Clostridium Subterminale Research Articles

Lysine‐2,3‐aminomutase and a newly discovered glutamate‐2,3‐aminomutase produce β‐amino acids to overcome salt stress in methanogenic archaea

Many methanogenic archaea synthesize N ε ‐acetyl‐β‐lysine and/or β‐glutamate as osmolytes that allow survival in high salinity environments. Methanococcus maripaludis C7 contains two genes, MmarC7_0106 and MmarC7_1783, encoding radical S‐adenosylmethionine(SAM) enzymes with sequence similarity to lysine‐2,3‐aminomutase (KAM). MmarC7_0106 is immediately upstream of an acetyltransferase and was expected to be the KAM involved in the biosynthesis of N ε ‐acetyl‐β‐lysine. To confirm this, the gene was cloned and expressed with a hexahistidine tag in E. coli, followed by anaerobic purification and biochemical characterization. Although the bacterial KAMs involved in lysine degradation in Clostridium subterminale and a post‐translational modification of elongation factor P in E. coli have been well‐characterized, the archaeal KAM involved in compatible solute biosynthesis had never been studied in vitro before. In the presence of L‐lysine, SAM, and dithionite at 37°C, this archaeal KAM had a k cat = 0.42 s ‐1 and a K m = 17.8 mM. The product was shown to be 3(S)‐β‐lysine, which is like the well‐characterized Clostridium KAM as opposed to the E. coli KAM that produces 3(R)‐β‐lysine. The archaeal KAM was highly specific for L‐lysine, with no activity observed with D‐lysine or most other amino acids. However, low aminomutase activity was observed with L‐arginine. We further report the function of MmarC7_1783, a putative radical SAM aminomutase with a ~160 amino acid extension at its N‐terminus. Bioinformatic analysis of possible substrate binding residues suggested a function as glutamate‐2,3‐aminomutase, which was confirmed here through heterologous expression in a methanogen followed by detection of β‐glutamate in cell extracts. Finally, we investigated the differences in amino acid compatible solute usage amongst M. maripaludis C7 (contains both aminomutase genes) and M. maripaludis S2 (contains only the canonical KAM gene) in different salt concentrations. Taken together, this work defines the biosynthetic routes for β‐amino acids in methanogenic archaea and expands the importance and diversity of radical SAM enzymes in all domains of life.

S2946 A Rare Case of COVID-19 Infection Presenting With Devastating Intestinal Perforation Requiring Emergent Surgical Intervention: A Case Report

Introduction: COVID-19 pandemic is believed to be a systemic disease that affects not only the lungs but also other vital organs however, current available literature mainly focuses on acute respiratory symptoms. Symptoms include fever, myalgia and dyspnea. However gastrointestinal (GI) symptoms were reported in 3-50% of cases, including nausea, vomiting, abdominal pain, and diarrhea. Here we report a case of COVID-19 presenting with intestinal perforation. Case Description/Methods: 52-year-old female with a remote history of Roux-en-Y gastric bypass surgery who was diagnosed with COVID-19 three days prior to admission presented with epigastric pain, nausea, vomiting, and constipation for 2 days. Vital signs were normal. Laboratory workup was remarkable for non-anion gap metabolic acidosis and elevated inflammatory markers. Chest X-Ray showed air under the diaphragm. Abdominal CT scan showed pneumoperitoneum in the right subdiaphragmatic and mid abdomen/pelvis. Surgery was consulted and patient underwent emergent diagnostic laparoscopy and subsequent exploratory laparotomy with findings of large perforation at gastrojejunostomy, Intra-abdominal abscess. Patient underwent revision of gastrojejunostomy, drainage of intra-abdominal abscess with perotinel lavage. Intraabdominal cultures isolated Klebsiella pneumonia, Citrobacter freundii, Prevotella melaninogenica,Enterococcus faecalis and several Candida. Blood cultures isolated Clostridium subterminale bacteremia. Patient was started on IV antibiotics. The patient was discharged to be followed by surgery for resolution of symptoms and drainage removal. Discussion: Gastrointestinal symptoms of COVID-19 disease were reported in accumulated literature. Moreover, COVID-19 virus has been isolated in stool specimens. The question becomes, are those GI manifestations considered part of systemic symptoms of an airborne infection, or COVID-19 itself can potentially be transmitted feco-orally. Pathophysiology is yet unclear, suggested hypothesis includes direct invasion of intestinal cells by the SARS-CoV-2 RNA, as GI cells express ACE2. Another theory is imbalance of autonomic innervation of GI tract given the neuroinvasive tendency of COVID-19. It is not clear whether patients with past GI diseases like inflammatory bowel disease, peptic ulcers, and prior GI surgery are more prone to GI complications. This case highlights GI complications in Covid-19-positive patients especially those with GI history for early diagnosis and prevention of possible complications.Figure 1.: Figure (A): CT shows free air, Figure (B): CT abdomen shows air-fluid level, Figure (C): Abdominal X-ray shows air under the diaphragm.

