Discrete Responses to Limitation for Iron and Manganese in Agrobacterium tumefaciens: Influence on Attachment and Biofilm Formation

ABSTRACTTransition metals such as iron and manganese are crucial trace nutrients for the growth of most bacteria, functioning as catalytic cofactors for many essential enzymes. Dedicated uptake and regulatory systems have evolved to ensure their acquisition for growth, while preventing toxicity. Transcriptomic analysis of the iron- and manganese-responsive regulons ofAgrobacterium tumefaciensrevealed that there are discrete regulatory networks that respond to changes in iron and manganese levels. Complementing earlier studies, the iron-responsive gene network is quite large and includes many aspects of iron-dependent metabolism and the iron-sparing response. In contrast, the manganese-responsive network is restricted to a limited number of genes, many of which can be linked to transport and utilization of the transition metal. Several of the target genes predicted to drive manganese uptake are required for growth under manganese-limited conditions, and anA. tumefaciensmutant with a manganese transport deficiency is attenuated for plant virulence. Iron and manganese limitation independently inhibit biofilm formation byA. tumefaciens, and several candidate genes that could impact biofilm formation were identified in each regulon. The biofilm-inhibitory effects of iron and manganese do not rely on recognized metal-responsive transcriptional regulators, suggesting alternate mechanisms influencing biofilm formation. However, under low-manganese conditions thedcpAoperon is upregulated, encoding a system that controls levels of the cyclic di-GMP second messenger. Mutation of this regulatory pathway dampens the effect of manganese limitation.IMPORTANCEResponses to changes in transition metal levels, such as those of manganese and iron, are important for normal metabolism and growth in bacteria. Our study used global gene expression profiling to understand the response of the plant pathogenAgrobacterium tumefaciensto changes of transition metal availability. Among the properties that are affected by both iron and manganese levels are those required for normal surface attachment and biofilm formation, but the requirement for each of these transition metals is mechanistically independent from the other.

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Spermidine Inversely Influences Surface Interactions and Planktonic Growth in Agrobacterium tumefaciens

Journal of Bacteriology ◽

10.1128/jb.00265-16 ◽

2016 ◽

Vol 198 (19) ◽

pp. 2682-2691 ◽

Cited By ~ 15

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.

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

Journal of Bacteriology ◽

10.1128/jb.00309-20 ◽

2020 ◽

Vol 202 (22) ◽

Cited By ~ 1

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.

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

mBio ◽

10.1128/mbio.00156-15 ◽

2015 ◽

Vol 6 (4) ◽

Cited By ~ 28

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.

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Identification of c-di-GMP/FleQ-Regulated New Target Genes, Including cyaA, Encoding Adenylate Cyclase, in Pseudomonas putida

mSystems ◽

10.1128/msystems.00295-21 ◽

2021 ◽

Vol 6 (3) ◽

Author(s):

Yujie Xiao ◽

Haozhe Chen ◽

Liang Nie ◽

Meina He ◽

Qi Peng ◽

...

Keyword(s):

Adenylate Cyclase ◽

Biofilm Formation ◽

Target Genes ◽

Second Messenger ◽

Transcriptional Level ◽

Camp Content ◽

Membrane Pore ◽

Content Type

ABSTRACT The bacterial second messenger cyclic diguanylate (c-di-GMP) modulates plankton-to-biofilm lifestyle transition of Pseudomonas species through its transcriptional regulatory effector FleQ. FleQ regulates transcription of biofilm- and flagellum-related genes in response to c-di-GMP. Through transcriptomic analysis and FleQ-DNA binding assay, this study identified five new target genes of c-di-GMP/FleQ in P. putida, including PP_0681, PP_0788, PP_4519 (lapE), PP_5222 (cyaA), and PP_5586. Except lapE encoding an outer membrane pore protein and cyaA encoding an adenylate cyclase, the functions of the other three genes encoding hypothetical proteins remain unknown. FleQ and c-di-GMP coordinately inhibit transcription of PP_0788 and cyaA and promote transcription of PP_0681, lapE, and PP_5586. Both in vitro and in vivo assays show that FleQ binds directly to promoters of the five genes. Further analyses confirm that LapE plays a central role of in the secretion of adhesin LapA and that c-di-GMP/FleQ increases lapE transcription, thereby promoting adhesin secretion and biofilm formation. The adenylate cyclase CyaA is responsible for synthesis of another second messenger, cyclic AMP (cAMP). FleQ and c-di-GMP coordinate to decrease the content of cAMP, suggesting that c-di-GMP and FleQ coregulate cAMP by modulating cyaA expression. Overall, this study adds five new members to the c-di-GMP/FleQ-regulated gene family and reveals the role of c-di-GMP/FleQ in LapA secretion and cAMP synthesis regulation in P. putida. IMPORTANCE c-di-GMP/FleQ promotes the plankton-to-biofilm lifestyle transition at the transcriptional level via FleQ in Pseudomonas species. Identification of new target genes directly regulated by c-di-GMP/FleQ helps to broaden the knowledge of c-di-GMP/FleQ-mediated transcriptional regulation. Regulation of lapE by c-di-GMP/FleQ guarantees highly efficient LapA secretion and biofilm formation. The mechanism of negative correlation between c-di-GMP and cAMP in both P. putida and P. aeruginosa remains unknown. Our result concerning transcriptional inhibition of cyaA by c-di-GMP/FleQ reveals the mechanism underlying the decrease of cAMP content by c-di-GMP in P. putida.

