About

Anath Das, PhD, is a professor affiliated with the departments of Biochemistry, Molecular Biology & Biophysics at the University of Minnesota. He holds a PhD from the University of Nebraska. His research focuses on the mechanism of polar assembly of the secretion apparatus and substrate translocation in Agrobacterium tumefaciens, a bacterium that incites crown gall tumor disease on plants. His work involves studying how the bacterium transfers a segment of its plasmid-borne DNA to plant cells using a type IV secretion apparatus assembled at a cell pole, contributing to the understanding of bacterial secretion systems and their role in plant pathology.

Research topics

• Biology
• Biochemistry
• Biophysics
• Cell biology

Selected publications

• Cryo-EM structure of the <i>Agrobacterium tumefaciens</i> type IV secretion system-associated T-pilus reveals stoichiometric protein-phospholipid assembly

  bioRxiv (Cold Spring Harbor Laboratory) · 2022 · 1 citations

  • Biology
  • Biophysics
  • Cell biology

  Abstract Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease by the horizontal transfer of oncogenic DNA that is integrated into the host’s genome. The conjugation is mediated by the conjugative VirB/D4 type 4 secretion system (T4SS). A. tumefaciens T4SS assembles an extracellular filament, the T-pilus, that is involved in the formation of a mating pair between A. tumefaciens and the recipient plant cell by a not fully understood mechanism. Here, we present a 3 Å cryo-EM structure of the T-pilus, solved by helical reconstruction. Our structure reveals that the T-pilus comprises the major pilin protein VirB2 and phosphatidylglycerol (PG) phospholipid at a 1:1 stoichiometric ratio with 5-start helical symmetry. We further show that PG-headgroups and the positively charged Arg 91 residues of VirB2 protomers form extensive electrostatic interactions in the lumen of the T-pilus. Mutagenesis of Arg 91 destabilized the VirB2 protein and completely abolished pilus formation. While our T-pilus structure shows architectural similarity with previously published conjugative pili structures, positively charged sidechains protrude into the lumen and the lumen is narrower, raising questions whether the T-pilus is a conduit for ssDNA transfer. We also show that the VirB2 subunits in T-pilus filament are not cyclic, as previously thought.

  PublisherDOI

• Identification of a Carboxy-Terminal Glutamine-Rich Domain in <i>Agrobacterium tumefaciens</i> Coupling Protein VirD4 Required for Recognition of T-Strand DNA and Not VirE2 as a Substrate for Transfer to Plant Cells

  Molecular Plant-Microbe Interactions · 2019-12-19 · 3 citations

  articleOpen access1st authorCorresponding

  Agrobacterium tumefaciens transfers DNA and proteins to a plant cell inciting crown gall tumor disease on most plants. VirD4 targets the DNA and protein substrates to a type IV secretion (T4S) apparatus for translocation into the plant cell. Several bacteria with VirD4 homologs use T4S for intercellular export of microbial macromolecules to eukaryotic and prokaryotic hosts. How the VirD4 proteins recognize the diverse substrates is not well understood. To identify functional domains of A. tumefaciens pTiA6 VirD4, we introduced random 19-codon and targeted 10-codon insertions throughout the coding region. Analysis of 21 mutants showed that only the carboxy-terminal end of VirD4 is tolerant of an insertion. Sequence comparison of VirD4 proteins of Agrobacterium spp. and their close relative, Rhizobium etli, showed that these proteins contain a highly conserved C-terminal end, but the immediate upstream regions share no discernible sequence similarity. The conserved region sequence is rich in the amino acid glutamine (6/13 Q). Using site-specific and deletion mutagenesis, we demonstrated that the conserved Q-rich region is required for VirD4 function and for the specific recognition of VirD2-linked T-strand DNA as a substrate for translocation to plants. The Q-rich region is not required for the transfer of a second A. tumefaciens substrate, VirE2, to plants or a promiscuous Escherichia coli IncQ plasmid to another A. tumefaciens strain. We identified Q-rich sequences at or near the C terminus of several VirD4 homologs, including the E. coli F plasmid TraD. In F TraD, the Q-rich sequence maps to a region required specifically for the conjugative transfer of the F plasmid.

