Assembly of Bacterial Capsular Polysaccharides and Exopolysaccharides.

Polysaccharides are dominant features of most bacterial surfaces and are displayed in different formats. Many bacteria produce abundant long-chain capsular polysaccharides, which can maintain a strong association and form a capsule structure enveloping the cell and/or take the form of exopolysaccharides that are mostly secreted into the immediate environment. These polymers afford the producing bacteria protection from a wide range of physical, chemical, and biological stresses, support biofilms, and play critical roles in interactions between bacteria and their immediate environments. Their biological and physical properties also drive a variety of industrial and biomedical applications. Despite the immense variation in capsular polysaccharide and exopolysaccharide structures, patterns are evident in strategies used for their assembly and export. This review describes recent advances in understanding those strategies, based on a wealth of biochemical investigations of select prototypes, supported by complementary insight from expanding structural biology initiatives. This provides a framework to identify and distinguish new systems emanating from genomic studies.

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.

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.

Structural characterization and physicochemical properties of the exopolysaccharide produced by the moderately halophilic bacterium Chromohalobacter salexigens, strain 3EQS1.

A strain, 3EQS1, was isolated from a salt sample taken from Lake Qarun (Fayoum Province, Egypt). On the basis of physiological, biochemical, and phylogenetic analyses, the strain was classified as Chromohalobacter salexigens. By 72 h of growth at 25 °C, strain 3EQS1 produced large amounts (15.1 g L-1) of exopolysaccharide (EPS) in a liquid mineral medium (initial pH 8.0) containing 10% sucrose and 10% NaCl. The EPS was precipitated from the cell-free culture medium with chilled ethanol and was purified by gel-permeation and anion-exchange chromatography. The molecular mass of the EPS was 0.9 × 106 Da. Chemical analyses, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy showed that the EPS was a linear ß-D-(2 → 6)-linked fructan (levan). In aqueous solution, the EPS tended to form supramolecular aggregates with a critical aggregation concentration of 240 µg mL-1. The EPS had high emulsifying activity (E24, %) against kerosene (31.2 ± 0.4%), sunflower oil (76.9 ± 1.3%), and crude oil (98.9 ± 0.8%), and it also had surfactant properties. A 0.1% (w/v) aqueous EPS solution reduced the surface tension of water by 11.9%. The levan of C. salexigens 3EQS1 may be useful in various biotechnological processes.

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.

Cloning and characterization of phosphoglucose isomerase in Lentinula edodes.

Phosphoglucose isomerase (PGI) is a key enzyme that participates in polysaccharide synthesis, which is responsible for the interconversion of glucose-6-phosphate (G-6-P) and fructose-6-phosphate (F-6-P), but there is little research focusing on its role in fungi, especially in higher basidiomycetes. The pgi gene was cloned from Lentinula edodes and named lepgi. Then, the lepgi-silenced strains were constructed by RNA interference. In this study, we found that lepgi-silenced strains had significantly less biomass than the wild-type (WT) strain. Furthermore, the extracellular polysaccharide (EPS) and intracellular polysaccharide (IPS) levels increased 1.5- to 3-fold and 1.5-fold, respectively, in lepgi-silenced strains. Moreover, the cell wall integrity in the silenced strains was also altered, which might be due to changes in the compounds and structure of the cell wall. The results showed that compared to WT, silencing lepgi led to a significant decrease of approximately 40% in the ß-1,3-glucan content, and there was a significant increase of 2-3-fold in the chitin content. These findings provide support for studying the biological functions of lepgi in L. edodes.

Enterococcus faecalis rnc gene modulates its susceptibility to disinfection agents: a novel approach against biofilm.

