Larger Than Life: Isolation and Genomic Characterization of a Jumbo Phage That Infects the Bacterial Plant Pathogen, Agrobacterium tumefaciens

ABSTRACTMechanistic studies of many processes inAgrobacterium tumefacienshave been hampered by a lack of genetic tools for characterization of essential genes. In this study, we used a Tn7-based method for inducible control of transcription from an engineered site on the chromosome. We demonstrate that this method enables tighter control of inducible promoters than plasmid-based systems and can be used for depletion studies. The method enables the construction of depletion strains to characterize the roles of essential genes inA. tumefaciens. Here, we used the strategy to deplete the alphaproteobacterial master regulator CtrA and found that depletion of this essential gene results in dramatic rounding of cells, which become nonviable.IMPORTANCEAgrobacterium tumefaciensis a bacterial plant pathogen and natural genetic engineer. Thus, studies of essential processes, including cell cycle progression, DNA replication and segregation, cell growth, and division, may provide insights for limiting disease or improving biotechnology applications.

Download Full-text

Identification and Biochemical Characterization of an Acid Sphingomyelinase-Like Protein from the Bacterial Plant Pathogen Ralstonia solanacearum that Hydrolyzes ATP to AMP but Not Sphingomyelin to Ceramide

PLoS ONE ◽

10.1371/journal.pone.0105830 ◽

2014 ◽

Vol 9 (8) ◽

pp. e105830 ◽

Cited By ~ 6

Author(s):

Michael V. Airola ◽

Jessica M. Tumolo ◽

Justin Snider ◽

Yusuf A. Hannun

Keyword(s):

Plant Pathogen ◽

Ralstonia Solanacearum ◽

Biochemical Characterization ◽

Acid Sphingomyelinase ◽

Bacterial Plant Pathogen

Download Full-text

Exploring the diverse functions of peptidoglycan hydrolases in the plant pathogen, Agrobacterium tumefaciens

10.32469/10355/75005 ◽

2019 ◽

Author(s):

◽

Wanda Melissa Figueroa-Cuilan

Keyword(s):

Cell Wall ◽

Cell Division ◽

Agrobacterium Tumefaciens ◽

Plant Pathogen ◽

Beta Lactam ◽

Peptidoglycan Hydrolases ◽

Cell Wall Hydrolases ◽

Cell Wall Hydrolysis

The plant pathogen Agrobacterium tumefaciens displays an atypical form of unipolar elongation, followed by incipient pole synthesis during cell division and cell separation. Currently, how polar growing bacteria modulate cell wall hydrolysis during growth and division remains largely unknown. This work includes the comprehensive analysis and characterization of the role of cell wall hydrolyses involved in bacterial growth, division, recycling and beta-lactam resistance in A. tumefaciens. First, we performed bioinformatic analyses and used reverse genetics to better understand the role cell wall hydrolases in A. tumefaciens. Inactivation of most cell wall hydrolases, led to no phenotypic defects suggesting a high degree of redundancy. However, inactivation of the amidase, AmiD, and the lytic transglycosylase Atu3779, revealed significant changes in beta-lactam resistance suggesting that these proteins are involved in the activation beta-lactamases and outer-membrane integrity. Next, we developed a tool (Figueroa-Cuilan et al., 2016) to dissect the role of essential genes, which enabled characterization of the essential regulator of cell division, DipM, a LytM-containing factor. Absence of DipM causes severe cell division defects, including increased cell length, mid-cell width and lysis. A cell wall composition analysis of cells devoid of DipM shows an increase in the activity of the PG hydrolases, lytic transglycosylases, suggesting that DipM may inhibit the activity of these enzymes. Lastly, we find that deletion of individual lytic transglycolsylases (LTs) from the DipM depletion strain delays the onset of the DipM depletion phenotype. Overall, this research provides mechanistic insights about the roles of peptidoglycan hydrolases and their regulators in cell growth and division. Understanding how bacterial cell wall hydrolysis is spatiotemporally regulated and coordinated with cell wall synthesis and cell division (Figueroa-Cuilan and Brown, 2018), will be applicable to other closely related polar-growing bacteria.

Download Full-text

Isolation and Characterization T4- and T7-Like Phages that Infect the Bacterial Plant Pathogen Agrobacterium tumefaciens

Viruses ◽

10.3390/v11060528 ◽

2019 ◽

Vol 11 (6) ◽

pp. 528 ◽

Cited By ~ 3

Author(s):

Hedieh Attai ◽

Pamela J.B. Brown

Keyword(s):

Waste Water ◽

Phylogenetic Analysis ◽

Agrobacterium Tumefaciens ◽

Plant Pathogen ◽

Open Reading Frames ◽

Whole Genome ◽

Plant Interactions ◽

Isolation And Characterization ◽

Phage P22 ◽

Bacterial Plant Pathogen

In the rhizosphere, bacteria–phage interactions are likely to have important impacts on the ecology of microbial communities and microbe–plant interactions. To better understand the dynamics of Agrobacteria–phage interactions, we have isolated diverse bacteriophages which infect the bacterial plant pathogen, Agrobacterium tumefaciens. Here, we complete the genomic characterization of Agrobacterium tumefaciens phages Atu_ph04 and Atu_ph08. Atu_ph04—a T4-like phage belonging to the Myoviridae family—was isolated from waste water and has a 143,349 bp genome that encodes 223 predicted open reading frames (ORFs). Based on phylogenetic analysis and whole-genome alignments, Atu_ph04 is a member of a newly described T4 superfamily that contains other Rhizobiales-infecting phages. Atu_ph08, a member of the Podoviridae T7-like family, was isolated from waste water, has a 59,034 bp genome, and encodes 75 ORFs. Based on phylogenetic analysis and whole-genome alignments, Atu_ph08 may form a new T7 superfamily which includes Sinorhizobium phage PCB5 and Ochrobactrum phage POI1126. Atu_ph08 is predicted to have lysogenic activity, as we found evidence of an integrase and several transcriptional repressors with similarity to proteins in transducing phage P22. Together, this data suggests that Agrobacterium phages are diverse in morphology, genomic content, and lifestyle.

Download Full-text

Biochemical Characteristics of the Novel Haloalkane Dehalogenase DatA, Isolated from the Plant PathogenAgrobacterium tumefaciensC58

Applied and Environmental Microbiology ◽

10.1128/aem.02109-10 ◽

2010 ◽

Vol 77 (5) ◽

pp. 1881-1884 ◽

Cited By ~ 26

Author(s):

Khomaini Hasan ◽

Andrea Fortova ◽

Tana Koudelakova ◽

Radka Chaloupkova ◽

Mayuko Ishitsuka ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Plant Pathogen ◽

Biochemical Characterization ◽

The Novel ◽

Haloalkane Dehalogenase ◽

Biochemical Characteristics ◽

Dimeric Structure ◽

Racemic Mixtures ◽

Cooperative Kinetics

ABSTRACTWe report the biochemical characterization of a novel haloalkane dehalogenase, DatA, isolated from the plant pathogenAgrobacterium tumefaciensC58. DatA possesses a peculiar pair of halide-stabilizing residues, Asn-Tyr, which have not been reported to play this role in other known haloalkane dehalogenases. DatA has a number of other unique characteristics, including substrate-dependent and cooperative kinetics, a dimeric structure, and excellent enantioselectivity toward racemic mixtures of chiral brominated alkanes and esters.

Download Full-text