ABrevibacterium linenspRBL1 Replicon Functional inCorynebacterium glutamicum

Ankri, Serge; Bouvier, Isabelle; Reyes, Oscar; Predali, Florence; Leblon, Gérard

doi:10.1006/plas.1996.0029

Cited by 24 publications

(29 citation statements)

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“…The high levels of homology between Orf1 and Orf2 of pRGO1 and the RepA and RepB proteins, which have been reported to play an important role in plasmid replication (2,27,33), led us to construct a shuttle vector for shuttling between Propionibacterium sp. and E. coli.…”

Section: Resultsmentioning

confidence: 99%

Characterization of pRGO1, a Plasmid from Propionibacterium acidipropionici , and Its Use for Development of a Host-Vector System in Propionibacteria

Kiatpapan

Hashimoto

Nakamura

et al. 2000

Appl Environ Microbiol

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The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6,868 bp long, and its G؉C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 ؋ 10 6 CFU/g of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10 4 to 10 7 CFU/g of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.Propionibacteria, which have a wide range of probiotic activity, are used in making dairy foods, such as cheese, for the production of vitamin B 12 , tetrapyrrole compounds, and propionic acid (8,15,21), in bread baking, as starters for ensilage, and in some pharmaceutical preparations (35). To elucidate the biosynthetic pathways of vitamin B 12 and siroheme in Propionibacterium, we previously identified several genes coding for the enzymes involved in production of tetrapyrrole derivatives (hemYHBXRL) (11,12) and vitamin B 12 (cobA, cbiO) (30).Development of genetic manipulation in propionibacteria has progressed slowly due to a lack of detailed information on the genetics of the bacteria and a lack of an appropriate plasmid that can serve as a possible transformation vector. A number of plasmids from Propionibacterium acidipropionici, P. freudenreichii, and P. jensenii, ranging in size from 4.4 to more than 119 MDa, have been described (19,24). However, neither analysis of a plasmid DNA sequence nor construction of a vector for propionibacteria has been reported. To establish a versatile vector system to facilitate genetic analysis and to allow the transfer of a gene of interest, we investigated the development of a host-vector system in propionibacteria.We succeeded in determining the co...

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Section: Resultsmentioning

confidence: 99%

Characterization of pRGO1, a Plasmid from Propionibacterium acidipropionici , and Its Use for Development of a Host-Vector System in Propionibacteria

Kiatpapan

Hashimoto

Nakamura

et al. 2000

Appl Environ Microbiol

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“…The proteins related to replication are encoded by orf6 and orf7. All the above-mentioned replication proteins show motifs that are typical of theta-replicating plasmids (34). Propionicin SM1, the first bacteriocin encoded by a Propionibacterium plasmid, is related to orf9 and orf10 (35).…”

Section: Resultsmentioning

confidence: 99%

Development of a Propionibacterium-Escherichia coli Shuttle Vector for Metabolic Engineering of Propionibacterium jensenii, an Efficient Producer of Propionic Acid

Zhuge

Liu

Shin

et al. 2013

Appl Environ Microbiol

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dPropionic acid (PA) is an important chemical building block and is widely applied for organic synthesis, food, feedstuff, and pharmaceuticals. To date, the strains that can efficiently produce PA have included Propionibacterium thoenii, P. freudenreichii, and P. acidipropionici. In this report, we show that P. jensenii ATCC 4868 is also able to produce PA in much higher yields than the previously reported strains. To further improve the production capacity, a P. jensenii-Escherichia coli shuttle vector was developed for the metabolic engineering of P. jensenii. Specifically, a 6.9-kb endogenous plasmid, pZGX01, was isolated from P. acidipropionici ATCC 4875 and sequenced. Since the sequencing analysis indicated that pZGX01 could encode 11 proteins, the transcriptional levels of the corresponding genes were also investigated. Then, a P. jensenii-Escherichia coli shuttle vector was constructed using the pZGX01 plasmid, the E. coli pUC18 plasmid, and a chloramphenicol resistance gene. Interestingly, not only could the developed shuttle vector be transformed into P. jensenii ATCC 4868 and 4870, but it also could be transformed into freudenreichii ATCC 6207 subspecies of P. freudenreichii. Finally, the glycerol dehydrogenase gene (gldA) from Klebsiella pneumoniae was expressed in P. jensenii ATCC 4868 with the constructed shuttle vector. In a 3-liter batch culture, the PA production by the engineered P. jensenii ATCC 4868 strain reached 28.23 ؎ 1.0 g/liter, which was 26.07% higher than that produced by the wild-type strain (22.06 ؎ 1.2 g/liter). This result indicated that the constructed vector can be used a useful tool for metabolic engineering of P. jensenii.