BACKGROUND: Enterococcus faecalis (E. faecalis) plays an important role in the failure of root canal treatment and refractory periapical periodontitis. As an important virulence factor of E. faecalis, extracellular polysaccharide (EPS) serves as a matrix to wrap bacteria and form biofilms. The homologous rnc gene, encoding Ribonuclease III, has been reported as a regulator of EPS synthesis. In order to develop novel anti-biofilm targets, we investigated the effects of the rnc gene on the biological characteristics of E. faecalis, and compared the biofilm tolerance towards the typical root canal irrigation agents and traditional Chinese medicine fluid Pudilan. METHODS: E. faecalis rnc gene overexpression (rnc+) and low-expression (rnc-) strains were constructed. The growth curves of E. faecalis ATCC29212, rnc+, and rnc- strains were obtained to study the regulatory effect of the rnc gene on E. faecalis. Scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and crystal violet staining assays were performed to evaluate the morphology and composition of E. faecalis biofilms. Furthermore, the wild-type and mutant biofilms were treated with 5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and Pudilan. The residual viabilities of E. faecalis biofilms were evaluated using crystal violet staining and colony counting assays. RESULTS: The results demonstrated that the rnc gene could promote bacterial growth and EPS synthesis, causing the EPS-barren biofilm morphology and low EPS/bacteria ratio. Both the rnc+ and rnc- biofilms showed increased susceptibility to the root canal irrigation agents. The 5% NaOCl group showed the highest biofilm removing effect followed by Pudilan and 2% CHX. The colony counting results showed almost complete removal of bacteria in the 5% NaOCl, 2% CHX, and Chinese medicine agents' groups. CONCLUSIONS: This study concluded that the rnc gene could positively regulate bacterial proliferation, EPS synthesis, and biofilm formation in E. faecalis. The rnc mutation caused an increase in the disinfectant sensitivity of biofilm, indicating a potential anti-biofilm target. In addition, Pudilan exhibited an excellent ability to remove E. faecalis biofilm.

Exopolysaccharide from Porphyridium cruentum (purpureum) is Not Toxic and Stimulates Immune Response against Vibriosis: The Assessment Using Zebrafish and White Shrimp Litopenaeus vannamei.

Exopolysaccharides, or extracellular polysaccharides (EPS, sPS), represent a valuable metabolite compound synthesized from red microalgae. It is a non-toxic natural agent and can be applied as an immunostimulant. The toxicity test of exopolysaccharides from Porphyridium has been done in vivo using zebrafish (Danio rerio) embryonic model, or the ZET (zebrafish embryotoxicity test). The administration of extracellular polysaccharides or exopolysaccharides (EPS) from microalgae Porphyridium cruentum (synonym: P. purpureum) to shrimps Litopenaeus vannamei was investigated to determine the effect of this immunostimulant on their non-specific immune response and to test if this compound can be used as a protective agent for shrimps in relation to Vibrio infection. For immune response, exopolysaccharides were given to shrimps via the immersion method on day 1 and booster on day 8. Shrimp hemocytes were taken on day 1 (EPS administration), day 7 (no treatment), day 8 (EPS booster) and day 9 (Vibrio infection) and tested for their immune response on each treatment. The result shows that the EPS is not toxic, as represented by the normal embryonic development and the mortality data. In the Pacific white shrimps, an increase in the values of all immune parameters was shown, in line with the increasing EPS concentration, except for the differential hemocyte count (DHC). In detail, an increase was noted in total hemocytes (THC) value, phagocytotic activity (PA) and respiratory burst (RB) in line with the EPS concentration increase. These results and other previous studies indicate that EPS from Porphyridium is safe, enhances immune parameters in shrimp rapidly, and has the ability to act as an immunostimulant or an immunomodulator. It is a good modulator for the non-specific immune cells of Pacific white shrimps, and it can be used as a preventive agent against vibriosis.

Enhancing robustness of activated sludge with Aspergillus tubingensis as a protective backbone structure under high-salinity stress.

High salt seriously destroys the stable interactions among key functional species of activated sludge, which in turn limits the performance of high-salinity wastewater biological treatment. In this study, pelletized Aspergillus tubingensis (AT) was used as a protective backbone structure for activated sludge under high-salinity stress, and a superior salt-tolerant AT-based aerobic granular sludge (AT-AGS) was developed. Results showed that the COD and NH4+-N removal efficiencies of salt-domesticated AT-AGS were 11.83% and 7.18% higher than those of salt-domesticated flocculent activated sludge (FAS) at 50 gNaCl/L salinity. Compared to the salt-domesticated FAS, salt-domesticated AT-AGS showed stronger biomass retention capacity (with a MLVSS concentration of 7.92 g/L) and higher metabolic activity (with a dehydrogenase activity of 48.06 mgTF/gVSS·h). AT modified the extracellular polymeric substances pattern of microbes, and the total extracellular polysaccharide content of AT-AGS (80.7 mg/gVSS) was nearly twice than that of FAS (46.3 mg/gVSS) after salt-domestication, which demonstrated that extracellular polysaccharide played a key role in keeping the system stable. The high-throughput sequencing analysis illustrated that AT contributed to maintain the microbial richness and diversity of AT-AGS in high-salt environment, and Marinobacterium (with a relative abundance of 32.04%) became the most predominant genus in salt-tolerant AT-AGS. This study provided a novel insight into enhancing the robustness of activated sludge under high-salinity stress.

