scholarly journals The Agrobacterium tumefaciens CheY-like protein ClaR regulates biofilm formation

Microbiology ◽  
2017 ◽  
Vol 163 (11) ◽  
pp. 1680-1691 ◽  
Author(s):  
Nathan Feirer ◽  
DohHyun Kim ◽  
Jing Xu ◽  
Nico Fernandez ◽  
Christopher M. Waters ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Biofilm Formation

scholarly journals Spermidine Inversely Influences Surface Interactions and Planktonic Growth in Agrobacterium tumefaciens

2016 ◽  
Vol 198 (19) ◽  
pp. 2682-2691 ◽  
Author(s):  
Yi Wang ◽  
Sok Ho Kim ◽  
Ramya Natarajan ◽  
Jason E. Heindl ◽  
Eric L. Bruger ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Biofilm Formation ◽  
Direct Synthesis ◽  
Wild Type ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Exogenous Addition ◽  
In The Wild ◽  
Growth Requirement ◽  
Transposon Insertions

ABSTRACTIn bacteria, the functions of polyamines, small linear polycations, are poorly defined, but these metabolites can influence biofilm formation in several systems. Transposon insertions in an ornithine decarboxylase (odc) gene inAgrobacterium tumefaciens, predicted to direct synthesis of the polyamine putrescine from ornithine, resulted in elevated cellulose. Null mutants forodcgrew somewhat slowly in a polyamine-free medium but exhibited increased biofilm formation that was dependent on cellulose production. Spermidine is an essential metabolite inA. tumefaciensand is synthesized from putrescine inA. tumefaciensvia the stepwise actions of carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase (CASDC). Exogenous addition of either putrescine or spermidine to theodcmutant returned biofilm formation to wild-type levels. Low levels of exogenous spermidine restored growth to CASDH and CASDC mutants, facilitating weak biofilm formation, but this was dampened with increasing concentrations. Norspermidine rescued growth for theodc, CASDH, and CASDC mutants but did not significantly affect their biofilm phenotypes, whereas in the wild type, it stimulated biofilm formation and depressed spermidine levels. Theodcmutant produced elevated levels of cyclic diguanylate monophosphate (c-di-GMP), exogenous polyamines modulated these levels, and expression of a c-di-GMP phosphodiesterase reversed the enhanced biofilm formation. Prior work revealed accumulation of the precursors putrescine and carboxyspermidine in the CASDH and CASDC mutants, respectively, but unexpectedly, both mutants accumulated homospermidine; here, we show that this requires a homospermidine synthase (hss) homologue.IMPORTANCEPolyamines are small, positively charged metabolites that are nearly ubiquitous in cellular life. They are often essential in eukaryotes and more variably in bacteria. Polyamines have been reported to influence the surface-attached biofilm formation of several bacteria. InAgrobacterium tumefaciens, mutants with diminished levels of the polyamine spermidine are stimulated for biofilm formation, and exogenous provision of spermidine decreases biofilm formation. Spermidine is also essential forA. tumefaciensgrowth, but the related polyamine norspermidine exogenously rescues growth and does not diminish biofilm formation, revealing that the growth requirement and biofilm control are separable. Polyamine control of biofilm formation appears to function via effects on the cellular second messenger cyclic diguanylate monophosphate, regulating the transition from a free-living to a surface-attached lifestyle.

scholarly journals Agrobacterium tumefaciens ExoR represses succinoglycan biosynthesis and is required for biofilm formation and motility

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2670-2681 ◽  
Author(s):  
Amelia D. Tomlinson ◽  
Bronwyn Ramey-Hartung ◽  
Travis W. Day ◽  
Peter M. Merritt ◽  
Clay Fuqua
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Biofilm Formation ◽  
Sinorhizobium Meliloti ◽  
Plant Tissues ◽  
Flagellar Motility ◽  
Regulatory Pathway ◽  
Flow Conditions ◽  
Two Component System ◽  
Form Complex ◽  
Abiotic Surfaces

