First cross-border outbreak of foodborne botulism in the European Union associated with the consumption of commercial dried roach (Rutilus rutilus).

Botulism outbreaks due to commercial products are extremely rare in the European Union. Here we report on the first international outbreak of foodborne botulism caused by commercial salt-cured, dried roach (Rutilus rutilus). Between November and December 2016, an outbreak of six foodborne botulism type E cases from five unrelated households was documented in Germany and Spain. The outbreak involved persons of Russian and Kazakh backgrounds, all consumed unheated salt-cured, dried roach-a snack particularly favored in Easter-European countries. The implicated food batches had been distributed by an international wholesaler and were recalled from Europe-wide outlets of a supermarket chain and other independent retailers. Of interest, and very unlike to other foodborne disease outbreaks which usually involves a single strain or virus variant, different Clostridium botulinum strains and toxin variants could be identified even from a single patient's sample. Foodborne botulism is a rare but potentially life-threatening disease and almost exclusively involves home-made or artisan products and thus, outbreaks are limited to individual or few cases. As a consequence, international outbreaks are the absolute exception and this is the first one within the European Union. Additional cases were likely prevented by a broad product recall, underscoring the importance of timely public health action. Challenges and difficulties on the diagnostic and epidemiological level encountered in the outbreak are highlighted.

Interlaboratory Validation of a Detection Method for Hepatitis E Virus RNA in Pig Liver.

BACKGROUND: In the last years, the number of notified hepatitis E cases in humans has continuously increased in Europe. Foodborne infection with the zoonotic hepatitis E virus (HEV) genotype 3 is considered the major cause of this disease. Undercooked liver and raw sausages containing the liver of pigs and wild boar are at high risk of containing HEV. However, so far, no standardized method for the detection of HEV-RNA in pig liver is available. METHODS: An international collaborative study on method reproducibility involving 11 laboratories was performed for an HEV-RNA detection method, which consists of steps of sample homogenization, RNA extraction and real-time RT-PCR detection, including a process control. Naturally contaminated pork liver samples containing two different amounts of HEV and a HEV-negative pork liver sample were tested by all laboratories using the method. RESULTS: Valid results were retrieved from 10 laboratories. A specificity of 100% and a sensitivity of 79% were calculated for the method. False negative results were only retrieved from the sample containing very low HEV amounts near the detection limit. CONCLUSIONS: The results show that the method is highly specific, sufficiently sensitive and robust for use in different laboratories. The method can, therefore, be applied to routine food control as well as in monitoring studies.

Large Nationwide Outbreak of Invasive Listeriosis Associated with Blood Sausage, Germany, 2018-2019.

Invasive listeriosis is a severe foodborne infection in humans and is difficult to control. Listeriosis incidence is increasing worldwide, but some countries have implemented molecular surveillance programs to improve recognition and management of listeriosis outbreaks. In Germany, routine whole-genome sequencing, core genome multilocus sequence typing, and single nucleotide polymorphism calling are used for subtyping of Listeria monocytogenes isolates from listeriosis cases and suspected foods. During 2018-2019, an unusually large cluster of L. monocytogenes isolates was identified, including 134 highly clonal, benzalkonium-resistant sequence type 6 isolates collected from 112 notified listeriosis cases. The outbreak was one of the largest reported in Europe during the past 25 years. Epidemiologic investigations identified blood sausage contaminated with L. monocytogenes highly related to clinical isolates; withdrawal of the product from the market ended the outbreak. We describe how epidemiologic investigations and complementary molecular typing of food isolates helped identify the outbreak vehicle.

Corynebacterium silvaticum sp. nov., a unique group of NTTB corynebacteria in wild boar and roe deer.

