Pseudoaneurysm of mitral-aortic intervalvular fibrosa with rupture: a case report.

BACKGROUND: Mitral-aortic intervalvular fibrosa (MAIVF) is a fibrous region connecting the anterior mitral leaflet (AML) and aortic valve. Pseudoaneurysm of the MAIVF is a rare condition that has been reported as a sequela of infective endocarditis (IE) and surgical trauma. Here, we report a case of a ruptured pseudoaneurysm of the MAIVF, along with some literature reviews. CASE PRESENTATION: A 65-year-old man diagnosed with moderate aortic regurgitation five years previously had a fever of unknown origin. He suddenly developed headache and apraxia and was transported to our hospital. He was diagnosed with intracranial hemorrhage and admitted. One week after admission, echocardiography revealed aorto-mitral discontinuity and protrusion with severe regurgitant flow from left ventricular outflow tract to the left atrium. The AML was suspected to have ruptured. However, intraoperatively, the AML structure was preserved. A ruptured pseudoaneurysm of the MAIVF was also observed. Therefore, we successfully performed pseudoaneurysm repair using a bovine pericardial patch, aortic valve replacement, and mitral annuloplasty. CONCLUSIONS: P-MAIVF is a rare but potentially life-threatening complication of IE, for which timely diagnosis and prompt appropriate therapeutic intervention are required. In the present case, although neither obvious active IE nor history of previous IE could be identified, healed IE was considered based on the clinical course. The patient had intracranial hemorrhage (ICH) with well-controlled heart failure and underwent elective surgical repair more than one month after the onset of ICH, while the clinical course after the surgical procedure was uneventful.

Perforation-free removal of gastric gastrointestinal stromal tumors: Endoscopic inversion and strangulation of muscle layer and resection (EISMR).

Endoscopic resection for GIST has become more widespread in recent years because it is less invasive than surgery. However, when endoscopic resection is performed, a full-layer resection of the gastric wall is often necessary, and extensive suturing is required if perforation occurs, which is a technically challenging procedure. Recently, we reported a new method called endoscopic inversion and strangulation of the muscle layer and resection (EISMR), which consists of endoscopically inverting the muscle layer into the gastric lumen and strangulating the muscle layer with a detachable snare, followed by resection. The study comprised five consecutive patients with gastric GIST ≤50 mm in diameter who underwent EISMR procedures. The main outcomes of the study were en bloc resection rate, R0 resection rate, procedure time, and complications. The results showed that all five patients successfully underwent complete resection without perforation, and the en bloc resection and R0 resection rates were 100%. The median procedure time was 93 min (range, 58-120 min), and there were no major complications. We concluded that EISMR would be a safe and effective technique for endoscopic resection of gastric GISTs and may be an alternative to surgery or endoscopic submucosal dissection.

Novel Concepts for Graphene-Based Nanomaterials Synthesis for Phenol Removal from Palm Oil Mill Effluent (POME).

In recent years, the global population has increased significantly, resulting in elevated levels of pollution in waterways. Organic pollutants are a major source of water pollution in various parts of the world, with phenolic compounds being the most common hazardous pollutant. These compounds are released from industrial effluents, such as palm oil milling effluent (POME), and cause several environmental issues. Adsorption is known to be an efficient method for mitigating water contaminants, with the ability to eliminate phenolic contaminants even at low concentrations. Carbon-based materials have been reported to be effective composite adsorbents for phenol removal due to their excellent surface features and impressive sorption capability. However, the development of novel sorbents with higher specific sorption capabilities and faster contaminant removal rates is necessary. Graphene possesses exceptionally attractive chemical, thermal, mechanical, and optical properties, including higher chemical stability, thermal conductivity, current density, optical transmittance, and surface area. The unique features of graphene and its derivatives have gained significant attention in the application of sorbents for water decontamination. Recently, the emergence of graphene-based adsorbents with large surface areas and active surfaces has been proposed as a potential alternative to conventional sorbents. The aim of this article is to discuss novel synthesis approaches for producing graphene-based nanomaterials for the adsorptive uptake of organic pollutants from water, with a special focus on phenols associated with POME. Furthermore, this article explores adsorptive properties, experimental parameters for nanomaterial synthesis, isotherms and kinetic models, mechanisms of nanomaterial formation, and the ability of graphene-based materials as adsorbents of specific contaminants.

