Extracellular Polysaccharide Biosynthesis Research Articles

Pathogenic and Comparative Genomic Analysis of Ralstonia pseudosolanacearum Isolated from Casuarina.

Casuarina equisetifolia is crucial in protecting coastal regions of China against typhoon attacks, but has faced a substantial challenge due to wilt disease caused by pathogens of the Ralstonia solanacearum species complex (RSSC). Although the initial outbreak of Casuarina wilt in 1970s was effectively controlled by disease-resistant C. equisetifolia varieties, the disease has recently re-emerged in coastal regions of Guangdong. In this study, we report the isolation, characterization, and comparative genomic analysis of 11 RSSC strains from diseased C. equisetifolia at various locations along the coast of Guangdong. Phylogenomic analysis showed that the strains were closely related and clustered with phylotype I strains previously isolated from peanuts. Single-gene based analysis further suggested these strains could be derived from strains present in Guangdong since the 1980s, indicating a historical context to their current pathogenicity. Casuarina-isolated strains exhibited notably higher virulence against C. equisetifolia and peanuts than representative RSSC strains GMI1000 and EP1, suggesting host-specific adaptations which possibly contributed to the recent outbreak. Comparative genomic analysis among RSSC strains revealed a largely conserved genome structure and high levels of conservation in gene clusters encoding extracellular polysaccharides biosynthesis, secretion systems, and quorum sensing regulatory systems. However, we also found a number of unique genes in the Casuarina-isolated strains that were absent in GMI1000 and EP1, and vice versa, pointing to potential genetic factors underpinning their differential virulence. These unique genes offer promising targets for future functional studies. Overall, our findings provide crucial insights into the RSSC pathogens causing Casuarina wilt in Guangdong, guiding future efforts in disease control and prevention.

Strain-Specific Anti-Inflammatory Effects of Faecalibacterium prausnitzii Strain KBL1027 in Koreans.

Faecalibacterium prausnitzii is one of the most dominant commensal bacteria in the human gut, and certain anti-inflammatory functions have been attributed to a single microbial anti-inflammatory molecule (MAM). Simultaneously, substantial diversity among F. prausnitzii strains is acknowledged, emphasizing the need for strain-level functional studies aimed at developing innovative probiotics. Here, two distinct F. prausnitzii strains, KBL1026 and KBL1027, were isolated from Korean donors, exhibiting notable differences in the relative abundance of F. prausnitzii. Both strains were identified as the core Faecalibacterium amplicon sequence variant (ASV) within the healthy Korean cohort, and their MAM sequences showed a high similarity of 98.6%. However, when a single strain was introduced to mice with dextran sulfate sodium (DSS)-induced colitis, KBL1027 showed the most significant ameliorative effects, including alleviation of colonic inflammation and restoration of gut microbial dysbiosis. Moreover, the supernatant from KBL1027 elevated the secretion of IL-10 cytokine more than that of KBL1026 in mouse bone marrow-derived macrophage (BMDM) cells, suggesting that the strain-specific, anti-inflammatory efficacy of KBL1027 might involve effector compounds other than MAM. Through analysis of the Faecalibacterium pan-genome and comparative genomics, strain-specific functions related to extracellular polysaccharide biosynthesis were identified in KBL1027, which could contribute to the observed morphological disparities. Collectively, our findings highlight the strain-specific, anti-inflammatory functions of F. prausnitzii, even within the same core ASV, emphasizing the influence of their human origin.

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.

Tradeoffs of microbial life history strategies drive the turnover of microbial-derived organic carbon in coastal saline soils.