The ubiquitous plant pathogen Agrobacterium tumefaciens attaches efficiently to plant tissues and abiotic surfaces and can form complex biofilms. A genetic screen for mutants unable to form biofilms on PVC identified disruptions in a homologue of the exoR gene. ExoR is a predicted periplasmic protein, originally identified in Sinorhizobium meliloti, but widely conserved among alphaproteobacteria. Disruptions in the A. tumefaciens exoR gene result in severely compromised attachment to abiotic surfaces under static and flow conditions, and to plant tissues. These mutants are hypermucoid due to elevated production of the exopolysaccharide succinoglycan, via derepression of the exo genes that direct succinoglycan synthesis. In addition, exoR mutants have lost flagellar motility, do not synthesize detectable flagellin and are diminished in flagellar gene expression. The attachment deficiency is, however, complex and not solely attributable to succinoglycan overproduction or motility disruption. A. tumefaciens ExoR can function independently of the ChvG–ChvI two component system, implicated in ExoR-dependent regulation in S. meliloti. Mutations that suppress the exoR motility defect suggest a branched regulatory pathway controlling succinoglycan synthesis, motility and biofilm formation.

scholarly journals Arginine-Rich Small Proteins with a Domain of Unknown Function, DUF1127, Play a Role in Phosphate and Carbon Metabolism of Agrobacterium tumefaciens

2020 ◽  
Vol 202 (22) ◽  
Author(s):  
Alexander Kraus ◽  
Mareen Weskamp ◽  
Jennifer Zierles ◽  
Miriam Balzer ◽  
Ramona Busch ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Carbon Metabolism ◽  
Unknown Function ◽  
Biofilm Formation ◽  
Nutrient Acquisition ◽  
Protein Profiling ◽  
Cell Physiology ◽  
Content Type ◽  
Small Proteins ◽  
Domain Of Unknown Function

ABSTRACT In any given organism, approximately one-third of all proteins have a yet-unknown function. A widely distributed domain of unknown function is DUF1127. Approximately 17,000 proteins with such an arginine-rich domain are found in 4,000 bacteria. Most of them are single-domain proteins, and a large fraction qualifies as small proteins with fewer than 50 amino acids. We systematically identified and characterized the seven DUF1127 members of the plant pathogen Agrobacterium tumefaciens. They all give rise to authentic proteins and are differentially expressed as shown at the RNA and protein levels. The seven proteins fall into two subclasses on the basis of their length, sequence, and reciprocal regulation by the LysR-type transcription factor LsrB. The absence of all three short DUF1127 proteins caused a striking phenotype in later growth phases and increased cell aggregation and biofilm formation. Protein profiling and transcriptome sequencing (RNA-seq) analysis of the wild type and triple mutant revealed a large number of differentially regulated genes in late exponential and stationary growth. The most affected genes are involved in phosphate uptake, glycine/serine homeostasis, and nitrate respiration. The results suggest a redundant function of the small DUF1127 paralogs in nutrient acquisition and central carbon metabolism of A. tumefaciens. They may be required for diauxic switching between carbon sources when sugar from the medium is depleted. We end by discussing how DUF1127 might confer such a global impact on cell physiology and gene expression. IMPORTANCE Despite being prevalent in numerous ecologically and clinically relevant bacterial species, the biological role of proteins with a domain of unknown function, DUF1127, is unclear. Experimental models are needed to approach their elusive function. We used the phytopathogen Agrobacterium tumefaciens, a natural genetic engineer that causes crown gall disease, and focused on its three small DUF1127 proteins. They have redundant and pervasive roles in nutrient acquisition, cellular metabolism, and biofilm formation. The study shows that small proteins have important previously missed biological functions. How small basic proteins can have such a broad impact is a fascinating prospect of future research.

scholarly journals A Pterin-Dependent Signaling Pathway Regulates a Dual-Function Diguanylate Cyclase-Phosphodiesterase Controlling Surface Attachment in Agrobacterium tumefaciens

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Nathan Feirer ◽  
Jing Xu ◽  
Kylie D. Allen ◽  
Benjamin J. Koestler ◽  
Eric L. Bruger ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Signaling Pathway ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Dominant Role ◽  
Second Messenger ◽  
Dual Function ◽  
Diguanylate Cyclase ◽  
Cyclic Diguanylate ◽  
Content Type