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“…SS9 chr2 is the second chromosome sequence of Photobacterium profundum. The (putative) origin sequences of the following plasmids have been presented previously: pRBL1 (1,11,32); pBLA8 (32); pB264 (33); pKA22 (31); pFJ2600 (11,32); pMB1 (11,32,40); pXZ10142 (11,32,41); pRC4 (20); pAL5000 (11,32,45); pRGO1 (25); pLME106 (36); and pLIM (37). Regions with partial homology further extend on both sides of the highly conserved sequences.…”

Section: Acknowledgmentsmentioning

confidence: 99%

Anatomy of the Replication Origin of Plasmid ColE2-P9

et al. 2006

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The plasmid ColE2-P9 origin is a 32-bp region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. We analyzed the structural and functional organization of the ColE2 origin by using various derivatives carrying deletions and single-base-pair substitutions. The origin may be divided into three subregions: subregion I, which is important for stable binding of the Rep protein; subregion II, which is important for binding of the Rep protein and for initiation of DNA replication; and subregion III, which is important for DNA replication but apparently not for binding of the Rep protein. The Rep protein might recognize three specific DNA elements in subregions I and II. The relative transformation frequency of the autonomously replicating plasmids carrying deletions in subregion I is lower, and nevertheless the copy numbers of these plasmids in host bacteria are higher than those of the wild-type plasmid. Efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. Subregion II might be essential for interaction with the catalytic domain of the Rep protein for primer RNA synthesis. The 8-bp sequence across the border of subregions II and III, including the primer sequence, is conserved in the (putative) origins of many plasmids, the putative Rep proteins of which are related to the ColE2-P9 Rep protein. Subregion III might be required for a step that is necessary after Rep protein binding has taken place.DNA replication initiates at a specific chromosomal region called the replication origin. The DNA sequence elements required for replication origin activity have been extensively defined and characterized (30). Typically, they consist of a recognition element for the initiator protein and an AT-rich region. In the process of initiation of replication, the initiator protein binds to the origin and induces local unwinding of the duplex DNA. Subsequent events are the loading of a DNA helicase onto the unwound region followed by recruitment of additional factors to initiate DNA synthesis. In the Escherichia coli replication system, the ATP-bound DnaA proteins bind to the 9-mer DnaA boxes in oriC, and then the DnaB helicase is loaded onto unwound 13-mer AT-rich sequences, followed by loading of proteins required for initiation of DNA synthesis, including the DnaG primase (30).The plasmid ColE2-P9 (ColE2) is a circular duplex DNA molecule of about 7 kb (18) and is present at about 10 to 15 copies per host chromosome (4, 21). The plasmid specifies an initiator protein (Rep protein, 35 kDa), which is the only plasmid-specified trans-acting factor essential for its replication (21,26,60). In addition, plasmid replication requires the host DNA polymerase I (28, 50) along with other host factors. The minimal cis-acting region (origin) (Fig. 1) required for plasmid replication consists of only 32 bp, where replication initiates (23, 60). The ColE2 Rep protein specifically binds to the ColE2 origin as ...

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ABrevibacterium linenspRBL1 Replicon Functional inCorynebacterium glutamicum

Cited by 24 publications

References 0 publications

Characterization of pRGO1, a Plasmid from Propionibacterium acidipropionici , and Its Use for Development of a Host-Vector System in Propionibacteria

Characterization of pRGO1, a Plasmid from Propionibacterium acidipropionici , and Its Use for Development of a Host-Vector System in Propionibacteria

Development of a Propionibacterium-Escherichia coli Shuttle Vector for Metabolic Engineering of Propionibacterium jensenii, an Efficient Producer of Propionic Acid

Anatomy of the Replication Origin of Plasmid ColE2-P9

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