A total of 34 Corynebacterium sp. strains were isolated from caseous lymph node abscesses of wild boar and roe deer in different regions of Germany. They showed slow growth on Columbia sheep blood agar and sparse growth on Hoyle's tellurite agar. Cellular fatty acid analysis allocated them in the C. diphtheriae group of genus Corynebacterium. MALDI-TOF MS using specific database extensions and rpoB sequencing resulted in classification as C. ulcerans. Their quinone system is similar to C. ulcerans, with major menaquinone MK-8(H2). Their complex polar lipid profile includes major lipids phosphatidylinositol, phosphatidylinositol-mannoside, diphosphatidylglycerol, but also unidentified glycolipids, distinguishing them clearly from C. ulcerans. They ferment glucose, ribose and maltose (like C. ulcerans), but do not utilise d-xylose, mannitol, lactose, sucrose and glycogen (like C. pseudotuberculosis). They showed activity of catalase, urease and phospholipase D, but variable results for alkaline phosphatase and alpha-glucosidase. All were non-toxigenic, tox gene bearing and susceptible to clindamycin, penicillin and erythromycin. In 16SrRNA gene and RpoB protein phylogenies the strains formed distinct brancheswith C. ulcerans as nearest relative.Whole genome sequencing revealed the unique sequence type 578, a distinctbranch in pangenomic core genome MLST, average nucleotide identities <91%, enhancedgenome sizes (2.55 Mbp) and G/C content (54.4 mol%) compared to related species.These results suggest that the strains represent a novel species, for which wepropose the name Corynebactriumsilvaticum sp. nov., based on their first isolation from forest-dwellinggame animals. The type strain isKL0182T (= CVUAS 4292T = DSM 109166T = LMG 31313T= CIP 111 672T).

Reliable differentiation of a non-toxigenic tox gene-bearing Corynebacterium ulcerans variant frequently isolated from game animals using MALDI-TOF MS.

Corynebacterium (C.) ulcerans is a zoonotic member of the C. diphtheriae group and is known to cause abscesses in humans and several animal species. Toxigenic strains, expressing the tox gene encoding diphtheria toxin, are also able to cause diphtheria in humans. In recent years, a non-toxigenic but tox gene-bearing (NTTB) variant of C. ulcerans has been identified that was frequently isolated from clinically healthy as well as from diseased wildlife animals, especially wild boars (Sus scrofa scrofa) in Germany and Austria. The described clinical cases showed similar signs of disease and the isolated corynebacteria displayed common genetic features as well as similar spectroscopic characteristics, therefore being assigned to a so called wild boar cluster (WBC). This study describes the establishment and validation of a method using MALDI-TOF mass spectrometry for a reliable differentiation between various members of the C. diphtheriae group at species level as well as a reliable sub-level identification of C. ulcerans isolates of the WBC variant. For this study 93 C. ulcerans isolates from wildlife animals, 41 C. ulcerans isolates from other animals and humans, and 53 isolates from further representatives of the C. diphtheriae group, as well as 26 non-diphtheriae group Corynebacteria collected via the MALDI user platform from seven MALDI users were used. By assigning 86 C. ulcerans isolates to the WBC the extensive geographical distribution of this previously less noticed variant in two Central European countries could be shown.

Interlaboratory Validation of a Method for Hepatitis E Virus RNA Detection in Meat and Meat Products.

Increasing numbers of hepatitis E cases are currently recognized in many European countries. The zoonotic hepatitis E virus (HEV) genotype 3 mainly circulates in domestic pigs and wild boars, and can be transmitted to humans via consumption of insufficiently heated meat or meat products produced from those animals. Here, a detailed protocol for detection of HEV RNA in meat products is provided, which is based on the method originally described by Szabo et al. (Intl J Food Microbiol 215:149-156, 2015). It consists of a TRI Reagent®/chloroform-based food matrix homogenization, a silica bead-based RNA extraction and a real-time RT-PCR-based RNA detection. The method was further validated in a ring trial with nine independent laboratories using pork liver sausage samples artificially contaminated with different amounts of HEV. The results indicate sufficient sensitivity, specificity, and accuracy of the method for its broad future use in survey studies, routine food control or outbreak investigations.