  PublisherOA PDFDOI

• Expression of Agrobacterium tumefaciens octopine Ti-plasmid virB8 gene is regulated by translational coupling

  Plasmid · 2012-09-16 · 9 citations

  articleSenior authorCorresponding
  PublisherDOI

• <i>Agrobacterium tumefaciens</i>Type IV Secretion Protein VirB3 Is an Inner Membrane Protein and Requires VirB4, VirB7, and VirB8 for Stabilization

  Journal of Bacteriology · 2010-03-27 · 60 citations

  articleSenior authorCorresponding

  Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus and a T-pilus for secretion of DNA and proteins into plant cells. The pilin-like protein VirB3, a membrane protein of unknown topology, is required for the assembly of the T-pilus and for T-DNA secretion. Using PhoA and green fluorescent protein (GFP) as periplasmic and cytoplasmic reporters, respectively, we demonstrate that VirB3 contains two membrane-spanning domains and that both the N and C termini of the protein reside in the cytoplasm. Fusion proteins with GFP at the N or C terminus of VirB3 were fluorescent and, like VirB3, localized to a cell pole. Biochemical fractionation studies demonstrated that VirB3 proteins encoded by three Ti plasmids, the octopine Ti plasmid pTiA6NC, the supervirulent plasmid pTiBo542, and the nopaline Ti plasmid pTiC58, are inner membrane proteins and that VirB4 has no effect on membrane localization of pTiA6NC-encoded VirB3 (pTiA6NC VirB3). The pTiA6NC and pTiBo542 VirB2 pilins, like VirB3, localized to the inner membrane. The pTiC58 VirB4 protein was earlier found to be essential for stabilization of VirB3. Stabilization of pTiA6NC VirB3 requires not only VirB4 but also two additional VirB proteins, VirB7 and VirB8. A binary interaction between VirB3 and VirB4/VirB7/VirB8 is not sufficient for VirB3 stabilization. We hypothesize that bacteria use selective proteolysis as a mechanism to prevent assembly of unproductive precursor complexes under conditions that do not favor assembly of large macromolecular structures.

  PublisherDOI

• Molecular characterization of the Agrobacterium tumefaciens DNA transfer protein VirB6

  Microbiology · 2005-11-01 · 33 citations

  articleSenior author

  The VirB proteins of Agrobacterium tumefaciens assemble a T-pilus and a type IV secretion (T4S) apparatus for the transfer of DNA and proteins to plant cells. VirB6 is essential for DNA transfer and is a polytopic integral membrane protein with at least four membrane-spanning domains. VirB6 is postulated to function in T-pilus biogenesis and to be a component of the T4S apparatus. To identify amino acids required for VirB6 function, random mutations were introduced into virB6, and mutants that failed to complement a deletion in virB6 in tumour formation assays were isolated. Twenty-one non-functional mutants were identified, eleven of which had a point mutation that led to a substitution in a single amino acid. Characterization of the mutants indicated that the N-terminal large periplasmic domain and the transmembrane domain TM3 are required for VirB6 function. TM3 has an unusual sequence feature in that it is rich in bulky hydrophobic amino acids. This feature is found conserved in the VirB6 family of proteins. Studies on the effect of VirB6 on other VirB proteins showed that the octopine Ti-plasmid VirB6, unlike its nopaline Ti-plasmid counterpart, does not affect accumulation of VirB3 and VirB5, but has a strong negative effect on the accumulation of the VirB7-VirB7 dimer. Using indirect immunofluorescence microscopy the authors recently demonstrated that VirB6 localizes to a cell pole in a VirB-dependent manner. Mutations identified in the present study did not affect polar localization of the protein or the formation of the VirB7-VirB7 dimer. A VirB6-GFP fusion that contained the entire VirB6 ORF did not localize to a cell pole in either the presence or the absence of the other VirB proteins. IMF studies using dual labelling demonstrated that VirB6 colocalizes with VirB3 and VirB9, and not with VirB4, VirB5 and VirB11. These results support the conclusion that VirB6 is a structural component of the T4S apparatus.