X-ray crystallographic and mutational analysis of the NylC precursor: catalytic mechanism of autocleavage and substrate hydrolysis of nylon hydrolase.

Nylon hydrolase (NylC), a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, is responsible for the degradation of various aliphatic nylons, including nylon-6 and nylon-66. NylC is initially expressed as an inactive precursor (36 kDa), but the precursor is autocatalytically cleaved at Asn266/Thr267 to generate an active enzyme composed of 27 and 9 kDa subunits. We isolated various mutants with amino acid changes at the catalytic centre. X-ray crystallographic analysis revealed that the NylC precursor forms a doughnut-shaped quaternary structure composed of four monomers (molecules A-D) with D2 symmetry. Catalytic residues in the precursor are covered by loop regions at the A/B interface (equivalent to the C/D interface). However, the catalytic residues are exposed to the solvent environment through autocleavage followed by movements of the loop regions. T267A, D306A and D308A mutations resulted in a complete loss of autocleavage. By contrast, in the T267S mutant, autocleavage proceeded slowly at a constant reaction rate (k = 2.8 × 10-5  s-1 ) until complete conversion, but the reaction was inhibited by K189A and N219A mutations. Based on the crystallographic and molecular dynamic simulation analyses, we concluded that the Asp308-Asp306-Thr267 triad, resembling the Glu-Ser-Ser triad conserved in Ntn-hydrolase family enzymes, is responsible for autocleavage and that hydrogen-bonding networks connecting Thr267 with Lys189 and Asn219 are required for increasing the nucleophilicity of Thr267-OH in both the water accessible and water inaccessible systems. Furthermore, we determined that NylC employs the Asp308-Asp306-Thr267 triad as catalytic residues for substrate hydrolysis, but the reaction requires Lys189 and Tyr146 as additional catalytic/substrate-binding residues specific for nylon hydrolysis.

Parametric and kinetic studies of activated sludge dewatering by cationic chitosan-like bioflocculant BF01314 produced from Citrobacter youngae.

Bacterial strains belonging to Citrobacter spp. were reported to produce polysaccharides consisting of N-acetylglucosamine and glucosamine like chitosan, with high flocculation activity. In this work, the flocculation dewatering performance of activated sludge conditioned by a novel cationic chitosan-like bioflocculant (BF) named BF01314, produced from Citrobacter youngae GTC 01314, was evaluated under the influences of flocculant dosage, pH, and temperature. At BF dosage as low as 0.5 kg/t DS, the sludge dewaterability was significantly enhanced in comparison to the raw (untreated) sludge, featuring well-flocculated characteristic (reduction in CST from 22.0 s to 9.4 s) and good sludge filterability with reduced resistance (reduction in SRF by one order from 7.42 × 1011 to 9.59 × 1010 m/kg) and increased compactness of sludge (increase in CSC from 15.2 to 23.2%). Besides, the BF demonstrated comparable high sludge dewatering performance within the pH range between 2 and 8, and temperature range between 25 °C and 80 °C. Comparison between the BF, the pristine chitosan and the commercial cationic copolymer MF 7861 demonstrated equivalent performance with enhanced dewaterability at the dosage between 2.0 and 3.0 kg/t DS. Besides, the BF demonstrated strong flocculation activity (>99%) when added to the sludge suspension using moderate to high flocculation speeds (100-200 rpm) with at least 3-min mixing time. The BF's reaction in sludge flocculation was best fitted with a pseudo first-order kinetic model. Electrostatic charge patching and polymer bridging mechanisms are believed to be the dominant mechanistic phenomena during the BF's sludge conditioning process (coagulation-flocculation).