Stable soil organic carbon (SOC) formation in coastal saline soils is important to improve arable land quality and mitigate greenhouse gas emissions. However, how microbial life-history strategies and metabolic traits regulate SOC turnover in coastal saline soils remains unknown. Here, we investigated the effects of microbial life history strategy tradeoffs on microbial carbon use efficiency (CUE) and microbial-derived SOC formation using metagenomic sequencing technology in different salinity soils. The results showed that high-salinity is detrimental to microbial CUE and microbial-derived SOC formation. Moreover, the regulation of nutrients stoichiometry could not mitigate adverse effects of salt stress on microbial CUE, which indicated that microbial-derived SOC formation is independent of stoichiometry in high-salinity soil. Low-salinity soil is dominated by a high growth yield (Y) strategy, such as higher microbial biomass carbon and metabolic traits which are related to amino acid metabolism, carbohydrate metabolism, and cell processes. However, high-salinity soil is dominated by stress tolerance (S) (e.g., higher metabolic functions of homologous recombination, base excision repair, biofilm formation, extracellular polysaccharide biosynthesis, and osmolytes production) and resource acquisition (A) strategies (e.g., higher alkaline phosphatase activity, transporters, and flagellar assembly). These trade-offs of strategies implied that resource reallocation took place. The high-salinity soil microbes diverted investments away from growth yield to microbial survival and resource capture, thereby decreasing biomass turnover efficiency and impeding microbial-derived SOC formation. Moreover, altering the stoichiometry in low-salinity soil caused more investment in the A-strategy, such as the production of more β-glucosidase and β-N-acetyl-glucosaminidase, and increasing bacterial chemotaxis, which thereby reduced microbial-derived SOC formation. Our research reveals that shift the microbial community from S- and A- strategies to the Y-strategy is important to increase the microbial CUE, and thus enhance SOC turnover in coastal saline soils.

AepG is a glucuronosyltransferase involved in acidic exopolysaccharide synthesis and contributes to environmental adaptation of Haloarcula hispanica

The attachment of a sugar to a hydrophobic lipid carrier is the first step in the biosynthesis of many glycoconjugates. In the halophilic archaeon Haloarcula hispanica, HAH_1206, renamed AepG, is a predicted glycosyltransferase belonging to the CAZy Group 2 family that shares a conserved amino acid sequence with dolichol phosphate mannose synthases. In this study, the function of AepG was investigated by genetic and biochemical approaches. We found that aepG deletion led to the disappearance of dolichol phosphate-glucuronic acid. Our biochemical assays revealed that recombinant cellulose-binding, domain-tagged AepG could catalyze the formation of dolichol phosphate-glucuronic acid in time- and dose-dependent manners. Based on the invivo and invitro analyses, AepG was confirmed to be a dolichol phosphate glucuronosyltransferase involved in the synthesis of the acidic exopolysaccharide produced by H.hispanica. Furthermore, lack of aepG resulted in hindered growth and cell aggregation in high salt medium, indicating that AepG is vital for the adaptation of H.hispanica to a high salt environment. In conclusion, AepG is the first dolichol phosphate glucuronosyltransferase identified in any of the three domains of life and, moreover, offers a starting point for further investigation into the diverse pathways used for extracellular polysaccharide biosynthesis in archaea.

Structural insight into a glucomannan-type extracellular polysaccharide produced by a marine Bacillus altitudinis SORB11 from Southern Ocean

Extracellular polysaccharide (EPS) produced by a deep-sea, psychrotolerant Bacillus altitudinis SORB11 was evaluated by considering physiochemical nature and structural constituents. The productivity of crude EPS was measured ~ 13.17 g L−1. The surface topography of the crude EPS showed a porous, webbed structure along with a branched coil-like configuration. The crystalline crude EPS contained a high amount of sulfur. Further, the crude EPS was subjected for purification. The molecular weight of purified EPS was determined ~ 9.8 × 104 Da. The purified EPS was appeared to show glucomannan-like configuration that is composed of → 4)-β-Manp-(1 → and → 4)-β-Glcp-(1 → residues. So, this polysaccharide was comparable to the structure of plant-derived glucomannan. Subsequently, EPS biosynthesis protein clusters like EpsC, EpsD, EpsE, and glycosyltransferase family proteins were predicted from the genome of strain SORB11, which may provide an insight into the production of glucomannan-type of polysaccharide. This low molecular weight linear form of glucomannan-type EPS might be involved to form a network-like unattached aggregation, and helps in cell-to-cell interaction in deep-sea microbial species.

