0088, strongly enhanced rugose colony morphotypes and strengthened Congo Red (CR) binding capacity, both of which are indicators of biofilm matrix overproduction. Furthermore, rugose enhanced colonies were accompanied by increased transcript levels of extracellular polysaccharide (EPS) biosynthesis genes and decreased expression of flagellar synthesis genes compared to smooth colonies (WTpBAD control), as demonstrated by overexpression strains WTp4033 and ∆VA4033p4033. Overall, the high abundance of c-di-GMP metabolic proteins in V. alginolyticus suggests that c-di-GMP signaling and regulatory system could play a key role in its response and adaptation to the ever-changing marine environment. This work provides a robust foundation for the study of the molecular mechanisms of c-di-GMP in the biofilm formation of V. alginolyticus.

Regulation of σB-Dependent Biofilm Formation in Staphylococcus aureus through Strain-Specific Signaling Induced by Diosgenin.

Staphylococcus aureus is a commensal skin bacterium and a causative agent of infectious diseases. Biofilm formation in S. aureus is a mechanism that facilitates the emergence of resistant strains. This study proposes a mechanism for the regulation of biofilm formation in S. aureus through strain-specific physiological changes induced by the plant steroid diosgenin. A comparison of diosgenin-induced changes in the expression of regulatory genes associated with physiological changes revealed the intracellular regulatory mechanisms involved in biofilm formation. Diosgenin reduced biofilm formation in S. aureus ATCC 6538 and methicillin-resistant S. aureus (MRSA) CCARM 3090 by 39% and 61%, respectively. Conversely, it increased biofilm formation in S. aureus ATCC 29213 and MRSA CCARM 3820 by 186% and 582%, respectively. Cell surface hydrophobicity and extracellular protein and carbohydrate contents changed in a strain-specific manner in response to biofilm formation. An assessment of the changes in gene expression associated with biofilm formation revealed that diosgenin treatment decreased the expression of icaA and spa and increased the expression of RNAIII, agrA, sarA, and sigB in S. aureus ATCC 6538 and MRSA CCARM 3090; however, contrasting gene expression changes were noted in S. aureus ATCC 29213 and MRSA CCARM 3820. These results suggest that a regulatory mechanism of biofilm formation is that activated sigB expression sequentially increases the expression of sarA, agrA, and RNAIII. This increased RNAIII expression decreases the expression of spa, a surface-associated adhesion factor. An additional regulatory mechanism of biofilm formation is that activated sigB expression decreases the expression of an unknown regulator that increases the expression of icaA. This in turn decreases the expression of icaA, which decreases the synthesis of polysaccharide intercellular adhesins and ultimately inhibits biofilm formation. By assessing strain-specific contrasting regulatory signals induced by diosgenin in S. aureus without gene mutation, this study elucidated the signal transduction mechanisms that regulate biofilm formation based on physiological and gene expression changes.

Extracellular polysaccharide of Lactobacillus plantarum enhance immune efficacy of oprH gene recombinant subunit vaccine from Pseudomonas aeruginosa.

To evaluate the immune enhancement effect of the extracellular polysaccharide of Lactobacillus plantarum on oprH recombinant subunit vaccine from Pseudomonas aeruginosa, a recombinant subunit vaccine of oprH (rOprH vaccine) was developed. The EP-rOprH vaccine was prepared with the extracellular polysaccharide of L. plantarum as an adjuvant. Mice were vaccinated with the rOprH and EP-rOprH vaccines, and the outer membrane protein (OMP) and inactivated vaccines were used as controls. The levels of serum antibody, interferon-γ (IFN-γ), interleukin (IL-2), and IL-4 were determined after vaccination. Finally, the protective efficacy of the vaccine was evaluated after challenge with virulent P. aeruginosa. Following vaccination, the serum antibody levels were significantly higher in mice vaccinated with the EP-rOprH vaccine than in those vaccinated with the rOprH vaccine (P<0.05). Moreover, the serum antibody levels detected in the EP-rOprH vaccine group were similar to those detected in the OMP vaccine group when P. aeruginosa suspension was used as the coating antigen. However, the levels in the EP-rOprH vaccine group were higher than those in the OMP vaccine and inactivated vaccine groups when the purified rOprH protein was used as the coating antigen (P<0.05). The level of IFN-γ, IL-2, and IL-4 in mice vaccinated with the EP-rOprH vaccine was significantly higher than that in mice vaccinated with the rOprH vaccine (P<0.05) and comparable to that in mice vaccinated with the OMP vaccine. The protective rates were 65%, 80%, 80%, and 95% with the rOprH, EP-rOprH, OMP, and inactivated vaccines, respectively. Thus, the extracellular polysaccharide of L. plantarum significantly enhanced the immune response and protection provided by the recombinant subunit vaccine of oprH.

Neutron Scattering Analysis of Cryptococcus neoformans Polysaccharide Reveals Solution Rigidity and Repeating Fractal-like Structural Patterns.