Viral Infection in Esophageal, Gastric, and Colorectal Cancer.

The human gastrointestinal tract, which constitutes the digestive system, contains a large number of virus particles that maintain organizational homeostasis and health. Conversely, viral pathogens have also attracted attention for their involvement in the pathogenesis of certain cancers, including gastrointestinal cancers. To aid prevention and treatment of these cancers, the relevance of gastrointestinal viral factors as potential risk factors needs to be carefully investigated. This review summarizes and discusses the available literature on the relationship between the development of esophageal, gastric, and colorectal cancers and their corresponding viruses. This review reveals that research on the association between colorectal cancer and viruses, in particular, is still in its infancy compared to the association between HPV and esophageal cancer and between EBV and gastric cancer.

Gel Immersion Endoscopic Mucosal Resection (EMR) for Superficial Nonampullary Duodenal Epithelial Tumors May Reduce Procedure Time Compared with Underwater EMR (with Video).

Materials and Methods: This was a retrospective cohort study conducted in two municipal hospitals. We identified 24 patients with SNADETs of 3-18 mm in diameter who underwent UEMR or GIEMR. One lesion was excluded from the analysis because it was found to be in the stomach after surgery. The primary outcome was procedure time. Results: GIEMR significantly reduced the procedure time compared with UEMR (5 min vs. 10 min, P = 0.016). There was no significant difference between the UEMR and GIEMR groups for en bloc resection rate (93% vs. 100%, P = 1.0) and R0 resection rate (57% vs. 80%, P = 0.39). No serious complications were observed in either group. Conclusions: GIEMR of SNADET has the potential to reduce procedure time compared with UEMR and may be particularly effective in areas where immersion in water is difficult.

Repeated Stimulation of Toll-Like Receptor 2 and Dectin-1 Induces Chronic Pancreatitis in Mice Through the Participation of Acquired Immunity.

BACKGROUND: Stimulation of Toll-like receptor 3 (TLR3) induces autoimmune-mediated pancreatitis in susceptible mice, whereas stimulation of TLR4 causes nonautoimmune-mediated pancreatitis. However, the effects of TLR2 stimulation on the pancreas are unknown. AIMS: We investigated the role of TLR2 stimulation on pancreatic damage by repeatedly stimulating mice with TLR2 ligands. METHODS: Wild-type (WT) and interleukin 10-deficient (IL-10-knockout (KO)) mice were administered zymosan and lipoteichoic acid (LTA) intraperitoneally at various doses twice weekly for 4 weeks. Syngeneic T-cell-deficient mice, B-cell-deficient mice, recombination activating gene 2-deficient (RAG2-KO) mice and RAG2-KO mice that had been reconstituted with CD4+ or CD8+ T cells isolated from WT mice were treated with zymosan similarly. Mice were killed, the severity of pancreatitis was graded histologically, and serum cytokine levels were measured. RESULTS: Repeated administration of zymosan induced pancreatitis dose dependently in both WT and IL-10-KO mice. Administration of LTA induced pancreatitis only in IL-10-KO mice. Adoptive transfer of splenocytes obtained from IL-10-KO mice with pancreatitis did not cause pancreatitis in recipient RAG2-KO mice. Pancreatitis was scarcely observed in RAG2-KO mice and was attenuated in T-cell-deficient and B-cell-deficient mice compared with WT mice. A single administration of zymosan significantly increased the serum level of monocyte chemoattractant protein 1 (MCP-1) in WT mice. CONCLUSIONS: Repeated stimulation of TLR2 and dectin-1 induced nonautoimmune-mediated pancreatitis in mice. Participation of acquired immunity seems to play an important role in the pathogenesis of pancreatitis in association with the increase in serum MCP-1 level.

