Andreas Burkovski

Sugar Transport Systems of Bifidobacterium longum NCC2705

Here we present the complement of the carbohydrate uptake systems of the strictly anaerobic probiotic Bifidobacterium longum NCC2705. The genome analysis of this bacterium predicts that it has 19 permeases for the uptake of diverse carbohydrates. The majority belongs to the ATP-binding cassette transporter family with 13 systems identified. Among them are permeases for lactose, maltose, raffinose, and fructooligosaccharides, a commonly used prebiotic additive. We found genes that encode a complete phosphotransferase system (PTS) and genes for three permeases of the major facilitator superfamily. These systems could serve for the import of glucose, galactose, lactose, and sucrose. Growth analysis of NCC2705 cells combined with biochemical characterization and microarray data showed that the predicted substrates are consumed and that the corresponding transport and catabolic genes are expressed. Biochemical analysis of the PTS, in which proteins are central in regulation of carbon metabolism in many bacteria, revealed that B. longum has a glucose-specific PTS, while two other species (Bifidobacterium lactis and Bifidobacterium bifidum) have a fructose-6-phosphate-forming fructose-PTS instead. It became obvious that most carbohydrate systems are closely related to those from other actinomycetes, with a few exceptions. We hope that this report on B. longum carbohydrate transporter systems will serve as a guide for further in-depth analyses on the nutritional lifestyle of this beneficial bacterium.

A Genomic View of Sugar Transport in Mycobacterium smegmatis and Mycobacterium tuberculosis

We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.

Characterization of Methionine Export in Corynebacterium glutamicum

Corynebacterium glutamicum is known for its effective excretion of amino acids under particular metabolic conditions. Concomitant activities of uptake and excretion systems would create an energy-wasting futile cycle; amino acid export systems are therefore tightly regulated. We have used a DNA microarray approach to identify genes for membrane proteins which are overexpressed under conditions of elevated cytoplasmic concentrations of methionine. One of these genes was brnF, coding for the larger subunit of BrnFE, a previously identified two-component isoleucine export system. By deletion, complementation, and overexpression of the brnFE genes in a C. glutamicum strain, in which the two uptake systems for methionine were inactivated, we identified BrnFE as being responsible for methionine export. In the presence of both substrates in the cytoplasm, BrnFE was found to transport isoleucine and methionine at similar rates. The expression of the brnFE gene cluster depends on an Lrp-type transcription factor and was shown to be strongly induced by increasing cytoplasmic methionine concentration. Methionine was a better inducer than isoleucine, indicating that methionine rather than isoleucine might be the native substrate of BrnFE. When the synthesis of BrnFE was blocked by chloramphenicol, fast methionine export was still observed, but only at greatly increased cytoplasmic levels of this amino acid. This indicates the presence of at least one other methionine export system, presumably with low affinity but high capacity. Under conditions where cytoplasmic methionine does not exceed a concentration of 50 mM, BrnFE is the dominant export system for this amino acid.

Comparative analysis of two complete Corynebacterium ulcerans genomes and detection of candidate virulence factors.

BackgroundCorynebacterium ulcerans has been detected as a commensal in domestic and wild animals that may serve as reservoirs for zoonotic infections. During the last decade, the frequency and severity of human infections associated with C. ulcerans appear to be increasing in various countries. As the knowledge of genes contributing to the virulence of this bacterium was very limited, the complete genome sequences of two C. ulcerans strains detected in the metropolitan area of Rio de Janeiro were determined and characterized by comparative genomics: C. ulcerans 809 was initially isolated from an elderly woman with fatal pulmonary infection and C. ulcerans BR-AD22 was recovered from a nasal sample of an asymptomatic dog.ResultsThe circular chromosome of C. ulcerans 809 has a total size of 2,502,095 bp and encodes 2,182 predicted proteins, whereas the genome of C. ulcerans BR-AD22 is 104,279 bp larger and comprises 2,338 protein-coding regions. The minor difference in size of the two genomes is mainly caused by additional prophage-like elements in the C. ulcerans BR-AD22 chromosome. Both genomes show a highly similar order of orthologous coding regions; and both strains share a common set of 2,076 genes, demonstrating their very close relationship. A screening for prominent virulence factors revealed the presence of phospholipase D (Pld), neuraminidase H (NanH), endoglycosidase E (EndoE), and subunits of adhesive pili of the SpaDEF type that are encoded in both C. ulcerans genomes. The rbp gene coding for a putative ribosome-binding protein with striking structural similarity to Shiga-like toxins was additionally detected in the genome of the human isolate C. ulcerans 809.ConclusionsThe molecular data deduced from the complete genome sequences provides considerable knowledge of virulence factors in C. ulcerans that is increasingly recognized as an emerging pathogen. This bacterium is apparently equipped with a broad and varying set of virulence factors, including a novel type of a ribosome-binding protein. Whether the respective protein contributes to the severity of human infections (and a fatal outcome) remains to be elucidated by genetic experiments with defined bacterial mutants and host model systems.

