RESUMO
The genus Corynebacterium includes species of great importance in medical, veterinary and biotechnological fields. The genus-specific families (PLfams) from PATRIC have been used to observe conserved proteins associated to all species. Our results showed a large number of conserved proteins that are associated with the cellular division process. Was not observe in our results other proteins like FtsA and ZapA that interact with FtsZ. Our findings point that SepF overlaps the function of this proteins explored by molecular docking, protein-protein interaction and sequence analysis. Transcriptomic analysis showed that these two (Sepf and FtsZ) proteins can be expressed in different conditions together. The work presents novelties on molecules participating in the cell division event, from the interaction of FtsZ and SepF, as new therapeutic targets.
Assuntos
Proteínas de Bactérias/fisiologia , Divisão Celular/fisiologia , Corynebacterium/citologia , Citocinese/fisiologia , Proteínas do Citoesqueleto/fisiologia , Proteínas de Bactérias/genética , Corynebacterium/fisiologia , Proteínas do Citoesqueleto/genética , Perfilação da Expressão Gênica , Simulação de Acoplamento Molecular , Mapeamento de Interação de ProteínasRESUMO
A Gram-stain-positive, facultatively anaerobic, short rod-shaped, oxidase-negative and non-motile novel strain, designated YIM 101505T, was isolated from the faeces of a primate, Assamese macaque, and was studied to determine its taxonomic position. The cell wall contained meso-diaminopimelic acid and short-chain mycolic acids. Whole cell sugars were mannose, galactose and arabinose as major components. The major fatty acids (>10 %) were C18 : 1ω9c, C16 : 0 and C17 : 1ω8c and the major menaquinone was MK-9(H2). The polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, glycolipid and six unidentified lipids. The new isolate shared most of the typical chemotaxonomic characteristics of members of the genus Corynebacterium. The closest related species was Corynebacterium efficiens based on 16S rRNA gene (98.1 % similarity) and partial rpoB gene (91.4 % similarity) sequences. Similarities with other species of this genus were below 97 % based on the 16S rRNA gene. The DNA-DNA hybridization value between YIM 101505T and C. efficiens DSM 44549T was 47.7±3.6 %. Moreover, the physiological and biochemical characteristics of YIM 101505T and C. efficiens DSM 44549T were different. Thus, strain YIM 101505T is considered to represent a novel member of the genus Corynebacterium, for which the name Corynebacterium faecale sp. nov. is proposed. The type strain is YIM 101505T (=DSM 45971T=CCTCC AB 2013226T).
Assuntos
Corynebacterium/classificação , Corynebacterium/isolamento & purificação , Fezes/microbiologia , Macaca/microbiologia , Animais , Técnicas de Tipagem Bacteriana , China , Corynebacterium/química , Corynebacterium/citologia , DNA Bacteriano/genética , Fosfolipídeos/análise , Filogenia , Análise de Sequência de DNARESUMO
Biochemical characterization of Corynebacterium ammoniagenes FADS (CaFADS) pointed to certain confusion about the stoichiometry of this bifunctional enzyme involved in the production of FMN and FAD in prokaryotes. Resolution of its crystal structure suggested that it might produce a hexameric ensemble formed by a dimer of trimers. We used atomic force microscopy (AFM) to direct imaging single CaFADS molecules bound to mica surfaces, while preserving their catalytic properties. AFM allowed solving individual CaFADS monomers, for which it was even possible to distinguish their sub-molecular individual N- and C-terminal modules in the elongated enzyme. Differences between monomers and higher stoichiometries were easily imaged, enabling us to detect formation of oligomeric species induced by ligand binding. The presence of ATP:Mg(2+) particularly induced the appearance of the hexameric assembly whose mean molecular volume resembles the crystallographic dimer of trimers. Finally, the AFM results are confirmed in cross-linking solution, and the presence of such oligomeric CaFADS species detected in cell extracts. All these results are consistent with the formation of a dimer of trimers during the enzyme catalytic cycle that might bear biological relevance.