A giant Brunner gland hamartoma successfully treated by endoscopic excision followed by transanal retrieval: A case report.

RATIONALE: Brunner gland hamartoma (BGH) is a rare tumor of the duodenum. Although BGH is a benign tumor, larger lesion with gastrointestinal symptoms requires tumor removal. We report a giant BGH, successfully treated by endoscopic excision followed by transanal retrieval. PATIENT CONCERNS: A 38-year-old woman complained of severe anemia, tarry stool, and vomiting. DIAGNOSES: Esophagogastroduodenoscopy (EGD) showed a pedunculated giant submucosal mass at the duodenal bulb. INTERVENTIONS: We attempted to remove it because the lesion seemed to be responsible for patient's anemia and vomiting. The lesion had clear but bulky stalk. We carefully cut the stalk using needle-knife and IT knife2. We tried to retrieve specimen, but the mass could not pass through the pyloric ring because of its size. Then we tried to obtain the specimen from anus. Polyethylene glycol solution was administered to accelerate rapid excretion. OUTCOMES: The mass was successfully removed and was histologically confirmed as a giant BGH, measuring 55 mm in size. LESSONS: Reports about endoscopic resection of giant BGH are rare. Moreover, our case is the first report of transanal retrieval of resected specimen using polyethylene glycol solution. Endoscopic resection of BGH is less-invasive but can be more challenging if the mass is large. Our case provides useful option for endoscopic treatment of giant BGH.

Structural and functional characterization of nylon hydrolases.

Biodegradation of synthetic polymers is recognized as a useful way to reduce their environmental load and pollution, loss of natural resources, extensive energy consumption, and generation of greenhouse gases. The potential use of enzymes responsible for the degradation of the targeted polymers is an effective approach which enables the conversion of the used polymers to original monomers and/or other useful compounds. In addition, the enzymes are expected to be applicable in industrial processes such as improving the surface structures of the polymers. Especially, conversion of the solid polymers to soluble oligomers/monomers is a key step for the biodegradation of the polymers. Regarding the hydrolysis of polyamides, three enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (NylA), 6-aminohexanoate-dimer hydrolase (NylB), and 6-aminohexanoate-oligomer endo-hydrolase (nylon hydrolase, NylC), are found in several bacterial strains. In this chapter, we describe our approach for the screening of microorganisms which degrade nylons and related compounds; preparation of substrates; assay of hydrolytic activity for soluble and insoluble substrates; and X-ray crystallographic and computational approaches for analysis of structure and catalytic mechanisms of the nylon-degrading enzymes.

Degradation Potential of the Nonylphenol Monooxygenase of Sphingomonas sp. NP5 for Bisphenols and Their Structural Analogs.

The nonylphenol-degrading bacterium Sphingomonas sp. strain NP5 has a very unique monooxygenase that can attack a wide range of 4-alkylphenols with a branched side chain. Due to the structural similarity, it can also attack bisphenolic compounds, which are very important materials for the synthesis of plastics and resins, but many of them are known to or suspected to have endocrine disrupting effects to fish and animals. In this study, to clarify the substrate specificity of the enzyme (NmoA) for bisphenolic compounds, degradation tests using the cell suspension of Pseudomonas putida harboring the nonylphenol monooxygenase gene (nmoA) were conducted. The cell suspension degraded several bisphenols including bisphenol F, bisphenol S, 4,4'-dihydroxybenzophenone, 4,4'-dihydroxydiphenylether, and 4,4'-thiodiphenol, indicating that this monooxygenase has a broad substrate specificity for compounds with a bisphenolic structure.

[Intussusception Due to Inflammatory Polyp in the Ileum after ESD for Rectal LST].