Cryptococcus neoformans is a fungal pathogen that can cause life-threatening brain infections in immunocompromised individuals. Unlike other fungal pathogens, it possesses a protective polysaccharide capsule that is crucial for its virulence. During infections, Cryptococcus cells release copious amounts of extracellular polysaccharides (exo-PS) that interfere with host immune responses. Both exo-PS and capsular-PS play pivotal roles in Cryptococcus infections and serve as essential targets for disease diagnosis and vaccine development strategies. However, understanding their structure is complicated by their polydispersity, complexity, sensitivity to sample isolation and processing, and scarcity of methods capable of isolating and analyzing them while preserving their native structure. In this study, we employ small-angle neutron scattering (SANS) and ultra-small angle neutron scattering (USANS) for the first time to investigate both fungal cell suspensions and extracellular polysaccharides in solution. Our data suggests that exo-PS in solution exhibits collapsed chain-like behavior and demonstrates mass fractal properties that indicate a relatively condensed pore structure in aqueous environments. This observation is also supported by scanning electron microscopy (SEM). The local structure of the polysaccharide is characterized as a rigid rod, with a length-scale corresponding to 3 to 4 repeating units. This research not only unveils insights into exo-PS and capsular-PS structures but also demonstrates the potential of USANS for studying changes in cell dimensions and the promise of contrast variation in future neutron scattering studies.

New strategy for bioplastic and exopolysaccharides production: Enrichment of field microbiomes with cyanobacteria.

Seven photosynthethic microbiomes were collected from field environmental samples to test their potential in polyhydroxybutyrate (PHB) and exopolysaccharides (EPS) production, both alternatives to chemical-based polymers. Microscope observations together with microbial sequence analysis revealed the microbiome enrichment in cyanobacteria after culture growth under phosphorus limitation. PHB and EPS production were studied under three culture factors (phototrophy, mixotrophy and heterotrophy) by evaluating and optimizing the effect of three parameters (organic and inorganic carbon and days under light:dark cycles) by Box-Behnken design. Results showed that optimal conditions for both biopolymers synthesis were microbiome-dependent; however, the addition of organic carbon boosted PHB production in all the tested microbiomes, producing up to 14 %dcw PHB with the addition of 1.2 g acetate·L-1 and seven days under light:dark photoperiods. The highest EPS production was 59 mg·L-1 with the addition of 1.2 g acetate·L-1 and four days under light:dark photoperiods. The methodology used is suitable for enriching microbiomes in cyanobacteria, and for testing the best conditions for bioproduct synthesis for further scale up.

An extracellular polysaccharide is involved in the aluminum tolerance of Pullulanibacillus sp. CA42, a newly isolated strain from the Chinese water chestnut growing in an actual acid sulfate soil area in Vietnam.

A novel aluminum-tolerant bacterial strain CA42 was isolated from the aquatic plant Eleocharis dulcis, which grows in a highly acidic swamp in Vietnam. Inoculation with CA42 allowed Oryza sativa to grow in the presence of 300 µM AlCl3 at pH 3.5, and biofilms were observed around the roots. Using 16S rRNA gene sequencing analysis, the strain was identified as Pullulanibacillus sp. CA42. This strain secreted large amounts of an extracellular polysaccharide (CA42 EPS). Results from structural analyses on CA42 EPS, namely methylation analysis and nuclear magnetic resonance (NMR), indicated that the chemical structure of CA42 EPS was a glycogen-like α-glucan. Purified CA42 EPS and the commercially available oyster glycogen adsorbed aluminum ions up to 15-30 µmol/g dry weight. Digestion treatments with α-amylase and pullulanase completely attenuated the aluminum ion-adsorbing activity of purified CA42 EPS and oyster glycogen, suggesting that the glycogen-like structure adsorbed aluminum ions and that its branching structure played an important role in its aluminum adsorbing activity. Furthermore, the aluminum tolerance of CA42 cells was attenuated by pullulanase treatment directly on the live CA42 cells. These results suggest that CA42 EPS adsorbs aluminum ions and is involved in the aluminum tolerance mechanism of Pullulanibacillus sp. CA42. Thus, this strain may be a potential plant growth-promoting bacterium in acidic soils. In addition, this study is the first to report a glycogen-like polysaccharide that adsorbs aluminum ions.

Role of two RpoN in Bradyrhizobium sp. strain DOA9 in symbiosis and free-living growth.

RpoN is an alternative sigma factor (sigma 54) that recruits the core RNA polymerase to promoters of genes. In bacteria, RpoN has diverse physiological functions. In rhizobia, RpoN plays a key role in the transcription of nitrogen fixation (nif) genes. The Bradyrhizobium sp. DOA9 strain contains a chromosomal (c) and plasmid (p) encoded RpoN protein. We used single and double rpoN mutants and reporter strains to investigate the role of the two RpoN proteins under free-living and symbiotic conditions. We observed that the inactivation of rpoNc or rpoNp severely impacts the physiology of the bacteria under free-living conditions, such as the bacterial motility, carbon and nitrogen utilization profiles, exopolysaccharide (EPS) production, and biofilm formation. However, free-living nitrogen fixation appears to be under the primary control of RpoNc. Interestingly, drastic effects of rpoNc and rpoNp mutations were also observed during symbiosis with Aeschynomene americana. Indeed, inoculation with rpoNp, rpoNc, and double rpoN mutant strains resulted in decreases of 39, 64, and 82% in the number of nodules, respectively, as well as a reduction in nitrogen fixation efficiency and a loss of the bacterium's ability to survive intracellularly. Taken together, the results show that the chromosomal and plasmid encoded RpoN proteins in the DOA9 strain both play a pleiotropic role during free-living and symbiotic states.