Angelina Ramos

Characterization and Use of Catabolite-Repressed Promoters from Gluconate Genes in Corynebacterium glutamicum

The genes involved in gluconate catabolism (gntP and gntK) in Corynebacterium glutamicum are scattered in the chromosome, and no regulatory genes are apparently associated with them, in contrast with the organization of the gnt operon in Escherichia coli and Bacillus subtilis. In C. glutamicum, gntP and gntK are essential genes when gluconate is the only carbon and energy source. Both genes contain upstream regulatory regions consisting of a typical promoter and a hypothetical cyclic AMP (cAMP) receptor protein (CRP) binding region but lack the expected consensus operator region for binding of the GntR repressor protein. Expression analysis by Northern blotting showed monocistronic transcripts for both genes. The expression of gntP and gntK is not induced by gluconate, and the gnt genes are subject to catabolite repression by sugars, such as glucose, fructose, and sucrose, as was detected by quantitative reverse transcription-PCR (qRT-PCR). Specific analysis of the DNA promoter sequences (PgntK and PgntP) was performed using bifunctional promoter probe vectors containing mel (involved in melanin production) or egfp2 (encoding a green fluorescent protein derivative) as the reporter gene. Using this approach, we obtained results parallel to those from qRT-PCR. An applied example of in vivo gene expression modulation of the divIVA gene in C. glutamicum is shown, corroborating the possible use of the gnt promoters to control gene expression. glxR (which encodes GlxR, the hypothetical CRP protein) was subcloned from the C. glutamicum chromosomal DNA and overexpressed in corynebacteria; we found that the level of gnt expression was slightly decreased compared to that of the control strains. The purified GlxR protein was used in gel shift mobility assays, and a specific interaction of GlxR with sequences present on PgntP and PgntK fragments was detected only in the presence of cAMP.

Improved vectors for transcriptional/translational signal screening in corynebacteria using the melC operon from Streptomyces glaucescens as reporter

The tyrosinase operon (melC) from Streptomyces glaucescens was cloned and functionally expressed in Brevibacterium lactofermentum and Corynebacterium glutamicum under the control of the promoter of the kan gene from Tn5. Recombinant corynebacterial cells containing the tyrosinase operon produced melanin on agar plates and in liquid culture when supplemented with copper and tyrosine. A conjugative bifunctional replacement vector for transcriptional/translational signal screening (pEMel-1) was constructed using expression of the melC operon from S. glaucescens, which can be used for cloning promoter sequences as EcoRI–NdeI fragments. When the DNA fragments with promoter activity such as cspBp or trpp were inserted into pEMel-1, B. lactofermentum harboring the chimeric plasmids produced melanin at different stages of growth, allowing temporal detection of promoter activity. The vector was also used to detect the activity of a Streptomyces promoter (xysAp), which was inactive in B. lactofermentum, after PCR mutagenesis. The melC operon can be used for the visual, inexpensive (compared to the high price of starch azure for amylase detection), and non-selective (in contrast to the kan or cat genes) screening of several thousand clones at high colony density without killing of the transformants due to the presence of iodine (as in the case of amylase assay).

Construction of a Xylanase-Producing Strain of Brevibacterium lactofermentum by Stable Integration of an Engineered xysA Gene from Streptomyces halstedii JM8

ABSTRACT A xylanolytic strain of Brevibacteriumlactofermentum containing theStreptomyceshalstedii His-taggedxysA gene was generated. The new strain contains DNA derived from S. halstedii, expresses xylanolytic activity, and was obtained by an integrative process mediated by a conjugative plasmid targeted to a dispensable chromosomal region located downstream from the essential cell division geneftsZ. The His-tagged Xys1 enzyme was constitutively expressed under the control of the kan promoter from Tn5 and was easily purified by use of Ni-nitrilotriacetic acid-agarose. The new strain is stable for more than 200 generations, lacks any known antibiotic resistance gene, and does not need any selective pressure to maintain the integrated gene. This strategy can be used to integrate any gene into theB. lactofermentum chromosome and to maintain it stably without the use of antibiotics for selection.

Modulation of Deoxysugar Transfer by the Elloramycin Glycosyltransferase ElmGT through Site-Directed Mutagenesis

The glycosyltransferase ElmGT from Streptomyces olivaceus is involved in the biosynthesis of the antitumor drug elloramycin, and it has been shown to possess a broad deoxysugar recognition pattern, being able to transfer different l- and d-deoxysugars to 8-demethyl-tetracenomycin C, the elloramycin aglycone. Site-directed mutagenesis in residues L309 and N312, located in the alpha/beta/alpha motif within the nucleoside diphosphate-sugar binding region, can be used to modulate the substrate flexibility of ElmGT, making it more precise for transfer of specific deoxysugars.

The cell division genes ftsQ and ftsZ, but not the three downstream open reading frames YFIH, ORF5 and ORF6, are essential for growth and viability in Brevibacterium lactofermentum ATCC 13869.

Abstract. The three ORFs (YFIH, ORF5 and ORF6) located downstream of the cell division genes ftsQ and ftsZ in Brevibacterium lactofermentum were disrupted by single homologous recombination events between internal fragments of the corresponding genes and the chromosomal sequences. The phenotypes of the disrupted mutants were similar to that of the wild type, suggesting that these genes are dispensable for growth and viability. However, using different plasmid constructs, it was not possible to obtain disrupted ftsZ or ftsQ mutants by single crossover events. When the ftsZ or ftsQ gene sequence was disrupted in vitro and used to replace the homologous chromosomal gene by double recombination, only single recombination events took place, and therefore no disruptants were obtained. It may be concluded therefore that, as in Escherichia coli, the cell division genes ftsQ and ftsZ are indispensable for growth and viability of B. lactofermentum. Northern hybridisation analyses performed using internal fragments of the genes coding for YFIH, ORF5 and ORF6 allowed us to dissect their transcriptional organization and to confirm the disruption of these genes.

Cloning and expression of the inorganic pyrophosphatase gene from the amino acid producer Brevibacterium lactofermentum ATCC 13869

A 20-kDa Brevibacterium lactofermentum protein was detected when purifying the His-tagged FtsZBL. The protein was identified by matrix-assisted laser desorption/ionisation time of flight as the inorganic pyrophosphatase encoded by the ppa gene, which is present as a single copy in the genome of Corynebacterium glutamicum. The ppa gene was cloned from B. lactofermentum chromosomal DNA by polymerase chain reaction; it seemed to be an essential gene and it might represent an attractive target for drug discovery. The cloned ppa gene complemented a ppa- Escherichia coli mutant and a ppa-gfp gene fusion revealed that the gene product mainly accumulated at the cell poles in both E. coli and B. lactofermentum.