Assuntos
Proteínas de Bactérias/química , Corynebacterium/enzimologia , Nucleotidiltransferases/química , Estrutura Quaternária de Proteína , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Biocatálise , Corynebacterium/citologia , Corynebacterium/metabolismo , Eletroforese em Gel de Poliacrilamida , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Ligantes , Microscopia de Força Atômica , Modelos Moleculares , Nucleotidiltransferases/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
Corynebacterium renale harbors four small cryptic plasmids, pCR1, pCR2, pCR3 and pCR4, and can be a good system for understanding host-plasmid interactions. In the present study, effect of plasmid loss and their subsequent introduction on various properties of the host was evaluated. Loss of plasmids caused a reduction in bacterial size and also slowed down their growth rate, µ, and respiratory rate, r. Both µ and r values were partially recovered in C. renale R, obtained by retransformation of the cured strain with all the four cryptic plasmids. Further delineation revealed that a 3153bp plasmid pCR2 alone is sufficient for the observed increase in µ in C. renale R. The advantages conferred by the remaining three plasmids may be are two subtle to be seen under laboratory conditions. Overall, the observations point to the gross metabolic crisis in the host partly as a result of loss of plasmids. Based on the findings, a mutualistic relationship between the host and the plasmids resulting from their coevolution is proposed.
Assuntos
Corynebacterium/genética , Corynebacterium/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Corynebacterium/citologia , Corynebacterium/ultraestrutura , Glucose/metabolismo , Mapeamento por RestriçãoRESUMO
RamA plays a regulatory role for acetate utilization and S-layer biosynthesis in Corynebacterium glutamicum. Looking for any additional role, the function of RamA was analyzed in Corynebacterium ammoniagenes, which is closely related to C. glutamicum. In this study, we showed that the DeltaramA mutant constructed by a markerless knockout strategy possessed increased cell surface hydrophobicity, leading to the formation of aggregated cell masses in liquid media. In addition, the mutant exhibited an elongated cell shape as observed by SEM, suggesting that cell wall-associated proteins might be influenced. Furthermore, cell surface proteome analysis revealed that the expression of cmytA gene encoding corynomycoloyl transferase required for cell wall biosynthesis was down-regulated in the mutant, supporting the regulatory role of RamA in cell wall assembly. These studies support a novel regulatory role of RamA in inducing the expression of proteins required for cell wall assembly.
Assuntos
Proteínas de Bactérias/fisiologia , Corynebacterium/citologia , Corynebacterium/fisiologia , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/fisiologia , Transferases/biossíntese , Aderência Bacteriana , Proteínas de Bactérias/genética , Sequência de Bases , Parede Celular/química , Corynebacterium/química , DNA Bacteriano/química , DNA Bacteriano/genética , Deleção de Genes , Técnicas de Inativação de Genes , Ordem dos Genes , Genes Bacterianos , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteoma/análise , Análise de Sequência de DNA , Fatores de Transcrição/genéticaRESUMO
Clinical and environmental-associated strains (n=17), genotypically related to Corynebacterium spp., yet distinct from any species of the genus Corynebacterium with validly published names, have been isolated during the last 20 years and tentatively identified as Corynebacterium sanguinis, although the combination, "Corynebacterium sanguinis" was never validly published. The comprehensive genotypic and phenotypic characterisations and genomic analyses in this study support the proposal for recognizing the species within the genus Corynebacterium, for which the name, Corynebacterium sanguinis sp. nov., is reaffirmed and proposed. Strains of Corynebacterium sanguinis are Gram-positive, non-motile, non-spore-forming, short, pleomorphic and coryneform bacilli, growing aerobically, with CO2. They contain mycolic acids, major respiratory menaquinones, MK-8 (II-H2) and MK-9 (II-H2), and polar lipids, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphoglycolipid, glycolipids and a novel lipid that remains to be characterized and identified. Strains of Corynebacterium sanguinis are genotypically most similar to Corynebacterium lipophiliflavum, with 16S rRNA gene sequence similarities of 98.3% and rpoB sequence similarities of 94.9-95.2%. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis were able to clearly differentiate Corynebacterium sanguinis from the most closely related species. The genome size of Corynebacterium sanguinis is 2.28-2.37Mbp with 65.1-65.5mol% G+C content. A total of 2202-2318 ORFs were predicted, comprising 2141-2251 protein-encoding genes. The type strain is CCUG 58655T (=CCM 8873T=NCTC 14287T).
Assuntos
Infecções por Corynebacterium/microbiologia , Corynebacterium/classificação , Microbiologia Ambiental , Proteínas de Bactérias/genética , Composição de Bases , Corynebacterium/química , Corynebacterium/citologia , Corynebacterium/fisiologia , DNA Bacteriano/genética , Ácidos Graxos/química , Tamanho do Genoma , Genoma Bacteriano/genética , Glicolipídeos/química , Humanos , Hibridização de Ácido Nucleico , Fosfolipídeos/química , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Especificidade da Espécie , Vitamina K 2/químicaRESUMO
In this study, we report the first synthesis of an alkyne-based trehalose monomycolate probe containing a ß-hydroxylated fatty acid and an α-branched chain similar to those of the natural mycolic acid. We demonstrate its utility for the labeling of the mycomembrane of Corynebacteria as well as for the study of mycoloyltransferases.
