Genomic analysis of a novel nontoxigenic Corynebacterium diphtheriae strain isolated from a cancer patient.

The genome of a novel nontoxigenic Corynebacterium diphtheriae, strain 5015, isolated from a patient with adenoid cystic carcinoma was sequenced and compared with 117 publically available genomes. This strain is phylogenetically distinct and lacks virulence genes encoding the toxin, BigA and Sdr-like adhesins. Strain 5015 possesses spaD-type and spaH-type pilus gene clusters with a loss of some gene functions, and 31 unique genes that need molecular characterization to understand their potential role in virulence characteristics.

Genomic analyses reveal two distinct lineages of Corynebacterium ulcerans strains.

Corynebacteriumulcerans is an important zoonotic pathogen which is causing diphtheria-like disease in humans globally. In this study, the genomes of three recently isolated C. ulcerans strains, 4940, 2590 and BR-AD 2649, respectively from an asymptomatic carrier, a patient with pharyngitis and a canine host, were sequenced to investigate their virulence potential. A comparative analysis was performed including the published genome sequences of 16 other C. ulcerans isolates. C. ulcerans strains belong to two lineages; 13 strains are grouped together in lineage 1, and six strains comprise lineage 2. Consistent with the zoonotic nature of C. ulcerans infections, isolates from both the human and canine hosts clustered in both the lineages. Most of the strains possessed spaDEF and spaBC gene clusters along with the virulence genes cpp, pld, cwlH, nanH, rpfI, tspA and vsp1. The gene encoding Shiga-like toxin was only present in one strain, and 11 strains carried the tox gene encoding the diphtheria-like toxin. However, none of strains 4940, 2590 and BR-AD 2649 carried any toxin genes. These strains varied in the number of prophages in their genomes, which suggests that they play an important role in introducing diversity in C. ulcerans. The pan-genomic analyses revealed a variation in the number of membrane-associated and secreted proteins that may contribute to the variation in pathogenicity among different strains.

GltS, the sodium-coupled L-glutamate uptake system of Corynebacterium glutamicum: identification of the corresponding gene and impact on L-glutamate production.

A screening procedure was established to identify Corynebacterium glutamicum transposon mutants with an altered L-glutamate excretion behaviour. By this microtiter plate-based approach seven non- or less excreting C. glutamicum strains and two hyper-excreters were found. The subsequently carried out molecular analysis of a hyper-producing clone led to the identification of the gltS gene, which codes for the sodium-coupled secondary L-glutamate uptake system in C. glutamicum. Characterization of a gltS deletion strain revealed that this transporter has a weak but significant impact on L-glutamate production induced by biotin limitation in the wild type. Obviously, GltS leads to the re-uptake of excreted L-glutamate causing a futile cycle. In accord with this hypothesis, the overexpression of gltS decreased L-glutamate production.

Influence of threonine exporters on threonine production in Escherichia coli.

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [(14)C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.

Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applications.

Transport processes play a pivotal role in cellular metabolism, e.g. for the uptake of nutrients or the excretion of metabolic waste products. Moreover, they are also important in biotechnological processes such as the production of various amino acids by the use of microorganisms. The focus of this review is on bacterial amino acid transport systems, in particular those of Corynebacterium glutamicum and Escherichia coli, with respect to their function and biotechnological significance.

Glutamate synthase of Corynebacterium glutamicum is not essential for glutamate synthesis and is regulated by the nitrogen status.

The Corynebacterium glutamicum gltB and gltD genes, encoding the large (alpha) and small (beta) subunit of glutamate synthase (GOGAT), were investigated in this study. Using RT-PCR, a common transcript of gltB and gltD was shown. Reporter gene assays and Northern hybridization experiments revealed that transcription of this operon depends on nitrogen starvation. The expression of gltBD is under control of the global repressor protein AmtR as demonstrated by gel shift experiments and analysis of gltB transcription in an amtR deletion strain. In contrast to other bacteria, in C. glutamicum GOGAT plays no pivotal role; e.g. gltB and gltD inactivation did not result in growth defects when cells were grown in standard minimal medium and only a slight increase in the doubling time of the corresponding mutant strains was observed in the presence of limiting amounts of ammonia or urea. Additionally, mutant analyses revealed that GOGAT has no essential function in glutamate production by C. glutamicum.

Glutamine synthetases of Corynebacterium glutamicum: transcriptional control and regulation of activity.

