Functional genomics and expression analysis of the Corynebacterium glutamicum fpr2-cysIXHDNYZ gene cluster involved in assimilatory sulphate reduction

BACKGROUND: Corynebacterium glutamicum is a high-GC Gram-positive soil bacterium of great biotechnological importance for the production of amino acids. To facilitate the rational design of sulphur amino acid-producing strains, the pathway for assimilatory sulphate reduction providing the necessary reduced sulfur moieties has to be known. Although this pathway has been well studied in Gram-negative bacteria like Escherichia coli and low-GC Gram-positives like Bacillus subtilis, little is known for the Actinomycetales and other high-GC Gram-positive bacteria. RESULTS: The genome sequence of C. glutamicum was searched for genes involved in the assimilatory reduction of inorganic sulphur compounds. A cluster of eight candidate genes could be identified by combining sequence similarity searches with a subsequent synteny analysis between C. glutamicum and the closely related C. efficiens. Using mutational analysis, seven of the eight candidate genes, namely cysZ, cysY, cysN, cysD, cysH, cysX, and cysI, were demonstrated to be involved in the reduction of inorganic sulphur compounds. For three of the up to now unknown genes possible functions could be proposed: CysZ is likely to be the sulphate permease, while CysX and CysY are possibly involved in electron transfer and cofactor biosynthesis, respectively. Finally, the candidate gene designated fpr2 influences sulphur utilisation only weakly and might be involved in electron transport for the reduction of sulphite. Real-time RT-PCR experiments revealed that cysIXHDNYZ form an operon and that transcription of the extended cluster fpr2 cysIXHDNYZ is strongly influenced by the availability of inorganic sulphur, as well as L-cysteine. Mapping of the fpr2 and cysIXHDNYZ promoters using RACE-PCR indicated that both promoters overlap with binding-sites of the transcriptional repressor McbR, suggesting an involvement of McbR in the observed regulation. Comparative genomics revealed that large parts of the extended cluster are conserved in 11 of 17 completely sequenced members of the Actinomycetales. CONCLUSION: The set of C. glutamicum genes involved in assimilatory sulphate reduction was identified and four novel genes involved in this pathway were found. The high degree of conservation of this cluster among the Actinomycetales supports the hypothesis that a different metabolic pathway for the reduction of inorganic sulphur compounds than that known from the well-studied model organisms E. coli and B. subtilis is used by members of this order, providing the basis for further biochemical studies

A systems biology approach reveals major metabolic changes in the thermoacidophilic archaeon Sulfolobus solfataricus in response to the carbon source L-fucose versus D-glucose

Archaea are characterised by a complex metabolism with many unique enzymes that differ from their bacterial and eukaryotic counterparts. The thermoacidophilic archaeon Sulfolobus solfataricus is known for its metabolic versatility and is able to utilize a great variety of different carbon sources. However, the underlying degradation pathways and their regulation are often unknown. In this work, we analyse growth on different carbon sources using an integrated systems biology approach. The comparison of growth on L-fucose and D-glucose allows first insights into the genome-wide changes in response to the two carbon sources and revealed a new pathway for L-fucose degradation in S. solfataricus. During growth on L-fucose we observed major changes in the central carbon metabolic network, as well as an increased activity of the glyoxylate bypass and the 3-hydroxypropionate/4-hydroxybutyrate cycle. Within the newly discovered pathway for L-fucose degradation the following key reactions were identified: (i) L-fucose oxidation to L-fuconate via a dehydrogenase, (ii) dehydration to 2-keto-3-deoxy-L-fuconate via dehydratase, (iii) 2-keto-3-deoxy-L-fuconate cleavage to pyruvate and L-lactaldehyde via aldolase and (iv) L-lactaldehyde conversion to L-lactate via aldehyde dehydrogenase. This pathway as well as L-fucose transport shows interesting overlaps to the D-arabinose pathway, representing another example for pathway promiscuity in Sulfolobus species

Oxidative Stickland reactions in an obligate aerobic organism – amino acid catabolism in the Crenarchaeon Sulfolobus solfataricus

The thermoacidophilic Crenarchaeon Sulfolobus solfataricus is a model organism for archaeal adaptation to extreme environments and renowned for its ability to degrade a broad variety of substrates. It has been well characterised concerning the utilisation of numerous carbohydrates as carbon source. However, its amino acid metabolism, especially the degradation of single amino acids, is not as well understood. In this work, we performed metabolic modelling as well as metabolome, transcriptome and proteome analysis on cells grown on caseinhydrolysate as carbon source in order to draw a comprehensive picture of amino acid metabolism in S. solfataricus P2. We found that 10 out of 16 detectable amino acids are imported from the growth medium. Overall, uptake of glutamate, methionine, leucine, phenylalanine and isoleucine was the highest of all observed amino acids. Our simulations predict an incomplete degradation of leucine and tyrosine to organic acids, and in accordance with this, we detected the export of branched-chain and aromatic organic acids as well as amino acids, ammonium and trehalose into the culture supernatants. The branched-chain amino acids as well as phenylalanine and tyrosine are degraded to organic acids via oxidative Stickland reactions. Such reactions are known for prokaryotes capable of anaerobic growth, but so far have never been observed in an obligate aerobe. Also, 3-methyl-2-butenoate and 2-methyl-2-butenoate are for the first time found as products of modified Stickland reactions for the degradation of branched-chain amino acids. This work presents the first detailed description of branched-chain and aromatic amino acid catabolism in S. solfataricus