Assuntos
Aciltransferases/análise , Membrana Celular/química , Corynebacterium/enzimologia , Corantes Fluorescentes/química , Ácidos Micólicos/química , Aciltransferases/metabolismo , Membrana Celular/metabolismo , Corynebacterium/citologia , Corantes Fluorescentes/síntese química , Estrutura Molecular , Ácidos Micólicos/síntese químicaRESUMO
Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for M. tuberculosis was determined to be 5.1x104 cfu per ml and for M. avium 1.5x104 cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries.
Assuntos
Hibridização in Situ Fluorescente , Complexo Mycobacterium avium/isolamento & purificação , Mycobacterium tuberculosis/isolamento & purificação , Bacillus subtilis/citologia , Bacillus subtilis/genética , Bacillus subtilis/isolamento & purificação , Bacillus subtilis/metabolismo , Corynebacterium/citologia , Corynebacterium/genética , Corynebacterium/isolamento & purificação , Corynebacterium/metabolismo , Corantes Fluorescentes , Genes de RNAr , Microscopia de Fluorescência , Complexo Mycobacterium avium/classificação , Complexo Mycobacterium avium/citologia , Complexo Mycobacterium avium/genética , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/genética , Nocardia/citologia , Nocardia/genética , Nocardia/isolamento & purificação , Nocardia/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Sensibilidade e EspecificidadeRESUMO
Forty-eight clinical strains that were tentatively identified as Corynebacterium minutissimum on the basis of standard biochemical reactions (Hollis-Weaver tables) as well as by the use of the API (RAPID) Coryne system were examined further. Two different groups of strains were observed. The first group (including the type strain of C minutissimum) contained 27 strains showing creamy colonies. These strains grew homogeneously in 6.5% NaCl broth, exhibited DNase activity, were susceptible to the vibriocidal compound O/129, produced succinic acid, and contained mycolic acids. The second group comprised 21 strains with dry colonies. They grew in clumps at the surface of 6.5% NaCl broth, DNase activity was not detected, they were resistant against O/129, produced large amounts of propionic acid, and mycolic acids were not detected. In combination with quantitative DNA-DNA hybridizations, it was demonstrated that strains of the second cluster belonged, in fact, to C amycolatum. Furthermore, it was observed that a few C minutissimum strains may also ferment mannitol. These data indicate that the clinical microbiologist must be careful not to misidentify C amycolatum strains as C minutissimum.
Assuntos
Infecções Bacterianas/microbiologia , Corynebacterium/isolamento & purificação , Corynebacterium/classificação , Corynebacterium/citologia , Corynebacterium/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Especificidade da EspécieRESUMO
L-forms from a Corynebacterium were induced in hyperosmolar media and gradually adapted to normal osmolarity over a period of two years. During this adaptation several morphologic L-form variants derived from the L-form cultures and were serologically identified as Acholeplasma laidlawii A. The possibility that the bacterial and L-form cultures were contaminated with acholeplasmas was carefully investigated; this was determined not to be the case. Membrane protein gel electrophoresis patterns of these L-form variants were identical to the ATCC A. laidlawii strain PG-8. Acholeplasma phage MVL-1 displayed no affinity for these L-form variants. Phage MVL-2 showed low affinity, but after virus enhancement in the specific host, high plaquing efficiency ensued. DNA hybridization experiments showed a high level of nucleotide sequence homology (greater than 90%) between the L-form-derived variants and PG-8. The homology between the diphtheroid L-forms and the PG-8 strain was 16.4% with te50 values of 86%; this indicates strong base pairing homology. These findings suggest that the L-form variants are acholeplasmas and that they are biologically and genetically related to the Corynebacterium L-forms.