ABSTRACTThe motile-to-sessile transition is an important lifestyle switch in diverse bacteria and is often regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). In general, high c-di-GMP concentrations promote attachment to surfaces, whereas cells with low levels of signal remain motile. In the plant pathogenAgrobacterium tumefaciens, c-di-GMP controls attachment and biofilm formation via regulation of a unipolar polysaccharide (UPP) adhesin. The levels of c-di-GMP inA. tumefaciensare controlled in part by the dual-function diguanylate cyclase-phosphodiesterase (DGC-PDE) protein DcpA. In this study, we report that DcpA possesses both c-di-GMP synthesizing and degrading activities in heterologous and native genetic backgrounds, a binary capability that is unusual among GGDEF-EAL domain-containing proteins. DcpA activity is modulated by a pteridine reductase called PruA, with DcpA acting as a PDE in the presence of PruA and a DGC in its absence. PruA enzymatic activity is required for the control of DcpA and through this control, attachment and biofilm formation. Intracellular pterin analysis demonstrates that PruA is responsible for the production of a novel pterin species. In addition, the control of DcpA activity also requires PruR, a protein encoded directly upstream of DcpA with a predicted molybdopterin-binding domain. PruR is hypothesized to be a potential signaling intermediate between PruA and DcpA through an as-yet-unidentified mechanism. This study provides the first prokaryotic example of a pterin-mediated signaling pathway and a new model for the regulation of dual-function DGC-PDE proteins.IMPORTANCEPathogenic bacteria often attach to surfaces and form multicellular communities called biofilms. Biofilms are inherently resilient and can be difficult to treat, resisting common antimicrobials. Understanding how bacterial cells transition to the biofilm lifestyle is essential in developing new therapeutic strategies. We have characterized a novel signaling pathway that plays a dominant role in the regulation of biofilm formation in the model pathogenAgrobacterium tumefaciens. This control pathway involves small metabolites called pterins, well studied in eukaryotes, but this is the first example of pterin-dependent signaling in bacteria. The described pathway controls levels of an important intracellular second messenger (cyclic diguanylate monophosphate) that regulates key bacterial processes such as biofilm formation, motility, and virulence. Pterins control the balance of activity for an enzyme that both synthesizes and degrades the second messenger. These findings reveal a complex, multistep pathway that modulates this enzyme, possibly identifying new targets for antibacterial intervention.

scholarly journals Biofilm Formation of Agrobacterium tumefaciens on Abiotic Surface is Independent of Cellulose Synthesizing Gene

2015 ◽  
Vol 15 (2) ◽  
pp. 41-46
Author(s):  
Reshma Tuladhar
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Biofilm Formation ◽  
Quantitative Estimation ◽  
Science And Technology ◽  
Extracellular Polysaccharide ◽  
Cellulose Synthesis ◽  
Normal Strain ◽  
Abiotic Surface ◽  
Surface Attachment ◽  
Cover Slip

Surface attachment is indispensable in the process of biofilm formation in Agrobacterium tumefaciens, which has been attributed to the production of extracellular polysaccharide. Adherence of bacteria on the plant was expected to be strong with cellulose synthesis. However, its role in the biofilm formation on abiotic surface is not known.  Biofilm cover-slip assay was carried to estimate the biofilm formation. Biofilm formation was not reduced by truncating ellulose synthesis gene. The celCD deleted strain produced biofilm slightly greater than normal strain NTL4 of A. tumefaciens. Besides, production of extracellular polysaccharide was not reduced in the cellulose gene deleted strain NTL4 celCD. Quantitative estimation showed increased exo-polysaccharide in celCD deleted strain. Abolishing the cellulose synthesis property by truncating the cellulose gene did not affect biofilm formation in abiotic surface. In  fact cellulose might be repressing biosynthesis of other polysaccharides responsible in biofilm formation.DOI: http://dx.doi.org/njst.v15i2.12112Nepal Journal of Science and Technology Vol. 15, No.2 (2014) 41-46

scholarly journals Motility and Chemotaxis in Agrobacterium tumefaciens Surface Attachment and Biofilm Formation

2007 ◽  
Vol 189 (22) ◽  
pp. 8005-8014 ◽  
Author(s):  
Peter M. Merritt ◽  
Thomas Danhorn ◽  
Clay Fuqua
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Wild Type ◽  
Swarming Motility ◽  
Surface Attachment ◽  
Directed Motility ◽  
Suppressor Mutants ◽  
Static Conditions ◽  
Specific Deletion