A 69-year-woman was admitted to the clinic in August 2018 because of general fatigue and low appetite.She had occult blood-positive and was referred to our hospital for further investigations.There was LST in the rectum for which colonoscopy and ESD were performed.She had abdominal pain and slight fever on postoperative day 1.Abdominal CT showed an intussusception in the ileum.We could not achieve endoscopic de-torsion and carried out laparotomy.The intussusception was found to be strangulated due to inflammatory polyp and mesenteric adhesion.The affected portion was resected.Although treatment for low hypoalbuminemia and neurogenic cystitis was required, she was discharged on postoperative day 28.

Draft Genome Sequence of a Bioflocculant-Producing Bacterium, Citrobacter freundii IFO 13545.

Here, we report the 5.2-Mb genome sequence of a bioflocculant-producing bacterial strain, Citrobacter freundii IFO 13545, which consists of 5,209,670 bp with a G+C content of 51.5% and 4,853 predicted coding sequences (CDSs). The genes related to the biosynthetic pathway of the bioflocculant were localized on the genome map.

Inhibition of the dephosphorylation of eukaryotic initiation factor 2α ameliorates murine experimental pancreatitis.

BACKGROUND: Endoplasmic reticulum (ER) stress in the pancreas is closely associated with the development of acute pancreatitis. However, the role of the protein kinase RNA-like ER kinase (PERK) in this disease is not fully understood. We investigated whether an inhibitor of the dephosphorylation of eukaryotic initiation factor 2α, salubrinal, could improve murine experimental pancreatitis through the amelioration of ER stress. METHODS: Acute pancreatitis was induced by the intraperitoneal administration of cerulein (50 µg/kg) six times at 1-h intervals followed by lipopolysaccharide (10 mg/kg). Salubrinal was administered intraperitoneally immediately after lipopolysaccharide injection and 3 h later. Mice were sacrificed 24 h after the first injection of cerulein, and serum amylase and proinflammatory cytokines were measured. The severity of pancreatitis was evaluated histologically using a scoring system. The expression levels of ER stress-related proteins were evaluated by Western blotting. RESULTS: The administration of salubrinal significantly attenuated the increase in serum amylase levels and improved histologically assessed pancreatitis. The serum levels of proinflammatory cytokines were significantly suppressed in salubrinal-treated mice, as was the expression of glucose-regulated protein 78, CCAAT/enhancer-binding protein homologous protein, and cleaved caspase-3. CONCLUSIONS: The amelioration of ER stress through augmentation of the PERK-signaling pathway may be a therapeutic target for the treatment of acute pancreatitis.

Preparation of 1L-myo-Inositol 1-Phosphate as a Substrate of Phosphatidylinositol Phosphate Synthase.

Novel drugs possessing a mechanism of action specific to pathogenic mycobacteria, including Mycobacterium tuberculosis, are needed. In 2010, we discovered that the biosynthetic pathway of phosphatidylinositol, which is a membrane phospholipid, differs between humans and mycobacteria. The key enzyme responsible for this difference is phosphatidylinositol phosphate (PIP) synthase, which is present only in a few bacteria belonging to the phylum Actinobacteria. Discovering compounds that inhibit the activity of this enzyme will lead to the development of new drugs specific to pathogenic mycobacteria. Measuring PIP synthase activity requires the isotope-labeled substrate 1l-myo-inositol 1-phosphate (1l-Ino-1P). Because this substrate is not commercially available, we synthesized it from [14C] glucose 6-phosphate ([14C] Glc-6P), using a crude enzyme solution isolated from the methanoarchaeon 1l-Ino-1P synthase. The activity of 1l-Ino-1P synthase in the crude enzyme mixture was low, and quantitative analysis of the synthesized 1l-Ino-1P was inaccurate due to impurities present in the crude enzyme mixture. In the present study, we describe a method for synthesizing 1l-Ino-1P using a solution containing recombinant 1l-Ino-1P synthase derived from the hyperthermophilic archaeon Aeropyrum pernix. In addition, we elucidate the conditions leading to the almost complete conversion of Glc-6P into 1l-Ino-1P using this enzyme. Quantitation of the synthesized 1l -Ino-1P was performed by colorimetry and gas liquid chromatography. Further, we confirmed that isotope-labeled 1l-Ino-1P, which is difficult to quantitate by gas liquid chromatography, can be accurately quantified by colorimetry. We also confirmed that 1d-inositol 1-phosphate cannot be a substrate for PIP synthase.