Enterotoxin Production of Bacillus thuringiensis Isolates From Biopesticides, Foods, and Outbreaks.

While the relevance of Bacillus (B.) cereus as a major cause of gastroenteritis is undisputed, the role of the closely related B. thuringiensis in foodborne disease is unclear. B. thuringiensis strains frequently harbor enterotoxin genes. However, the organism has only very rarely been associated with foodborne outbreaks, possibly due to the fact that during outbreak investigations, B. cereus is routinely not differentiated from B. thuringiensis. A recent EFSA scientific opinion stresses the urgent need for further data allowing for improved risk assessment, in particular as B. thuringiensis is a commonly used biopesticide. Therefore, the aim of this study was to gain further insights into the hazardous potential of B. thuringiensis. To this end, 39 B. thuringiensis isolates obtained from commercially used biopesticides, various food sources, as well as from foodborne outbreaks were characterized by panC typing, panC-based SplitsTree analysis, toxin gene profiling, FTIR spectroscopic analysis, a cytotoxicity assay screening for enterotoxic activity, and a sphingomyelinase assay. The majority of the tested B. thuringiensis isolates exhibited low (23%, n = 9) or mid level enterotoxicity (74%, n = 29), and produced either no (59%, n = 23) or low levels (33%, n = 13) of sphingomyelinase, which is reported to act synergistically with enterotoxins Nhe and Hbl. One strain isolated from rosemary was however classified as highly enterotoxic surpassing the cytotoxic activity of the high-level reference strain by a factor of 1.5. This strain also produced vast amounts of sphingomyelinase. Combining all results obtained in this study into a fingerprint pattern, several enterotoxic biopesticide strains were indistinguishable from those of isolates from foods or collected in association with outbreaks. Our study shows that many B. thuringiensis biopesticide strains exhibit mid-level cytotoxicity in a Vero cell assay and that some of these strains cannot be differentiated from isolates collected from foods or in association with outbreaks. Thus, we demonstrate that the use of B. thuringiensis strains as biopesticides can represent a food safety risk, underpinning the importance of assessing the hazardous potential of each strain and formulation used.

Phenotypic and Genotypic Characteristics of Members of the Genus Streptobacillus.

The genus Streptobacillus (S.) remained monotypic for almost 90 years until two new species were recently described. The type species, S. moniliformis, is one of the two etiological agents of rat bite fever, an under-diagnosed, worldwide occurring zoonosis. In a polyphasic approach field isolates and reference strains of S. moniliformis, S. hongkongensis, S. felis as well as divergent isolates were characterized by comparison of molecular data (n = 29) and from the majority also by their physiological as well as proteomic properties (n = 22). Based on growth-independent physiological profiling using VITEK2-compact, API ZYM and the Micronaut system fastidious growth-related difficulties could be overcome and streptobacilli could definitively be typed despite generally few differences. While differing in their isolation sites and dates, S. moniliformis isolates were found to possess almost identical spectra in matrix-assisted laser desorption ionization-time of flight mass spectrometry and Fourier transform infrared spectroscopy. Spectroscopic methods facilitated differentiation of S. moniliformis, S. hongkongensis and S. felis as well as one divergent isolate. Sequencing of 16S rRNA gene as well as functional genes groEL, recA and gyrB revealed only little intraspecific variability, but generally proved suitable for interspecies discrimination between all three taxa and two groups of divergent isolates.

Toxigenic Corynebacterium ulcerans isolated from a free-roaming red fox (Vulpes vulpes).

Corynebacterium (C.) ulcerans could be isolated from the spleen of a red fox (Vulpes vulpes) that had been found dead in the state of Baden-Württemberg, Germany. Pathohistological examination suggested that the fox had died of distemper, as was confirmed by PCR. The isolate was identified biochemically, by MALDI-TOF MS, FT-IR and by partial 16S rRNA, rpoB and tox gene sequencing. Using the Elek test the C. ulcerans isolate demonstrated diphtheria toxin production. FT-IR and sequencing data obtained from the C. ulcerans isolate from the red fox showed higher similarity to isolates from humans than to those from wild game.

