Amino acid-derived defense metabolites from plants: A potential source to facilitate novel antimicrobial development.

For millennia, humanity has relied on plants for its medicines, and modern pharmacology continues to reexamine and mine plant metabolites for novel compounds and to guide improvements in biological activity, bioavailability, and chemical stability. The critical problem of antibiotic resistance and increasing exposure to viral and parasitic diseases has spurred renewed interest into drug treatments for infectious diseases. In this context, an urgent revival of natural product discovery is globally underway with special attention directed toward the numerous and chemically diverse plant defensive compounds such as phytoalexins and phytoanticipins that combat herbivores, microbial pathogens, or competing plants. Moreover, advancements in "omics," chemistry, and heterologous expression systems have facilitated the purification and characterization of plant metabolites and the identification of possible therapeutic targets. In this review, we describe several important amino acid-derived classes of plant defensive compounds, including antimicrobial peptides (e.g., defensins, thionins, and knottins), alkaloids, nonproteogenic amino acids, and phenylpropanoids as potential drug leads, examining their mechanisms of action, therapeutic targets, and structure-function relationships. Given their potent antibacterial, antifungal, antiparasitic, and antiviral properties, which can be superior to existing drugs, phytoalexins and phytoanticipins are an excellent resource to facilitate the rational design and development of antimicrobial drugs.

One More Decade of Agrobacterium Taxonomy.

This chapter presents a historical overview of the development and changes in scientific approaches to classifying members of the Agrobacterium genus. We also describe the changes in the inference of evolutionary relationships among Agrobacterium biovars and Agrobacterium strains from using the 16S rRNA marker to recA genes and to the use of multilocus sequence analysis (MLSA). Further, the impacts of the genomic era enabling low cost and rapid whole genome sequencing on Agrobacterium phylogeny are reviewed with a focus on the use of new and sophisticated bioinformatics approaches to refine phylogenetic inferences. An updated genome-based phylogeny of ninety-seven Agrobacterium tumefaciens complex isolates representing ten known genomic species is presented, providing additional support to the monophyly of the Agrobacterium clade. Additional taxon sampling within Agrobacterium genomovar G3 indicates potential exceptions to interpretation of the concept of bacterial genomics species as ecological species because the genomovar G3 genomic cluster, which initially includes clinical strains, now also includes plant-associated and cave isolates.

Whole-genome sequencing of four culturable endophytic bacteria from German hardneck garlic cloves (Allium sativum L.).

We present the whole-genome sequence of four bacterial endophytes associated with German hardneck garlic cloves (Allium sativum L.). Among them, Agrobacterium fabrum and Pantoea agglomerans are associated with plant protection, while Rahnella perminowiae and Stenotrophomonas lactitubi are pathogens. These data will facilitate the identification of genes to improve garlic.

Comparative genomic analysis of six bacteria belonging to the genus Novosphingobium: insights into marine adaptation, cell-cell signaling and bioremediation.

BACKGROUND: Bacteria belonging to the genus Novosphingobium are known to be metabolically versatile and occupy different ecological niches. In the absence of genomic data and/or analysis, knowledge of the bacteria that belong to this genus is currently limited to biochemical characteristics. In this study, we analyzed the whole genome sequencing data of six bacteria in the Novosphingobium genus and provide evidence to show the presence of genes that are associated with salt tolerance, cell-cell signaling and aromatic compound biodegradation phenotypes. Additionally, we show the taxonomic relationship between the sequenced bacteria based on phylogenomic analysis, average amino acid identity (AAI) and genomic signatures. RESULTS: The taxonomic clustering of Novosphingobium strains is generally influenced by their isolation source. AAI and genomic signature provide strong support the classification of Novosphingobium sp. PP1Y as Novosphingobium pentaromaticivorans PP1Y. The identification and subsequent functional annotation of the unique core genome in the marine Novosphingobium bacteria show that ectoine synthesis may be the main contributing factor in salt water adaptation. Genes coding for the synthesis and receptor of the cell-cell signaling molecules, of the N-acyl-homoserine lactones (AHL) class are identified. Notably, a solo luxR homolog was found in strain PP1Y that may have been recently acquired via horizontal gene transfer as evident by the presence of multiple mobile elements upstream of the gene. Additionally, phylogenetic tree analysis and sequence comparison with functionally validated aromatic ring hydroxylating dioxygenases (ARDO) revealed the presence of several ARDOs (oxygenase) in Novosphingobium bacteria with the majority of them belonging to the Groups II and III of the enzyme. CONCLUSIONS: The combination of prior knowledge on the distinctive phenotypes of Novosphingobium strains and meta-analysis of their whole genomes enables the identification of several genes that are relevant in industrial applications and bioremediation. The results from such targeted but comprehensive comparative genomics analysis have the potential to contribute to the understanding of adaptation, cell-cell communication and bioremediation properties of bacteria belonging to the genus Novosphingobium.