Screening and Characterization of Two Extracellular Polysaccharide-Producing Bacteria from the Biocrust of the Mu Us Desert.

The extracellular polysaccharide (EPS) matrix embedding microbial cells and soil particles plays an important role in the development of biological soil crusts (BSCs), which is widely recognized as beneficial to soil fertility in dryland worldwide. This study examined the EPS-producing bacterial strains YL24-1 and YL24-3 isolated from sandy soil in the Mu Us Desert in Yulin, Shaanxi province, China. The strains YL24-1 and YL24-3 were able to efficiently produce EPS; the levels of EPS were determined to be 257.22 µg/mL and 83.41 µg/mL in cultures grown for 72 h and were identified as Sinorhizobium meliloti and Pedobacter sp., respectively. When the strain YL24-3 was compared to Pedobacter yulinensis YL28-9T using 16S rRNA gene sequencing, the resemblance was 98.6% and the strain was classified as Pedobacter sp. using physiological and biochemical analysis. Furthermore, strain YL24-3 was also identified as a subspecies of Pedobacter yulinensis YL28-9T on the basis of DNA-DNA hybridization and polar lipid analysis compared with YL28-9T. On the basis of the EPS-related genes of relevant strains in the GenBank, several EPS-related genes were cloned and sequenced in the strain YL24-1, including those potentially involved in EPS synthesis, assembly, transport, and secretion. Given the differences of the strains in EPS production, it is possible that the differences in gene sequences result in variations in the enzyme/protein activities for EPS biosynthesis, assembly, transport, and secretion. The results provide preliminary evidence of various contributions of bacterial strains to the formation of EPS matrix in the Mu Us Desert.

Intra-species variation within Lactobacillus rhamnosus correlates to beneficial or harmful outcomes: lessons from the oral cavity.

BACKGROUND: The origin of most of the Lactobacillus rhamnosus genome sequences lodged in NCBI can be traced to food and faecal isolates followed by blood and tissue sites but with minimal representation from oral and vaginal isolates. However, on the L. rhamnosus phylogenetic tree no apparent clade is linked to the origin of isolation or to the relevant clinical source, except for a distinct clade exclusively shared by L. rhamnosus isolates from early stages of dental pulp infection (LRHMDP2 and LRHMDP3) and from bronchoalveolar lavage (699_LRHA and 708_LRHA) from a critical care patient. These L. rhamnosus strains, LRHMDP2, LRHMDP3, 699_LRHA and 708_LRHA isolated from different continents, display closest genome neighbour gapped identity of 99.95%. The aim of this study was to define a potentially unique complement of genes of clinical relevance shared between these L. rhamnosus clinical isolates in comparison to probiotic L. rhamnosus strains. RESULTS: In this analysis we used orthologous protein identification tools such as ProteinOrtho followed by tblastn alignments to identify a novel tyrosine protein phosphatase (wzb)-tyrosine-protein kinase modulator EpsC (wzd)- synteny exopolysaccharide (EPS) cluster. This EPS cluster was specifically conserved in a clade of 5 clinical isolates containing the four L. rhamnosus clinical isolates noted above and Lactobacillus spp. HMSC077C11, a clinical isolate from a neck abscess. The EPS cluster was shared with only two other strains, L. rhamnosus BPL5 and BPL15, which formed a distant clade on the L. rhamnosus phylogenetic tree, with a closest genome neighbour gapped identity of 97.51% with L. rhamnosus LRHMDP2 and LRHMDP3. Exclusivity of this EPS cluster (from those identified before) was defined by five EPS genes, which were specifically conserved between the clade of 5 clinical isolates and L. rhamnosus BPL5 and BPL15 when compared to the remaining L. rhamnosus strains. Comparative genome analysis between the clade of 5 clinical isolates and L. rhamnosus BPL5 and BPL15 showed a set of 58 potentially unique genes characteristic of the clade of 5. CONCLUSION: The potentially unique functional protein orthologs associated with the clade of 5 clinical isolates may provide understanding of fitness under selective pressure.

Effects of resveratrol on cariogenic virulence properties of Streptococcus mutans.

BACKGROUND: Streptococcus mutans is the principal etiological agent of human dental caries. The major virulence factors of S. mutans are acid production, acid tolerance, extracellular polysaccharide (EPS) synthesis and biofilm formation. The aim of this study is to evaluate the effect of resveratrol, a natural compound, on virulence properties of S. mutans. RESULTS: Resveratrol at sub-MIC levels significantly decreased acid production and acid tolerance, inhibited synthesis of water-soluble polysaccharide and water-insoluble polysaccharide, compromised biofilm formation. Related virulence gene expression (ldh, relA, gtfC, comDE) was down-regulated with increasing concentrations of resveratrol. CONCLUSIONS: Resveratrol has an inhibitory effect on S. mutans cariogenic virulence properties and it represents a promising anticariogenic agent.