Antimicrobial activity against Staphylococcus aureus and genome features of Lactiplantibacillus plantarum LR-14 from Sichuan pickles.

The persistence of Staphylococcus aureus within biofilm can lead to contamination of medical devices and life-threatening infections. Luckily, lactic acid bacteria (LAB) have an inhibitory effect on the growth of these bacteria. This study aims to select LAB strains from fermented vegetables, and analyze their potential inhibition activities against S. aureus. In total, 45 isolates of LAB were successfully isolated from Sichuan pickles, and the CFS of Lactiplantibacillus plantarum LR-14 exerted the strongest inhibitory effect against S. aureus. Moreover, S. aureus cells in planktonic and biofilm states both wrinkled and damaged when treated with the CFS of L. plantarum LR-14. In addition, whole genome sequencing analysis indicates that L. plantarum LR-14 contains various functional genes, including predicted extracellular polysaccharides (EPS) biosynthesis genes, and genes participating in the synthesis and metabolism of fatty acid, implying that L. plantarum LR-14 has the potential to be used as a probiotic with multiple functions.

Bioinformatics analysis of Klebsiella pneumoniae M1, from microbial flocculation resources

The synthesis of a microbial flocculant is strictly controlled by its genetic genes, which may be the result of the expression of flocculant genes in the microbial genome. The whole genome of Klebsiella pneumoniae M1 was sequenced for obtaining the mechanism of flocculant synthesis and exploring the mechanism of flocculant production by flocculant bacteria. Therefore, it provided a basis for molecular genetics and functional genomic analysis of flocculants production bacteria. In this way, the mechanism of flocculant production by flocculating bacteria can be better explored. The whole genome sequence of flocculant strain M1 was determined using advanced second generation (Illumina) and third generation (PacBio) sequencing, which was screened from wheat alcohol wastewater. The genes related to flocculant characteristics produced by strain M1 were analyzed for combining with the analysis of flocculant structure characteristics. According to the de novo assembly, a total of 5,511,794 bp clean reads were generated and assembled into 24 contigs. The GC content was up to 58.39%. The genome contained approximately 5383 genes, but 5348 genes had obvious biological functions. A total of 437 genes were involved in carbohydrate metabolism and had coding genes of five carbohydrate-related enzymes. This result indicated that there were functional genes closely related to polysaccharide production in M1 genome. The main metabolic process of flocculant strain Klebsiella pneumoniae M1 was closely related to the potential pathway of extracellular polysaccharide biosynthesis, in which five kinds of carbohydrate synthase genes were involved.

Xanthomonas oryzae Pv. oryzicola Response Regulator VemR Is Co-opted by the Sensor Kinase CheA for Phosphorylation of Multiple Pathogenicity-Related Targets

Two-component systems (TCSs) (cognate sensor histidine kinase/response regulator pair, HK/RR) play a crucial role in bacterial adaptation, survival, and productive colonization. An atypical orphan single-domain RR VemR was characterized by the non-vascular pathogen Xanthomonas oryzae pv. oryzicola (Xoc) is known to cause bacterial leaf streak (BLS) disease in rice. Xoc growth and pathogenicity in rice, motility, biosynthesis of extracellular polysaccharide (EPS), and the ability to trigger HR in non-host tobacco were severely compromised in the deletion mutant strain RΔvemR as compared to the wild-type strain RS105. Site-directed mutagenesis and phosphotransfer experiments revealed that the conserved aspartate (D56) residue within the stand-alone phosphoacceptor receiver (REC) domain is essential for phosphorelay and the regulatory activity of Xoc VemR. Yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) data identified CheA as the HK co-opting the RR VemR for phosphorylation. Affinity proteomics identified several downstream VemR-interacting proteins, such as 2-oxoglutarate dehydrogenase (OGDH), DNA-binding RR SirA, flagellar basal body P-ring formation protein FlgA, Type 4a pilus retraction ATPase PilT, stress-inducible sensor HK BaeS, septum site-determining protein MinD, cytoskeletal protein CcmA, and Type III and VI secretion system proteins HrpG and Hcp, respectively. Y2H and deletion mutant analyses corroborated that VemR interacted with OGDH, SirA, FlgA, and HrpG; thus, implicating multi-layered control of diverse cellular processes including carbon metabolism, motility, and pathogenicity in the rice. Physical interaction between VemR and HrpG suggested cross-talk interaction between CheA/VemR- and HpaS/HrpG-mediated signal transduction events orchestrating the hrp gene expression.

