Streptococcus zooepidemicus is an important opportunistic pathogen of several species including humans. This organism is also well known as the main producing strain in industrial production of hyaluronic acid (HA), which is the component of its capsule polysaccharide. How its virulence and capsule polysaccharide production is regulated remains poorly understood. Intercellular chemical signaling among bacteria provides communities of microbes the opportunity to coordinate gene expression to facilitate group behavior, such as pathogenicity, capsule polysaccharide production, etc. Yet no conserved cell-to-cell signaling system has been elucidated in S. zooepidemicus. Encoded within the genome of S. zooepidemicus is one Rgg regulator encoding gene (rgg) with low similarity to both rgg2 and rgg3 from Streptococcus pyogenes. A small ORF (named as shp) encoding a novel short hydrophobic peptide (SHP) was found in the vicinity of rgg. We found that the active form of pheromone is short and hydrophobic (LLLLKLA), corresponding to the C terminal 7 amino acids of the pre-peptide Shp, which shows divergent sequence to all peptide pheromones reported in streptococci. In response to active SHP, Rgg functions as a transcriptional activator to induce the expression of shp, forming a positive feedback circuit. Bacteria social behaviors, such as capsule polysaccharide production and biofilm formation, were significantly affected when the rgg-shp pathway was inactivated. These data provide the first demonstration that Rgg/Shp signaling pathway comprises an active quorum sensing system in S. zooepidemicus.

Biosynthesis of polysaccharide hyaluoronic acid (HA) by Streptococcus zooepidemicus is a carbon-intensive process. The carbon flux and factor(s) restricting HA yield were not well understood. Here, we investigated the function of genes involved in sucrose metabolism and identified targets limiting HA yield, which were exploited to construct efficient S. zooepidemicus strains for HA production. The sucrose uptake was addressed by deletion of scrA and scrB, which encodes sucrose-PTS permease and sucrose-6-phosphate hydrolase, respectively. We found that scrB was essential for the growth of S. zooepidemicus and HA biosynthesis, and accumulation of sucrose-6-phosphate was toxic. ΔscrB could not grow in THY-sucrose medium, while ΔscrA and ΔscrAΔscrB showed negligible growth defects. Overexpression of scrA significantly reduced biomass and HA production, while overexpression of scrB resulted in 26% increase of biomass and 30% increase of HA yield. We revealed that fructose-6-phosphate for HA biosynthesis mainly originates from glucose-6-phosphate. Deletion of scrK, a gene encoding hexokinase, led to 11% reduction of biomass and 12% decrease of HA yield, while deletion of hasE, a gene encoding phosphoglucoisomerase, resulted in the abolishment of HA biosynthesis and a significantly slow growth. We found that HA biosynthesis could be improved by directing carbon flux to fructose-6-phosphate. Deletion of fruA encoding the EII of fructose-PTS and fruK encoding phosphofructokinase showed no apparent effect on cell growth, but resulted in 22 and 27% increase of HA yield, respectively. Finally, a strain with 55% increase of HA was constructed by overexpression of scrB in ΔfruK. These results provide a solid foundation for further metabolic engineering of S. zooepidemicus for highly efficient HA production.

Citric acid is widely used in the food, chemical and pharmaceutical industries. Aspergillus niger is the workhorse used for citric acid production in industry. A canonical citrate biosynthesis that occurred in mitochondria was well established; however, some research suggested that the cytosolic citrate biosynthesis pathway may play a role in this chemical production. Here, the roles of cytosolic phosphoketolase (PK), acetate kinase (ACK) and acetyl-CoA synthetase (ACS) in citrate biosynthesis were investigated by gene deletion and complementation in A. niger. The results indicated that PK, ACK and ACS were important for cytosolic acetyl-CoA accumulation and had significant effects on citric acid biosynthesis. Subsequently, the functions of variant PKs and phosphotransacetylase (PTA) were evaluated, and their efficiencies were determined. Finally, an efficient PK-PTA pathway was reconstructed in A. niger S469 with Ca-PK from Clostridium acetobutylicum and Ts-PTA from Thermoanaerobacterium saccharolyticum. The resultant strain showed an increase of 96.4% and 88% in the citrate titer and yield, respectively, compared with the parent strain in the bioreactor fermentation. These findings indicate that the cytosolic citrate biosynthesis pathway is important for citric acid biosynthesis, and increasing the cytosolic acetyl-CoA level can significantly enhance citric acid production.

