Structural basis for a bacterial Pip system plant effector recognition protein.

A number of plant-associated proteobacteria have LuxR family transcription factors that we refer to as PipR subfamily members. PipR proteins play roles in interactions between bacteria and their plant hosts, and some are important for bacterial virulence of plants. We identified an ethanolamine derivative, N-(2-hydroxyethyl)-2-(2-hydroxyethylamino) acetamide (HEHEAA), as a potent effector of PipR-mediated gene regulation in the plant endophyte Pseudomonas GM79. HEHEAA-dependent PipR activity requires an ATP-binding cassette-type active transport system, and the periplasmic substrate-binding protein (SBP) of that system binds HEHEAA. To begin to understand the molecular basis of PipR system responses to plant factors we crystallized a HEHEAA-responsive SBP in the free- and HEHEAA-bound forms. The SBP, which is similar to peptide-binding SBPs, was in a closed conformation. A narrow cavity at the interface of its two lobes is wide enough to bind HEHEAA, but it cannot accommodate peptides with side chains. The polar atoms of HEHEAA are recognized by hydrogen-bonding interactions, and additional SBP residues contribute to the binding site. This binding mode was confirmed by a structure-based mutational analysis. We also show that a closely related SBP from the plant pathogen Pseudomonas syringae pv tomato DC3000 does not recognize HEHEAA. However, a single amino acid substitution in the presumed effector-binding pocket of the P. syringae SBP converted it to a weak HEHEAA-binding protein. The P. syringae PipR depends on a plant effector for activity, and our findings imply that different PipR-associated SBPs bind different effectors.

A plant-responsive bacterial-signaling system senses an ethanolamine derivative.

Certain plant-associated Proteobacteria sense their host environment by detecting an unknown plant signal recognized by a member of a LuxR subfamily of transcription factors. This interkingdom communication is important for both mutualistic and pathogenic interactions. The Populus root endophyte Pseudomonas sp. GM79 possesses such a regulator, named PipR. In a previous study we reported that PipR activates an adjacent gene (pipA) coding for a proline iminopeptidase in response to Populus leaf macerates and peptides and that this activation is dependent on a putative ABC-type transporter [Schaefer AL, et al. (2016) mBio 7:e01101-16]. In this study we identify a chemical derived from ethanolamine that induces PipR activity at picomolar concentrations, and we present evidence that this is the active inducer present in plant leaf macerates. First, a screen of more than 750 compounds indicated ethanolamine was a potent inducer for the PipR-sensing system; however, ethanolamine failed to bind to the periplasmic-binding protein (PBP) required for the signal response. This led us to discover that a specific ethanolamine derivative, N-(2-hydroxyethyl)-2-(2-hydroxyethylamino) acetamide (HEHEAA), binds to the PBP and serves as a potent PipR-dependent inducer. We also show that a compound, which coelutes with HEHEAA in HPLC and induces pipA gene expression in a PipR-dependent manner, can be found in Populus leaf macerates. This work sheds light on how plant-associated bacteria can sense their environment and on the nature of inducers for a family of plant-responsive LuxR-like transcription factors found in plant-associated bacteria.

An aryl-homoserine lactone quorum-sensing signal produced by a dimorphic prosthecate bacterium.

Many species of Proteobacteria produce acyl-homoserine lactone (AHL) compounds as quorum-sensing (QS) signals for cell density-dependent gene regulation. Most known AHL synthases, LuxI-type enzymes, produce fatty AHLs, and the fatty acid moiety is derived from an acyl-acyl carrier protein (ACP) intermediate in fatty acid biosynthesis. Recently, a class of LuxI homologs has been shown to use CoA-linked aromatic or amino acid substrates for AHL synthesis. By using an informatics approach, we found the CoA class of LuxI homologs exists primarily in α-Proteobacteria. The genome of Prosthecomicrobium hirschii, a dimorphic prosthecate bacterium, possesses a luxI-like AHL synthase gene that we predicted to encode a CoA-utilizing enzyme. We show the P. hirschii LuxI homolog catalyzes synthesis of phenylacetyl-homoserine lactone (PA-HSL). Our experiments show P. hirschii obtains phenylacetate from its environment and uses a CoA ligase to produce the phenylacetyl-CoA substrate for the LuxI homolog. By using an AHL degrading enzyme, we showed that PA-HSL controls aggregation, biofilm formation, and pigment production in P. hirschii These findings advance a limited understanding of the CoA-dependent AHL synthases. We describe how to identify putative members of the class, we describe a signal synthesized by using an environmental aromatic acid, and we identify phenotypes controlled by the aryl-HSL.

Burkholderia dipogonis sp. nov., isolated from root nodules of Dipogon lignosus in New Zealand and Western Australia.