Assuntos
Acholeplasma laidlawii/fisiologia , Corynebacterium/citologia , Formas L/fisiologia , Acholeplasma laidlawii/classificação , Acholeplasma laidlawii/citologia , Acholeplasma laidlawii/genética , Proteínas de Bactérias/análise , Bacteriófagos , Composição de Bases , Sequência de Bases , Corynebacterium/crescimento & desenvolvimento , DNA Bacteriano/análise , Humanos , Formas L/classificação , Formas L/genética , Proteínas de Membrana/análise , Hibridização de Ácido Nucleico , Concentração Osmolar , Testes Sorológicos , Cloreto de Sódio/farmacologiaRESUMO
Mycolic acids, the major lipid constituents of Corynebacterineae, play an essential role in maintaining the integrity of the bacterial cell envelope. We have previously characterized a corynebacterial mycoloyltransferase (PS1) homologous in its N-terminal part to the three known mycobacterial mycoloyltransferases, the so-called fibronectin-binding proteins A, B and C. The genomes of Corynebacterium glutamicum (ATCC13032 and CGL2005) and Corynebacterium diphtheriae were explored for the occurrence of other putative corynebacterial mycoloyltransferase-encoding genes (cmyt). In addition to csp1 (renamed cmytA), five new cmyt genes (cmytB-F) were identified in the two strains of C. glutamicum and three cmyt genes in C. diphtheriae. In silico analysis showed that each of the putative cMyts contains the esterase domain, including the three key amino acids necessary for the catalysis. In C. glutamicum CGL2005 cmytE is a pseudogene. The four new cmyt genes were disrupted in this strain and overexpressed in the inactivated strains. Quantitative analyses of the mycolate content of all these mutants demonstrated that each of the new cMyt-defective strains, except cMytC, accumulated trehalose monocorynomycolate and exhibited a lower content of covalently bound corynomycolate than did the parent strain. For each mutant, the mycolate content was fully restored by complementation with the corresponding wild-type gene. Finally, complementation of the cmytA-inactivated mutant by the individual new cmyt genes established the existence of two classes of mycoloyltransferases in corynebacteria.
Assuntos
Aciltransferases/genética , Aciltransferases/metabolismo , Adesinas Bacterianas , Parede Celular/enzimologia , Corynebacterium/enzimologia , Ácidos Micólicos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Corynebacterium/citologia , Corynebacterium/genética , Corynebacterium diphtheriae/enzimologia , Corynebacterium diphtheriae/genética , Genoma Bacteriano , Metabolismo dos Lipídeos , Dados de Sequência Molecular , Mutagênese , FenótipoRESUMO
PS1 is a major exported protein of Corynebacterium glutamicum homologous to mycobacterial antigen 85. It is largely associated with the mycolic acid-containing cell wall and acts as a mycoloyl transferase. The transport of PS1 to the cell wall is slow and occurs through two energetically distinct steps: the first one, which includes processing by signal peptidase, is rapid and inhibited by sodium azide or carbonyl cyanide m-chlorophenylhydrazone. This step is probably associated with translocation across the cytoplasmic membrane. The kinetics of the second step depend on the size of the polypeptide chain to be transported but neither ATP nor proton motive force is required. This step may correspond to the diffusion of PS1 across the cell wall to its final location.
Assuntos
Aciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Corynebacterium/citologia , Corynebacterium/metabolismo , Proteínas de Membrana , Trifosfato de Adenosina/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/enzimologia , Corynebacterium/enzimologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Cinética , Potenciais da Membrana/efeitos dos fármacos , Ácidos Micólicos/metabolismo , Transporte Proteico/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Azida Sódica/farmacologiaRESUMO
The response of the L-lysine producing Corynebacterium glutamicum strain MH20-22B to osmotic stress was studied in batch cultures. To mimic the conditions during a fermentation process the long term adaptation of cells subjected to a constant osmotic stress between 1.0 and 2.5 osM was investigated. Cytoplasmic water content and volume of C. glutamicum cells were found to depend on growth phase, extent of osmotic stress and availability of betaine. The maximal cytoplasmic volumes, which were highest at maximal growth rate, were linearily related to osmotic stress, whereas in stationary cells no active volume regulation was observed. Under severe osmotic stress proline was the prominent compatible solute in growing cells. Uptake of betaine, if available in the medium, reduced the concentration of proline from 750 to 300 mM, indicating that uptake of compatible solutes is preferred to synthesis. Furthermore, betaine was shown to have a higher efficiency to counteract osmotic stress, since the overall concentration of compatible solutes was lower in the presence of betaine. Under severe osmotic stress, the addition of betaine shifted L-lysine production in MH20-22B to earlier fermentation times and increased both product concentration and yield in these phases, but did not improve the final L-lysine yield.