The Utility of MALDI-TOF-Mass Spectrometry, Analytical Profile Index (API) and Conventional-PCR for the Detection of Foodborne Pathogens from Meat

The rich nutrient composition of meat and its sufficient water activity supports the growth of both spoilage and pathogenic bacteria. The microbial quality of 150 raw meat samples (50 chicken, 50 pork and 50 beef) were evaluated for foodborne pathogens using Analytical Profile Index (API Staph-Ident kit, API-20E strips and API-20A), MALDI-TOF-MS, and conventional PCR. Thirty isolates (30) were identified using biochemical tests and Confirmatory biochemical test (API). All 30 isolates were confirmed by 16S rDNA PCR and sequencing. Detected isolates are Macrococcus caseolyticus 57%, Enterococcus faecalis 23%, Bacillus cereus 3%, Enterococcus mundtii 3%, Escherichia coli 3%, Citrobacter freundii 3%, and Clostridium subterminale 3%. Seven (23%) isolates were correctly identified using MALDI-TOF MS, whilst 18 (60%) isolates were misidentified, and 5 isolates were not detected (No peaks). Seventy three percent 73% (22) of the organisms showed high degree of resistance to tetracycline, followed by sulphonamide with 13% (4/30), and ciprofloxacin 3% (1/30). However, Bacillus cereus, Citrobacter freundii were not resistant to any of the antibiotics. All the isolates were susceptible to streptomycin. Furthermore, some bacteria exhibited multiple antibiotic resistances with tetracycline. Due to the presence and potential hazard of pathogens in meat samples, the detection of these pathogens in different types of meat is vital to maintain public health.

Clostridium culturomicium sp. nov. and Clostridium jeddahitimonense sp. nov., novel members of the Clostridium genus isolated from the stool of an obese Saudi Arabian.

Taxono-genomics is an innovative concept coined for the description of new bacterial species. Phenotypic characteristics were combined with a genomic approach to describe two new species within the Clostridium senso stricto genus: Clostridium culturomicium strain CL-6T and Clostridium jeddahitimonense strain CL-2T, both isolated from the gut microbiota of an obese man from Saudi Arabia. Strains CL-6T and CL-2T shared a similarity of 98.4% with the 16S rRNA gene of Clostridium subterminale strain JCM 1417T (accession number NR113027) and 98% with that of Clostridium disporicum strain DS1T (accession number NR026491), respectively. The highest OrthoANI values were shared with Clostridium punense for strain CL-6T (70.8%) and with Clostridium disporicum for strain CL-2T (87.1%). Additionally, strain CL-6T and strain CL-2T shared a 16S rRNA similarity of 91.4%. Both strains were anaerobic, spore-forming and Gram-stain-positive non-motile bacilli. The genome of Clostridium culturomicium strain CL-6T is 4,325,182bp long with 32.2% GC content. As for Clostridium jeddahitimonense strain CL-2T, the genome is 4,074,758bp long with 29.2% GC content.

MALDI-TOF Mass Spectrometry and 16S rRNA Gene Sequence Analysis for the Identification of Foodborne Clostridium Spp.