Whole-Genome Sequence of Endophytic Bacteria Associated with Poison Ivy Vine (Toxicodendron radicans).

Here, we report the genome assemblies of 11 endophytic bacteria, isolated from poison ivy vine (Toxicodendron radicans). Five species belonging to the genus Pseudomonas, two species of Curtobacterium, one strain of Pantoea agglomerans, and one species from the Bacillus, Cellulomonas, and Enterobacter genera were isolated from the interior tissue of poison ivy.

Endophytic bacteria associated with wild-type banana seed (Musa balbisiana): whole genome sequencing.

We present the whole-genome sequences of five endophytic bacteria isolated from Musa balbisiana seeds. These strains represent five different genera: Bacillus, Brachybacterium, Enterobacter, Enterococcus, and Pantoea. Among these, three genera (Bacillus, Pantoea, and Enterobacter) were previously recognized for their antagonistic effects against Fusarium wilt, a highly destructive disease that affects banana plants.

Genome sequence of Novosphingobium sp. strain Rr 2-17, a nopaline crown gall-associated bacterium isolated from Vitis vinifera L. grapevine.

Novosphingobium sp. strain Rr 2-17 is an N-acyl homoserine lactone (AHL)-producing bacterium isolated from the crown gall tumor of a grapevine. To our knowledge, this is the first draft genome announcement of a plant-associated strain from the genus Novosphingobium.

Genome sequence of Methylobacterium sp. strain GXF4, a xylem-associated bacterium isolated from Vitis vinifera L. grapevine.

Methylobacterium sp. strain GXF4 is an isolate from grapevine. Here we present the sequence, assembly, and annotation of its genome, which may shed light on its role as a grapevine xylem inhabitant. To our knowledge, this is the first genome announcement of a plant xylem-associated strain of the genus Methylobacterium.

Whole-genome sequence of Enterobacter sp. strain SST3, an endophyte isolated from Jamaican sugarcane (Saccharum sp.) stalk tissue.

Enterobacter sp. strain SST3 is an endophytic bacterium isolated from Saccharum spp. Here we present its annotated draft genome that may shed light on its role as a bacterial endophyte of sugarcane. To our knowledge, this is the first genome announcement of a sugarcane-associated bacterium from the genus Enterobacter.

LasR receptor for detection of long-chain quorum-sensing signals: identification of N-acyl-homoserine lactones encoded by the avsI locus of Agrobacterium vitis.