Streptobacillus felis sp. nov., isolated from a cat with pneumonia, and emended descriptions of the genus Streptobacillus and of Streptobacillus moniliformis.

A pleomorphic, Gram-stain-negative, rod-shaped, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile bacterium (strain 131000547(T)) was isolated from the lungs of a cat with pneumonia. On the basis of 16S rRNA gene sequence analyses the strain was assigned to the genus Streptobacillus with 97.6% sequence similarity to the type strain of Streptobacillus moniliformis and 94.6% to that of Streptobacillus hongkongensis. The clear differentiation of strain 131000547(T) from Streptobacillus moniliformis and Streptobacillus hongkongensis was also supported by gyrB, groEL, and recA nucleotide and amino acid sequence analysis. DNA-DNA hybridization demonstrated ≤ 19.9% (reciprocal 28.7%) DNA-DNA relatedness between strain 131000547(T) and Streptobacillus moniliformis DSM 12112(T). Physiological data confirmed the allocation of strain 131000547(T) to the family Leptotrichiaceae. Strain 131000547(T) has a unique profile of enzyme activities allowing differentiation from the most closely related species. Within the genus Streptobacillus, isolate 131000547(T) could also unambiguously be separated from Streptobacillus moniliformis and Streptobacillus hongkongensis by both matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Fourier transform-infrared spectroscopy. On the basis of these data, a novel species of the genus Streptobacillus, Streptobacillus felis sp. nov., is proposed with the type strain 131000547(T) ( = DSM 29248(T) = CCUG 66203(T) = CCM 8542(T)). Emended descriptions of the genus Streptobacillus and of Streptobacillus moniliformis are also given.

Outbreak with clonally related isolates of Corynebacterium ulcerans in a group of water rats.

BACKGROUND: The zoonotic bacterium Corynebacterium ulcerans may be pathogenic both in humans and animals: toxigenic strains can cause diphtheria or diphtheria-like disease in humans via diphtheria toxin, while strains producing the dermonecrotic exotoxin phospholipase D may lead to caseous lymphadenitis primarily in wild animals. Diphtheria toxin-positive Corynebacterium ulcerans strains have been isolated mainly from cattle, dogs and cats. RESULTS: Here, we report a series of ten isolations of Corynebacterium ulcerans from a group of water rats (Hydromys chrysogaster) with ulcerative skin lesions, which were kept in a zoo. The isolates were clearly assigned to species level by biochemical identification systems, Fourier-transform infrared-spectroscopy, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and partial rpoB sequencing, respectively. All ten isolates turned out to represent the same sequence type, strongly indicating a cluster of infections by clonally-related isolates as could be demonstrated for the first time for this species using multilocus sequence typing. Unequivocal demonstration of high relatedness of the isolates could also be demonstrated by Fourier-transform infrared-spectroscopy. All isolates were lacking the diphtheria toxin encoding tox-gene, but were phospholipase D-positive. CONCLUSIONS: Our results indicate that water rats represent a suitable new host species that is prone to infection and must be regarded as a reservoir for potentially zoonotic Corynebacterium ulcerans. Furthermore, the applied methods demonstrated persistent infection as well as a very close relationship between all ten isolates.

Nontoxigenic tox-bearing Corynebacterium ulcerans infection among game animals, Germany.

Corynebacterium ulcerans may cause diphtheria in humans and caseous lymphadenitis in animals. We isolated nontoxigenic tox-bearing C. ulcerans from 13 game animals in Germany. Our results indicate a role for game animals as reservoirs for zoonotic C. ulcerans.

Isolation of Bacillus cytotoxicus from various commercial potato products.