The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence

BackgroundCorynebacterium pseudotuberculosis is generally regarded as an important animal pathogen that rarely infects humans. Clinical strains are occasionally recovered from human cases of lymphadenitis, such as C. pseudotuberculosis FRC41 that was isolated from the inguinal lymph node of a 12-year-old girl with necrotizing lymphadenitis. To detect potential virulence factors and corresponding gene-regulatory networks in this human isolate, the genome sequence of C. pseudotuberculosis FCR41 was determined by pyrosequencing and functionally annotated.ResultsSequencing and assembly of the C. pseudotuberculosis FRC41 genome yielded a circular chromosome with a size of 2,337,913 bp and a mean G+C content of 52.2%. Specific gene sets associated with iron and zinc homeostasis were detected among the 2,110 predicted protein-coding regions and integrated into a gene-regulatory network that is linked with both the central metabolism and the oxidative stress response of FRC41. Two gene clusters encode proteins involved in the sortase-mediated polymerization of adhesive pili that can probably mediate the adherence to host tissue to facilitate additional ligand-receptor interactions and the delivery of virulence factors. The prominent virulence factors phospholipase D (Pld) and corynebacterial protease CP40 are encoded in the genome of this human isolate. The genome annotation revealed additional serine proteases, neuraminidase H, nitric oxide reductase, an invasion-associated protein, and acyl-CoA carboxylase subunits involved in mycolic acid biosynthesis as potential virulence factors. The cAMP-sensing transcription regulator GlxR plays a key role in controlling the expression of several genes contributing to virulence.ConclusionThe functional data deduced from the genome sequencing and the extended knowledge of virulence factors indicate that the human isolate C. pseudotuberculosis FRC41 is equipped with a distinct gene set promoting its survival under unfavorable environmental conditions encountered in the mammalian host.

Molecular Identification of the Urea Uptake System and Transcriptional Analysis of Urea Transporter- and Urease-Encoding Genes in Corynebacterium glutamicum

The molecular identification of the Corynebacterium glutamicum urea uptake system is described. This ABC-type transporter is encoded by the urtABCDE operon, which is transcribed in response to nitrogen limitation. Expression of the urt genes is regulated by the global nitrogen regulator AmtR, and an amtR deletion strain showed constitutive expression of the urtABCDE genes. The AmtR repressor protein also controls transcription of the urease-encoding ureABCEFGD genes in C. glutamicum. The ure gene cluster forms an operon which is mainly transcribed in response to nitrogen starvation. To confirm the increased synthesis of urease subunits under nitrogen limitation, proteome analyses of cytoplasmic protein extracts from cells grown under nitrogen surplus and nitrogen limitation were carried out, and five of the seven urease subunits were identified.

Corynebacterium diphtheriae invasion-associated protein (DIP1281) is involved in cell surface organization, adhesion and internalization in epithelial cells

BackgroundCorynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated the function of surface-associated protein DIP1281, previously annotated as hypothetical invasion-associated protein.ResultsMicroscopic inspection of DIP1281 mutant strains revealed an increased size of the single cells in combination with an altered less club-like shape and formation of chains of cells rather than the typical V-like division forms or palisades of growing C. diphtheriae cells. Cell viability was not impaired. Immuno-fluorescence microscopy, SDS-PAGE and 2-D PAGE of surface proteins revealed clear differences of wild-type and mutant protein patterns, which were verified by atomic force microscopy. DIP1281 mutant cells were not only altered in shape and surface structure but completely lack the ability to adhere to host cells and consequently invade these.ConclusionsOur data indicate that DIP1281 is predominantly involved in the organization of the outer surface protein layer rather than in the separation of the peptidoglycan cell wall of dividing bacteria. The adhesion- and invasion-negative phenotype of corresponding mutant strains is an effect of rearrangements of the outer surface.