Bacterial biosensor strains have greatly facilitated the rapid discovery, isolation, and study of quorum-sensing systems. In this study, we determined the relative sensitivity of a LasR-based E. coli bacterial bioluminescence biosensor JM109 (pSB1075) for 13 diverse long-chain N-acyl-homoserine lactones (AHLs) including oxygen-substituted and -unsubstituted AHLs containing 14, 16, and 18 carbons and with and without double bonds. Furthermore, we show by bioassay, HPLC, and GC/MS that four long-chain AHLs of the C16-HSL family are encoded by the avsI gene of Agrobacterium vitis strain F2/5, a non-tumorigenic strain that inhibits pathogenic strains of A. vitis from causing crown gall on grape. The four C16-HSLs include: C16-HSL, N-hexadecanoyl homoserine lactone; 3-oxo-C16-HSL, N-(3-oxohexadecanoyl)homoserine lactone; C16:1-HSL, N-(cis-9-octadecenoyl)homoserine lactone; and 3-oxo-C16:1-HSL, N-(3-oxo-cis-11-hexadecenoyl)homoserine lactone. Thus, the LasR-based bioluminescent biosensor tested in this study should serve as a useful tool for the detection of various long-chain AHLs with and without double bonds as well as those oxylated at the third carbon from uninvestigated species.

Genomic characterization of bacteria from the ultra-oligotrophic Madison aquifer: insight into the archetypical LuxI/LuxR and identification of novel LuxR solos.

OBJECTIVES: To characterize the bacterial community of Wind Cave's Madison aquifer through whole-genome sequencing, and to better understand the bacterial ecology by identifying genes involved in acyl-homoserine lactone (AHL) based quorum-sensing (QS) systems. RESULTS: Genome-based taxonomic classification revealed the microbial richness present in the pristine Madison aquifer. The strains were found to span eleven genera and fourteen species, of which eight had uncertain taxonomic classifications. The genomes of strains SD129 and SD340 were found to contain the archetypical AHL QS system composed of two genes, luxI and luxR. Surprisingly, the genomes of strains SD115, SD129, SD274 and SD316 were found to contain one to three luxR orphans (solos). Strain SD129, besides possessing an archetypical AHL QS luxI-luxR pair, also contained two luxR solos, while strain SD316 contained three LuxR solos and no luxI-luxR pairs. The ligand-binding domain of two LuxR solos, one each from strains SD129 and SD316, were found to contain novel substitutions not previously reported, thus may represent two LuxR orphans that detection and response to unknown self-produced signal(s), or to signal(s) produced by other organisms.

Insight into the resistome and quorum sensing system of a divergent Acinetobacter pittii isolate from an untouched site of the Lechuguilla Cave.

Acinetobacter are Gram-negative bacteria belonging to the sub-phyla Gammaproteobacteria, commonly associated with soils, animal feeds and water. Some members of the Acinetobacter have been implicated in hospital-acquired infections, with broad-spectrum antibiotic resistance. Here we report the whole-genome sequence of LC510, an Acinetobacter species isolated from deep within a pristine location of the Lechuguilla Cave. Pairwise nucleotide comparison to three type strains within the genus Acinetobacter assigned LC510 as an Acinetobacter pittii isolate. Scanning of the LC510 genome identified two genes coding for b-lactamase resistance, despite the fact that LC510 was isolated from a portion of the cave not previously visited by humans and protected from anthropogenic input. The ability to produce acyl-homoserine lactone (AHL) signal in culture medium, an observation that is consistent with the identification of the luxI and luxR homologues in its genome, suggests that cell-to-cell communication remains important in an isolated cave ecosystem.

Whole-Genome Sequencing of Pantoea sp. Strain RIT388, a Potential Oral Opportunistic Pathogen Isolated from a Chewing Stick (Distemonanthus benthamianus).

In this study, we report the isolation, identification, characterization, and whole-genome sequence of the endophyte Pantoea sp. strain RIT388, isolated from Distemonanthus benthamianus, a plant known for its antifungal and antibacterial properties that is commonly used for chewing sticks.

Aeromonas hydrophila RIT668 and Citrobacter portucalensis RIT669-Potential Zoonotic Pathogens Isolated from Spotted Turtles.