Bacillus (B.) cytotoxicus is a newly described thermotolerant member of the Bacillus cereus group. This potential foodborne pathogen had so far only been isolated from vegetable products, including mashed potatoes. Here we report the detection of B. cytotoxicus in a variety of potato products taken on retail level or from catering establishments (n=151). Identification of isolates as B. cytotoxicus was performed after enrichment at 50°C, followed by differentiation using Fourier transform-infrared spectroscopy and detection of the specific cytK-1 gene by PCR. Thirty-five percent of all samples were positive for B. cytotoxicus. Highest prevalence was found in dehydrated potato products (44/62=71%) such as powder for mashed potatoes and products made thereof. B. cytotoxicus was not detected in products that were evidently made directly from potatoes (n=24) but in one sample of raw potatoes (n=10; 10%). The high prevalence of this thermotolerant pathogen in potato products could pose a risk for consumers, especially if prepared foods are held at improper holding temperatures.

Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus Group occasionally associated with food poisoning.

An aerobic endospore-forming bacillus (NVH 391-98(T)) was isolated during a severe food poisoning outbreak in France in 1998, and four other similar strains have since been isolated, also mostly from food poisoning cases. Based on 16S rRNA gene sequence similarity, these strains were shown to belong to the Bacillus cereus Group (over 97% similarity with the current Group species) and phylogenetic distance from other validly described species of the genus Bacillus was less than 95%. Based on 16S rRNA gene sequence similarity and MLST data, these novel strains were shown to form a robust and well-separated cluster in the B. cereus Group, and constituted the most distant cluster from species of this Group. Major fatty acids (iso-C(15:0), C(16:0), iso-C(17:0), anteiso-C(15 : 0), iso-C(16:0), iso-C(13:0)) supported the affiliation of these strains to the genus Bacillus, and more specifically to the B. cereus Group. NVH 391-98(T) taxon was more specifically characterized by an abundance of iso-C(15:0) and low amounts of iso-C(13:0) compared with other members of the B. cereus Group. Genome similarity together with DNA-DNA hybridization values and physiological and biochemical tests made it possible to genotypically and phenotypically differentiate NVH 391-98(T) taxon from the six current B. cereus Group species. NVH 391-98(T) therefore represents a novel species, for which the name Bacillus cytotoxicus sp. nov. is proposed, with the type strain NVH 391-98(T) (= DSM 22905(T) = CIP 110041(T)).

[Corynebacterium ulcerans infection in roe deer (Capreolus capreolus)]. / Corynebacterium ulcerans-Infektion bei einem Reh (Capreolus capreolus).

This is the first report of a Corynebacterium (C) ulcerans-infection in European roe deer (Capreolus capreolus). The bacterium was isolated from a grapefruit sized abscess of an animal that had been shot. In addition to biochemical tests, the isolate was identified by Fourier transform infrared spectroscopy (FT-IR) and partial sequencing of the rpoB gene. The isolated bacteria showed phospholipase D activity that could be demonstrated by reverse CAMP-test. A tox-gene could be detected by PCR but the Elek-test specific for diphtheria toxin failed.The isolate was compared to two C. ulcerans-strains isolated from wild boar (Sus scrofa) from the state of Baden-Wuerttemberg described recently.

Detection of rotavirus in food associated with a gastroenteritis outbreak in a mother and child sanatorium.

In the course of a rotavirus outbreak in a mother and child sanatorium 74 food samples from the sanatorium kitchen were taken and tested for rotavirus. Rotavirus particles were isolated from 25 g food samples by a simple method including ultrafiltration, originally designed for the detection of norovirus in various food matrices. Rotavirus was successfully detected in a sample of potato stew by conventional RT-seminested-PCR. Sequence comparison of the amplification products obtained from the potato stew and a stool sample from an infected child verified that the two viruses were identical.