Looking for non-coding RNAs in Corynebacterium glutamicum

Gkogkoglou K, Mentz A, Albersmeier A, Kalinowski J, Giegerich R. Looking for non-coding RNAs in Corynebacterium glutamicum. FEBS JOURNAL. 2008;275(Suppl. 1):153

Updating benchtop sequencing performance comparison

In April 2012, your journal published a study by Loman et al.1 that systematically compared desktop next-generation sequencers (NGS) from three instrument providers. Using the custom scripts supplied by the authors, the same software and the same draft genome (with 153 remaining gaps within several scaffolds) as the reference, we reproduced their results with their data of the enterohemorrhagic Escherichia coli (EHEC) strain found in the 2011 outbreak in Germany. However, we wish to bring readers’ attention to some shortcomings in the report from Loman et al.1, focusing particularly on its discussion of read-level error analysis. NGS is a rapidly changing market, which clearly complicates the comparisons such as that made by Loman et al. Since the original study1, Illumina (San Diego) has launched the MiSeq sequencer officially and has released Nextera library construction kits and 2 × 250–base-pair (250-bp) paired-end (PE) sequencing chemistry. Furthermore, Life Technologies (Carlsbad, California), has made 200-bp and 300-bp kits available for the Ion Torrent Personal Genome Machine (PGM). Roche (Basel, Switzerland) has updated the Sequencing System software for its 454 GS Junior (GSJ) from version 2.6 to 2.7. In this report, we provide an up-to-date snapshot of how benchtop platforms have evolved since the previous study1

Dermabacter hominis: a usually daptomycin-resistant gram-positive organism infrequently isolated from human clinical samples.

During a 12-year period, Dermabacter hominis was isolated from 21 clinical samples belonging to 14 patients attending a tertiary hospital in León, Spain. Samples included blood cultures (14), peritoneal dialysis catheter exit sites (three), cutaneous abscesses (two), an infected vascular catheter (one) and a wound swab (one). Identification was made by API Coryne™ V2.0, Biolog™ GP2 and 16S rRNA gene amplification. Six febrile patients had positive blood cultures (one, two or three sets) and all of them were treated with teicoplanin (two patients), vancomycin, ampicillin plus gentamicin, amoxicillin/clavulanic acid and ciprofloxacin (one each). An additional patient with a single positive blood culture was not treated, the finding being considered non-significant. In the remaining seven patients the organism was isolated from a single specimen and three of them received antimicrobial treatment (ciprofloxacin, ceftriaxone plus vancomycin and amoxicillin/clavulanic acid). At least ten patients had several underlying diseases and conditions, and no direct mortality was observed in relation to the isolated organism. All isolates were susceptible to vancomycin, rifampin and linezolid. Resistance to other antibiotics varied: erythromycin (100%), clindamycin (78.5%), ciprofloxacin (21.4%) and gentamicin, quinupristin-dalfopristin, benzylpenicillin and imipenem 7.1% each. Thirteen isolates were highly resistant to daptomycin with MICs ranging from 8 to 48 (MIC90 = 32 mg/L); only one was daptomycin-sensitive (MIC = 0.19 mg/L).Research Project GRS 698/A/11. Gerencia Regional de Salud. Junta de Castilla y León, Spain.S

Control in the Label: Self-Declared, Certified, Accredited? : On-Pack Consumer Communication About Compliance Control in Voluntary Food Schemes from a Legal Perspective

In the EU, the number of voluntary food schemes has grown rampantly over the past 20 years. From a consumer perspective, what was initially praised as a solution to the information asymmetry problem of credence goods soon became a problem of itself: consumers do not know what the various food scheme labels mean, and especially whether and how they are subject to compliance control (self-declaration, certification, and accreditation). In this contribution, we argue that it is necessary to examine how messages about compliance control are communicated to the consumer on pack and how they affect consumers, and show that on pack communication of food scheme labels offers significant opportunities for influencing consumer perceptions. Consequently, food businesses are under a legal duty not to mislead consumers and to ensure that information is sufficiently clear, including elements indicating the control a scheme is subject to