A cell-free transcription-translation pipeline for recreating methylation patterns boosts DNA transformation in bacteria.

The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic biological chassis. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here, we report imitating methylation patterns rapidly in TXTL (IMPRINT), a generalized, rapid, and scalable approach based on cell-free transcription-translation (TXTL) to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from a bacterium's restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the bacterium's DNA methylation pattern, circumventing restriction and enhancing transformation. With IMPRINT, we efficiently multiplex methylation by diverse DNA methyltransferases and enhance plasmid transformation in gram-negative and gram-positive bacteria. We also develop a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to screen a ribosome-binding site library in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling the use of sophisticated genetic manipulation tools across the bacterial world.

Expanding the flexibility of base editing for high-throughput genetic screens in bacteria.

Genome-wide screens have become powerful tools for elucidating genotype-to-phenotype relationships in bacteria. Of the varying techniques to achieve knockout and knockdown, CRISPR base editors are emerging as promising options. However, the limited number of available, efficient target sites hampers their use for high-throughput screening. Here, we make multiple advances to enable flexible base editing as part of high-throughput genetic screening in bacteria. We first co-opt the Streptococcus canis Cas9 that exhibits more flexible protospacer-adjacent motif recognition than the traditional Streptococcus pyogenes Cas9. We then expand beyond introducing premature stop codons by mutating start codons. Next, we derive guide design rules by applying machine learning to an essentiality screen conducted in Escherichia coli. Finally, we rescue poorly edited sites by combining base editing with Cas9-induced cleavage of unedited cells, thereby enriching for intended edits. The efficiency of this dual system was validated through a conditional essentiality screen based on growth in minimal media. Overall, expanding the scope of genome-wide knockout screens with base editors could further facilitate the investigation of new gene functions and interactions in bacteria.

Systematic interrogation of CRISPR antimicrobials in Klebsiella pneumoniae reveals nuclease-, guide- and strain-dependent features influencing antimicrobial activity.

CRISPR-Cas systems can be utilized as programmable-spectrum antimicrobials to combat bacterial infections. However, how CRISPR nucleases perform as antimicrobials across target sites and strains remains poorly explored. Here, we address this knowledge gap by systematically interrogating the use of CRISPR antimicrobials using multidrug-resistant and hypervirulent strains of Klebsiella pneumoniae as models. Comparing different Cas nucleases, DNA-targeting nucleases outperformed RNA-targeting nucleases based on the tested targets. Focusing on AsCas12a that exhibited robust targeting across different strains, we found that the elucidated modes of escape varied widely, restraining opportunities to enhance killing. We also encountered individual guide RNAs yielding different extents of clearance across strains, which were linked to an interplay between improper gRNA folding and strain-specific DNA repair and survival. To explore features that could improve targeting across strains, we performed a genome-wide screen in different K. pneumoniae strains that yielded guide design rules and trained an algorithm for predicting guide efficiency. Finally, we showed that Cas12a antimicrobials can be exploited to eliminate K. pneumoniae when encoded in phagemids delivered by T7-like phages. Altogether, our results highlight the importance of evaluating antimicrobial activity of CRISPR antimicrobials across relevant strains and define critical parameters for efficient CRISPR-based targeting.

The PRMT5/WDR77 complex restricts hepatitis E virus replication.

Hepatitis E virus (HEV) is one of the main pathogenic agents of acute hepatitis in the world. The mechanism of HEV replication, especially host factors governing HEV replication is still not clear. Here, using HEV ORF1 trans-complementation cell culture system and HEV replicon system, combining with stable isotope labelling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we aimed to identify the host factors regulating HEV replication. We identified a diversity of host factors associated with HEV ORF1 protein, which were putatively responsible for viral genomic RNA replication, in these two cell culture models. Of note, the protein arginine methyltransferase 5 (PRMT5)/WDR77 complex was identified in both cell culture models as the top hit. Furthermore, we demonstrated that PRMT5 and WDR77 can specifically inhibit HEV replication, but not other viruses such as HCV or SARS-CoV-2, and this inhibition is conserved among different HEV strains and genotypes. Mechanistically, PRMT5/WDR77 can catalyse methylation of ORF1 on its R458, impairing its replicase activity, and virus bearing R458K mutation in ORF1 relieves the restriction of PRMT5/WDR77 accordingly. Taken together, our study promotes more comprehensive understanding of viral infections but also provides therapeutic targets for intervention.