ABSTRACT Bacterial motility mechanisms, including swimming, swarming, and twitching, are known to have important roles in biofilm formation, including colonization and the subsequent expansion into mature structured surface communities. Directed motility requires chemotaxis functions that are conserved among many bacterial species. The biofilm-forming plant pathogen Agrobacterium tumefaciens drives swimming motility by utilizing a small group of flagella localized to a single pole or the subpolar region of the cell. There is no evidence for twitching or swarming motility in A. tumefaciens. Site-specific deletion mutations that resulted in either aflagellate, flagellated but nonmotile, or flagellated but nonchemotactic A. tumefaciens derivatives were examined for biofilm formation under static and flowing conditions. Nonmotile mutants were significantly deficient in biofilm formation under static conditions. Under flowing conditions, however, the aflagellate mutant rapidly formed aberrantly dense, tall biofilms. In contrast, a nonmotile mutant with unpowered flagella was clearly debilitated for biofilm formation relative to the wild type. A nontumbling chemotaxis mutant was only weakly affected with regard to biofilm formation under nonflowing conditions but was notably compromised in flow, generating less adherent biomass than the wild type, with a more dispersed cellular arrangement. Extragenic suppressor mutants of the chemotaxis-impaired, straight-swimming phenotype were readily isolated from motility agar plates. These mutants regained tumbling at a frequency similar to that of the wild type. Despite this phenotype, biofilm formation by the suppressor mutants in static cultures was significantly deficient. Under flowing conditions, a representative suppressor mutant manifested a phenotype similar to yet distinct from that of its nonchemotactic parent.

scholarly journals Enzymatic and Mutational Analysis of the PruA Pteridine Reductase Required for Pterin-Dependent Control of Biofilm Formation in Agrobacterium tumefaciens

2020 ◽  
Vol 202 (16) ◽  
Author(s):  
Monica Labine ◽  
Lisa DePledge ◽  
Nathan Feirer ◽  
Jennifer Greenwich ◽  
Clay Fuqua ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Signaling Pathway ◽  
Plant Pathogen ◽  
Biofilm Formation ◽  
Mutational Analysis ◽  
Reductase Activity ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Control Surface ◽  
Content Type

ABSTRACT Pterins are ubiquitous biomolecules with diverse functions, including roles as cofactors, pigments, and redox mediators. Recently, a novel pterin-dependent signaling pathway that controls biofilm formation was identified in the plant pathogen Agrobacterium tumefaciens. A key player in this pathway is a pteridine reductase, termed PruA, where its enzymatic activity has been shown to control surface attachment and limit biofilm formation. Here, we biochemically characterized PruA to investigate the catalytic properties and the substrate specificity of this pteridine reductase. PruA demonstrated maximal catalytic efficiency with dihydrobiopterin and comparable activities with the stereoisomers dihydromonapterin and dihydroneopterin. Since A. tumefaciens does not synthesize or utilize biopterins, the likely physiological substrate is dihydromonapterin or dihydroneopterin or both. Notably, PruA did not exhibit pteridine reductase activity with dihydrofolate or fully oxidized pterins. Site-directed mutagenesis studies of a conserved tyrosine residue, the key component of a putative catalytic triad, indicated that this tyrosine is not directly involved in PruA catalysis but may be important for substrate or cofactor binding. Additionally, mutagenesis of the arginine residue in the N-terminal TGX3RXG motif significantly reduced the catalytic efficiency of PruA, supporting its proposed role in pterin binding and catalysis. Finally, we report on the enzymatic characterization of PruA homologs from Pseudomonas aeruginosa and Brucella abortus, thus expanding the roles and potential significance of pteridine reductases in diverse bacteria. IMPORTANCE Biofilms are complex multicellular communities that are formed by diverse bacteria. In the plant pathogen Agrobacterium tumefaciens, the transition from a free-living motile state to a nonmotile biofilm state is governed by a novel signaling pathway involving small molecules called pterins. The involvement of pterins in biofilm formation is unexpected and prompts many questions about the molecular details of this pathway. This work biochemically characterized the PruA pteridine reductase involved in the signaling pathway to reveal its enzymatic properties and substrate preference, thus providing important insight into pterin biosynthesis and its role in A. tumefaciens biofilm control. Additionally, the enzymatic characteristics of related pteridine reductases from mammalian pathogens were examined to uncover potential roles of these enzymes in other bacteria.

