The cryptic multicopy plasmid pGA1 (4,826 bp) from Corynebacterium glutamicum LP-6 belongs to the fifth group of rolling-circle-replicating plasmids. A determinant, which negatively controls pGA1 replication, was localized in the leader region of the rep gene coding for the initiator of plasmid replication. This region, when cloned into the compatible vector pEC6, was found to cause decrease of segregational stability of the pGA1 derivative pKG48. A promoter and a single transcriptional start site were found in the rep leader region in orientation opposite to the rep gene. These results suggest that a small countertranscribed RNA (ctRNA) (ca. 89 nucleotides in length), which might inhibit translation of pGA1 rep gene, is formed. Analysis of predicted secondary structure of the pGA1-encoded ctRNA revealed features common with the known ctRNAs in bacteria. Inactivation of the promoter P-ctRNA caused a dramatic increase of copies of the respective plasmid, which proved a negative role of the ctRNA in control of pGA1 copy number. A region between the promoters Prep and P-ctRNA with a potential to form secondary structures on both ctRNA and rep mRNA was found to cause low activity of the rep promoter even when promoter P-ctRNA was deleted. Thus, the sequence within the rep leader region itself seems to act, in addition to the ctRNA, as a second regulatory element of a novel type, negatively influencing expression of the pGA1 rep gene.

• MeSH
• bakteriální RNA MeSH
• Corynebacterium genetika   MeSH
• DNA bakterií biosyntéza   genetika   MeSH
• konformace nukleové kyseliny MeSH
• molekulární sekvence - údaje MeSH
• plazmidy biosyntéza   genetika   MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u bakterií * MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální RNA MeSH
• DNA bakterií MeSH

The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheriae). This function was confirmed by deletion mapping of the minimal replicon of pGA1 (1.7 kb) which contains only ORFC. Deletion derivatives of pGA1 devoid of ORFA exhibited significant decreases in the copy number in C. glutamicum cells and displayed segregational instability. Introduction of ORFA in trans into the cells harboring these deletion plasmids dramatically increased their copy number and segregational stability. The ORFA gene product thus positively influences plasmid copy number. This is the first report on such activity associated with a nonintegrating bacterial plasmid. The related plasmids pGA1, pSR1, and pNG2 lacking significant homology with any other plasmid seem to be representatives of a new group of plasmids replicating in the rolling-circle mode.

• MeSH
• bakteriální proteiny chemie   genetika   fyziologie   MeSH
• Corynebacterium genetika   MeSH
• DNA vazebné proteiny * MeSH
• DNA-helikasy chemie   genetika   MeSH
• molekulární sekvence - údaje MeSH
• otevřené čtecí rámce MeSH
• plazmidy genetika   MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u bakterií MeSH
• replikon genetika   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• trans-aktivátory chemie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA vazebné proteiny * MeSH
• DNA-helikasy MeSH
• ORFA protein, Corynebacterium MeSH Prohlížeč
• replication initiator protein MeSH Prohlížeč
• trans-aktivátory MeSH