  PublisherDOI

• The type IV secretion apparatus protein VirB6 of <i>Agrobacterium tumefaciens</i> localizes to a cell pole

  Molecular Microbiology · 2004-11-25 · 58 citations

  articleOpen accessSenior authorCorresponding

  Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus for the transfer of DNA and proteins to plant cells. To study the role of the VirB6 protein in the assembly and function of the type IV apparatus, we determined its subcellular location by immunofluorescence microscopy. In wild-type bacteria VirB6 localized to the cell poles but in the absence of the tumour-inducing plasmid it localized to random sites on the cell membranes. Five of the 11 VirB proteins, VirB7-VirB11, are required for the polar localization of VirB6. We identified two regions of VirB6, a conserved tryptophan residue at position 197 and the extreme C-terminus, that are essential for its polar localization. Topology determination by PhoA fusion analysis placed both regions in the cell cytoplasm. Alteration of tryptophan 197 or the deletion of the extreme C-terminus led to the mislocalization of the mutant protein. The mutations abolished the DNA transfer function of the protein as well. The C-terminus of VirB6, in silico, can form an amphipathic helix that may encode a protein-protein interaction domain essential for targeting the protein to a cell pole. We previously reported that another DNA transfer protein, VirD4, localizes to a cell pole. To determine whether VirB6 and VirD4 localize to the same pole, we performed colocalization experiments. Both proteins localized to the same pole indicating that VirB6 and VirD4 are in close proximity and VirB6 is probably a component of the transport apparatus.

  PublisherOA PDFDOI

• Polar location and functional domains of the <i>Agrobacterium tumefaciens</i> DNA transfer protein VirD4

  Molecular Microbiology · 2002-03-01 · 93 citations

  articleSenior authorCorresponding

  Agrobacterium tumefaciens VirD4 is essential for DNA transfer to plants. VirD4 presumably functions as a coupling factor that facilitates communication between a substrate and the transport pore. To serve as a coupling protein, VirD4 may be required to localize near the transport apparatus. In a previous study, we observed that several constituents of the transport apparatus localize to the cell membranes. In this study, we demonstrate that VirD4 has a unique cellular location. In immunofluorescence microscopy, cells probed with anti-VirD4 antibodies had foci of fluorescence primarily at the cell poles, indicating that VirD4 localizes to the cell pole. Polar location of VirD4 was not dependent on T-DNA processing, the formation of the transport apparatus and the presence of other Vir proteins. VirD4 is an integral membrane protein with one periplasmic domain. The large cytoplasmic region contains a nucleotide-binding domain. To investigate the role of these domains in DNA transfer, we introduced mutations in virD4 and studied the effect of a mutation on substrate transfer. A deletion of most of the periplasmic domain as well as the alterations of glycine 151 to serine and lysine 152 to alanine led to the complete loss of DNA transfer, indicating that both domains are essential for substrate transfer. Subcellular localization of the mutant proteins indicated that both the periplasmic and the nucleotide-binding domains are required for polar localization of VirD4. The periplasmic domain mutant VirD4Delta36-61 was distributed throughout the cell membrane, whereas the nucleotide binding site mutant VirD4G151S localized to sites other than the cell poles. Polar location of VirD4 suggests a role for the cell pole in DNA transfer.

  PublisherDOI

• Functional Analysis of the<i>Agrobacterium tumefaciens</i>T-DNA Transport Pore Protein VirB8

  Journal of Bacteriology · 2001-06-15 · 33 citations

  articleOpen accessSenior author

  The VirB8 protein of Agrobacterium tumefaciens is essential for DNA transfer to plants. VirB8, a 237-residue polypeptide, is an integral membrane protein with a short N-terminal cytoplasmic domain. It interacts with two transport pore proteins, VirB9 and VirB10, in addition to itself. To study the role of these interactions in DNA transfer and to identify essential amino acids of VirB8, we introduced random mutations in virB8 by the mutagenic PCR method. The putative mutants were tested for VirB8 function by the ability to complement a virB8 deletion mutant in tumor formation assays. After multiple rounds of screening 13 mutants that failed to complement the virB8 deletion mutation were identified. Analysis of the mutant strains by DNA sequence analysis, Western blot assays, and reconstruction of new point mutations led to the identification of five amino acid residues that are essential for VirB8 function. The substitution of glycine-78 to serine, serine-87 to leucine, alanine-100 to valine, arginine-107 to proline or alanine, and threonine-192 to methionine led to the loss of VirB8 activity. When introduced into the wild-type strain, virB8(S87L) partially suppressed the tumor forming ability of the wild-type protein. Analysis of protein-protein interaction by the yeast two-hybrid assay indicated that VirB8(R107P) is defective in interactions with both VirB9 and VirB10. A second mutant VirB8(S87L) is defective in interaction with VirB9.