Clostridium is a genus of Gram-positive, spore-forming, anaerobic bacteria comprising approximately 100 species. Some Clostridium spp. (C. botulinum, C. perfringens, C. tetani, and C. difficile) have been recognized to cause acute food poisoning, botulism, tetanus, and diarrheal illness in humans. Thus, rapid identification of Clostridium spp. is critical for source-tracking of contaminated food and to understand the transmission dynamics of these foodborne pathogens. This study was carried out to rapidly identify Clostridium-like isolates by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS and rRNA sequencing methods. Thirty-three Clostridium-like isolates were recovered from various baby food and surveillance samples. Species identification of these isolates was accomplished using the VITEK MS system. Sequence characterization of the 16S rRNA region was done on an ABI 3500xL Genetic Analyzer. The VITEK MS system identified 28 of the 33 Clostridium-like isolates with a high confidence value (99.9%); no identification was observed for the remaining five isolates. Nucleotide sequencing of the 16S rRNA region identified all 33 Clostridium-like isolates. Furthermore, while characterizing the 16S rRNA gene, 11 distinct Clostridium spp. (Clostridium aciditolerans, Clostridium aerotolerans, Clostridium argentinense, Clostridium beijerinckii, Clostridium bifermentans, Clostridium butyricum, Clostridium cochlearium, Clostridium difficile, Clostridium perfringens, Clostridium sporogenes, and Clostridium subterminale) were recognized among the 33 Clostridium-like isolates. One of the Clostridium-like isolates was identified as Citrobacter amalonaticus by both diagnostic methods. The generated 16S rRNA sequences matched completely (100%) with sequences available in GenBank for Clostridium and Citrobacter species. Species identification attained using the VITEK MS for the Clostridium-like isolates was comparable to that from the 16S rRNA sequencing-based data. The VITEK MS and 16S rRNA sequence analysis can be implemented in the species identification of Clostridium spp. isolates of public health importance. MALDI-TOF MS and 16S rRNA sequencing can be used in the species identification of Clostridium species.

Biochemical Characterization of Aminomutases Involved in Salt Tolerance in Methanogenic Archaea

Lysine-2,3-aminomutase (LAM) catalyzes the conversion of L-α-lysine to L-β-lysine, which entails the transfer of an α-amino group to the b-carbon alongside the migration of a β-hydrogen to the α-carbon. This enzyme plays a role in combating salt stress in methanogenic archaea by carrying out the first step in the biosynthesis of Ne-acetyl-β-lysine, a well-studied osmolyte synthesized and accumulated in methanogens. LAM is of special interest as it belongs to the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes which perform diverse and complex chemistry that often produces valuable compounds. Radical SAM enzymes utilize a reduced [4Fe-4S] cluster to initiate the homolytic cleavage of SAM, yielding a highly reactive 5’-deoxyadenosyl radical that then abstracts a hydrogen atom from an otherwise unreactive substrate. Although the bacterial LAM involved in lysine degradation in Clostridium subterminale has been well-characterized, the archaeal LAM involved in osmolyte biosynthesis has never been studied in vitro. Here, we report on the recombinant expression, purification, and enzymatic properties of LAM from Methanococcus maripaludis. The gene encoding LAM from M. maripaludis C7 (MmarC7_0106) was cloned and the his-tagged protein was overexpressed in Escherichia coli. After purification by metal-affinity chromatography under strictly anaerobic conditions, the protein was brown in color, had UV-Vis spectrum characteristics of a [4Fe-4S] cluster that was capable of being reduced to an active state, and had 3.9 mol of iron per monomer. A wide ranged temperature profile study revealed that enzymatic activity was highest at 37°C. The enzyme reaction in the presence of L-lysine, SAM, and dithionite at 37°C had a Kcat = 0.42 s-1 and a Km = 17.8 mM. Finally, alternative substrates for the aminomutation reaction were investigated. There exists another gene annotated as LAM in M. maripaludis C7 due to its close sequence similarity with the bacterial LAM; however, this gene (MmarC7_1783) lacks lysine binding residues and has a 164 amino acid N-terminal extension and as such is referred to as a “long-LAM”. The presence of a “long-LAM” such as MmarC7_1783 in a methanogen is not uncommon as there are several annotated in thermophilic methanogenic species that are known to solely use β-glutamate, another beta-amino acid, as their osmolyte. Given that the only known pathway for the biosynthesis of β-glutamate is through the use of a glutamate-2,3-aminomutase (GAM) and that these thermophilic methanogens do not have an annotated GAM, we proposed that these long-LAMs were in fact GAM which we have now confirmed through heterologous expression studies. Thus, in addition to the biochemical characterization of an archaeal LAM (MmarC7_0106) described above, we have determined that long-LAMs are in fact GAMs required in the biosynthesis of an alternative osmolyte used for survival in high salt concentrations by halotolerant methanogenic archaea.