A epsB mutant of Ralstonia solanacearum as novel biocontrol agent of tobacco bacterial wilt via activating salicylic acid signalling

Extracellular polysaccharide (EPS) is the most important virulence factor of R. solanacearum and promotes rapid systemic colonization and induces plant wilting symptoms. In this study, biological control mechanism and effect of EPS biosynthesis genes mutants (ΔepsB, ΔepsC and ΔwecC) on bacterial wilt were evaluated. The expression level of virulence-associated genes, such as xpsR, epsE, vsrB, vsrC and phcA, were significantly decreased in the Δeps strains. Furthermore, application of ΔepsB strain was observed to delay the development of wilt progress for two days and the WT population in rhizosphere soil was significantly reduced by 46.77-fold after ΔepsB strain treatment. If the ΔepsB was applied earlier than WT population, the control efficiencies of ΔepsB were 84.67%, 72.74%, 60.68%, 56.38% and 53.29% at 8, 10, 12, 14, and 16 d after inoculation, respectively. What ‘s more, the ΔepsB mutant significantly induced the expression of plant defence associated genes involved in salicylic acid pathway, but did not affect the expression of genes associated with the jasmonic acid and ethylene pathways. Compared with plant inoculated with WT treatment, the expression of PR1a/c, PR2 and NPR1 was induced 4.5-fold, 6.9-fold and 2.2-fold, respectively, in the plant colonized by ΔepsB mutant. Meanwhile, application of ΔepsB strain in tobacco rhizosphere significantly induced the accumulation of salicylic acid, with increases of 1.58-fold compared with the control. Taken together, the results suggested that the ΔepsB mutant suppressed bacterial wilt disease by activating salicylic acid signalling and has the potential to be used as biocontrol agents for controlling bacterial wilt caused by R. solanacearum.

Sucrose selectively regulates Streptococcus mutans polysaccharide by GcrR.

Streptococcus mutans (S. mutans) is the principal etiological agent in cariogenesis because of its ability to metabolize sucrose into extracellular polysaccharides (EPS). The response regulators GcrR and VicR could regulate EPS metabolism, but with opposing regulatory functions. However, the cooperative interactions between gcrR and vicR regulating sucrose-selective EPS metabolism have not been fully elucidated. First, we constructed several dual-mutant strains (vicR+ gcrR+, vicR and gcrR overexpression; vicR+gcrR-, vicR overexpression and gcrR deficient; ASvicRgcrR+, vicR low-expression and gcrR overexpression; ASvicRgcrR-, vicR low-expression and gcrR deficient) to clarify gtfB/gtfC expression levels were modulated by gcrR regardless of the vicR gene expression levels. Next, we found gcrR deletion mutant (SmugcrR) displayed obvious auto-aggregation and bacterial cells were densely packed in enriched EPS induced by sucrose. In the contrast, SmugcrR biofilm showed very little carbohydrate-dependent aggregation in the absence of sucrose. The presence of sucrose amplifies the negative regulation of gcrR acting as a 'switch-off'. After sucrose induction, dexA gene expression was significantly enhanced in gcrR overexpression mutant (SmugcrR+). Furthermore, GcrR was shown to directly bind to the promoter region of the dexA gene. Taken together, our results reveal that GcrR interacts with VicR to block EPS biosynthesis via polysaccharide digestion by DexA, and that this process is induced in a sucrose-selective manner. Hence, targeting GcrR is a potential strategy for the management of dental caries.

