Construction and characterization of promoter-probe vectors for Corynebacteria using the kanamycinresistance reporter gene

Cadenas, Rosa F.; Martı́n, Juan F.; Gil, José A.

doi:10.1016/0378-1119(91)90113-p

Cited by 47 publications

(20 citation statements)

References 8 publications

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“…The transcriptional initiation ability in both micro-organisms indicates that these two promoter regions are recognized by the RNA polymerases of E. coli and C. glutamicum. These promoters belong to the type I corynebacteria promoters (Martín et al, 1990;Cadenas et al, 1991), also known as CEP ('Corynebacteria-E. coli promoters'), described in several genes of C. glutamicum (Pátek et al, 2003a;Barreiro et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH

et al. 2006

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Corynebacterium glutamicum, a soil Gram-positive bacterium used for industrial amino acid production, was found to grow optimally at pH 7?0-9?0 when incubated in 5 litre fermenters under pH-controlled conditions. The highest biomass was accumulated at pH 9?0. Growth still occurred at pH 9?5 but at a reduced rate. The expression of the pH-regulated F 0 F 1 ATPase operon (containing the eight genes atpBEFHAGDC) was induced at alkaline pH. A 7?5 kb transcript, corresponding to the eight-gene operon, was optimally expressed at pH 9?0. The same occurred with a 1?2 kb transcript corresponding to the atpB gene. RT-PCR studies confirmed the alkaline pH induction of the F 0 F 1 operon and the existence of the atpI gene. The atpI gene, located upstream of the F 0 F 1 operon, was expressed at a lower level than the polycistronic 7?5 kb mRNA, from a separate promoter (P-atp1). Expression of the major promoter of the F 0 F 1 operon, designated P-atp2, and the P-atp1 promoter was quantified by coupling them to the pET2 promoter-probe vector. Both P-atp1 and P-atp2 were functional in C. glutamicum and Escherichia coli. Primer extension analysis identified one transcription start point inside each of the two promoter regions. The P-atp1 promoter fitted the consensus sequence of promoters recognized by the vegetative s factor of C. glutamicum, whereas the "35 and "10 boxes of P-atp2 fitted the consensus sequence for s H -recognized Mycobacterium tuberculosis promoters C C / G GG A / G AC 17-22 nt C / G GTT C / G , known to be involved in expression of heat-shock and other stress-response genes. These results suggest that the F 0 F 1 operon is highly expressed at alkaline pH, probably using a s H RNA polymerase.

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Section: Discussionmentioning

confidence: 99%

Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH

et al. 2006

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“…The Pkan promoter of the kan gene from Tn5 is efficiently expressed in corynebacteria (Cadenas et al, 1991). C. glutamicum transformed with pNV4A vector (MAPF strain) contains a wild-type copy of ftsI in addition to the gfp-ftsI cassette, which is under the control of the Pkan promoter and inserted as a single copy in the chromosome (Fig.…”

Section: Visualization Of Ftsi Cg -Gfp Fusionsmentioning

confidence: 99%

Morphological changes and proteome response of Corynebacterium glutamicum to a partial depletion of FtsI

et al. 2006

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In Corynebacterium glutamicum, as in many Gram-positive bacteria, the cell division gene ftsI is located at the beginning of the dcw cluster, which comprises cell division-and cell wall-related genes. Transcriptional analysis of the cluster revealed that ftsI is transcribed as part of a polycistronic mRNA, which includes at least mraZ, mraW, ftsL, ftsI and murE, from a promoter that is located upstream of mraZ. ftsI appears also to be expressed from a minor promoter that is located in the intergenic ftsL-ftsI region. It is an essential gene in C. glutamicum, and a reduced expression of ftsI leads to the formation of larger and filamentous cells. A translational GFP-FtsI fusion protein was found to be functional and localized to the mid-cell of a growing bacterium, providing evidence of its role in cell division in C. glutamicum. This study involving proteomic analysis (using 2D SDS-PAGE) of a C. glutamicum strain that has partially depleted levels of FtsI reveals that at least 20 different proteins were overexpressed in the organism. Eight of these overexpressed proteins, which include DivIVA, were identified by MALDI-TOF. Overexpression of DivIVA was confirmed by Western blotting using anti-DivIVA antibodies, and also by fluorescence microscopy analysis of a C. glutamicum RESF1 strain expressing a chromosomal copy of a divIVA-gfp transcriptional fusion. Overexpression of DivIVA was not observed when FtsI was inhibited by cephalexin treatment or by partial depletion of FtsZ.

