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The Corynebacterium species C. diphtheriae, C. ulcerans, and C. pseudotuberculosis form the C. diphtheriae group, as shown by 16S rRNA gene sequence analysis (1). Strains of this group carrying lysogenic [...]

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.sub.cat]/[K.sub.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.

Hydrogenophaga intermedia strain S1 and Agrobacterium radiobacter strain $2 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 pcaB2Sl 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-2en-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.

The 2,3-dihydroxybiphenyl dioxygenase from Sphingomonas sp. strain BN6 (BphC1-BN6) is different from most other extradiol dioxygenases because it can oxidize 3-chlorocatechol to 3-chloro-2-hydroxymuconic semialdehyde by a distal cleavage mechanism. The turnover of various substrates and the effects of a number of inhibitors on BphC1-BN6 were contrasted with those of another 2,3-dihydroxybiphenyl dioxygenase from the same strain (BphC2-BN6) as well as with those of the archetypical catechol 2,3-dioxygenase (C23O-mt2) encoded by the TOL plasmid.

The use of the sulfonated azo compound 4-carboxy-4'-sulfoazobenzene (CSAB) as the sole source of carbon and energy by a bacterial strain was described. The bacterial strain S5 was isolated by continuous adaptation of 'Hydrogenophaga palleronii' S1 while the model CSAB was synthesized by nitro-amine condensation. Results showed a reductive cleavage in CSAB with sulfanilate as one of the reduction products. Furthermore, strain S5 displayed the ability to mineralize CSAB, indicating the possibility of degrading sulfonated azo dyes under aerobic conditions.

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 Å 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) Biochemistry 43, 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. [ABSTRACT FROM AUTHOR]

Presents information on a study which isolated a bacterial strain with the ability to sulfonate azo compound 4-carboxy-4'-sulfoazobenzene, in relation to its sole source of carbon and energy. Adaptation of the Hydrogenophaga palleronii S1; Reference to strain S5; Methodology used to conduct the study; Results of the study.

The article discusses the genes of 3-carboxy-cis, cis-muconate-lactonizing enzymes of pcaB2S1 and pcaB2S2 that were cloned from the Hydrogenophaga intermedia strain S1 and Agrobacterium radiobater strain S2. The genes were located close to the genes encoding the 4-sulfocatechol-converting enzymes wherein gene products of pcaB2S1 and pcaB2S2 were shown to convert 4-sulfomuconolactone, 3-sulfomuconate and others that was identified by HPLC-MS.

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.

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.

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.

The identification of thermotolerant campylobacters in official food control in the state of Baden-Wuerttemberg has been traditionally performed using the cultural procedure as described in the ISO-Norm 10272:1995. Analysis thus took 5-6 days to complete. Additionally diagnostic problems caused by the accompanying flora as well as the resistance to nalidixic acid occured. Within the scope of this study these problems could be solved by introducing a filtration step for the reduction of the accompanying flora and by performing the indoxyl acetate-hydrolysis-test in addition to the antibiotic-resistance-test. Besides various PCR protocols for the identification of thermotolerant campylobacters from food were established as an alternative to the cultural procedure, providing reliable results within two days. Furthermore, infrared spectroscopy was tested for the identification of Campylobacter isolates. Using this technique and with the help of a suitable data base, bacterial pure cultures can be differentiated within 2 hours. Among others 356 samples of raw poultry meat were tested with the newly established procedures as well as with the classical cultural method, showing that 32% of the samples were Campylobacter spp. positive. 37% of these isolates were resistant against nalidixic acid. This indicates that the development of resistances in Campylobacter spp. in Germany follows the same trend described for other European countries.