The actinomycete Corynebacterium glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVA Cg ) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVA Cg -depleted C. glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVA proteins from Bacillus subtilis or Streptococcus pneumoniae, which localized at the septum of C. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVA Cg localized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that in C. glutamicum DivIVA is not involved in cell division or chromosome segregation.

Time-lapse imaging of Streptomyces hyphae revealed foci of the essential protein DivIVA at sites where lateral branches will emerge. Overexpression experiments showed that DivIVA foci can trigger establishment of new zones of cell wall assembly, suggesting a key role of DivIVA in directing peptidoglycan synthesis and cell shape in Streptomyces.Gram-positive bacteria of the genus Streptomyces grow by tip extension and form branched hyphae and mycelia (8,11,12). This polarized cell wall growth is strikingly different from the mode of growth of, e.g., Escherichia coli and Bacillus subtilis, which like most rod-shaped bacteria extend the cell and acquire their rod shape by intercalatory insertion of new peptidoglycan units along the lateral wall (5, 6). This is dependent on the actin-like MreB proteins, which form helical filaments extending along the cell and acting via interaction with membrane proteins to organize the cell wall assembly (1,2,16,19). In contrast, Streptomyces tip extension appears to occur by an mreB-independent mechanism (22) and is also independent of FtsZ and cell division (23). The Streptomyces coelicolor genome contains two mreB genes, but they are involved primarily in sporulation and have no overt impact on tip extension in the vegetative mycelium (22; G. Muth, University of Tübingen, Germany, personal communication). In fact, most rod-shaped relatives of Streptomyces within the phylum Actinobacteria, like mycobacteria and corynebacteria, lack mreB genes and assemble their cell walls at the cell poles (3,5,15,24).This mreB-independent and polarized growth in Actinobacteria involves the coiled-coil protein DivIVA. In S. coelicolor, DivIVA is essential for growth and accumulates at growing hyphal tips, and the effects of partial depletion and ectopic overexpression revealed a strong impact on tip extension and cell shape determination (10). Among other Actinobacteria, the DivIVA orthologues, also named antigen 84 and Wag31, in Mycobacterium tuberculosis, Mycobacterium smegmatis, and Corynebacterium glutamicum are polarly localized and appear to be essential and, when overproduced, have a very similar effect on cell shape to that seen in S. coelicolor (17,24,25). Recently, DivIVA was found to be required for polar cell elongation and acquisition of rod shape in C. glutamicum and M. smegmatis (17,20). Furthermore, Streptomyces and Mycobacterium DivIVA could restore polar growth to a C. glutamicum strain depleted for DivIVA, while orthologues from the phylum Firmicutes (e.g., Bacillus subtilis) could not (20) and are known to be associated with different cellular functions (9,21,26,29). While these findings suggest a role for Streptomyces DivIVA in tip extension, its exact function is not known. In this study, we have investigated the subcellular targeting of S. coelicolor DivIVA and its involvement in the establishment of tip extension during hyphal branching.DivIVA is a molecular marker of new branch sites in S. coelicolor. Apart from the striking apical localization of S. coelicolor DivIVA, o...

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.

Over the past 15 years the biosynthetic gene clusters for numerous bioactive polyketides have been intensively studied and recently this work has been extended to the antifungal polyene macrolides. These compounds consist of large macrolactone rings that have a characteristic series of conjugated double bonds, as well as an exocyclic carboxyl group and an unusual mycosamine sugar. The biosynthetic gene clusters for nystatin, pimaricin, amphotericin and candicidin have been investigated in detail. These clusters contain the largest modular polyketide synthase genes reported to date. This body of work also provides insights into the enzymes catalysing the unusual post-polyketide modifications, and the genes regulating antibiotic biosynthesis. The sequences also provide clues about the evolutionary origins of polyene biosynthetic genes. Successful genetic manipulation of the producing organisms leading to production of polyene analogues indicates good prospects for generating improved antifungal compounds via genetic engineering.

We identified the first enzymes that use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two arsenate reductases from Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key role in the defense against arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol biosynthesis pathway confer arsenate resistance. With steady-state kinetics, arsenite analysis, and theoretical reactivity analysis, we unraveled the catalytic mechanism for the reduction of arsenate to arsenite in C. glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct formation between arsenate and mycothiol. Mycoredoxin, a redox enzyme for which the function was never shown before, reduces the thiol-arseno bond and forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that, in its turn, is reduced by the NADPHdependent mycothione reductase. Cg_ArsCs show a low specificity constant of ϳ5 M ؊1 s ؊1 , typically for a thiol/disulfide cascade with nucleophiles on three different molecules. With the in vitro reconstitution of this novel electron transfer pathway, we have paved the way for the study of redox mechanisms in actinobacteria.