Post translational modifications of Trifolitoxin: a blue fluorescent peptide antibiotic*

Trifolitoxin (TFX, C41H63N15O15S) is a selective, ribosomally-synthesized, post-translationally modified, peptide antibiotic, produced by Rhizobium leguminosarum bv. trifolii T24. TFX specifically inhibits α-proteobacteria, including the plant symbiont Rhizobium spp., the plant pathogen Agrobacterium spp. and the animal pathogen Brucella abortus. TFX-producing strains prevent legume root nodulation by TFX-sensitive rhizobia. TFX has been isolated as a pair of geometric isomers, TFX1 and TFX2, which are derived from the biologically inactive primary amino acid sequence: Asp-Ile-Gly-Gly-Ser-Arg-Gln-Gly-Cys-Val-Ala. Gly-Cys is present as a thiazoline ring and the Arg-Gln-Gly sequence is extensively modified to a UV absorbing, blue fluorescent chromophore. The chromophore consists of a conjugated, 5-membered heterocyclic ring and side chain of modified glutamine.

Hopanoids confer robustness to physicochemical variability in the niche of the plant symbiont Bradyrhizobium diazoefficiens

Climate change poses a threat to soil health and agriculture, but the potential effects of climate change on soil bacteria that can help maintain soil health are understudied. Rhizobia are a group of bacteria that increase soil nitrogen content through a symbiosis with legume plants. The soil and symbiosis are potentially stressful environments, and the soil will likely become even more stressful as the climate changes. Many rhizobia within the bradyrhizobia clade, like Bradyrhizobium diazoefficiens, possess the genetic capacity to synthesize hopanoids, steroid-like lipids similar in structure and function to cholesterol. Hopanoids are known to protect against stresses relevant to the niche of B. diazoefficiens. Paradoxically, mutants unable to synthesize the extended class of hopanoids participate in similarly successful symbioses compared to the wild type, despite being delayed in root nodule initiation. Here, we show that in B. diazoefficiens, the in vitro growth defects of extended hopanoid deficient mutants can be at least partially compensated for by the physicochemical environment, specifically by optimal osmotic and divalent cation concentrations. Through biophysical measurements, we show that extended hopanoids confer robustness to environmental variability. These results help explain the discrepancy between previous in vitro and in planta results and indicate that hopanoids may provide a greater fitness advantage to rhizobia in the variable soil environment than the more controlled environment within root nodules. To improve the legume-rhizobia symbiosis through either bioengineering or strain selection, it will be important to consider the full lifecycle of rhizobia, from the soil to the symbiosis.

Transposon sequencing analysis of Bradyrhizobium diazoefficiens 110spc4

Bradyrhizobium diazoefficiens USDA110 is one of the most effective nitrogen-fixing symbionts of soybeans. Here we carried out a large-scale transposon insertion sequencing (Tn-seq) analysis of strain Bd110spc4, which is derived from USDA110, with the goal of increasing available resources for identifying genes crucial for the survival of this plant symbiont under diverse conditions. We prepared two transposon (Tn) insertion libraries of Bd110spc4 with 155,042 unique Tn insertions when the libraries were combined, which is an average of one insertion every 58.7 bp of the reference USDA110 genome. Application of bioinformatic filtering steps to remove genes too small to be expected to have Tn insertions, resulted in a list of genes that were classified as putatively essential. Comparison of this gene set with genes putatively essential for the growth of the closely related alpha-proteobacterium, Rhodopseudomonas palustris, revealed a small set of five genes that may be collectively essential for closely related members of the family Bradyrhizobiaceae. This group includes bacteria with diverse lifestyles ranging from plant symbionts to animal-associated species to free-living species.

A class I hydrophobin in Trichoderma virens influences plant-microbe interactions through enhancement of enzyme activity and MAMP recognition

The filamentous fungus, Trichoderma virens, is a well-known mycoparasitic plant symbiont, valued for its biocontrol capabilities. T. virens initiates a symbiotic relationship with a plant host through the colonization of its roots. To achieve colonization, the fungus must communicate with the host and evade its innate defenses. Hydrophobins from Trichoderma spp. have previously been demonstrated to be involved in colonization of host roots. In this study, the class I hydro-phobin, HFB9A from T. virens was characterized for a potential role in root colonization. Δhfb9a gene deletion mutants colonized less than the wild-type strain, were unable to induce systemic resistance against Colletotrichum graminicola, and showed a reduction in the activity of its cell wall degrading enzymes. The purified HFB9A protein was able to complement the enzyme activity of mutant culture filtrates as well as enhance the activity of commercially sourced cellulase. When exogenously applied to Arabidopsis plants, HFB9A protein induced phosphorylation of AtMAPK3/6, suggesting that it functions as a microbe-associated molecular pattern.