CLOSTRIDIAL MYCOTIC ANEURYSM LEADING TO EMPHYSEMATOUS AORTITIS: A CASE REPORT

SESSION TITLE: Medical Student/Resident Chest Infections Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Emphysematous aortitis (EA) is a rare consequence of mycotic aneurysm associated with high morbidity and mortality. Most cases have been linked to infection with Staphylococcus species. This case reports clostridial bacteremia leading to arterial myonecrosis and subsequent aortitis. CASE PRESENTATION: The patient was a 66-year-old male with a past medical history of end stage renal disease with an arterio-venous fistula, hypertension, and diabetes who presented with tachycardia, tachypnea, and hypertension for one week. He also had leukocytosis of 23,000/µL with elevated inflammatory markers including a D-dimer of 4700 ng/mL. Computed tomography showed air within the walls of the thoracic aorta and in the periaortic soft tissue with extension from the aortic arch to the mid descending thoracic aorta consistent with aortitis. There was also involvement in the periaortic mediastinal fat and a loculated large left pleural effusion concerning for empyema. Blood cultures revealed Clostridium subterminale, however, gastrointestinal work up did not reveal esophageal or colonic malignancy, or mucosal trauma. He then underwent ascending aorta and distal arch reconstruction. Unfortunately, his post-operative course was complicated by hypotension unresponsive to multiple vasopressors and asystole. Post-mortem aortic tissue cultures were positive for Clostridium innocuum. DISCUSSION: EA is a rare but often fatal sequelae of mycotic aneurysms and has been hypothesized to occur through arterial injury (trauma), bacteremic seeding, contiguous infection, or septic emboli. Risk factors include arterial injury (Intravenous drug use/iatrogenic interventions), infection, immunosuppression, atherosclerosis, and pre-existing aneurysm. Mycotic aneurysms comprise only 0.5% to 1.3% of all aneurysms, and Clostridium species only comprise 0.7% of those cases. C. innocuum aortitis appears to present radiographically similar to other types of Clostridium aortic aneurysms, in which periaortic gas was found to be present in 92.6% of cases. Curiously, the patient was found to have bacteremia with a separate Clostridium species (C. subterminale) from the one found in the aortic wall (C. innocuum). Some reported cases of C. subterminale bacteremia was associated with mucosal damage. The patient’s negative endoscopy findings may suggest a different source of infection for both Clostridium species, possibly via the patient's arterio-venous fistula leading to infection within the atherosclerotic plaques of the aorta. CONCLUSIONS: Emphysematous aortitis is a rare complication of anaerobic infection. A high suspicion should be present in the differential based on radiographic and clinical findings as well as patient risk factors. Reference #1: Sörelius K, Budtz-Lilly J. Systematic Review of the Management of Mycotic Aortic Aneurysms. Eur J Vasc Endovasc Surg. 2019;58(3):426-435. doi:10.1016/j.ejvs.2019.05.004 Reference #2: Ito F, Inokuchi R, Matsumoto A, et al. Presence of periaortic gas in Clostridium septicuminfected aortic aneurysm AIDS in early diagnosis: A case report and systematic review of the literature. J Med Case Rep. 2017;11(1). doi:10.1186/s13256-017-1422-0 Reference #3: Seder CW, Kramer M, Long G, Uzieblo MR, Shanley CJ, Bove P. Clostridium septicum aortitis: Report of two cases and review of the literature. J Vasc Surg. 2009;49(5):1304- 1309. doi:10.1016/j.jvs.2008.11.058 DISCLOSURES: No relevant relationships by Thomas Ng, source=Web Response No relevant relationships by Christopher Pham, source=Web Response

Microbiome of root vegetables\u2014a source of gluten-degrading bacteria

Gluten is a cereal protein that is incompletely digested by human proteolytic enzymes that create immunogenic peptides that accumulate in the gastrointestinal tract (GIT). Although both environmental and human bacteria have been shown to expedite gluten hydrolysis, gluten intolerance is a growing concern. Here we hypothesize that together with food, we acquire environmental bacteria that could impact our GIT with gluten-degrading bacteria. Using in vitro gastrointestinal simulation conditions, we evaluated the capacity of endophytic bacteria that inhabit root vegetables, potato (Solanum tuberosum), carrot (Daucus sativus), beet (Beta vulgaris), and topinambur (Jerusalem artichoke) (Helianthus tuberosus), to resist these conditions and degrade gluten. By 16S rDNA sequencing, we discovered that bacteria from the families Enterobacteriaceae, Bacillaceae, and Clostridiaceae most effectively multiply in conditions similar to the human GIT (microoxic conditions, 37 °C) while utilizing vegetable material and gluten as nutrients. Additionally, we used stomach simulation (1 h, pH 3) and intestinal simulation (1 h, bile salts 0.4%) treatments. The bacteria that survived this treatment retained the ability to degrade gluten epitopes but at lower levels. Four bacterial strains belonging to species Bacillus pumilus, Clostridium subterminale, and Clostridium sporogenes isolated from vegetable roots produced proteases with postproline cleaving activity that successfully neutralized the toxic immunogenic epitopes.Key points• Bacteria from root vegetables can degrade gluten.• Some of these bacteria can resist conditions mimicking gastrointestinal tract.