[Cereulide forming presumptive Bacillus cereus strains from food--differentiating analyses using cultural methods, LC-MS/MS, PCR, and infrared spectroscopy in consideration of thermotolerant isolates]. / Cereulidbildende präsumtive Bacillus cereus aus Lebensmitteln--Differenzierende Untersuchungen mittels kultureller Methoden, LC-MS/MS, PCR und Infrarotspektroskopie unter Berücksichtigung thermotoleranter Vertreter.

Pathogenic Bacillus cereus (B. cereus) cause two types of foodborne diseases: the diarrhoeal type and, after production of a heat stable toxin called cereulide, an emetic type. The identification of B. cereus in official food monitoring has been traditionally performed using the cultural procedure as described in method 00.00-25 according to section 64 of the German Food and Feed Law (LFGB). Strains isolated by this method are called "presumptive B. cereus" a collective name for B. cereus sensu strictu, B. thuringiensis and closely related Bacilli. Some potentially pathogenic thermotolerant isolates ("B. cytotoxicus") are not covered by this method. In this work Fourier-Transform-infrared spectroscopy (FT-IR) in combination with artificial neural network based data analysis was tested and verified for the further differentiation of "presumptive B. cereus" isolates and closely related Bacilli. For this purpose 122 Bacillus strains were, in addition to the section 64 LFGB method, assayed for formation of parasporal crystals, thermotolerant growth, PCR and LC-MS/MS. Based on this data a further FT-IR-method was developed for the differentiation of emetic B. cereus. Exemplarily, these methods were applied in a B. cereus related foodborne outbreak. In addition, the obtained FT-IR-spectra visualize the chain of infection.

4-sulfomuconolactone hydrolases from Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2.

The 4-carboxymethylen-4-sulfo-but-2-en-olide (4-sulfomuconolactone) hydrolases from Hydrogenophaga intermedia strain S1 and Agrobacterium radiobacter strain S2 are part of a modified protocatechuate pathway responsible for the degradation of 4-sulfocatechol. In both strains, the hydrolase-encoding genes occur downstream of those encoding the enzymes that catalyze the lactonization of 3-sulfomuconate. The deduced amino acid sequences of the 4-sulfomuconolactone hydrolases demonstrated the highest degree of sequence identity to 2-pyrone-4,6-dicarboxylate hydrolases, which take part in the meta cleavage pathway of protocatechuate. The 4-sulfomuconolactone hydrolases did not convert 2-pyrone-4,6-dicarboxylate, and the 2-pyrone-4,6-dicarboxylate hydrolase from Sphingomonas paucimobilis SYK-6 did not convert 4-sulfomuconolactone. Nevertheless, the presence of highly conserved histidine residues in the 4-sulfomuconolactone and the 2-pyrone-4,6-dicarboxylate hydrolases and some further sequence similarities suggested that both enzymes belong to the metallo-dependent hydrolases (the "amidohydrolase superfamily"). The 4-sulfomuconolactone hydrolases were heterologously expressed as His-tagged enzyme variants. Gel filtration experiments suggested that the enzymes are present as monomers in solution, with molecular weights of approximately 33,000 to 35,000. 4-Sulfomuconolactone was converted by sulfomuconolactone hydrolases to stoichiometric amounts of maleylacetate and sulfite. The 4-sulfomuconolactone hydrolases from both strains showed pH optima at pH 7 to 7.5 and rather similar catalytic constant (k(cat)/K(M))values. The suggested 4-sulfocatechol pathway from 4-sulfocatechol to maleylacetate was confirmed by in situ nuclear magnetic resonance analysis using the recombinantly expressed enzymes.

Characterization of the genes encoding the 3-carboxy-cis,cis-muconate-lactonizing enzymes from the 4-sulfocatechol degradative pathways of Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2.

