RESUMO
A protein with an apparent molecular mass of 46 kDa was detected as the major polypeptide in the culture medium of the biotechnologically important methanotrophic bacterium Methylococcus capsulatus (Bath). The protein cross-reacted with polyclonal antibodies raised against the outer-membrane-associated protein MopE. The antiserum was used to identify a positive clone from a lambda gt11 library. The nucleotide sequence determined for the clone demonstrated that MopE and the secreted protein are encoded by the same gene, and that the secreted protein represents an N-terminally truncated form of MopE. By using monospecific antibodies against MopE in immunogold electron microscopy, the protein was localized at the cell surface and cell periphery. The mopE gene was expressed in Escherichia coli. The MopE protein synthesized was found in the periplasmic space of E. coli. No protein with sequence similarity over the entire length of MopE was detected in the databases, but some sequence similarity to the copper-repressible CorA protein of the methanotroph Methylomicrobium albus (Berson and Lidstrom 1997) was observed for the C-terminal region of MopE.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Methylococcus capsulatus/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Parede Celular/química , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos , Methylococcus capsulatus/genética , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Alinhamento de SequênciaRESUMO
The 40 kDa-outer membrane protein FomA of Fusobacterium periodonticum ATCC 33693 was found to exhibit heat modifiable properties, typical for a porin, and N-terminal sequencing indicated a close relationship to the porin FomA of Fusobacterium nucleatum. A polymerase chain reaction approach was therefore applied for sequencing the fomA gene of F. periodonticum, and nucleotide and deduced amino acid sequences were aligned and compared with the corresponding sequences of different strains of F. nucleatum. In all strains we found a common protein upstream of the fomA gene. The noncoding area upstream of the putative -35 region of the F. periodonticum fomA gene exhibited little sequence similarity with the F. nucleatum gene. The transcriptional unit of FomA, on the other hand, was very similar, with the similarities concentrated in domains that were interspersed with hypervariable regions. A topology model was made and compared with those made for F. nucleatum. This indicated that the great similarities reside in the membrane-spanning segments of the protein, while most cell surface exposed loops were hypervariable. The results strongly support the proposed model for FomA and also indicate that these taxa are related but on a lower level than the subspecies level. The codon usage of F. periodonticum is comparable to that of F. nucleatum, and the triplet AGA is the only codon used for arginine.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Fusobacterium/química , Fusobacterium/genética , Genes Bacterianos/genética , Sequência de Aminoácidos , Sequência de Bases , Códon , Códon de Terminação , Fusobacterium nucleatum/química , Fusobacterium nucleatum/genética , Genes Reguladores/genética , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Porinas/química , Porinas/genética , Conformação Proteica , Sinais Direcionadores de Proteínas , Estrutura Secundária de Proteína , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F1 F3, F6, and Fev1 were grown in different media. The influence of growth conditions on the outer membrane proteins (OMPs) was analyzed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). There were no apparent differences in the outer membrane protein profiles when cells in the same phase of growth in various rich media were compared. Differences, however, were observed between early logarithmic phase and stationary phase cells. Thus several proteins were only synthesized during late logarithmic and stationary phase. Synthesis of some of these proteins, in particular a 65K and a 14K protein, seemed to depend on the presence of peptides in the medium. In a complete medium, these proteins were synthesized after depletion of some amino acids, and peptides were then utilized.
Assuntos
Proteínas da Membrana Bacteriana Externa/análise , Fusobacterium/crescimento & desenvolvimento , Autorradiografia , Proteínas da Membrana Bacteriana Externa/biossíntese , Meios de Cultura , Eletroforese em Gel de PoliacrilamidaRESUMO
Fusobacterium nucleatum strains ATCC 10953, Fevl, F1, F3, and F6 utilized amino acids, in particular glutamate, histidine, and aspartate were common to all strains. Strain differences were observed in the utilization of threonine, serine, lysine, tyrosine, and methionine, and only strain ATCC 10953 utilized all these amino acids. The glutamate and histidine pools were in all cases fully depleted before the other amino acids were attacked and at the same time all strains except 10953 started to utilize peptides at a noticeable rate. For strain Fevl, glutamyl- and aspartyl-containing peptides seemed to be of considerable nutritional importance, and this strain did not grow on a medium based on amino acids alone. On the other hand, strain 10953 did not utilize any peptides to a noticeable extent, and it could grow on an amino acid based medium.