Regulation of glnA expression and glutamine synthetase I activity was analyzed in Corynebacterium glutamicum. Transcription is regulated by the global repressor protein AmtR, essential for derepression of glnA transcription are GlnK and uridylyltransferase, key proteins of the C. glutamicum nitrogen regulatory system. Glutamine synthetase I activity is controlled by adenylylation/deadenylylation via adenylyltransferase. The gene encoding this bifunctional enzyme, glnE, was isolated and its function was characterized by deletion analysis. Upstream of glnE, a second gene encoding a GSI-type protein in C. glutamicum was isolated. This gene, designated glnA2, forms an operon with glnE, its transcription is not regulated and neither its deletion or overexpression showed any effect. Therefore, the physiological role of glnA2 remains unclear.

Detection of fluorescence dye-labeled proteins in 2-D gels using an Arthur 1442 Multiwavelength Fluoroimager.

Labeling of proteins with SYPRO Orange, SYPRO Red, and SYPRO Ruby after 2-D polyacrylamide gel electrophoresis (PAGE) using plastic-backed immobilized pH gradient (IPG) strips and precast SDS polyacrylamide gels was tested. Protein spots were detected using an Arthur 1442 Multiwavelength Fluoroimager. The labeling methods described allow detection of proteins both after isoelectric focusing (IEF) and PAGE with a sensitivity higher than or comparable to standard silver staining methods. In addition to the post-labeling methods mentioned above, pre-labeling with the cysteine-specific fluorophore monobromobimane before 2-D PAGE is a sensitive, fast, and cost-effective alternative to existing staining protocols.

Proteome analysis of Corynebacterium glutamicum.

By the use of different Corynebacterium glutamicum strains more than 1.4 million tons of amino acids, mainly L-glutamate and L-lysine, are produced per year. A project was started recently to elucidate the complete DNA sequence of this bacterium. In this communication we describe an approach to analyze the C. glutamicum proteome, based on this genetic information, by a combination of two-dimensional (2-D) gel electrophoresis and protein identification via microsequencing or mass spectrometry. We used these techniques to resolve proteins of C. glutamicum with the aim to establish 2-D protein maps as a tool for basic microbiology and for strain improvement. In order to analyze the C. glutamicum proteome, methods were established to fractionate the C. glutamicum proteins according to functional entities, i.e., cytoplasm, membranes, and cell wall. Protein spots of the cytoplasmic and membrane fraction were identified by N-terminal sequencing, immunodetection, matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-mass spectrometry (ESI-MS). Additionally, a protocol to analyze proteins secreted by C. glutamicum was established. Approximately 40 protein spots were observed on silver-stained 2-D gels, 12 of which were identified.

Corynebacterium glutamicum: a dissection of the PTS.

The high-GC Gram-positive actinomycete Corynebacterium glutamicum is commercially exploited as a producer of amino acids that are used as animal feed additives and flavor enhancers. Despite its beneficial role, carbon metabolism and its possible influence on amino acid metabolism is poorly understood. We have addressed this issue by analyzing the phosphotransferase system (PTS), which in many bacteria controls the flux of nutrients and therefore regulates carbon metabolism. The general PTS phosphotransferases enzyme I (EI) and HPr were characterized by demonstration of PEP-dependent phosphotransferase activity. An EI mutant exhibited a pleiotropic negative phenotype in carbon utilization. The role of the PTS as a major sugar uptake system was further demonstrated by the finding that glucose and fructose negative mutants were deficient in the respective enzyme II PTS permease activities. These carbon sources also caused repression of glutamate uptake, which suggests an involvement of the PTS in carbon regulation. The observation that no HPr kinase/phosphatase could be detected suggests that the mechanism of carbon regulation in C. glutamicum is different to the one found in low-GC Gram-positive bacteria.

Multiplicity of ammonium uptake systems in Corynebacterium glutamicum: role of Amt and AmtB.

In Corynebacterium glutamicum, a Gram-positive soil bacterium widely used in the industrial production of amino acids, two genes encoding (putative) ammonium uptake carriers have been described. The isolation of amt was the first report of the sequence of a gene encoding a bacterial ammonium uptake system combined with the characterization of the corresponding protein. Recently, a second amt gene, amtB, with so far unknown function, was isolated. The isolation of this gene and the suggestion of a new concept for ammonium acquisition prompted the reinvestigation of ammonium transport in C. glutamicum. In this study it is shown that Amt mediates uptake of (methyl)ammonium into the cell with high affinity and strictly depending on the membrane potential. As shown by the determination of K:(m) at different pH values, ammonium/methylammonium, but not ammonia/methylamine, are substrates of Amt. AmtB exclusively accepts ammonium as a transport substrate. In addition, hints of another, until now unknown, low-affinity, ammonium-specific uptake system were found.

The low-molecular-mass subunit of the cell wall channel of the Gram-positive Corynebacterium glutamicum. Immunological localization, cloning and sequencing of its gene porA.