  PublisherOA PDFDOI

• Genetic Transformation of<i>Coccidioides immitis</i>Facilitated by<i>Agrobacterium tumefaciens</i>

  The Journal of Infectious Diseases · 2000-06-01 · 125 citations

  articleOpen access

  Agrobacterium tumefaciens was used to facilitate genetic transformation of Coccidioides immitis. A gene cassette containing the gene encoding hygromycin phosphotransferase (hph) was cloned into a T-DNA vector plasmid and introduced into A. tumefaciens, and the resultant strain was used for cocultivation with germinated arthroconidia. This procedure produced numerous colonies 60- to >500-fold more resistant to hygromycin than untransformed mycelia. Both polymerase chain reaction and Southern blot analysis of the transformants indicated that all contained hph, usually as a single genomic copy. A transformation frequency of 1 per 10(5) arthroconidia was obtained by varying the germination time prior to cocultivation and altering the bacterium: fungus ratio. This approach requires no special equipment that might complicate biocontainment. Furthermore, transformation does not require digestion of fungal cell walls, further simplifying this procedure. A. tumefaciens-facilitated transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for C. immitis and perhaps other fungi of medical importance.

  PublisherOA PDFDOI

• The<i>Agrobacterium</i>T-DNA Transport Pore Proteins VirB8, VirB9, and VirB10 Interact with One Another

  Journal of Bacteriology · 2000-02-01 · 123 citations

  articleOpen access1st authorCorresponding

  The VirB proteins of Agrobacterium tumefaciens form a transport pore to transfer DNA from bacteria to plants. The assembly of the transport pore will require interaction among the constituent proteins. The identification of proteins that interact with one another can provide clues to the assembly of the transport pore. We studied interaction among four putative transport pore proteins, VirB7, VirB8, VirB9 and VirB10. Using the yeast two-hybrid assay, we observed that VirB8, VirB9, and VirB10 interact with one another. In vitro studies using protein fusions demonstrated that VirB10 interacts with VirB9 and itself. These results suggest that the outer membrane VirB7-VirB9 complex interacts with the inner membrane proteins VirB8 and VirB10 for the assembly of the transport pore. Fusions that contain small, defined segments of the proteins were used to define the interaction domains of VirB8 and VirB9. All interaction domains of both proteins mapped to the N-terminal half of the proteins. Two separate domains at the N- and C-terminal ends of VirB9 are involved in its homotypic interaction, suggesting that VirB9 forms a higher oligomer. We observed that the alteration of serine at position 87 of VirB8 to leucine abolished its DNA transfer function. Studies on the interaction of the mutant protein with the other VirB proteins showed that the VirB8S87L mutant is defective in interaction with VirB9. The mutant, however, interacted efficiently with VirB8 and VirB10, suggesting that the VirB8-VirB9 interaction is essential for DNA transfer.

  PublisherOA PDFDOI

Frequent coauthors

• Renu B. Kumar

  University of Minnesota

  8 shared

• Gregory J. Pazour

  University of Massachusetts Chan Medical School

  8 shared

• A M Vogel

  University of Minnesota

  4 shared

• John N. Galgiani

  University of Arizona

  3 shared

• Raed AbuOdeh

  University of Sharjah

  3 shared

• Paul K. Judd

  University of Minnesota

  3 shared

• Yong-Hong Xie

  Institute of Molecular Biology and Biophysics

  2 shared

• Christopher N. Ta

  Smith-Kettlewell Eye Research Institute

  2 shared

Labs

• Viral Innovation CorePI

Awards & honors

• Dr. James E. Rubin Medical Memorial Award
• Graduating Medical Student Research Award
• Veneziale-Steer Award
• Dr. Marvin and Hadassah Bacaner Research Awards
• Schmidt Steer Award