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.

Genome Reduction and Secondary Metabolism of the Marine Sponge-Associated Cyanobacterium Leptothoe.

Sponges form symbiotic relationships with diverse and abundant microbial communities. Cyanobacteria are among the most important members of the microbial communities that are associated with sponges. Here, we performed a genus-wide comparative genomic analysis of the newly described marine benthic cyanobacterial genus Leptothoe (Synechococcales). We obtained draft genomes from Le. kymatousa TAU-MAC 1615 and Le. spongobia TAU-MAC 1115, isolated from marine sponges. We identified five additional Leptothoe genomes, host-associated or free-living, using a phylogenomic approach, and the comparison of all genomes showed that the sponge-associated strains display features of a symbiotic lifestyle. Le. kymatousa and Le. spongobia have undergone genome reduction; they harbored considerably fewer genes encoding for (i) cofactors, vitamins, prosthetic groups, pigments, proteins, and amino acid biosynthesis; (ii) DNA repair; (iii) antioxidant enzymes; and (iv) biosynthesis of capsular and extracellular polysaccharides. They have also lost several genes related to chemotaxis and motility. Eukaryotic-like proteins, such as ankyrin repeats, playing important roles in sponge-symbiont interactions, were identified in sponge-associated Leptothoe genomes. The sponge-associated Leptothoe stains harbored biosynthetic gene clusters encoding novel natural products despite genome reduction. Comparisons of the biosynthetic capacities of Leptothoe with chemically rich cyanobacteria revealed that Leptothoe is another promising marine cyanobacterium for the biosynthesis of novel natural products.

Light Signaling Regulates Aspergillus niger Biofilm Formation by Affecting Melanin and Extracellular Polysaccharide Biosynthesis.

ABSTRACTLight is an important signal source in nature, which regulates the physiological cycle, morphogenetic pathways, and secondary metabolites of fungi. As an external pressure on Aspergillus niger, light signaling transmits stress signals into the cell via the mitogen-activated protein kinase (MAPK) signaling pathway. Studying the effect of light on the biofilm of A. niger will provide a theoretical basis for light in the cultivation of filamentous fungi and industrial applications. Here, the characterization of A. niger biofilm under different light intensities confirmed the effects of light signaling. Our results indicated that A. niger intensely accumulated protective mycelial melanin under light illumination. We also discovered that the RlmA transcription factor in the MAPK signaling pathway is activated by light signaling to promote the synthesis of melanin, chitin, and other exopolysaccharides. However, the importance of melanin to A. niger biofilm is rarely reported; therefore, we knocked out key genes of the melanin biosynthetic pathway—Abr1 and Ayg1. Changes in hydrophobicity and electrostatic forces resulted in the decrease of biofilm caused by the decrease of melanin in mutants.

Impact of olive mill wastewaters on the physiological behavior of a wild-type new Ganoderma resinaceum isolate.

A new wild-type Ganoderma resinaceum isolate was cultivated on glucose-enriched liquid cultures with olive mill wastewaters (OMWs) in initial phenolic compounds concentrations 0.0 (control), 0.5, 0.8, and 1.5g/l. The effect of the fungus on the reduction of phenolics and color was assessed, whereas biomass production, glucose consumption, intra-cellular (IPS) and extra-cellular (EPS) polysaccharides biosynthesis, antioxidant activity of the biomass, and laccase synthesis were monitored. Results showed that significant phenolic reduction (94.5%) and decolorization (76.5%) occurred, 14.6g/l of biomass was produced, glucose was almost totally consumed, EPS were produced in sufficient amounts (0.79g/l), whereas the presence of OMWs enhanced the synthesis of IPS (maximum absolute values 4.0-5.2g/l corresponding to 35-42% w/w). Kinetic analysis demonstrated that EPS and IPS values fluctuated with time, regardless of the available amount of glucose in the media, showing a maximum in the 17th day of culture. Laccase was highly synthesized in the middle of the fermentation, reaching the maximum value of 14U/ml. Little growth was however observed at 1.5g/l phenolics. Strong correlation between total phenolic content and free radical scavenging activity has been noticed in the methanolic extracts of the mycelium. Results strongly suggest the potentiality of G. resinaceum utilization in the OMW waste treatment.

