This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Raghunand, T. R. Articles by Bishai, W. R. Search for Related Content PubMed PubMed Citation Articles by Raghunand, T. R. Articles by Bishai, W. R.

 Previous Article  |  Next Article 

Journal of Bacteriology, October 2006, p. 6966-6976, Vol. 188, No. 19
0021-9193/06/$08.00+0     doi:10.1128/JB.00384-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Mapping Essential Domains of Mycobacterium smegmatis WhmD: Insights into WhiB Structure and Function

Tirumalai R. Raghunand and William R. Bishai^*

Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231

Received 17 March 2006/ Accepted 19 June 2006

A growing body of evidence suggests that the WhiB-like proteins exclusive to the GC-rich actinomycete genera play significant roles in pathogenesis and cell division. Each of these proteins contains four invariant cysteine residues and a conserved helix-turn-helix motif. whmD, the Mycobacterium smegmatis homologue of Streptomyces coelicolor whiB, is essential in M. smegmatis, and the conditionally complemented mutant M. smegmatis 628-53 undergoes filamentation under nonpermissive conditions. To identify residues critical to WhmD function, we developed a cotransformation-based assay to screen for alleles that complement the filamentation phenotype of M. smegmatis 628-53 following inducer withdrawal. Mycobacterium tuberculosis whiB2 and S. coelicolor whiB complemented the defect in M. smegmatis 628-53, indicating that these genes are true functional orthologues of whmD. Deletion analysis suggested that the N-terminal 67 and C-terminal 12 amino acid residues are dispensable for activity. Site-directed mutagenesis indicated that three of the four conserved cysteine residues (C₉₀, C₉₃, and C₉₉) and a conserved aspartate (D₇₁) are essential. Mutations in a predicted loop glycine (G₁₁₁) and an unstructured leucine (L₁₁₆) were poorly tolerated. The region essential for WhmD activity encompasses 6 of the 10 residues conserved in all seven M. tuberculosis WhiBs, as well as in most members of the WhiB family identified thus far. WhmD structure was found to be sensitive to the presence of a reducing agent, suggesting that the cysteine residues are involved in coordinating a metal ion. Iron-specific staining strongly suggested that WhmD contains a bound iron atom. With this information, we have now begun to comprehend the functional significance of the conserved sequence and structural elements in this novel family of proteins.

---

* Corresponding author. Mailing address: Center for TB Research, Johns Hopkins University School of Medicine, 1550 Orleans Street, Baltimore, MD 21231-1002. Phone: (410) 955-3507. Fax: (410) 614-8173. E-mail: wbishai{at}jhmi.edu.

---

Journal of Bacteriology, October 2006, p. 6966-6976, Vol. 188, No. 19
0021-9193/06/$08.00+0     doi:10.1128/JB.00384-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Hett, E. C., Rubin, E. J. (2008). Bacterial Growth and Cell Division: a Mycobacterial Perspective. Microbiol. Mol. Biol. Rev. 72: 126-156 [Abstract] [Full Text]  
• Ventura, M., Canchaya, C., Tauch, A., Chandra, G., Fitzgerald, G. F., Chater, K. F., van Sinderen, D. (2007). Genomics of Actinobacteria: Tracing the Evolutionary History of an Ancient Phylum. Microbiol. Mol. Biol. Rev. 71: 495-548 [Abstract] [Full Text]