scholarly journals The Effect of Cellulose Overproduction on Binding and Biofilm Formation on Roots by Agrobacterium tumefaciens

2005 ◽  
Vol 18 (9) ◽  
pp. 1002-1010 ◽  
Author(s):  
Ann G. Matthysse ◽  
Mazz Marry ◽  
Leonard Krall ◽  
Mitchell Kaye ◽  
Bronwyn E. Ramey ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Biofilm Formation ◽  
Root Colonization ◽  
Viable Cell ◽  
Bacterial Attachment ◽  
Chemical Fractionation ◽  
Wild Type ◽  
Ciona Savignyi ◽  
Cellulose Synthase Gene ◽  
Plant Surfaces

Agrobacterium tumefaciens growing in liquid attaches to the surface of tomato and Arabidopsis thaliana roots, forming a biofilm. The bacteria also colonize roots grown in sterile quartz sand. Attachment, root colonization, and biofilm formation all were markedly reduced in celA and chvB mutants, deficient in production of cellulose and cyclic β-(1,2)-D-glucans, respectively. We have identified two genes (celG and celI) in which mutations result in the overproduction of cellulose as judged by chemical fractionation and methylation analysis. Wild-type and chvB mutant strains carrying a cDNA clone of a cellulose synthase gene from the marine urochordate Ciona savignyi also overproduced cellulose. The overproduction in a wild-type strain resulted in increased biofilm formation on roots, as evaluated by light microscopy, and levels of root colonization intermediate between those of cellulose-minus mutants and the wild type. Overproduction of cellulose by a nonattaching chvB mutant restored biofilm formation and bacterial attachment in microscopic and viable cell count assays and partially restored root colonization. Although attachment to plant surfaces was restored, overproduction of cellulose did not restore virulence in the chvB mutant strain, suggesting that simple bacterial binding to plant surfaces is not sufficient for pathogenesis.

scholarly journals CelR, an Ortholog of the Diguanylate Cyclase PleD of Caulobacter, Regulates Cellulose Synthesis in Agrobacterium tumefaciens

2013 ◽  
Vol 79 (23) ◽  
pp. 7188-7202 ◽  
Author(s):  
D. Michael Barnhart ◽  
Shengchang Su ◽  
Brenna E. Baccaro ◽  
Lois M. Banta ◽  
Stephen K. Farrand
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Cellulose Synthesis ◽  
Diguanylate Cyclase ◽  
Cellulose Biosynthesis ◽  
Plant Host ◽  
Cellulose Production ◽  
Signal Molecule ◽  
Content Type

ABSTRACTCellulose fibrils play a role in attachment ofAgrobacterium tumefaciensto its plant host. While the genes for cellulose biosynthesis in the bacterium have been identified, little is known concerning the regulation of the process. The signal molecule cyclic di-GMP (c-di-GMP) has been linked to the regulation of exopolysaccharide biosynthesis in many bacterial species, includingA. tumefaciens. In this study, we identified two putative diguanylate cyclase genes,celR(atu1297) andatu1060, that influence production of cellulose inA. tumefaciens. Overexpression of either gene resulted in increased cellulose production, while deletion ofcelR, but notatu1060, resulted in decreased cellulose biosynthesis.celRoverexpression also affected other phenotypes, including biofilm formation, formation of a polar adhesion structure, plant surface attachment, and virulence, suggesting that the gene plays a role in regulating these processes. Analysis ofcelRand Δcelmutants allowed differentiation between phenotypes associated with cellulose production, such as biofilm formation, and phenotypes probably resulting from c-di-GMP signaling, which include polar adhesion, attachment to plant tissue, and virulence. Phylogenetic comparisons suggest that species containing bothcelRandcelA, which encodes the catalytic subunit of cellulose synthase, adapted the CelR protein to regulate cellulose production while those that lackcelAuse CelR, called PleD, to regulate specific processes associated with polar localization and cell division.

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