Ammonium nitrate regulated the color characteristic changes of pigments in Monascus purpureus M9

Monascus pigments (MPs) with different color characteristics, produced by submerged fermentation of Monascus purpureus M9, have potential application in food industry. In the present study, the effects and regulatory mechanisms of ammonium nitrate (AN) on the color characteristics of MPs were investigated. The concentration of intracellular pigments was significantly decreased when subjected to AN. The hue and lightness value indicated AN altered the total pigments appearance from original red to orange. The HPLC analysis for six major components of MPs showed that the production of rubropunctatin or monascorubrin, was significantly reduced to the undetectable level, whereas the yields of monascin, ankaflavin, rubropunctamine and monascorubramine, were apparently increased with AN supplement. To be noted, via real-time quantitative PCR strategy, the expression level of mppG, closely relative to orange pigments biosynthesis, was significantly down-regulated. However, the expression of mppE, involved in yellow pigments pathway, was up-regulated. Moreover, the broth pH value was dropped to 2.5–3.5 in the fermentation process resulted from AN treatment, along with the increased extracellular polysaccharide biosynthesis. Taken together, the change of MPs categories and amounts by AN might be the driving force for the color characteristics variation in M. purpureus M9. The present study provided useful data for producing MPs with different compositions and modified color characteristics.

PSII-7 Complete genome analysis of a novel mucolytic bacterium, Prevotella mucinisolvens sp. nov., isolated from bovine rumen epithelium

Abstract We report the complete genome sequence of a novel mucolytic bacterium, Prevotella mucinisolvens sp. nov. Mucolytic bacteria were isolated from rumen epithelium of the dorsal sac of Korean cattle steer using a targeted cultivation on a mucin defined medium as a sole carbon source in anaerobic conditions. The genome of P. mucinisolvens was sequenced by means of both the Illumina HiSeq™ X and PacBio RSII platforms. The genome (2,730,135 bp) was found to contain 2,445 genes, 2,374 coding sequences, 61 transfer RNA, 1 transfer-messenger RNA, and 9 ribosomal RNA. The P. mucinisolvens had a total 51 glycoside hydrolases (GHs), of which 14 GHs, including β-galactosidases (GH2, GH20), α-N-acetylgalactosaminidases (GH101), α-N-acetylglucosaminidase (GH89), sialidase (GH33), and fucosidases (GH29, GH95), were identified as enzymes involved in mucin degradation. Following the KEGG pathways, the putative mucolytic pathway was constructed, including the metabolism of carbon sources such as galactose, N-acetylglucosamine, sialic acid (N-acetylneuraminic acid), and mannose. The presence of putative extracellular polysaccharide biosynthesis pathways, including Wzx/Wzy-dependent pathway (4 putative glycosyltransferases, 3 acetyltransferases, 1 flippase, 1 polymerase, 1 polysaccharide co-polymerase, and 1 outer membrane transport protein) and synthase-dependent pathway (1 putative synthase, 3 precursors of synthesis), was confirmed in P. mucinisolvens. Twelve putative virulence factors associated with adherence (hasB, KpsF, and htpB), stress reactions (clpP and clpC), antiphagocytosis (hasB and bcs1), O-antigen (gmd, fcl, and galE), and metabolic adaption (panD) were identified. This study contributes to a better understanding of epimural bacteria in putative mucin-degrading ability.

Screening virulence factors of porcine extraintestinal pathogenic Escherichia coli (an emerging pathotype) required for optimal growth in swine blood.