The crosstalk between epilepsy and dementia: A systematic review and meta-analysis.

BACKGROUND: Epilepsy and dementia are bidirectional. The purpose of this review was to investigate the epidemiological characteristics of and to identify the risk factors for epilepsy in patients with dementia and dementia in patients with epilepsy. METHODS: We retrieved the PubMed, Embase, Cochrane and Web of Science databases through January 2023. Two individuals screened the articles, extracted the data, and used a random effects model to pool the estimates and 95% confidence intervals (CIs). RESULTS: From 3475 citations, 25 articles were included. The prevalence of seizures/epilepsy was 4% among dementia patients and 3% among Alzheimer's disease (AD) patients. For vascular dementia, Lewy body dementia, and frontotemporal dementia, the pooled period prevalence of seizures/epilepsy was 6%, 3%, and 2%, respectively. Baseline early-onset AD was associated with the highest risk of 5-year epilepsy (pooled hazard ratios: 4.06; 95% CI: 3.25-5.08). Dementia patients had a 2.29-fold greater risk of seizures/epilepsy than non-dementia patients (95% CI: 1.37-3.83). Moreover, for baseline epilepsy, the pooled prevalence of dementia was 17% (95% CI: 10-25%), and that of AD was 15% (95% CI: 9-21%). The pooled results suggested that epilepsy is associated with a greater risk of dementia (risk ratio: 2.83, 95% CI: 1.64-4.88). CONCLUSIONS: There are still gaps in epidemiology regarding the correlation between dementia types and epilepsy, vascular risk factors, and the impact of antiseizure medication or cognitive improvement drugs on epilepsy and AD comorbidity.

Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption.

COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as "Cluster 5" that contained mutations in the spike protein. However, the functional properties of these "Cluster 5" mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry. Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.

SARS-CoV-2 NSP8 suppresses type I and III IFN responses by modulating the RIG-I/MDA5, TRIF, and STING signaling pathways.

SARS-CoV-2 has developed a variety of approaches to counteract host innate antiviral immunity to facilitate its infection, replication and pathogenesis, but the molecular mechanisms that it employs are still not been fully understood. Here, we found that SARS-CoV-2 NSP8 inhibited the production of type I and III interferons (IFNs) by acting on RIG-I/MDA5 and the signaling molecules TRIF and STING. Overexpression of NSP8 downregulated the expression of type I and III IFNs stimulated by poly (I:C) transfection and infection with SeV and SARS-CoV-2. In addition, NSP8 impaired IFN expression triggered by overexpression of the signaling molecules RIG-I, MDA5, and MAVS, instead of TBK1 and IRF3-5D, an active form of IRF3. From a mechanistic view, NSP8 interacts with RIG-I and MDA5, and thereby prevents the assembly of the RIG-I/MDA5-MAVS signalosome, resulting in the impaired phosphorylation and nuclear translocation of IRF3. NSP8 also suppressed the TRIF- and STING- induced IFN expression by directly interacting with them. Moreover, ectopic expression of NSP8 promoted virus replications. Taken together, SARS-CoV-2 NSP8 suppresses type I and III IFN responses by disturbing the RIG-I/MDA5-MAVS complex formation and targeting TRIF and STING signaling transduction. These results provide new insights into the pathogenesis of COVID-19.

Chromosome evolution of Escherichia coli Nissle 1917 for high-level production of heparosan.