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“…Plasmids pBL1 and pAM330 and the cryptic plasmid described by Filpula et al (6) have the same restriction maps, and their nucleotide sequences are almost identical. Plasmid pBL1 is one of the most frequently used coryneform replicons for construction of cloning vectors, shuttle vectors, or promoter-probe vectors (3,13,21,22,25). In our laboratory, several antibiotic resistance genes have been successfully used for developing a pBL1-based cloning system for B. lactofermentum: the kanamycin resistance gene (kan) from TnS, the hygromycin resistance gene (hyg) from Streptomyces hygroscopicus, and the chloramphenicol resistance gene (cat) from Streptomyces acrimycini (20,21).…”

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Characterization of a region of plasmid pBL1 of Brevibacterium lactofermentum involved in replication via the rolling circle model

et al. 1994

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The minimal region for autonomous replication of pBL1, a 4.5-kb cryptic plasmid of Brevibacterium lactofermentum ATCC 13869 that has been used to construct a variety of corynebacterium vectors, was shown to be contained on a 1.8-kb HindII-SphI DNA fragment. This region contains two open reading frames (ORFs) (ORF1 and ORF5) which are essential for pBL1 replication in B. lactofermentum. Accumulation of single-strand intermediates in some of the constructions indicates that plasmid pBL1 replicates via the rolling circle replication model; its plus strand and minus strand were identified by hybridization with two synthetic oligonucleotide probes complementary to each pBL1 strand. ORF1 seems to encode the Rep protein and showed partial homology with sequences for Rep proteins from Streptomyces plasmids which replicate via rolling circle replication such as pUJ101, pSB24, and pJVl.pBL1 is a multicopy plasmid isolated from Brevibacterium lactofermentum ATCC 13869 (19) and later described as pAM330 (14) Streptomyces lividans. pUL61 was unstable in B. lactofermentum, and two stable deletion derivatives (pUL330 and pUL340) appeared after B. lactofermentum had been transformed with pUL61. A similar instability has also been observed in most other small plasmids from gram-positive bacteria. The main characteristic of plasmids showing instability is their mode of replication via the rolling circle replication (RCR) mechanism. RCR plasmids were classified into four families according to homologies of their replication proteins (Rep) and the position of the double-stranded origin (DSO) (7). RCR plasmids have the information to synthesize their own Rep protein. The Rep protein initiates replication by nicking the DNA at the DSO sequence, after which replication of the plus strand begins. The newly generated single-stranded DNA (ssDNA) intermediates (plus strand) serve as a template for minus strand synthesis. The conversion of the ssDNA intermediates to duplex forms requires the recognition of another sequence, different and separated from the DSO sequence, called single-stranded origin (SSO). The absence or nonfunctionality of SSO results in the accumulation of ssDNA. The SSO sequence is rather specific, and it is usually recognized inefficiently in hosts different from the parental one (7, 15).We describe in this paper the identification and characterization of the region involved in replication functions of pBL1. Our results indicate that pBL1 belongs to the group of plasmids which replicate via the RCR mechanism. MATERIALS AND METHODSPlasmids, bacterial strains, and transformation conditions. Plasmids used in this study are listed in Table 1. B. lactofermentum BL31 (20), a pBL1-cured strain, and C. glutamicum ATCC 13032 were grown in Trypticase soy broth (TSB) medium and transformed by electroporation as described by Dunican and Shivnan (5). E. coli DH5ao was grown in L broth and transformed by the method of Hanahan (8).

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Construction and characterization of promoter-probe vectors for Corynebacteria using the kanamycinresistance reporter gene

Cited by 47 publications

References 8 publications

Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH

Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH

Morphological changes and proteome response of Corynebacterium glutamicum to a partial depletion of FtsI

Characterization of a region of plasmid pBL1 of Brevibacterium lactofermentum involved in replication via the rolling circle model

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