Wheat (Triticum aestivum L.) is the predominant grain crop and plays a pivotal role in grain production in Xinjiang Uygur Autonomous Region (XUAR), China. Its cultivated area constitutes approximat...

Fusarium pseudograminearum is an important plant pathogen that invades many crops (Zhang et al. 2018). Since it was first discovered in Australia in 1951, F. pseudograminearum has been reported in many countries and regions and caused huge economic losses (Burgess et al. 2001). In 2012, crown rot of wheat caused by F. pseudograminearum was discovered for the first time in Henan Province, China (Li et al. 2012). Wheat (Triticum aestivum L.) is one of the most important food crops in Xinjiang Uygur Autonomous Region (XUAR), with 1.07 million hectares cultivated in 2020. In June 2023, a survey of crown rot disease was carried out in winter wheat cv. Xindong 20 in Hotan area, XUAR, China (80.148907°E, 37.051474°N). About 5% of wheat plants showed symptoms of crown rot such as browning of the stem base and white head. The disease was observed in 85% of wheat fields. In order to identify the pathogens, 36 pieces of diseased stem basal tissue, 0.5 cm in length, were collected and sterilized with 75% alcohol for 30s and 5% NaOCl solution for 2 min, then rinsed three times with sterile water and placed on potato dextrose agar (PDA) medium at 25°C. A total of 27 isolates with consistent morphological characteristics were obtained using single-spore technique (Leslie and Summerell. 2006), and the isolation rate was 75%. The isolates grew rapidly on PDA, produced large numbers of fluffy white hyphae, and pink pigment accumulated in the medium. The isolates were grown on 2% mung bean flour medium and identified by morphological and molecular methods. Macroconidia were abundant, relatively slender, curved to almost straight, commonly two to seven septate, and averaged 22 to 72 × 1.8 to 4.9 μm. Microconidia were not observed. The morphological characters are consistent with Fusarium (Aoki and O'Donnell. 1999). Two isolates (LP-1 and LP-3) were selected for molecular identification. Primers EF1/EF2 (5'-ATGGGTAAGGARGACAAGAC-3'/5'-GGARGTACCAGTSATCATG-3') were used to amplify a portion of the EF-1α gene (O'Donnell et al. 1998). The two 696 bp PCR products were sequenced and submitted to GenBank. The EF-1α gene sequences (GenBank Accession No: PP062794 and PP062795) shared 99.9% identity (695/696) with published F.pseudograminearum sequences (e.g., OP105187, OP105184, OP105179, OP105173). The identification was further confirmed by F. pseudograminearum species-specific PCR primers Fp1-1/Fp1-2 (Aoki and O'Donnell. 1999). The expected PCR products of 518 bp were produced only in F. pseudograminearum. Pathogenicity tests of LP-1 and LP-3 isolates were performed on 7-day-old seedlings of winter wheat cv. Xindong 20 using the drip inoculation method with a 10-μl of a 106 macroconidia ml-1 suspension near the stem base (Xu et al. 2017). The experiment was repeated five times in a 20 to 25°C greenhouse. Control seedlings were treated with sterile water. After 4 weeks, wheat seedling death and crown browning occurred in the inoculated plants with over 90% incidence. No symptoms were observed in the control plants. The pathogen was reisolated from the inoculated plants by the method described above and identified by morphological and PCR amplification using F. pseudograminearum species-specific primers Fp1-1/Fp1-2. No F. pseudograminearum was isolated from the control plants, fulfilling Koch's postulates. To our knowledge, this is the first report of F. pseudograminearum causing crown rot of winter wheat in XUAR of China. Since F. pseudograminearum can cause great damage to wheat, one of the most important food crops in China, necessary measures should be taken to prevent the spread of F. pseudograminearum to other regions.