Heparosan is a crucial-polysaccharide precursor for the chemoenzymatic synthesis of heparin, a widely used anticoagulant drug. Presently, heparosan is mainly extracted with the potential risk of contamination from Escherichia coli strain K5, a pathogenic bacterium causing urinary tract infection. Here, a nonpathogenic probiotic, E. coli strain Nissle 1917 (EcN), was metabolically engineered to carry multiple copies of the 19-kb kps locus and produce heparosan to 9.1 g/L in fed-batch fermentation. Chromosome evolution driven by antibiotics was employed to amplify the kps locus, which governed the synthesis and export of heparosan from EcN at 21 mg L-1 OD-1 . The average copy number of kps locus increased from 1 to 24 copies per cell, which produced up to 104 mg L-1 OD-1 of heparosan in the shaking flask cultures of engineered strains. The following in-frame deletion of recA stabilized the recombinant duplicates of chromosomal kps locus and the productivity of heparosan in continuous culture for at least 56 generations. Fed-batch fermentation of the engineered strain EcN8 was carried out to bring the yield of heparosan up to 9.1 g/L. Heparosan from the fermentation culture was further purified at a 75% overall recovery. The structure of purified heparosan was characterized and further modified by N-sulfotransferase with 3'-phosphoadenosine-5'-phosphosulfate as the sulfo-donor. The analysis of element composition showed that heparosan was N-sulfated by over 80%. These results indicated that duplicating large DNA cassettes up to 19-kb, followed by high-cell-density fermentation, was promising in the large-scale preparation of chemicals and could be adapted to engineer other industrial-interest bacteria metabolically.

Bortezomib plus dexamethasone as first-line therapy for patients with POEMS syndrome.

POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes) syndrome is a rare plasma cell dyscrasia without standard front-line treatment. Merely, few studies have reported the responses and outcomes of bortezomib plus dexamethasone (BDex) in POEMS syndrome. In this study, a total of 69 patients (40 males) treated with front-line BDex were included. The median age at diagnosis was 50 years (range, 30-78 years). After a median of 9 cycles BDex (range 1-9), fifty-two (88.1%), thirty-two (46.4%), and forty-seven (71.2%) patients achieved the best neurologic response, hematological complete response, and serum vascular endothelial growth factor (VEGF) response, respectively. The extravascular overload, pulmonary hypertension, and renal impairment also substantially improved. No treatment-related death occurred. Two patients developed grade-1 bortezomib-induced peripheral neuropathy and were reversible after drug withdrawal. After a median follow-up of 22.5 months, the estimated 2-year overall survival and time to next treatment were 95.7% and 65.6%, respectively. In conclusion, the combination of bortezomib and dexamethasone is effective, with a high response rate and safety profile for patients with newly diagnosed POEMS syndrome.

Dietary diversity and mental health in preschoolers in rural China.

OBJECTIVE: To investigate the prevalence of mental health problems among preschoolers in rural China and examine the relationship between dietary diversity and mental health. DESIGN: A cross-sectional survey analysis was performed. Child mental health was assessed with the Strengths and Difficulties Questionnaire (SDQ). Child dietary diversity was assessed with the dietary diversity score (DDS), which was calculated based on nine food groups using a 24-h recall method. Data were analysed using unadjusted and adjusted logistic regression models. SETTING: Two nationally designated poverty counties in Hunan Province of China. PARTICIPANTS: Preschoolers (n 1334) aged 3-5 years, preschools (n 26). RESULTS: Of 950 preschoolers with data on both dietary diversity and mental health, 663 (70 %) were classified as having at least one kind of mental health problem. The prevalences of emotional symptoms, conduct problems, symptoms of hyperactive/inattention, peer relationship problems and poor prosocial behaviour were 39, 27, 23, 12 and 26 %, respectively. Male preschoolers showed higher risks of having mental health problems than their female counterparts on each SDQ subscale except for conduct problems. Moreover, a higher DDS was significantly associated with a lower likelihood of having symptoms of hyperactivity/inattention, peer relationship problems and prosocial behaviour problems after adjustment for confounders (preschoolers' age, gender, cognitive ability, parental migration status, primary caregiver's education and household socio-economic status). CONCLUSIONS: The prevalence of mental health problems was high among preschoolers in rural China. Improving child dietary diversity might be an important strategy to consider in the design of interventions to improve child mental health.