Hydrogenophaga intermedia strain S1 and Agrobacterium radiobacter strain S2 form a mixed bacterial culture which degrades sulfanilate (4-aminobenzenesulfonate) by a novel variation of the beta-ketoadipate pathway via 4-sulfocatechol and 3-sulfomuconate. It was previously proposed that the further metabolism of 3-sulfomuconate is catalysed by modified 3-carboxy-cis,cis-muconate-lactonizing enzymes (CMLEs) and that these 'type 2' enzymes were different from the conventional CMLEs ('type 1') from the protocatechuate pathway in their ability to convert 3-sulfomuconate in addition to 3-carboxy-cis,cis-muconate. In the present study the genes for two CMLEs (pcaB2S1 and pcaB2S2) were cloned from H. intermedia S1 and A. radiobacter S2, respectively. In both strains, these genes were located close to the previously identified genes encoding the 4-sulfocatechol-converting enzymes. The gene products of pcaB2S1 and pcaB2S2 were therefore tentatively identified as type 2 enzymes involved in the metabolism of 3-sulfomuconate. The genes were functionally expressed and the gene products were shown to convert 3-carboxy-cis,cis-muconate and 3-sulfomuconate. 4-Carboxymethylene-4-sulfo-but-2-en-olide (4-sulfomuconolactone) was identified by HPLC-MS as the product, which was enzymically formed from 3-sulfomuconate. His-tagged variants of both CMLEs were purified and compared with the CMLE from the protocatechuate pathway of Pseudomonas putida PRS2000 for the conversion of 3-carboxy-cis,cis-muconate and 3-sulfomuconate. The CMLEs from the 4-sulfocatechol pathway converted 3-sulfomuconate with considerably higher activities than 3-carboxy-cis,cis-muconate. Also the CMLE from P. putida converted 3-sulfomuconate, but this enzyme demonstrated a clear preference for 3-carboxy-cis,cis-muconate as substrate. Thus it was demonstrated that in the 4-sulfocatechol pathway, distinct CMLEs are formed, which are specifically adapted for the preferred conversion of sulfonated substrates.

Structure and function of the 3-carboxy-cis,cis-muconate lactonizing enzyme from the protocatechuate degradative pathway of Agrobacterium radiobacter S2.

3-carboxy-cis,cis-muconate lactonizing enzymes participate in the protocatechuate branch of the 3-oxoadipate pathway of various aerobic bacteria. The gene encoding a 3-carboxy-cis,cis-muconate lactonizing enzyme (pcaB1S2) was cloned from a gene cluster involved in protocatechuate degradation by Agrobacterium radiobacter strain S2. This gene encoded for a 3-carboxy-cis,cis-muconate lactonizing enzyme of 353 amino acids - significantly smaller than all previously studied 3-carboxy-cis,cis-muconate lactonizing enzymes. This enzyme, ArCMLE1, was produced in Escherichia coli and shown to convert not only 3-carboxy-cis,cis-muconate but also 3-sulfomuconate. ArCMLE1 was purified as a His-tagged enzyme variant, and the basic catalytic constants for the conversion of 3-carboxy-cis,cis-muconate and 3-sulfomuconate were determined. In contrast, Agrobacterium tumefaciens 3-carboxy-cis,cis-muconate lactonizing enzyme 1 could not, despite 87% sequence identity to ArCMLE1, use 3-sulfomuconate as substrate. The crystal structure of ArCMLE1 was determined at 2.2 A resolution. Consistent with the sequence, it showed that the C-terminal domain, present in all other members of the fumarase II family, is missing in ArCMLE1. Nonetheless, both the tertiary and quaternary structures, and the structure of the active site, are similar to those of Pseudomonas putida 3-carboxy-cis,cis-muconate lactonizing enzyme. One principal difference is that ArCMLE1 contains an Arg, as opposed to a Trp, in the active site. This indicates that activation of the carboxylic nucleophile by a hydrophobic environment is not required for lactonization, unlike earlier proposals [Yang J, Wang Y, Woolridge EM, Arora V, Petsko GA, Kozarich JW & Ringe D (2004) Biochemistry43, 10424-10434]. We identified citrate and isocitrate as noncompetitive inhibitors of ArCMLE1, and found a potential binding pocket for them on the enzyme outside the active site.