Seven strains, ICMP 19430T, ICMP 19429, ICMP 19431, WSM4637, WSM4638, WSM4639 and WSM4640, were isolated from nitrogen-fixing nodules on roots of the invasive South African legume Dipogon lignosus (subfamily Papilionoideae, tribe Phaseoleae) in New Zealand and Western Australia, and their taxonomic positions were investigated by using a polyphasic approach. All seven strains grew at 10-37 °C (optimum, 25-30 °C), at pH 4.0-9.0 (optimum, pH 6.0-7.0) and with 0-2 % (w/v) NaCl (optimum growth in the absence of NaCl). On the basis of 16S rRNA gene sequence analysis, the strains showed 99.0-99.5 % sequence similarity to the closest type strain, Burkholderia phytofirmans PsJNT, and 98.4-99.7 % sequence similarity to Burkholderia caledonica LMG 19076T. The predominant fatty acids were C18 : 1ω7c (21.0 % of the total fatty acids in strain ICMP 19430T), C16 : 0 (19.1 %), C17 : 0 cyclo (18.9 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 10.7 %) and C19 : 0 cyclov ω8c (7.5 %). The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized aminophospholipids and phospholipids. The major isoprenoid quinone was Q-8 and the DNA G+C content of strain ICMP 19430T was 63.2 mol%. The DNA­DNA relatedness of the novel strains with respect to the closest neighbouring members of the genus Burkholderia was 55 % or less. On the basis of 16S rRNA and recA gene sequence similarities and chemotaxonomic and phenotypic data,these strains represent a novel symbiotic species in the genus Burkholderia, for which the name Burkholderia dipogonis sp. nov. is proposed, with the type strain ICMP 19430T (=LMG28415T=HAMBI 3637T).

Plant-Influenced Gene Expression in the Rice Endophyte Burkholderia kururiensis M130.

Burkholderia kururiensis M130 is one of the few rice endophytic diazotrophic bacteria identified thus far which is able to enhance growth of rice. To date, very little is known of how strain M130 and other endophytes enter and colonize plants. Here, we identified genes of strain M130 that are differentially regulated in the presence of rice plant extract. A genetic screening of a promoter probe transposon mutant genome bank and RNAseq analysis were performed. The screening of 10,100 insertions of the genomic transposon reporter library resulted in the isolation of 61 insertions displaying differential expression in response to rice macerate. The RNAseq results validated this screen and indicated that this endophytic bacterium undergoes major changes in the presence of plant extract regulating 27.7% of its open reading frames. A large number of differentially expressed genes encode membrane transporters and secretion systems, indicating that the exchange of molecules is an important aspect of bacterial endophytic growth. Genes related to motility, chemotaxis, and adhesion were also overrepresented, further suggesting plant­bacteria interaction. This work highlights the potential close signaling taking place between plants and bacteria and helps us to begin to understand the adaptation of an endophyte in planta.

Regulon studies and in planta role of the BraI/R quorum-sensing system in the plant-beneficial Burkholderia cluster.

The genus Burkholderia is composed of functionally diverse species, and it can be divided into several clusters. One of these, designated the plant-beneficial-environmental (PBE) Burkholderia cluster, is formed by nonpathogenic species, which in most cases have been found to be associated with plants. It was previously established that members of the PBE group share an N-acyl-homoserine lactone (AHL) quorum-sensing (QS) system, designated BraI/R, that produces and responds to 3-oxo-C14-HSL (OC14-HSL). Moreover, some of them also possess a second AHL QS system, designated XenI2/R2, producing and responding to 3-hydroxy-C8-HSL (OHC8-HSL). In the present study, we performed liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis to determine which AHL molecules are produced by each QS system of this group of bacteria. The results showed that XenI2/R2 is mainly responsible for the production of OHC8-HSL and that the BraI/R system is involved in the production of several different AHLs. This analysis also revealed that Burkholderia phymatum STM815 produces greater amounts of AHLs than the other species tested. Further studies showed that the BraR protein of B. phymatum is more promiscuous than other BraR proteins, responding equally well to several different AHL molecules, even at low concentrations. Transcriptome studies with Burkholderia xenovorans LB400 and B. phymatum STM815 revealed that the BraI/R regulon is species specific, with exopolysaccharide production being the only common phenotype regulated by this system in the PBE cluster. In addition, BraI/R was shown not to be important for plant nodulation by B. phymatum strains or for endophytic colonization and growth promotion of maize by B. phytofirmans PsJN.

Common features of environmental and potentially beneficial plant-associated Burkholderia.

The genus Burkholderia comprises more than 60 species isolated from a wide range of niches. Although they have been shown to be diverse and ubiquitously distributed, most studies have thus far focused on the pathogenic species due to their clinical importance. However, the increasing number of recently described Burkholderia species associated with plants or with the environment has highlighted the division of the genus into two main clusters, as suggested by phylogenetical analyses. The first cluster includes human, animal, and plant pathogens, such as Burkholderia glumae, Burkholderia pseudomallei, and Burkholderia mallei, as well as the 17 defined species of the Burkholderia cepacia complex, while the other, more recently established cluster comprises more than 30 non-pathogenic species, which in most cases have been found to be associated with plants, and thus might be considered to be potentially beneficial. Several species from the latter group share characteristics that are of use when associating with plants, such as a quorum sensing system, the presence of nitrogen fixation and/or nodulation genes, and the ability to degrade aromatic compounds. This review examines the commonalities in this growing subgroup of Burkholderia species and discusses their prospective biotechnological applications.