Cell-wall associated polysaccharide from the psychrotolerant bacterium Psychrobacter arcticus 273-4: isolation, purification and structural elucidation.

In this paper, the structure of the capsular polysaccharide isolated from the psychrotolerant bacterium Psychrobacter arcticus 273-4 is reported. The polymer was purified by gel filtration chromatography and the structure was elucidated by means of one- and two-dimensional NMR spectroscopy, in combination with chemical analyses. The polysaccharide consists of a trisaccharidic repeating unit containing two residues of glucose and a residue of a N,N-diacetyl-pseudaminic acid.

Bioremediation potential of a halophilic Halobacillus sp. strain, EG1HP4QL: exopolysaccharide production, crude oil degradation, and heavy metal tolerance.

A halophilic bacterial strain, EG1HP4QL, was isolated from a salt sample from Lake Qarun, Fayoum Province, Egypt. Morphological, physiological, biochemical, and phylogenetic analyses indicated that the strain belonged to the genus Halobacillus. Strain EG1HP4QL produced an extracellular polysaccharide (EPS), with production peaking (5.9 g L-1) during growth on medium S-G containing 2% (w/v) sucrose at 35 °C (pH 8.0). The EPS had significant emulsifying activity (E24 %) against kerosene (65.7 ± 0.8%), o-xylene (64.0 ± 1%), and sunflower oil (44.7 ± 0.5%). The composition of the EPS included two polymers-a negatively charged and a neutral one (~ 3:1)-in which mannose and glucose were the main neutral monosaccharide constituents. Strain EG1HP4QL was able to utilize crude oil (35.3%) as the sole carbon source within 12 days. The minimum inhibitory concentrations of heavy metals [Zn(II), Cd(II), Pb(II), Ni(II), and Cu(II)] for strain EG1HP4QL were 1.0, 2.0, 2.0, 2.5, and 5 mM, respectively.

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production.

Cyclic dimeric GMP (c-di-GMP) has emerged as a key regulatory player in the transition between planktonic and sedentary biofilm-associated bacterial lifestyles. It controls a multitude of processes including production of extracellular polysaccharides (EPSs). The PilZ domain, consisting of an N-terminal "RxxxR" motif and a ß-barrel domain, represents a prototype c-di-GMP receptor. We identified a class of c-di-GMP-responsive proteins, represented by the AraC-like transcription factor CuxR in plant symbiotic α-proteobacteria. In Sinorhizobium meliloti, CuxR stimulates transcription of an EPS biosynthesis gene cluster at elevated c-di-GMP levels. CuxR consists of a Cupin domain, a helical hairpin, and bipartite helix-turn-helix motif. Although unrelated in sequence, the mode of c-di-GMP binding to CuxR is highly reminiscent to that of PilZ domains. c-di-GMP interacts with a conserved N-terminal RxxxR motif and the Cupin domain, thereby promoting CuxR dimerization and DNA binding. We unravel structure and mechanism of a previously unrecognized c-di-GMP-responsive transcription factor and provide insights into the molecular evolution of c-di-GMP binding to proteins.

Discovery of novel bis-sulfoxide derivatives bearing acylhydrazone and benzothiazole moieties as potential antibacterial agents.

On the basis of the active substructure combination principle, 24 novel synthesis of novel bis-sulfoxide derivatives bearing acylhydrazone and benzothiazole moieties as potential antibacterial agents were designed and synthesized. The bioactivity assay results showed that many compounds had significant in vitro inhibitory effects against Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas citri pv. citri (Xac). Notably, compound 4b had the best in vitro antibacterial activity against Xoo at an half-maximal effective concentration value of 11.4 µg/mL, which was superior to those of thiodiazole copper (TDC) and bismerthiazol (BMT). Compared with TDC and BMT, compound 4b was more effective in vivo controlling rice bacterial leaf blight with curative and protection activities of 42.5% and 40.3%, respectively. In addition, compound 4b can influence biofilm formation, inhibit extracellular polysaccharide production, and ultimately reduce the pathogenicity of Xoo. All the results indicated that bis-sulfoxide derivatives bearing acylhydrazone and benzothiazole moieties can be used for the development of small-molecule pesticides with high antibacterial activity.

Production and characterization of a bioactive extracellular homopolysaccharide produced by Haloferax sp. BKW301.