The 5-kDa protein PorA of the Gram-positive bacterium Corynebacterium glutamicum is the subunit of the cell wall channel. Antibodies raised against PorA specifically detected the protein on the cell surface. PorA was sequenced using Edman degradation and a gas phase sequencer. The primary sequence was used to create degenerate oligonucleotide primers. The gene of the channel-forming protein and its flanking regions were obtained by PCR followed by inverse PCR. The gene porA comprises 138 bp and encodes a 45-amino-acid-long acidic polypeptide with an excess of four negatively charged amino acids in agreement with the high cation selectivity of the PorA cell wall channel. PorA does not contain an N-terminal extension. A ribosomal-binding site was recognized 6 bp before the start codon ATG of porA. It codes for the smallest subunit of a membrane channel known so far and for the first cell wall channel protein of a corynebacterium. Southern blots demonstrated that only the chromosomes of corynebacteria contain homologous sequences to porA; no hybridization could be detected with DNA from other mycolata.

Sensing nitrogen limitation in Corynebacterium glutamicum: the role of glnK and glnD.

A novel nitrogen control system regulating the transcription of genes expressed in response to nitrogen starvation in Corynebacterium glutamicum was identified by us recently. In this communication, we also show that the nitrogen regulation cascade in C. glutamicum functions by a new mechanism, although components highly similar to sensor and signal transmitter proteins of Escherichia coli are used, namely uridylyltransferase and a PII-type GlnK protein. The genes encoding these key components of the nitrogen regulation cascade, glnD and glnK, are organized in an operon together with amtB, which codes for an ammonium permease. Using a combination of site-directed mutagenesis, RNA hybridization experiments, reporter gene assays, transport measurements and non-denaturing gel electrophoresis followed by immunodetection, we showed that GlnK is essential for nitrogen control and that signal transduction is transmitted by uridylylation of this protein. As a consequence of the latter, a glnD deletion strain lacking uridylyltransferase is impaired in its response to nitrogen shortage. The glnD mutant revealed a decreased growth rate in the presence of limiting amounts of ammonium or urea; additionally, changes in its protein profile were observed, as shown by in vivo labelling and two-dimensional PAGE. In contrast to E. coli, expression of glnD is upregulated upon nitrogen limitation in C. glutamicum. This indicates that the glnD gene product is probably not the primary sensor of nitrogen status in C. glutamicum as shown for enterobacteria. In accordance with this hypothesis, we found a deregulated nitrogen control as a result of the overexpression of glnD. Furthermore, quantification of cytoplasmic amino acid pools excluded the possibility that a fall in glutamine concentration is perceived as the signal for nitrogen starvation by C. glutamicum, as is found in enterobacteria. Direct measurements of the intracellular ammonium pool indicated that the concentration of this compound might indicate the cellular nitrogen status. Deduced from glnK and glnD expression patterns and the genetic organization of these genes, this regulatory mechanism is also present in Corynebacterium diphtheriae, the causative agent of diphtheria.

Polyamine transport and role of potE in response to osmotic stress in Escherichia coli.

When transport of polyamines in Escherichia coli was examined, putrescine excretion was observed under two different physiological conditions: (i) strictly correlated to growth and (ii) following a hyperosmotic shock. Spermidine was not excreted. Characterization of a deletion mutant showed that PotE is not involved in these transport processes.

AmtR, a global repressor in the nitrogen regulation system of Corynebacterium glutamicum.

The uptake and assimilation of nitrogen sources is effectively regulated in bacteria. In the Gram-negative enterobacterium Escherichia coli, the NtrB/C two-component system is responsible for the activation of transcription of different enzymes and transporters, depending on the nitrogen status of the cell. In this study, we investigated regulation of ammonium uptake in Corynebacterium glutamicum, a Gram-positive soil bacterium closely related to Mycobacterium tuberculosis. As shown by Northern blot hybridizations, regulation occurs on the level of transcription upon nitrogen starvation. In contrast to enterobacteria, a repressor protein is involved in regulation, as revealed by measurements of methylammonium uptake and beta-galactosidase activity in reporter strains. The repressor-encoding gene, designated amtR, was isolated and sequenced. Deletion of amtR led to deregulation of transcription of amt coding for the C. glutamicum (methyl)ammonium uptake system. E. coli extracts from amtR-expressing cells were applied in gel retardation experiments, and binding of AmtR to the amt upstream region was observed. By deletion analyses, a target motif for AmtR binding was identified, and binding of purified AmtR protein to this motif, ATCTATAGN1-4ATAG, was shown. Furthermore, the binding of AmtR to this sequence was proven in vivo using a yeast one-hybrid system. Subsequent studies showed that AmtR not only regulates transcription of the amt gene but also of the amtB-glnK-glnD operon encoding an amt paralogue, the signal transduction protein PII and the uridylyltransferase/uridylyl-removing enzyme, key components of the nitrogen regulatory cascade. In summary, regulation of ammonium uptake and assimilation in the high G+C content Gram-positive bacterium C. glutamicum differs significantly from the mechanism found in the low G+C content Gram-positive model organism Bacillus subtilis and from the paradigm of nitrogen control in the Gram-negative enterobacteria.