Biochemical Characterization of an Arginine 2,3‐Aminomutase with Dual Substrate Specificity

Summary of main observation and conclusionThe radical S‐adenosylmethionine (SAM) aminomutases represent an important pathway for the biosynthesis of β‐amino acids. In this study, we report biochemical characterization of BlsG involved in blasticidin S biosynthesis as a radical SAM arginine 2,3‐aminomutase. We showed that BlsG acts on both L‐arginine and L‐lysine with comparable catalytic efficiencies. Similar dual substrate specificity was also observed for the lysine 2,3‐aminomutase from Escherichia coli (LAMEC). The catalytic efficiency of LAMEC is similar to that of BlsG, but is significantly lower than that of the enzyme from Clostridium subterminale (LAMCS), which acts only on L‐lysine rather than on L‐arginine. Moreover, we showed that enzymes can be grouped into two major phylogenetic clades, each corresponding to a certain C3 stereochemistry of the β‐amino acid product. Our study expands the radical SAM aminomutase members and provides insights into enzyme evolution, supporting a trade‐off between substrate promiscuity and catalytic efficiency.

The Antioxidants Glutathione, Ascorbic Acid and Uric Acid Maintain Butyrate Production by Human Gut Clostridia in The Presence of Oxygen In Vitro

Uncontrolled oxidative stress, reported in Salmonella and HIV infections, colorectal cancer or severe acute malnutrition, has been associated with anaerobic gut microbiome alteration, impaired butyrate production, mucosal immunity dysregulation and disruption of host-bacterial mutualism. However, the role of major antioxidant molecules in the human body, such as glutathione, ascorbic acid and uric acid, has been neglected in this context. Here, we performed an in vitro metabolomics study of the 3 most odorous anaerobic microbes isolated from the human gut in our laboratory (Clostridium sporogenes, Clostridium subterminale and Romboutsia lituseburensis) when grown in anaerobiosis or in aerobiosis with these 3 antioxidant molecules via gas and liquid chromatography-mass spectrometry (GC/MS and LC/MS). There was no growth or volatile organic compound production in aerobic cultures without the 3 antioxidant molecules. In anaerobiosis, the major metabolic products of the bacteria were thiols, alcohols and short-chain fatty acid esters. The production of alkanes, cycloheptatriene and, paradoxically, increased butyrate production, was observed in the cultures grown in aerobiosis with the 3 antioxidant molecules. The qualitative shift suggests specific molecular mechanisms that remain to be elucidated. The increased production of butyrate, but also isobutyrate and isovalerate in vitro suggests that these 3 antioxidant molecules contributed to the maintenance and active resilience of host-bacterial mutualism against mucosal oxygen and uncontrolled oxidative stress in vivo.

Wound botulism caused by Clostridium subterminale after a heroin injection.

Botulism is caused by toxin production from many species of Clostridium, most commonly Clostridium botulinum as well as C. baratii and C. butyricum. Development of wound botulism is associated with injection drug users but has also been described in traumatic injuries with exposure to soil. A patient presented to the emergency department with a complaint of descending, progressive weakness. He recently reported skin popping with heroin injections. Heptavalent botulinum antitoxin was obtained from the [Center for Disease Control and Prevention (CDC)]. On hospital day seven, the anaerobic wound cultures resulted with growth of Clostridium subterminale.

Isolation of six novel 7-oxo- or urso-type secondary bile acid-producing bacteria from rat cecal contents

Understanding the dynamics of secondary bile acid (SBA) formation in the gut by SBA-producing bacteria is important for host health, as SBAs have been shown to affect host pathophysiology and gut microbiota composition. However, our knowledge of SBA producers is limited in light of the diversity of gut microbes. Here, we isolated six novel SBA-producing bacteria from rat cecal contents, all of which were members of known species of gut microbes. Anaerostipes caccae D10, Bacteroides nordii C5, Clostridioides difficile D7, and Clostridium cadaveris G11 were capable of oxidizing cholic acid and chenodeoxycholic acid into 7-oxo-derivatives with varying yields. B.nordii C5 and its type strain JCM 12987T had the highest molar yield, ∼90%. Clostridium disporicum F4 and Clostridium subterminale C4 epimerized cholic acid into ursocholic acid with yields of ∼85%; the corresponding type strains lacked epimerization activity. Furthermore, although not novel as an SBA producer, Clostridium scindens G10 that produced deoxycholic acid from cholic acid was isolated for the first time from rodents. These findings will contribute to elucidation of SBA formation in the gut.