Structural basis of the correct subunit assembly, aggregation, and intracellular degradation of nylon hydrolase.

Nylon hydrolase (NylC) is initially expressed as an inactive precursor (36 kDa). The precursor is cleaved autocatalytically at Asn266/Thr267 to generate an active enzyme composed of an α subunit (27 kDa) and a ß subunit (9 kDa). Four αß heterodimers (molecules A-D) form a doughnut-shaped quaternary structure. In this study, the thermostability of the parental NylC was altered by amino acid substitutions located at the A/D interface (D122G/H130Y/D36A/L137A) or the A/B interface (E263Q) and spanned a range of 47 °C. Considering structural, biophysical, and biochemical analyses, we discuss the structural basis of the stability of nylon hydrolase. From the analytical centrifugation data obtained regarding the various mutant enzymes, we conclude that the assembly of the monomeric units is dynamically altered by the mutations. Finally, we propose a model that can predict whether the fate of the nascent polypeptide will be correct subunit assembly, inappropriate protein-protein interactions causing aggregation, or intracellular degradation of the polypeptide.

Characterization of the 3-methyl-4-nitrophenol degradation pathway and genes of Pseudomonas sp. strain TSN1.

3-Methyl-4-nitrophenol (3M4NP) is formed in soil as a hydrolysis product of fenitrothion, one of the major organophosphorus pesticides. A Pseudomonas strain was isolated as a 3M4NP degrader from a crop soil and designated TSN1. This strain utilized 3M4NP as a sole carbon and energy source. To elucidate the biodegradation pathway, we performed transposon mutagenesis with pCro2a (mini-Tn5495) and obtained three mutants accumulating a dark pink compound(s) from 3M4NP. Rescue cloning and sequence analysis revealed that in all mutants, the transposon disrupted an identical aromatic compound meta-cleaving dioxygenase gene, and a monooxygenase gene was located just downstream of the dioxygenase gene. These two genes were designated mnpC and mnpB, respectively. The gene products showed high identity with the methylhydroquinone (MHQ) monooxygenase (58%) and the 3-methylcatechol 2,3-dioxygenase (54%) of a different 3M4NP degrader Burkholderia sp. NF100. The transposon mutants converted 3M4NP or MHQ into two identical metabolites, one of which was identified as 2-hydroxy-5-methyl-1,4-benzoquinone (2H5MBQ) by GC/MS analysis. Furthermore, two additional genes (named mnpA1 and mnpA2), almost identical to the p-nitrophenol monooxygenase and the p-benzoquinone reductase genes of Pseudomonas sp. WBC-3, were isolated from the total DNA of strain TSN1. Disruption of mnpA1 resulted in the complete loss of the 3M4NP degradation activity, demonstrating that mnpA1 encodes the initial monooxygenase for 3M4NP degradation. The purified mnpA2 gene product could efficiently reduce methyl p-benzoquinone (MBQ) into MHQ. These results suggest that strain TSN1 degrades 3M4NP via MBQ, MHQ, and 2H5MBQ in combination with mnpA1A2 and mnpCB, existing at different loci on the genome.

Metabolic pathway of 6-aminohexanoate in the nylon oligomer-degrading bacterium Arthrobacter sp. KI72: identification of the enzymes responsible for the conversion of 6-aminohexanoate to adipate.