Response to nitrogen starvation in Corynebacterium glutamicum.

Proteins strongly synthesized in Corynebacterium glutamicum during nitrogen restriction were examined by two-dimensional gel electrophoresis and microsequencing. Two main groups of enzymes were identified beside miscellaneous proteins, enzymes involved (i) in protein synthesis, and (ii) in carbon metabolism. Biochemical measurements revealed an increase of oxygen consumption during nitrogen starvation, indicating an enhanced energy demand of the cells. By Northern hybridizations, an increased transcription for the gap and fda genes upon nitrogen deprivation was shown.

Two-dimensional electrophoretic analysis of Corynebacterium glutamicum membrane fraction and surface proteins.

An improved protocol for the two-dimensional analysis of proteins of the Corynebacterium glutamicum cytoplasmic membrane fraction is described. By use of increased 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) concentrations (2-4%) and an optimized electrophoresis protocol, horizontal streaking of proteins of the cytoplasmic membrane fraction was almost completely avoided. More important, in contrast to a previously published method, both a sample tray and IPG-phor isoelectric focusing unit can be used for the in-gel application of proteins. The described protocol was also found to be suitable for hydrophilic cytoplasmic proteins. Additionally, the preparation and analysis of C. glutamicum cell surface proteins is described. Proteins were extracted with lauroyl sarcosinate and 100-120 spots were separated on two-dimensional (2-D) gels in comparison to 18-20 spots observed previously by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). C. glutamicum proteins can now be separated into three distinct fractions resembling different functional units of the bacterial cell.

Nitrogen regulation in Corynebacterium glutamicum: isolation of genes involved and biochemical characterization of corresponding proteins.

The regulation of nitrogen assimilation was investigated in the Gram-positive actinomycete Corynebacterium glutamicum. Biochemical studies and site-directed mutagenesis revealed that glutamine synthetase activity is regulated via adenylylation in this organism. The genes encoding the central signal transduction protein PH (glnB) and the primary nitrogen sensor uridylyltransferase (glnD) were isolated and sequenced. Additionally, genes putatively involved in the degradation of ornithine (ocd) and sarcosine (soxA), ammonium uptake (amtP) and protein secretion (ftsY, srp) were identified in C. glutamicum. Based on these observations, the mechanism of N regulation in C. glutamicum is similar to that of the Gram-negative Escherichia coli. As deduced from data base searches, the described regulation may also hold true for the important pathogen Mycobacterium glutamicum.

Corynebacterium glutamicum is equipped with four secondary carriers for compatible solutes: identification, sequencing, and characterization of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine betaine carrier, EctP.

Gram-positive soil bacterium Corynebacterium glutamicum uses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in C. glutamicum, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A DeltabetP DeltaputP DeltaproP DeltaectP mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (Km, 48 microM) to ectoine (Km, 132 microM). The proP gene could not be expressed from its own promoter in C. glutamicum; however, expression was observed in Escherichia coli. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed ectP codes for a 615-residue transporter. EctP preferred ectoine (Km, 63 microM) to betaine (Km, 333 microM) and proline (Km, 1,200 microM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP.

Biochemical and biophysical characterization of the cell wall porin of Corynebacterium glutamicum: the channel is formed by a low molecular mass polypeptide.

The cell wall of the Gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes. The channel-forming protein was identified, by lipid bilayer experiments, in the cell envelope fractions isolated by sucrose-density centrifugations and in organic solvent of whole cells. It was purified to homogeneity by fast-protein liquid chromatography across a Mono-Q column. The pure protein had a rather low molecular mass of about 5 kDa as judged by SDS-PAGE, which suggested that the cell wall channel is formed by a protein oligomer. The monomer has according to partial sequencing no significant homology to known protein sequences. The purified protein formed large ion-permeable channels in lipid bilayer membranes from phosphatidylcholine/phosphatidylserine mixtures with a single-channel conductance of 5.5 nS in 1 M KCl. Experiments with different salts suggested that the cell wall channel of C. glutamicum was highly cation-selective caused by negative charges localized at the channel mouth. The analysis of the single-channel conductance data using the Renkin correction factor suggested that the diameter of the cell wall channel is about 2.2 nm. Channel-forming properties of the cell wall channel of C. glutamicum were compared with those of mycobacteria. These channels share common features because they form large and water-filled channels that contain point net charges.