Assuntos
Betaína/metabolismo , Técnicas de Cultura de Células/métodos , Corynebacterium/crescimento & desenvolvimento , Corynebacterium/metabolismo , Citoplasma/metabolismo , Lisina/biossíntese , Equilíbrio Hidroeletrolítico/fisiologia , Água/metabolismo , Adaptação Fisiológica/fisiologia , Corynebacterium/citologia , Meios de Cultura/metabolismo , SoluçõesRESUMO
The effect of acetate as a glucose co-substrate on growth, lysine synthesis and experimental lysine yield from carbon substrates by Corynebacterium glutamicum RC 115 was investigated. It was found that low amounts of acetate, injected with a glucose-acetate pulse into the steady-state continuous culture in bioreactor, caused a slight decrease in the specific rates of glucose uptake and bacterial growth, but a significant increase in the cell specific rate of lysine synthesis and an increase in lysine yield. In contrast, acetate injected in high amounts was followed by a drastic decrease in the values of these parameters. A strong increase in experimental lysine yield under the latter conditions was reached in the response to pyruvate addition. Therefore it was shown that acetate in low concentrations can be used as a glucose co-substrate to increase the cell specific rate of lysine synthesis and lysine yield by C. glutamicum RC 115. Pyruvate supplementation was found as a promising method to enhance lysine synthesis by bacterial cells grown in glucose-acetate media with an increased concentration of acetate.
Assuntos
Acetatos/metabolismo , Reatores Biológicos/microbiologia , Técnicas de Cultura de Células/métodos , Corynebacterium/crescimento & desenvolvimento , Corynebacterium/metabolismo , Glucose/metabolismo , Homeostase/fisiologia , Lisina/biossíntese , Adaptação Fisiológica/fisiologia , Divisão Celular/fisiologia , Corynebacterium/citologia , Retroalimentação/fisiologia , Análise de Injeção de FluxoRESUMO
A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.
Assuntos
Corynebacterium/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Melhoramento Genético/métodos , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Lisina/biossíntese , Fenótipo , Engenharia de Proteínas/métodos , Divisão Celular/fisiologia , Corynebacterium/citologia , Lisina/genética , Mutagênese Sítio-Dirigida , Mutação , NADP/metabolismo , Consumo de Oxigênio , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
A DNA microarray was developed to analyse global gene expression of the amino acid-producing bacterium Corynebacterium glutamicum. PCR products representing 93.4% of the predicted C. glutamicum genes were prepared and spotted in quadruplicate onto 3-aminopropyltrimethoxysilane-coated glass slides. The applicability of the C. glutamicum DNA microarray was demonstrated by co-hybridisation with fluorescently labelled cDNA probes. Analysis of the technical variance revealed that C. glutamicum genes detected with different intensities resulting in ratios greater than 1.52 or smaller than -1.52 can be regarded as differentially expressed with a confidence level of greater than 95%. In a validation example, we measured changes of the mRNA levels during growth of C. glutamicum with acetate and propionate as carbon sources. Acetate-grown C. glutamicum cultures were used as reference. At the 95% confidence interval, 117 genes revealed increased transcript levels in the presence of propionate, while 43 genes showed a decreased expression compared with the acetate-grown culture. Global expression profiling confirmed the induction of the prpD2B2C2 gene cluster already known to be essential for propionate degradation via the 2-methylcitrate cycle. Besides many genes of unknown function, the paralogous prpD1B1C1 gene cluster as well as fasI-B (encoding fatty-acid synthase IB), dtsR1 and dtsR2 (components of acyl-CoA carboxylases), gluABCD (glutamate transport system), putP (proline transport system), and pyc (pyruvate carboxylase) showed significantly increased expression levels. Differential expression of these genes was confirmed by real-time reverse transcription (RT) PCR assays.
Assuntos
Mapeamento Cromossômico/instrumentação , Corynebacterium/fisiologia , Perfilação da Expressão Gênica/instrumentação , Regulação Bacteriana da Expressão Gênica/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Propionatos/metabolismo , Carbono/metabolismo , Divisão Celular/fisiologia , Mapeamento Cromossômico/métodos , Corynebacterium/citologia , Desenho de Equipamento , Análise de Falha de Equipamento , Perfilação da Expressão Gênica/métodos , Genoma Bacteriano , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Especificidade da EspécieRESUMO
We report here on the utility of on-line culture redox potential and dissolved oxygen measurements to identify metabolic changes in fermentation by Corynebacterium glutamicum under aerobic conditions. Metabolic changes were identified by observing discrepancies in the profile of culture redox potential and dissolved oxygen. On the basis of these measurements, we can identify the end of the lag phase, threonine exhaustion, and glucose exhaustion during fermentation.