Identification of an anion-specific channel in the cell wall of the Gram-positive bacterium Corynebacterium glutamicum

A cation‐selective channel (porin), designated PorA, facilitates the passage of hydrophilic solutes across the cell wall of the mycolic acid‐containing actinomycete Corynebacterium glutamicum. Biochemical and electrophysiological investigations of the cell wall of the mutant strain revealed the presence of an alternative channel‐forming protein. This porin was purified to homogeneity and studied in lipid bilayer membranes. It forms small anion‐selective channels with a diameter of about 1.4 nm and an average single‐channel conductance of about 700 pS in 1 M KCl. The PorBCglut channel could be blocked by citrate in a dose‐dependent manner. This result was in agreement with growth experiments in citrate as sole carbon source where growth in citrate was impaired as compared with growth in other carbon sources. The PorBCglut protein was partially sequenced and based on the resulting amino acid sequence of the corresponding gene, which was designated as porB, was identified as an unannotated 381 bp long open reading frame (ORF) in the published genome sequence of C. glutamicum ATCC13032. PorBCglut contains 126 amino acids with an N‐terminal extension of 27 amino acids. One hundred and thirty‐eight base pairs downstream of porB, we found an ORF that codes for a protein with about 30% identity to PorBCglut, which was named PorCCglut. The arrangement of porB and porC on the chromosome suggested that both genes belong to the same cluster. RT‐PCR from overlapping regions between genes from wild‐type C. glutamicum ATCC 13032 and its ATCC 13032ΔporA mutant demonstrated that this is the case and that porB and porC are cotranscribed. The gene products PorBCglut and PorCCglut represent obviously other permeability pathways for the transport of hydrophilic compounds through the cell wall of C. glutamicum.

The role of the Corynebacterium glutamicum rel gene in (p)ppGpp metabolism

To investigate the metabolism of (p)ppGpp in amino-acid-producing coryneform bacteria, a PCR-based strategy using degenerate consensus oligonucleotides was applied to isolate the rel gene of Corynebacterium glutamicum ATCC 13032. The gene consists of 2283 nucleotides and encodes a protein of 760 amino acids with a molecular mass of 84.4 kDa. The amino acid sequence revealed extensive similarities to the related proteins RelA and SpoT of Escherichia coli, which are known to be involved in (p)ppGpp biosynthesis and degradation. The C. glutamicum rel gene is located downstream of the apt gene encoding an adenine phosphoribosyltransferase, and an ORF with similarities to dciAE, which represents part of a dipeptide transport system in E. coli. A C. glutamicum mutant strain carrying a defined deletion in the rel gene was constructed. This mutant failed to accumulate (p)ppGpp in response to amino acid starvation. When overexpressed in E. coli, the C. glutamicum rel gene was able to reverse growth defects caused by an overexpressed relA gene. It is proposed that the C. glutamicum rel gene encodes a bifunctional enzyme with (p)ppGpp synthetase and (p)ppGpp-degrading activities.

A genomic view on nitrogen metabolism and nitrogen control in mycobacteria.

Knowledge about nitrogen metabolism and control in the genus Mycobacterium is sparse, especially compared to the state of knowledge in related actinomycetes like Streptomyces coelicolor or the close relative Corynebacterium glutamicum. Therefore, we screened the published genome sequences of Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium ssp. paratuberculosis and Mycobacterium leprae for genes encoding proteins for uptake of nitrogen sources, nitrogen assimilation and nitrogen control systems, resulting in a detailed comparative genomic analysis of nitrogen metabolism-related genes for all completely sequenced members of the genus. Transporters for ammonium, nitrate, and urea could be identified, as well as enzymes crucial for assimilation of these nitrogen sources, i.e. glutamine synthetase, glutamate dehydrogenase, glutamate synthase, nitrate reductase, nitrite reductase, and urease proteins. A reduction of genes encoding proteins for nitrogen transport and metabolism was observed for the pathogenic mycobacteria, especially for M. leprae. Signal transduction components identified for the different species include adenylyl- and uridylyltransferase and a PII-type signal transduction protein. Exclusively for M. smegmatis, two homologs of putative nitrogen regulatory proteins were found, namely GlnR and AmtR, while in other mycobacteria, AmtR was absent and GlnR seems to be the nitrogen transcription regulator protein.