The production of extracellular polysaccharides (EPS) by haloarchaeal members, with novel and unusual physicochemical properties, is of special importance and has the potential for extensive biotechnological exploitation. An extremely halophilic archaeon, Haloferax sp. BKW301 (GenBank Accession No. KT240044) isolated from a solar saltern of Baksal, West Bengal, India has been optimized for the production of EPS under batch culture. It produced a considerable amount (5.95 g/L) of EPS in the medium for halophiles with 15% NaCl, 3% glucose, 0.5% yeast extract, and 6% inoculum under shake flask culture at 120 rpm. The purified EPS, a homopolymer of galactose as revealed by chromatographic methods and Fourier-transform infrared spectroscopy, is noncrystalline (CIxrd , 0.82), amorphous, and could emulsify hydrocarbons like kerosene, petrol, xylene, and so forth. Moreover, the polymer is highly thermostable (up to 420°C) and displayed pseudoplastic rheology. Biologically, the EPS was able to scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl) radical efficiently and inhibit the proliferation of the Huh-7 cell line at an IC50 value of 6.25 µg/ml with a Hill coefficient of 0.844. Large-scale production of this thermostable, pseudoplastic homopolysaccharide, therefore, could find suitable applications in industry and biotechnology.

Structure and Anti-Tumor Activities of Exopolysaccharides from Alternaria mali Roberts.

In this study, an extracellular polysaccharide from Alternaria mali Roberts (AMEP) was extracted, and its structure was characterized, in addition to its antitumor activity in vitro. Neutral polysaccharide AMEP-1 and anionic polysaccharide AMEP-2 were isolated from AMEP, and their monosaccharide compositions consisted of mannose (Man), glucose (Glc), and galactose (Gal) but at different ratios. The linking mode of both AMEP-1 and AMEP-2 is Manp-(1→4) and Glcp-(1→6), and the branched chains are connected to the main chain through O-6. AMEP-2 inhibited the proliferation of BGC-823 cells in a time- and concentration-dependent manner. AMEP-2 also induced the apoptosis of BGC-823 cells, and showed anti-tumor effects by inducing cell cycle arrest in the S phase, reactive oxygen species production, and mitochondrial membrane potential reduction in BGC-823 cells. Therefore, AMEP-2 shows potential for further development as a novel anti-tumor agent.

Elevated level of arsenic negatively influences nifH gene expression of isolated soil bacteria in culture condition as well as soil system.

Comprehensive studies on the effect of arsenic (As) on free-living diazotrophs that play a crucial role in soil fertility by nitrogen fixation are still scanty. Here, we isolated three free-living bacteria from rice field with potential nitrogen-fixing ability and investigated the impact of As on their nifH gene expression and extracellular polysaccharide (EPS) production in culture condition and soil system. 16S rRNA sequence analysis showed that the isolated bacteria were affiliated to ß-Proteobacteria, γ-Proteobacteria and Firmicutes. As(III) exposure to bacterial isolates followed by RT-qPCR analysis revealed that elevated levels of As reduced the expression of nifH gene in selective bacteria, both in culture medium and soil condition. We also noticed reduced production of EPS under higher concentration of As. All the three bacteria showed high tolerance to As(III), able to oxidize As and exhibited significant plant growth-promoting traits. This investigation indicated that an environment exposed with higher concentration of As might perturbed the activity of free-living diazotrophs in agricultural soil system.

Aspergillus fumigatus Afssn3-Afssn8 Pair Reverse Regulates Azole Resistance by Conferring Extracellular Polysaccharide, Sphingolipid Pathway Intermediates, and Efflux Pumps to Biofilm.

Antifungal treatment is often ineffectual, partly because of biofilm formation. In this study, by using a combined forward and reverse genetic strategy, we identified that nucleus-localized AfSsn3 and its partner AfSsn8, which constitute a Cdk8-cyclin pair, are required for azole resistance in Aspergillus fumigatus Deletion of Afssn3 led to increased absorption and utilization of glucose and amino acids. Interestingly, absorption and utilization of glucose accelerated the extracellular polysaccharide formation, while utilization of the amino acids serine, threonine, and glycine increased sphingolipid pathway intermediate accumulation. In addition, the absence of Afssn3 induced the activity of the efflux pump proteins. These factors indicate the mature biofilm is responsible for the major mechanisms of A. fumigatus resistance to azoles in the ΔAfssn3 mutant. Collectively, the loss of Afssn3 led to two "barrier" layers between the intracellular and extracellular spaces, which consequently decreased drug penetration into the cell.