Arthrobacter sp. strain KI72 grows on a 6-aminohexanoate oligomer, which is a by-product of nylon-6 manufacturing, as a sole source of carbon and nitrogen. We cloned the two genes, nylD 1 and nylE 1 , responsible for 6-aminohexanoate metabolism on the basis of the draft genomic DNA sequence of strain KI72. We amplified the DNA fragments that encode these genes by polymerase chain reaction using a synthetic primer DNA homologous to the 4-aminobutyrate metabolic enzymes. We inserted the amplified DNA fragments into the expression vector pColdI in Escherichia coli, purified the His-tagged enzymes to homogeneity, and performed biochemical studies. We confirmed that 6-aminohexanoate aminotransferase (NylD1) catalyzes the reaction of 6-aminohexanoate to adipate semialdehyde using α-ketoglutarate, pyruvate, and glyoxylate as amino acceptors, generating glutamate, alanine, and glycine, respectively. The reaction requires pyridoxal phosphate (PLP) as a cofactor. For further metabolism, adipate semialdehyde dehydrogenase (NylE1) catalyzes the oxidative reaction of adipate semialdehyde to adipate using NADP+ as a cofactor. Phylogenic analysis revealed that NylD1 should be placed in a branch of the PLP-dependent aminotransferase sub III, while NylE1 should be in a branch of the aldehyde dehydrogenase superfamily. In addition, we established a NylD1/NylE1 coupled system to quantify the aminotransferase activity and to enable the conversion of 6-aminohexaoate to adipate via adipate semialdehyde with a yield of > 90%. In the present study, we demonstrate that 6-aminohexanoate produced from polymeric nylon-6 and nylon oligomers (i.e., a mixture of 6-aminohexaoate oligomers) by nylon hydrolase (NylC) and 6-aminohexanoate dimer hydrolase (NylB) reactions are sequentially converted to adipate by metabolic engineering technology.

Correction to: Metabolic pathway of 6-aminohexanoate in the nylon oligomer-degrading bacterium Arthrobacter sp. KI72: identification of the enzymes responsible for the conversion of 6-aminohexanoate to adipate.

The original publication of this paper contains mistakes for Tables 1 and 2 legends as well as the sublabels in Figs. 2, 4, 5, 6, and 7.

Biosynthetic Pathway and Genes of Chitin/Chitosan-Like Bioflocculant in the Genus Citrobacter.

Chitin/chitosan, one of the most abundant polysaccharides in nature, is industrially produced as a powder or flake form from the exoskeletons of crustaceans such as crabs and shrimps. Intriguingly, many bacterial strains in the genus Citrobacter secrete a soluble chitin/chitosan-like polysaccharide into the culture medium during growth in acetate. Because this polysaccharide shows strong flocculation activity for suspended solids in water, it can be used as a bioflocculant (BF). The BF synthetic pathway of C. freundii IFO 13545 is expected from known bacterial metabolic pathways to be as follows: acetate is metabolized in the TCA cycle and the glyoxylate shunt via acetyl-CoA. Next, fructose 6-phosphate is generated from the intermediates of the TCA cycle through gluconeogenesis and enters into the hexosamine synthetic pathway to form UDP-N-acetylglucosamine, which is used as a direct precursor to extend the BF polysaccharide chain. We conducted the draft genome sequencing of IFO 13545 and identified all of the candidate genes corresponding to the enzymes in this pathway in the 5420-kb genome sequence. Disruption of the genes encoding acetyl-CoA synthetase and isocitrate lyase by homologous recombination resulted in little or no growth on acetate, indicating that the cell growth depends on acetate assimilation via the glyoxylate shunt. Disruption of the gene encoding glucosamine 6-phosphate synthase, a key enzyme for the hexosamine synthetic pathway, caused a significant decrease in flocculation activity, demonstrating that this pathway is primarily used for the BF biosynthesis. A gene cluster necessary for the polymerization and secretion of BF, named bfpABCD, was also identified for the first time. In addition, quantitative RT-PCR analysis of several key genes in the expected pathway was conducted to know their expression in acetate assimilation and BF biosynthesis. Based on the data obtained in this study, an overview of the BF synthetic pathway is discussed.