Porcine extraintestinal pathogenic Escherichia coli (ExPEC) is occurring with increasing frequency in China, which causes acute septicemia and sudden death in pigs leading to significant economic losses. Bacterial survival and even proliferation within host bloodstream are a common manifestation of a number of bacterial septicemias, including porcine ExPEC diseases. However, the underlying pathogenesis for this novel pathotype of ExPEC has not been explored deeply. Here, we used a conjunction with transposon mutagenesis to identify the mechanisms of bacterial fitness involved in optimal growth of porcine ExPEC in swine serum ex vivo under static culture. Our work identified 28 genes involved in nucleotide biosynthesis, extracellular polysaccharide biosynthesis, regulators Fur and FNR, acid/zinc resistance, and Deley-Douderoff carbon metabolism that are required for the serum fitness. Subsequent functional analyses revealed that either interruption of de novo nucleotide biosynthesis or blocking of several extracellular polysaccharide biosynthesis including O2-antigen, Lipid A-core, and ECA significantly affect porcine ExPEC's growth in swine serum and proliferation in host bloodstream. Furthermore, the reasonable regulations of ironand anaerobic metabolisms in response to host stimuli by global regulators Fur and FNR also play key roles during systemic infection of porcine ExPEC.These findings provide compelling evidences that de novo nucleotide biosynthesis may enable porcine ExPEC to adapt to swine blood-specific nutrient availability, and the effective assembly of O-antigen, lipid A-core, and ECA is required to resist the bactericidal activity of swine serum. These studies contribute to better understand the underlying mechanisms employed by porcine ExPEC to survive, grow in the swine bloodstream, and cause disease. These related factors may serve as therapeutic targets for countering or preventing ExPEC serum resistance in the clinic.

Eliminating the capsule-like layer to promote glucose uptake for hyaluronan production by engineered Corynebacterium glutamicum

Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many interests. Here, we describe the metabolic engineering of Corynebacterium glutamicum and application of a fermentation strategy to manufacture hyaluronan with different molecular weights. C. glutamicum is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways and attenuation of extracellular polysaccharide biosynthesis. The engineered strain produces 34.2 g L−1 hyaluronan in fed-batch cultures. We find secreted hyaluronan encapsulates C. glutamicum, changes its cell morphology and inhibits metabolism. Disruption of the encapsulation with leech hyaluronidase restores metabolism and leads to hyper hyaluronan productions of 74.1 g L−1. Meanwhile, the molecular weight of hyaluronan is also highly tunable. These results demonstrate combinatorial optimization of cell factories and the extracellular environment is efficacious and likely applicable for the production of other biopolymers.

Phosphoglycerate Kinase Is Involved in Carbohydrate Utilization, Extracellular Polysaccharide Biosynthesis, and Cell Motility of Xanthomonas axonopodis pv. glycines Independent of Clp.

Phosphoglycerate kinase (Pgk), catalyzing the reversible conversions between glycerate-1.3-2P and glycerate-3P, plays an important role in carbohydrate metabolism. Here, we show that a Pgk-deficient mutant (NΔpgk) of Xanthomonas axonopodis pv. glycines (Xag) could grow in medium with glucose, galactose, fructose, mannose, or sucrose, as the sole carbon source, suggesting that Xag may employ Entner-Doudoroff (ED) and pentose phosphate pathway (PPP), but not glycolysis, to catabolize glucose. NΔpgk could not utilize pyruvate, suggesting that Pgk might be essential for gluconeogenesis. Mutation in pgk led to a reduction of extracellular polysaccharide (EPS) biosynthesis, cell motility, and intracellular ATP. As a result, the virulence of NΔpgk was significantly compromised in soybean. NΔpgk could be fully complemented by the wild-type pgk, but not by clp (encoding Crp-like protein). qRT-PCR analyses demonstrated that pgk is regulated by the HrpG/HrpX cascade, but not by Clp. These results suggest that Pgk is involved in carbohydrate utilization, EPS biosynthesis, and cell motility of Xag independent of Clp.