Clostridium punense sp. nov., an obligate anaerobe isolated from healthy human faeces.

An obligately anaerobic, rod-shaped (0.5-1.0 × 2.0-10.0 μm), Gram-stain-positive bacterium, occurring mainly singly or in pairs, and designated BLPYG-8T, was isolated from faeces of a healthy human volunteer aged 56 years. Cells were non-motile. Oval, terminal spores were formed that swell the cells. The strain was affiliated with the genus Clostridium sensu stricto (Clostridium rRNA cluster I) as revealed by 16S rRNA gene sequence analysis. Strain BLPYG-8T showed 97.3 to 97.4 % 16S rRNA gene sequence similarity with Clostridium sulfidigenes DSM 18982T, Clostridium subterminale DSM 6970T and Clostridium thiosulfatireducens DSM 13105T. DNA-DNA hybridization and phenotypic analysis showed that the strain was distinct from its closest relatives, C. sulfidigenes DSM 18982T, C. subterminale DSM 6970T, C. thiosulfatireducens DSM 13105T with 54.2, 53.9 and 53.3 % DNA-DNA relatedness, respectively. Strain BLPYG-8T grew in PYG broth at temperatures between 20 and 40 °C (optimum 37 °C). The strain utilized a range of amino acids as well as carbohydrates as a source of carbon and energy. Glucose fermentation resulted in the formation of volatile fatty acids mainly acetic acid, n-butyric acid and organic acids such as succinic and lactic acid. The DNA G+C content of strain BLPYG-8T was 44.1 mol%. The major fatty acids (>10 %) were C14 : 0, iso-C15 : 0, C16 : 1ω7c and C16 : 0. Phylogenetic analysis and specific phenotypic characteristics and/or DNA G+C content differentiated the strain from its closest relatives. On the basis of these data, strain BLPYG-8T represents a novel species of the genus Clostridium, for which the name Clostridium punense sp. nov. is proposed. The type strain is BLPYG-8T ( = DSM 28650T = CCUG 64195T = MCC 2737T).

Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5'-dAdo• "Free Radical" Is Never Free.

Lysine 2,3-aminomutase (LAM) is a radical S-adenosyl-L-methionine (SAM) enzyme and, like other members of this superfamily, LAM utilizes radical-generating machinery comprising SAM anchored to the unique Fe of a [4Fe-4S] cluster via a classical five-membered N,O chelate ring. Catalysis is initiated by reductive cleavage of the SAM S-C5' bond, which creates the highly reactive 5'-deoxyadenosyl radical (5'-dAdo•), the same radical generated by homolytic Co-C bond cleavage in B12 radical enzymes. The SAM surrogate S-3',4'-anhydroadenosyl-L-methionine (anSAM) can replace SAM as a cofactor in the isomerization of L-α-lysine to L-β-lysine by LAM, via the stable allylic anhydroadenosyl radical (anAdo•). Here electron nuclear double resonance (ENDOR) spectroscopy of the anAdo• radical in the presence of (13)C, (2)H, and (15)N-labeled lysine completes the picture of how the active site of LAM from Clostridium subterminale SB4 "tames" the 5'-dAdo• radical, preventing it from carrying out harmful side reactions: this "free radical" in LAM is never free. The low steric demands of the radical-generating [4Fe-4S]/SAM construct allow the substrate target to bind adjacent to the S-C5' bond, thereby enabling the 5'-dAdo• radical created by cleavage of this bond to react with its partners by undergoing small motions, ∼0.6 Å toward the target and ∼1.5 Å overall, that are controlled by tight van der Waals contact with its partners. We suggest that the accessibility to substrate and ready control of the reactive C5' radical, with "van der Waals control" of small motions throughout the catalytic cycle, is common within the radical SAM enzyme superfamily and is a major reason why these enzymes are the preferred means of initiating radical reactions in nature.

Clostridium vulturis sp. nov., isolated from the intestine of the cinereous vulture (Aegypius monachus).

