Overexpression of the ftsZ gene from Corynebacterium glutamicum (Brevibacterium lactofermentum) in Escherichia coli

Our goal in this work was to overexpress the essential cell division FtsZ protein from Corynebacterium glutamicum (Brevibacterium lactofermentum) (FtsZCG) in Escherichia coli to produce anti-FtsZCG polyclonal antibodies. Previous results from our laboratory showed that ftsZCG was not expressed in E. coli in a sufficient amount to purify FtsZCG. However, when ftsZCG (without upstream sequences) was transcriptionally fused to the T7 promoter, different truncated FtsZCG proteins (28–32 kDa) were overexpressed in E. coli, and in all cases, stop codons were created because of DNA deletions or rearrangements. Nevertheless, we were able to overexpress and purify an N-terminally hexa-His-tagged FtsZCG from uninduced E. coli cells carrying a pET-28a(+) derivative, yielding about 5 mg of 98% pure protein per 100-mL culture.Key words: Brevibacterium lactofermentum, Corynebacterium glutamicum, FtsZ overexpresssion, hexa-His-tagged FtsZ.

Involvement of DivIVA in the morphology of the rod-shaped actinomycete Brevibacterium lactofermentum

In Brevibacterium lactofermentum, as in many Gram-positive bacteria, a divIVA gene is located downstream from the dcw cluster of cell-division- and cell-wall-related genes. This gene (divIVABL ) is mostly expressed during exponential growth, and the protein encoded, DivIVABL, bears some sequence similarity to antigen 84 (Ag84) from mycobacteria and was detected with monoclonal antibodies against Ag84. Disruption experiments using an internal fragment of the divIVABL gene or a disrupted divIVABL cloned in a suicide conjugative plasmid were unsuccessful, suggesting that the divIVABL gene is needed for cell viability in Brev. lactofermentum. Transformation of Brev. lactofermentum with a multicopy plasmid containing divIVABL drastically altered the morphology of the corynebacterial cells, which became larger and bulkier, and a GFP fusion to DivIVABL mainly localized to the ends of corynebacterial cells. This localization pattern, together with the overproduction phenotype, suggests that DivIVA may be important in regulating the apical growth of daughter cells.