Assuntos
Corynebacterium/metabolismo , Aerobiose , Biotecnologia , Divisão Celular , Corynebacterium/citologia , Fermentação , Glucose/metabolismo , Sistemas On-Line , Oxirredução , Consumo de Oxigênio , Treonina/metabolismoRESUMO
Since conventional cancer treatments cannot guarantee the destruction of all the tumour cells present within the patient, a relatively new form of therapy has been introduced whereby bacteria are used to stimulate immune responses, which if properly generated have the potential to eliminate the few remaining tumour cells that escaped the conventional treatments. This review deals with the results of clinical trials of immunotherapy and demonstrates that although it could form the basis of an effective therapy, it still requires more experiments to increase its efficiency and to decrease its side effects, the most serious of which is to accelerate tumour growth in some instances. The review concentrates on the two main agents currently in use, Bacillus Calmette-Guérin and Corynebacterium parvum.
Assuntos
Bacillus/imunologia , Corynebacterium/imunologia , Imunoterapia , Neoplasias/terapia , Vacina BCG/uso terapêutico , Bacillus/citologia , Bactérias/imunologia , Infecções Bacterianas/etiologia , Infecções Bacterianas/imunologia , Fenômenos Biomecânicos , Corynebacterium/citologia , Previsões , Humanos , Modelos Biológicos , Neoplasias/tratamento farmacológico , Neoplasias/mortalidade , Neoplasias/cirurgia , Complicações Pós-OperatóriasRESUMO
Corynebacterium crenatum SYPA 5-5 is an aerobic and industrial L: -arginine producer. It was proved that the Corynebacterium glutamicum/Escherichia coli shuttle vector pJC1 could be extended in C. crenatum efficiently when using the chloramphenicol acetyltransferase gene (cat) as a reporter under the control of promoter tac. The expression system was applied to over-express the gene vgb coding Vitreoscilla hemoglobin (VHb) to further increase the dissolved oxygen in C. crenatum. As a result, the recombinant C. crenatum containing the pJC-tac-vgb plasmid expressed VHb at a level of 3.4 nmol g(-1), and the oxygen uptake rates reached 0.25 mg A(562)(-1) h(-1) which enhanced 38.8% compared to the wild-type strain. Thus, the final L: -arginine concentration of the batch fermentation reached a high level of 35.9 g L(-1), and the biomass was largely increased to 6.45 g L(-1), which were 17.3% and 10.5% higher than those obtained by the wild-type strain, respectively. To our knowledge, this is the first report that the efficient expression system was constructed to introduce vgb gene increasing the oxygen and energy supply for L: -arginine production in C. crenatum, which supplies a good strategy for the improvement of amino acid products.
Assuntos
Arginina/biossíntese , Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Corynebacterium/metabolismo , Expressão Gênica , Hemoglobinas Truncadas/metabolismo , Cloranfenicol O-Acetiltransferase/metabolismo , Corynebacterium/citologia , Corynebacterium/crescimento & desenvolvimento , Oxigênio/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Recombinação Genética/genética , Análise Espectral , Transformação GenéticaRESUMO
This study was conducted to investigate the effects of fertilizers and biosurfactants on biodegradation of crude oil by three marine bacterial isolates; Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa. Five sets of experiments were carried out in shake flask and microcosm conditions with crude oil as follows: Set 1-only bacterial cells added (no fertilizer and biosurfactant), Set 2-with additional fertilizer only, Set 3-with additional biosurfactant only, Set 4-with added biosurfactant+fertilizer, Set 5-with no bacterial cells added (control), all the above experimental sets were incubated for 168 h. The biosurfactant+fertilizer added Set 4, resulted in maximum crude oil degradation within shake flask and microcosm conditions. Among the three bacterial isolates, P. aeruginosa and biosurfactant produced by this strain resulted in maximum crude oil degradation compared to the other two bacterial strains investigated. Interestingly, when biosurfactant and bacterial cells were used (Set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in Set 4 with added fertilizer+biosurfactant were only 4-5% higher degradation level in shake flask and 3.2-7% in microcosm experiments for all three bacterial strains used. It is concluded that, biosurfactants alone capable of promoting biodegradation to a large extent without added fertilizers, which will reduce the cost of bioremediation process and minimizes the dilution or wash away problems encountered when water soluble fertilizers used during bioremediation of aquatic environments.