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.

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.

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.

Members of the Acr3 family of arsenite permeases confer resistance to trivalent arsenic by extrusion from cells, with members in every phylogenetic domain. In this study bacterial Acr3 homologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum were cloned and expressed in Escherichia coli. Modification of a single cysteine residue that is conserved in all analyzed Acr3 homologues resulted in loss of transport activity, indicating that it plays a role in Acr3 function. The results of treatment with thiol reagents suggested that the conserved cysteine is located in a hydrophobic region of the permease. A scanning cysteine accessibility method was used to show that Acr3 has 10 transmembrane segments, and the conserved cysteine would be predicted to be in the fourth transmembrane segment.

Arsenic, a toxic metalloid, is currently and has always been ranked first on the Superfund List of Hazardous Substances (available on the World Wide Web), in part because of its environmental ubiquity. As a consequence, nearly all organisms have genes that confer resistance to arsenic. Environmental arsenic is sensed by members of the ArsR/SmtB family of metalloregulatory proteins (1-3). These winged helix repressor proteins specifically bind to arsenic and other toxic metals. Consequently, they control expression of genes involved in arsenic biotransformation and efflux. For example, the ArsR repressor encoded by Escherichia coli plasmid R773 binds to the promoter region of its respective ars operon in the absence of As(III) or Sb(III) (4). This homodimeric repressor has the sequence Cys 32 -Val-Cys 34 -Asp-Leu-Cys 37 in the DNA binding domain of each monomer (5, 6). The three sulfur thiolates of the cysteine residues form a very specific three-coordinate binding site for the trivalent metalloids As(III) and Sb(III). Binding of metalloids to R773 ArsR is presumed to induce a conformational change, leading to dissociation from the DNA and hence derepression. The Staphylococcus aureus CadC is a Cd(II)/Pb(II)/Zn(II)-responsive member of the ArsR/SmtB family that has four cysteine residues in the inducer binding domain (7). Of these four cysteine residues, two come from one subunit, whereas the other two come from the other subunit of the homodimer (8, 9). The position of this metal binding site in CadC is congruent to that of the R773 ArsR but is formed between two monomers. CadC also has a second type of metal binding site (DXHX 10 HX 2 E) for Zn(II) at the dimer interface that is not a regulatory site. This site, however, is identical to the regulatory Zn(II) site of SmtB from Synechococcus PCC 7942 (10). Another member of the ArsR/SmtB family is the ArsR from Acidithiobacillus ferrooxidans (AfArsR), 3 which has three cysteine residues (Cys 95 , Cys 96 , and Cys 102 ) at the dimer interface rather than in or near the DNA binding domain (11). These three cysteine residues form a three-coordinate or S 3 binding site for trivalent metalloids (12). Although both the As(III) binding site of AfArsR and the Zn(II) binding site of SmtB are at the C-terminal dimerization domain, the former is formed of three cysteine residues within a single subunit (two sites per dimer), whereas the latter is formed by four residues, two residues from one monomer and two from the other. Thus, metal(loid) binding sites appear to arise by convergent evolution, even in homologous proteins.