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1.  d-Alanine Modification of a Protease-Susceptible Outer Membrane Component by the Bordetella pertussis dra Locus Promotes Resistance to Antimicrobial Peptides and Polymorphonuclear Leukocyte-Mediated Killing 
Journal of Bacteriology  2013;195(22):5102-5111.
Bordetella pertussis is the causative agent of pertussis, a highly contagious disease of the human respiratory tract. Despite very high vaccine coverage, pertussis has reemerged as a serious threat in the United States and many developing countries. Thus, it is important to pursue research to discover unknown pathogenic mechanisms of B. pertussis. We have investigated a previously uncharacterized locus in B. pertussis, the dra locus, which is homologous to the dlt operons of Gram-positive bacteria. The absence of the dra locus resulted in increased sensitivity to the killing action of antimicrobial peptides (AMPs) and human phagocytes. Compared to the wild-type cells, the mutant cells bound higher levels of cationic proteins and peptides, suggesting that dra contributes to AMP resistance by decreasing the electronegativity of the cell surface. The presence of dra led to the incorporation of d-alanine into an outer membrane component that is susceptible to proteinase K cleavage. We conclude that dra encodes a virulence-associated determinant and contributes to the immune resistance of B. pertussis. With these findings, we have identified a new mechanism of surface modification in B. pertussis which may also be relevant in other Gram-negative pathogens.
doi:10.1128/JB.00510-13
PMCID: PMC3811601  PMID: 24013634
2.  BpsR Modulates Bordetella Biofilm Formation by Negatively Regulating the Expression of the Bps Polysaccharide 
Journal of Bacteriology  2012;194(2):233-242.
Bordetella bacteria are Gram-negative respiratory pathogens of animals, birds, and humans. A hallmark feature of some Bordetella species is their ability to efficiently survive in the respiratory tract even after vaccination. Bordetella bronchiseptica and Bordetella pertussis form biofilms on abiotic surfaces and in the mouse respiratory tract. The Bps exopolysaccharide is one of the critical determinants for biofilm formation and the survival of Bordetella in the murine respiratory tract. In order to gain a better understanding of regulation of biofilm formation, we sought to study the mechanism by which Bps expression is controlled in Bordetella. Expression of bpsABCD (bpsA-D) is elevated in biofilms compared with levels in planktonically grown cells. We found that bpsA-D is expressed independently of BvgAS. Subsequently, we identified an open reading frame (ORF), BB1771 (designated here bpsR), that is located upstream of and in the opposite orientation to the bpsA-D locus. BpsR is homologous to the MarR family of transcriptional regulators. Measurement of bpsA and bpsD transcripts and the Bps polysaccharide levels from the wild-type and the ΔbpsR strains suggested that BpsR functions as a repressor. Consistent with enhanced production of Bps, the bpsR mutant displayed considerably more structured biofilms. We mapped the bpsA-D promoter region and showed that purified BpsR protein specifically bound to the bpsA-D promoter. Our results provide mechanistic insights into the regulatory strategy employed by Bordetella for control of the production of the Bps polysaccharide and biofilm formation.
doi:10.1128/JB.06020-11
PMCID: PMC3256665  PMID: 22056934
3.  Interaction of Chandipura Virus N and P Proteins: Identification of Two Mutually Exclusive Domains of N Involved in Interaction with P 
PLoS ONE  2012;7(4):e34623.
The nucleocapsid protein (N) and the phosphoprotein (P) of nonsegmented negative-strand (NNS) RNA viruses interact with each other to accomplish two crucial events necessary for the viral replication cycle. First, the P protein binds to the aggregation prone nascent N molecules maintaining them in a soluble monomeric (N0) form (N0-P complex). It is this form that is competent for specific encapsidation of the viral genome. Second, the P protein binds to oligomeric N in the nucleoprotein complex (N-RNA-P complex), and thereby facilitates the recruitment of the viral polymerase (L) onto its template. All previous attempts to study these complexes relied on co-expression of the two proteins in diverse systems. In this study, we have characterised these different modes of N-P interaction in detail and for the first time have been able to reconstitute these complexes individually in vitro in the chandipura virus (CHPV), a human pathogenic NNS RNA virus. Using a battery of truncated mutants of the N protein, we have been able to identify two mutually exclusive domains of N involved in differential interaction with the P protein. An unique N-terminal binding site, comprising of amino acids (aa) 1–180 form the N0-P interacting region, whereas, C-terminal residues spanning aa 320–390 is instrumental in N-RNA-P interactions. Significantly, the ex-vivo data also supports these observations. Based on these results, we suggest that the P protein acts as N-specific chaperone and thereby partially masking the N-N self-association region, which leads to the specific recognition of viral genome RNA by N0.
doi:10.1371/journal.pone.0034623
PMCID: PMC3317646  PMID: 22485180
4.  Biochemical characterization of L1 repressor mutants with altered operator DNA binding activity 
Bacteriophage  2012;2(2):79-88.
A mycobacteriophage-specific repressor with the enhanced operator DNA binding activity at 32°C and no activity at 42°C has not been generated yet though it has potential in developing a temperature-controlled expression vector for mycobacterial system. To create such an invaluable repressor, here we have characterized four substitution mutants of mycobacteriophage L1 repressor by various probes. The W69C repressor mutant displayed no operator DNA binding activity, whereas, P131L repressor mutant exhibited very little DNA binding at 32°C. In contrast, both E36K and E39Q repressor mutants showed significantly higher DNA binding activity at 32°C, particularly, under in vivo conditions. Various mutations also had different effects on the structure, stability and the dimerization ability of L1 repressor. While the W69C mutant possessed a distorted tertiary structure, the P131L mutant dimerized poorly in solution at 32°C. Interestingly, both these mutants lost their two-domain structure and aggregated rapidly at 42°C. Of the native and mutant L1 repressor proteins, W69C and E36K mutants appeared to be the least stable at 32°C. Studies together suggest that the mutants, particularly P131L and E39Q mutants, could be used for creating a high affinity temperature-sensitive repressor in the future.
PMCID: PMC3442829  PMID: 23050218
mycobacteriophage L1; repressor; early promoter; operator DNA; mutant repressor and expression vector
5.  Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity 
Virology Journal  2007;4:64.
Background
Lysogenic mode of life cycle of a temperate bacteriophage is generally maintained by a protein called 'repressor'. Repressor proteins of temperate lambdoid phages bind to a few symmetric operator DNAs in order to regulate their gene expression. In contrast, repressor molecules of temperate mycobacteriophages and some other phages bind to multiple asymmetric operator DNAs. Very little is known at present about the structure-function relationship of any mycobacteriophage repressor.
Results
Using highly purified repressor (CI) of temperate mycobacteriophage L1, we have demonstrated here that L1 CI harbors an N-terminal domain (NTD) and a C-terminal domain (CTD) which are separated by a small hinge region. Interestingly, CTD is more compact than NTD at 25°C. Both CTD and CI contain significant amount of α-helix at 30°C but unfold partly at 42°C. At nearly 200 nM concentration, both proteins form appreciable amount of dimers in solution. Additional studies reveal that CI binds to O64 and OL types of asymmetric operators of L1 with variable affinity at 25°C. Interestingly, repressor – operator interaction is affected drastically at 42°C. The conformational change of CI is most possibly responsible for its reduced operator binding affinity at 42°C.
Conclusion
Repressors encoded by mycobacteriophages differ significantly from the repressor proteins of λ and related phages at functional level but at structural level they are nearly similar.
doi:10.1186/1743-422X-4-64
PMCID: PMC1934351  PMID: 17598887

Results 1-5 (5)