Heparin stimulates biofilm formation of Escherichia coli strain Nissle 1917.

OBJECTIVES: Escherichia coli strain Nissle 1917 (EcN), a gut probiotic competing with pathogenic bacteria, has been used to attenuate various intestinal dysfunctions. Heparin is a sulfated glycosaminoglycan enriched in the human and animal intestinal mucosa, which has a close connection with bacterial biofilm formation. However, the characteristics of heparin affecting bacterial biofilm formation remain obscure. In this study, we investigated the influence of heparin and its derivatives on EcN biofilm formation. RESULTS: Here, we found that heparin stimulated EcN biofilm formation in a dose-dependent manner. With the addition of native heparin, the EcN biofilm formation increased 6.9- to 10.8-fold than that without heparin, and was 1.4-, 3.1-, 3.0-, and 3.8-fold higher than that of N-desulfated heparin (N-DS), 2-O-desulfated heparin (2-O-DS), 6-O-desulfated heparin (6-O-DS), and N-/2-O-/6-O-desulfated heparin (N-/2-O-/6-O-DS), respectively. Depolymerization of heparin produced chain-shortened heparin fragments with decreased molecular weight. The depolymerized heparins did not stimulate EcN biofilm formation. The OD570 value of EcN biofilm with the addition of chain-shortened heparin fragments was 8.7-fold lower than that of the native heparin. Furthermore, the biofilm formation of Salmonella enterica serovar Typhimurium was also investigated with the addition of heparin derivatives, and the results were consistent with that of EcN biofilm formation. CONCLUSIONS: We conclude that heparin stimulated EcN biofilm formation. Both the sulfation and chain-length of heparin contributed to the enhancement of EcN biofilm formation. This study increases the understanding of how heparin affects biofilm formation, indicating the potential role of heparin in promoting intestinal colonization of probiotics that antagonize pathogen infections.

The construction of a dual-functional strain that produces both polysaccharides and sulfotransferases.

OBJECTIVES: Heparosan is used as the starting polysaccharide sulfated using sulfotransferase to generate fully elaborate heparin, a widely used clinical drug. However, the preparation of heparosan and enzymes was considered tedious since such material must be prepared in separate fermentation batches. In this study, a commonly admitted probiotic, Escherichia coli strain Nissle 1917 (EcN), was engineered to intracellularly express sulfotransferases and, simultaneously, secreting heparosan into the culture medium. RESULTS: The engineered strain EcN::T7M, carrying the λDE3 region of BL21(DE3) encoding T7 RNA polymerase, expressed the sulfotransferase domain (NST) of human N-deacetylase/N-sulfotransferase-1 (NDST-1) and the catalytic domain of mouse 3-O-sulfotransferase-1 (3-OST-1) in a flask. The fed-batch fermentation of EcN::T7M carrying the plasmid expressing NST was carried out, which brought the yield of NST to 0.21 g/L and the yield of heparosan to 0.85 g/L, respectively. Furthermore, the heparosan was purified, characterized by 1H nuclear magnetic resonance (NMR), and sulfated by NST using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the sulfo donor. The analysis of element composition showed that over 80% of disaccharide repeats of heparosan were N-sulfated. CONCLUSIONS: These results indicate that EcN::T7M is capable of preparing sulfotransferase and heparosan at the same time. The EcN::T7M strain is also a suitable host for expressing exogenous proteins driven by tac promoter and T7 promoter.