A Gram-stain positive, strict anaerobe, spore-forming, motile rod-shaped bacterial strain with peritrichous flagella, designated YMB-57(T), was isolated from the intestine of a cinereous vulture (Aegypius monachus) in Korea. Strain YMB-57(T) was found to show optimal growth at 37 °C, pH 7.5 and 1.0 % (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain YMB-57(T) belongs to the genus Clostridium and is most closely related to the type strains of Clostridium subterminale (96.9 % sequence similarity), Clostridium thiosulfatireducens (96.7 %) and Clostridium sulfidigenes (96.6 %). The main fermentation end-products identified following growth in PYG medium were acetate, butyrate, ethanol, propanol, carbon dioxide and hydrogen. Peptone was converted to ethanol, and butanol, whereas glucose was fermented to ethanol. The major cellular fatty acids were identified as C16:0, C18:1 ω9c, and C18:1 ω9c DMA and the DNA G+C content was determined to be 34.0 mol%. Phenotypic and phylogenetic differences indicate that strain YMB-57(T) is distinct from other Clostridium species. It is proposed that strain YMB-57(T) be classified as the type strain of a novel species of the genus Clostridium, with the name Clostridium vulturis sp. nov. The type strain is YMB-57(T) (=KCTC 15114(T) = JCM 17998(T)).

Detection of pathogenic clostridia in biogas plant wastes.

As the number of biogas plants has grown rapidly in the last decade, the amount of potentially contaminated wastes with pathogenic Clostridium spp. has increased as well. This study reports the results from examining 203 biogas plant wastes (BGWs). The following Clostridium spp. with different frequencies could be isolated via a new enrichment medium (Krüne medium) and detected by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS): Clostridium perfringens (58%) then Clostridium bifermentans (27%), Clostridium tertium (23%) and Clostridium butyricum (19%), Clostridium cadaveris (15%), Clostridium parapurificum (6%), Clostridium glycolicum (5%), Clostridium baratii (4%), Clostridium sporogenes (2 %), Clostridium sordellii (1%) and Clostridium subterminale (0.5%). The mean most probable number (MPN) count of sulfite reducing bacteria was between 10(3) and 10(4)/mL, and the higher the MPN, the more pathogenic Clostridium spp. were present. Also, real-time PCR was used to be compared with culture method for C. perfringens, C. bifermentans, C. butyricum, C. sporogenes/Clostridium botulinum and C. sordellii. Although real-time PCR was more sensitive than the culture method, both systems improve the recovery rate but in different ways and are useful to determine pathogenic clostridia in biogas plants. In conclusion, BGWs could present a biohazard risk of clostridia for humans and animals.

Clostridium sulfidigenes sp. nov., a mesophilic, proteolytic, thiosulfate- and sulfur-reducing bacterium isolated from pond sediment

A novel strictly anaerobic, endospore-forming, rod-shaped, Gram-positive bacterium, designated strain SGB2(T), was isolated from a mixed culture from a pond sediment during screening for sulfate-reducing bacteria capable of degrading cyanophycin (CGP). In this study, the taxonomic characterization of this mesophilic, proteolytic Clostridium isolate and the role which it, and its phylogenetic relatives, may play in peptide degradation and in the sulfur cycle are reported. Strain SGB2(T) was a commensal strain, utilizing CGP degradation products produced by other micro-organisms. Cells were motile until sporulation, forming oval, terminal spores that swell the cells. It showed optimum growth at 34 degrees C, pH 6.6 and in the absence of NaCl. Strain SGB2(T) utilized proteinaceous compounds such as peptone, Casamino acids, gelatin and trypticase soy, in addition to several amino acids and pyruvate. Utilization of many of these compounds was enhanced in the presence of thiosulfate. The isolate was unable to use any of the carbohydrates or alcohols investigated or CGP as carbon and energy sources. Thiosulfate and elemental sulfur were used as terminal electron acceptors. Phylogenetic analysis revealed that strain SGB2(T) belongs to the low-G+C-containing Clostridiales group. It exhibited 99 % 16S rRNA gene sequence similarity to its closest relatives Clostridium thiosulfatireducens Lup21(T) and Clostridium subterminale DSM 6970(T). DNA-DNA hybridization values with these two strains were 39.4 and 42.1 %, respectively. Based on phenotypic, genotypic and phylogenetic characteristics, we conclude that the isolate represents a novel species of the genus Clostridium, Clostridium sulfidigenes sp. nov. The type strain is SGB2(T) (=DSM 18982(T) =ATCC BAA-1538(T)).