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

Journal of Bacteriology ◽

10.1128/jb.00098-20 ◽

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02148-13 ◽

2013 ◽

Vol 79 (23) ◽

pp. 7188-7202 ◽

Cited By ~ 23

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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Intercalation of First Row Transition Metals inside Covalent-Organic Frameworks (COF): a Strategy to Fine Tune the Electronic Properties of Porous Crystalline Materials

10.26434/chemrxiv.6712826 ◽

2018 ◽

Author(s):

Srimanta Pakhira ◽

Jose Mendoza-Cortes

Keyword(s):

Transition Metal ◽

Transition Metals ◽

Electronic Properties ◽

High Surface ◽

Electronic Devices ◽

High Surface Area ◽

Main Role ◽

Covalent Organic Frameworks ◽

Porous Network ◽

Crystalline Materials

<div>Covalent organic frameworks (COFs) have emerged as an important class of nano-porous crystalline materials with many potential applications. They are intriguing platforms for the design of porous skeletons with special functionality at the molecular level. However, despite their extraordinary properties, it is difficult to control their electronic properties, thus hindering the potential implementation in electronic devices. A new form of nanoporous material, COFs intercalated with first row transition metal is proposed to address this fundamental drawback - the lack of electronic tunability. Using first-principles calculations, we have designed 31 new COF materials <i>in-silico</i> by intercalating all of the first row transition metals (TMs) with boroxine-linked and triazine-linked COFs: COF-TM-x (where TM=Sc-Zn and x=3-5). This is a significant addition considering that only 187 experimentally COFs structures has been reported and characterized so far. We have investigated their structure and electronic properties. Specifically, we predict that COF's band gap and density of states (DOSs) can be controlled by intercalating first row transition metal atoms (TM: Sc - Zn) and fine tuned by the concentration of TMs. We also found that the $d$-subshell electron density of the TMs plays the main role in determining the electronic properties of the COFs. Thus intercalated-COFs provide a new strategy to control the electronic properties of materials within a porous network. This work opens up new avenues for the design of TM-intercalated materials with promising future applications in nanoporous electronic devices, where a high surface area coupled with fine-tuned electronic properties are desired.</div>

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Bond-length distributions for ions bonded to oxygen: Metalloids and post-transition metals

10.26434/chemrxiv.5410921.v2 ◽

2017 ◽

Cited By ~ 1

Author(s):

Olivier Charles Gagné ◽

Frank Christopher Hawthorne

Keyword(s):

Transition Metal ◽

Transition Metals ◽

Metal Ions ◽

Bond Length ◽

Lone Pair ◽

Transition Metal Ions ◽

Oxygen Lone Pair

Bond-length distributions are examined for thirty-three configurations of the metalloid ions and fifty-six configurations of the post-transition-metal ions bonded to oxygen. Lone-pair stereoactivity is discussed.

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Bond-length distributions for ions bonded to oxygen: Metalloids and post-transition metals

10.26434/chemrxiv.5410921 ◽

2017 ◽

Cited By ~ 1

Author(s):

Olivier Charles Gagné ◽

Frank Christopher Hawthorne

Keyword(s):

Transition Metal ◽

Transition Metals ◽

Metal Ions ◽

Bond Length ◽

Lone Pair ◽

Transition Metal Ions ◽

Oxygen Lone Pair

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Electronic properties of quasi two-dimensional transition metals chalcogenides with low-dimensional magnetism

Doklady BGUIR ◽

10.35596/1729-7648-2020-18-7-87-95 ◽

2020 ◽

Vol 18 (7) ◽

pp. 87-95

Author(s):

M. S. Baranava ◽

P. A. Praskurava

Keyword(s):

Transition Metal ◽

Transition Metals ◽

Exchange Interaction ◽

Electronic Properties ◽

Metal Atom ◽

Transition Metal Atom ◽

Two Dimensional ◽

Transition Metal Atoms ◽

Ground Magnetic ◽

Low Dimensional

The search for fundamental physical laws which lead to stable high-temperature ferromagnetism is an urgent task. In addition to the already synthesized two-dimensional materials, there remains a wide list of possible structures, the stability of which is predicted theoretically. The article suggests the results of studying the electronic properties of MAX3 (M = Cr, Fe, A = Ge, Si, X = S, Se, Te) transition metals based compounds with nanostructured magnetism. The research was carried out using quantum mechanical simulation in specialized VASP software and calculations within the Heisenberg model. The ground magnetic states of twodimensional MAX3 and the corresponding energy band structures are determined. We found that among the systems under study, CrGeTe3 is a semiconductor nanosized ferromagnet. In addition, one is a semiconductor with a bandgap of 0.35 eV. Other materials are antiferromagnetic. The magnetic moment in MAX3 is localized on the transition metal atoms: in particular, the main one on the d-orbital of the transition metal atom (and only a small part on the p-orbital of the chalcogen). For CrGeTe3, the exchange interaction integral is calculated. The mechanisms of the formation of magnetic order was established. According to the obtained exchange interaction integrals, a strong ferromagnetic order is formed in the semiconductor plane. The distribution of the projection density of electronic states indicates hybridization between the d-orbital of the transition metal atom and the p-orbital of the chalcogen. The study revealed that the exchange interaction by the mechanism of superexchange is more probabilistic.

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