Whole genome sequencing of Thaumetopoea pityocampa revealed putative pesticide targets.

Insect neuropeptides play a major role in the regulation of the physiological processes. Due to their versatile effects on the development of insects, their corresponding receptors, which are mostly G-protein coupled receptors, are considered as ideal targets for designing next-generation pesticides. In this study, we aimed to find neuropeptide receptors of pine processionary moth (Thaumetopoea pityocampa), a pest in the Mediterranean countries, that feeds on the needles of pine trees. To this aim, Whole Genome Shotgun sequencing technique was used. de novo assembly of the genome was performed using two different assemblers, SGA and MaSuRCA. The results of two assemblers were compared, and MaSuRCA assembler showed higher N50 length. To find some target GPCRs, sequences of Drosophila melanogaster and evolutionarily close species were used as blast queries in the assembled data. Five GPCRs were chosen from the genome and their expression was confirmed in the larval stage of the insect.

Long-term outcomes of conservative treatment and endovascular treatment in patients with symptomatic spontaneous isolated superior mesenteric artery dissection: a single-center experience.

BACKGROUND: Spontaneous isolated superior mesenteric artery dissection (SISMAD) is a rare vascular disorder, and the treatment strategies remain controversial. This study aimed to compare outcomes of conservative and endovascular treatments in symptomatic patients with SISMAD. METHODS: Forty-two consecutive SISMAD patients who were admitted to a single center between October 2009 and May 2018 were enrolled in this study. Based on their symptoms, 15 had conservative treatment, and 27 had endovascular treatment. The baseline characteristics, treatments, and follow-up results of the conservative group and endovascular group were analysed. RESULTS: The rates of symptom relief were 93.3% in the conservative group and 96.3% in the endovascular group. The procedure-related complications in the endovascular group included one case of pseudoaneurysm formation in the left brachial artery. During the follow-up period (median 28.5 months), a higher proportion of patients in the conservative group had symptom recurrence (42.9% in the conservative group versus 4.8% in the endovascular group, p < 0.001). Four patients in the conservative group and one patient in the endovascular group had additional endovascular intervention during follow-up. Compared with the conservative group, patients in the endovascular group had statistically significantly longer symptom-free survival (p = 0.014) and a higher rate of superior mesenteric artery (SMA) remodeling (p < 0.001). CONCLUSIONS: For symptomatic SISMAD, endovascularly treated patients had a lower rate of symptom recurrence and a higher rate of SMA remodeling in the long term. Prospective, multi-center studies are needed to confirm the long-term outcomes of both treatments.

Clinical outcomes of radiation-induced carotid stenosis: A systematic review and meta-analysis.

OBJECTIVES: Clinical outcomes of radiation-induced carotid stenosis are still unclear. Therefore, a systematic review and meta-analysis is needed to evaluate the short- and long-term outcomes after interventions to treat radiation-induced carotid stenosis. METHODS: PubMed, EMBASE, the Cochrane Library and Web of Science were searched from 1 January 2000 for relevant RCTs and observational studies which reported outcomes after carotid endarterectomy (CEA) and carotid angioplasty and stenting (CAS) for carotid stenosis induced by radiation. Risk of bias were assessed through different scales according to study design. I2 statistic were used to evaluate the heterogeneity, and meta-regression were performed to investigate the source of heterogeneity. Visual inspection of funnel plots was used to judge publication bias. RESULTS: A total of 26 studies with 1002 patients were included. CEA was performed in 364 patients and CAS in 638 patients. The overall estimated rate of short-term stroke was 0.19% (95% CI: 0-0.90%), and the rate of long-term stroke was 2.68 % (95% CI: 1.19-4.57%). The rate of cranial nerve injury in CEA group was significantly higher than that in CAS group [risk ratio (RR): 6.03, 95% CI: 1.63-22.22, P = .007]. The univariate regression analysis showed that the risk of stroke in CAS group were significantly higher than CEA group in both short- and long-term [incidence rate ratio (IRR): 3.62, 95% CI: 1.21-10.85, P = 0.22; IRR: 2.95, 95% CI: 1.02-8.59, P = .046, respectively]. CONCLUSIONS: This systematic review provided the worldwide profile of outcome of treatment for radiation-induced carotid stenosis, and also found that CEA can yield better results for these patients than CAS. Nonetheless, as large-scale studies have not yet been conducted, and there is a definite need for further studies in the future.