Purification and characterization of an extracellular protease produced by psychrotolerant Clostridium sp. LP3 from lake sediment of Leh, India

An anaerobic, proteolytic bacterium isolated from lake sediments of Leh, India, was characterized with respect to morphology, biochemical characteristics, and 16S rRNA sequence and was identified as Clostridium species, with closest similarity to Clostridium subterminale. Isolate LP3 was psychrophilic, forming maximum cell mass between 10 and 20 degrees C, and produced extracellular protease. Growth was observed in the pH range of 7.0-8.5, with optimum at pH 7.5. Protease was purified 62.4-fold with a total yield of 17.5%. The effects of temperature, pH, and salt concentration on enzyme activity were studied. Protease was found to be a serine-type metallo-enzyme, active in a broad range of pHs. It was thermolabile and resistant to sodium dodecyl sulfate. Enzyme kinetics showed a tendency to increase Km with an increase in temperature for casein substrate.

Glutamate 2,3-aminomutase: A new member of the radical SAM superfamily of enzymes

A gene eam in Clostridium difficile encodes a protein that is homologous to lysine 2,3-aminomutase (LAM) in many other species but does not have the lysyl-binding residues Asp293 and Asp330 in LAM from Clostridium subterminale SB4. The C. difficile protein has Lys and Asn, respectively, in the sequence positions of the essential Asp residues in LAM. The C. difficile gene has been cloned into an E. coli expression vector, expressed in E. coli, and the protein purified and characterized. The recombinant protein displays excellent activity as a glutamate 2,3-aminomutase and no activity toward l-lysine. The PLP-, iron-, and sulfide-content and ultraviolet/visible spectrum are similar to LAM, and the enzyme requires SAM and dithionite as activators, as does LAM. Freeze-quench EPR experiments in the presence of l-glutamate reveal a glutamate-based free radical in the steady state of the reaction. A number of other bacterial genomes include genes encoding proteins homologous to the glutamate 2,3-aminomutase from C. difficile, and four of these proteins display the activity of glutamate 2,3-aminomutase when produced in E. coli. All of the homologous proteins have the cysteine motif CSMY CRH C corresponding to the motif CxxxCxxC characteristic of radical SAM enzymes. It is concluded that glutamate 2,3-aminomutase from C. difficile is a representative of a family found in a number of bacteria. It is likely that the β-glutamate found in a few bacterial and archeal species as an osmolyte arises from the action of glutamate 2,3-aminomutase.

Enantiomeric Free Radicals and Enzymatic Control of Stereochemistry in a Radical Mechanism: The Case of Lysine 2,3-Aminomutases

The product of yjeK in Escherichia coli is a homologue of lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4, and both enzymes catalyze the isomerization of (S)- but not (R)-alpha-lysine by radical mechanisms. The turnover number for LAM from E. coli is 5.0 min(-1), 0.1% of the value for clostridial LAM. The reaction of E. coli LAM with (S)-alpha-[3,3,4,4,5,5,6,6-(2)H8]lysine proceeds with a kinetic isotope effect (kH/kD) of 1.4, suggesting that hydrogen transfer is not rate-limiting. The product of the E. coli enzyme is (R)-beta-lysine, the enantiomer of the clostridial product. Beta-lysine-related radicals are observed in the reactions of both enzymes by electron paramagnetic resonance (EPR). The radical in the reaction of clostridial LAM has the (S)-configuration, whereas that in the reaction of E. coli LAM has the (R)-configuration. Moreover, the conformations of the beta-lysine-related radicals at the active sites of E. coli and clostridial LAM are different. The nuclear hyperfine splitting between the C3 hydrogen and the unpaired electron at C2 shows the dihedral angle to be 6 degrees, unlike the value of 77 degrees reported for the analogous radical bound to the clostridial enzyme. Reaction of (S)-4-thialysine produces a substrate-related radical in the steady state of E. coli LAM, as in the action of the clostridial enzyme. While (S)-beta-lysine is not a substrate for E. coli LAM, it undergoes hydrogen abstraction to form an (S)-beta-lysine-related radical with the same stereochemistry of hydrogen transfer from C2 of (S)-beta-lysine to the 5'-deoxyadenosyl radical as in the action of the clostridial enzyme. The resulting beta-lysyl radical has a conformation different from that at the active site of clostridial LAM. All evidence indicates that the opposite stereochemistry displayed by E. coli LAM is determined by the conformation of the lysine side chain in the active site. Stereochemical models for the actions of LAM from C. subterminale and E. coli are presented.