Serendipita sacchari sp. nov. from a sugarcane rhizosphere in southern China

We isolated a new species, proposed here as Serendipita sacchari, from a sugarcane rhizosphere in Guangxi Province, China. This species is characterized by its unstable nucleus numbers (1–15) in its chlamydospores versus their regular distribution in hyphal cells. ITS rDNA and combined LSU+ TEF1-α sequence analyses also support the uniqueness of this new plant symbiont.

A central role for the transcriptional regulator VtlR in small RNA-mediated gene regulation in Agrobacterium tumefaciens

LysR-type transcriptional regulators (LTTRs) are the most common type of transcriptional regulators in prokaryotes and function by altering gene expression in response to environmental stimuli. In the class Alphaproteobacteria, a conserved LTTR named VtlR is critical to the establishment of host-microbe interactions. In the mammalian pathogen Brucella abortus, VtlR is required for full virulence in a mouse model of infection, and VtlR activates the expression of abcR2, which encodes a small regulatory RNA (sRNA). In the plant symbiont Sinorhizobium meliloti, the ortholog of VtlR, named LsrB, is involved in the symbiosis of the bacterium with alfalfa. Agrobacterium tumefaciens is a close relative of both B. abortus and S. meliloti, and this bacterium is the causative agent of crown gall disease in plants. In the present study, we demonstrate that VtlR is involved in the ability of A. tumefaciens to grow appropriately in artificial medium, and an A. tumefaciens vtlR deletion strain is defective in motility, biofilm formation, and tumorigenesis of potato discs. RNA-sequencing analyses revealed that more than 250 genes are dysregulated in the ∆vtlR strain, and importantly, VtlR directly controls the expression of three sRNAs in A. tumefaciens. Taken together, these data support a model in which VtlR indirectly regulates hundreds of genes via manipulation of sRNA pathways in A. tumefaciens, and moreover, while the VtlR/LsrB protein is present and structurally conserved in many members of the Alphaproteobacteria, the VtlR/LsrB regulatory circuitry has diverged in order to accommodate the unique environmental niche of each organism.

The Leader Peptide peTrpL Forms Antibiotic-Containing Ribonucleoprotein Complexes for Posttranscriptional Regulation of Multiresistance Genes

ABSTRACT Bacterial ribosome-dependent attenuators are widespread posttranscriptional regulators. They harbor small upstream open reading frames (uORFs) encoding leader peptides, for which no functions in trans are known yet. In the plant symbiont Sinorhizobium meliloti, the tryptophan biosynthesis gene trpE(G) is preceded by the uORF trpL and is regulated by transcription attenuation according to tryptophan availability. However, trpLE(G) transcription is initiated independently of the tryptophan level in S. meliloti, thereby ensuring a largely tryptophan-independent production of the leader peptide peTrpL. Here, we provide evidence for a tryptophan-independent role of peTrpL in trans. We found that peTrpL increases the resistance toward tetracycline, erythromycin, chloramphenicol, and the flavonoid genistein, which are substrates of the major multidrug efflux pump SmeAB. Coimmunoprecipitation with a FLAG-peTrpL suggested smeR mRNA, which encodes the transcription repressor of smeABR, as a peptide target. Indeed, upon antibiotic exposure, smeR mRNA was destabilized and smeA stabilized in a peTrpL-dependent manner, showing that peTrpL acts in the differential regulation of smeABR. Furthermore, smeR mRNA was coimmunoprecipitated with peTrpL in antibiotic-dependent ribonucleoprotein (ARNP) complexes, which, in addition, contained an antibiotic-induced antisense RNA complementary to smeR. In vitro ARNP reconstitution revealed that the above-mentioned antibiotics and genistein directly support complex formation. A specific region of the antisense RNA was identified as a seed region for ARNP assembly in vitro. Altogether, our data show that peTrpL is involved in a mechanism for direct utilization of antimicrobial compounds in posttranscriptional regulation of multiresistance genes. Importantly, this role of peTrpL in resistance is conserved in other Alphaproteobacteria. IMPORTANCE Leader peptides encoded by transcription attenuators are widespread small proteins that are considered nonfunctional in trans. We found that the leader peptide peTrpL of the soil-dwelling plant symbiont Sinorhizobium meliloti is required for differential, posttranscriptional regulation of a multidrug resistance operon upon antibiotic exposure. Multiresistance achieved by efflux of different antimicrobial compounds ensures survival and competitiveness in nature and is important from both evolutionary and medical points of view. We show that the leader peptide forms antibiotic- and flavonoid-dependent ribonucleoprotein complexes (ARNPs) for destabilization of smeR mRNA encoding the transcription repressor of the major multidrug resistance operon. The seed region for ARNP assembly was localized in an antisense RNA, whose transcription is induced by antimicrobial compounds. The discovery of ARNP complexes as new players in multiresistance regulation opens new perspectives in understanding bacterial physiology and evolution and potentially provides new targets for antibacterial control.