Candida auris in Germany and Previous Exposure to Foreign Healthcare.

The emerging yeast Candida auris has disseminated worldwide. We report on 7 cases identified in Germany during 2015-2017. In 6 of these cases, C. auris was isolated from patients previously hospitalized abroad. Whole-genome sequencing and epidemiologic analyses revealed that all patients in Germany were infected with different strains.

Chemoenzymatic quantification for monitoring unpurified polysaccharide in rich medium.

The heparosan polysaccharide serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The previous quantification methods for heparosan rely on time-consuming purification or expensive instruments not readily available for many labs. Here, a chemoenzymatic approach is developed to monitor the production of heparosan in rich medium without purification. After removing the interfering small molecules by ultrafiltration, heparosan was decomposed into oligosaccharides using heparin lyase III. The oligosaccharides were separated from large molecules by ultrafiltration and quantitatively determined by the anthrone-sulfuric acid assay using a spectrophotometer. Based on the different substrate specificity of heparin lyases, the study showed that the concentration of heparosan and heparin in a mixture was discriminatively determined by the two-step chemoenzymatic assay. Furthermore, the anthrone-sulfuric acid assay was observed to be more reliable than the phenol-sulfuric acid assay under these conditions. Besides heparosan and heparin, the chemoenzymatic assay may be adapted to quantify other types of polysaccharides if the specific lyases were available.

Molecular docking and QSAR analyses of aromatic heterocycle thiosemicarbazone analogues for finding novel tyrosinase inhibitors.

A collection of 36 thiosemicarbazone analogues possessed a broad span of tyrosinase inhibitory activities was designed and obtained. Robust and reliable CoMFA and CoMSIA models were gained to predict the structure-activity relationship and the new modifier direction. Inhibitory activities of the compounds were found to greatly depend upon molecular shape, size, and charge. The sterically bulky group at the C-4 position of the thiophene ring contributed a high capacity for biological activity. Some bulky substituents at the C1-position and C12-position, and electron-negative groups at the C3-position, helped to improve the activity of these analogues. The molecular docking results provided visual evidence for QSAR analysis and detailed information about binding mode, affinity, and the principal mechanism between the ligands and tyrosinase. Based on these, a prospective structure modification and optimization of the most potent compound, T32, was suggested for further research.

Cyclic AMP (cAMP) Receptor Protein-cAMP Complex Regulates Heparosan Production in Escherichia coli Strain Nissle 1917.

Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is a significant concern for the cost-effective synthesis of bioengineered heparin. The carbon source is known as the pivotal factor affecting heparosan production. However, the mechanism by which carbon sources control the biosynthesis of heparosan is unclear. In this study, we found that the biosynthesis of heparosan was influenced by different carbon sources. Glucose inhibits the biosynthesis of heparosan, while the addition of either fructose or mannose increases the yield of heparosan. Further study demonstrated that the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex binds to the upstream region of the region 3 promoter and stimulates the transcription of the gene cluster for heparosan biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its capability of binding CRP and eliminated the stimulative effect on transcription. (1)H nuclear magnetic resonance (NMR) analysis was further performed to determine the Escherichia coli strain Nissle 1917 (EcN) heparosan structure and quantify extracellular heparosan production. Our results add to the understanding of the regulation of heparosan biosynthesis and may contribute to the study of other exopolysaccharide-producing strains.