Antimicrobial resistance (AMR) is one of the biggest challenges of the 21st century, and biofilm formation enables bacteria to resist antibiotic at much higher concentrations than planktonic cells. Earlier, we showed that the Gram-negative Aeromonas hydrophila RIT668 and Citrobacter portucalensis RIT669 (closely related to C. freundii NBRC 12681) from infected spotted turtles (Clemmys guttata), formed biofilms and upregulated toxin expression on plastic surfaces, and were predicted to possess multiple antibiotic resistance genes. Here, we show that they each resist several antibiotics in the planktonic phase, but were susceptible to neomycin, and high concentrations of tetracycline and cotrimoxazole. The susceptibility of their biofilms to neomycin and cotrimoxazole was tested using the Calgary device. For A. hydrophila, the minimum inhibitory concentration (MIC) = 500-1000, and the minimum biofilm eradication concentration (MBEC) > 1000 µg/mL, using cotrimoxazole, and MIC = 32.3-62.5, and MBEC > 1000 µg/mL, using neomycin. For C. freundii MIC = 7.8-15.6, and, MBEC > 1000 µg/mL, using cotrimoxazole, and MIC = 7.8, and MBEC > 1000 µg/mL, using neomycin. Both A. hydrophila and C. portucalensis activated an acyl homoserine lactone (AHL) dependent biosensor, suggesting that quorum sensing could mediate biofilm formation. Their multidrug resistance in the planktonic form, and weak biofilm eradication even with neomycin and cotrimoxazole, indicate that A. hydrophila and C. portucalensis are potential zoonotic pathogens, with risks for patients living with implants.

Identification of an rsh gene from a Novosphingobium sp. necessary for quorum-sensing signal accumulation.

The stringent response is a mechanism by which bacteria adapt to environmental stresses and nutritional deficiencies through the synthesis and hydrolysis of (p)ppGpp by RelA/SpoT enzymes. Alphaproteobacteria and plants contain a single Rsh enzyme (named for RelA/SpoT homolog) that is bifunctional. Here we report the identification of a new species of bacteria belonging to the genus Novosphingobium and characterization of an rsh mutation in this plant tumor-associated isolate. Isolate Rr 2-17, from a grapevine crown gall tumor, is a member of the Novosphingobium genus that produces the N-acyl-homoserine lactone (AHL) quorum-sensing (QS) signals. A Tn5 mutant, Hx 699, deficient in AHL production was found to have an insertion in an rsh gene. The Rsh protein showed significant percent sequence identity to Rsh proteins of alphaproteobacteria. The Novosphingobium sp. rsh gene (rsh(Nsp)) complemented the multiple amino acid requirements of the Escherichia coli relA spoT double mutant by restoring the growth on selection media. Besides QS signal production, the rsh mutation also affects soluble polysaccharide production and cell aggregation. Genetic complementation of the Hx 699 mutant with the rsh(Nsp) gene restored these phenotypes. This is the first discovery of a functional rsh gene in a member of the Novosphingobium genus.

Improved genome of Agrobacterium radiobacter type strain provides new taxonomic insight into Agrobacterium genomospecies 4.

The reported Agrobacterium radiobacter DSM 30174T genome is highly fragmented, hindering robust comparative genomics and genome-based taxonomic analysis. We re-sequenced the Agrobacterium radiobacter type strain, generating a dramatically improved genome with high contiguity. In addition, we sequenced the genome of Agrobacterium tumefaciens B6T, enabling for the first time, a proper comparative genomics of these contentious Agrobacterium species. We provide concrete evidence that the previously reported Agrobacterium radiobacter type strain genome (Accession Number: ASXY01) is contaminated which explains its abnormally large genome size and fragmented assembly. We propose that Agrobacterium tumefaciens be reclassified as Agrobacterium radiobacter subsp. tumefaciens and that Agrobacterium radiobacter retains it species status with the proposed name of Agrobacterium radiobacter subsp. radiobacter. This proposal is based, first on the high pairwise genome-scale average nucleotide identity supporting the amalgamation of both Agrobacterium radiobacter and Agrobacterium tumefaciens into a single species. Second, maximum likelihood tree construction based on the concatenated alignment of shared genes (core genes) among related strains indicates that Agrobacterium radiobacter NCPPB3001 is sufficiently divergent from Agrobacterium tumefaciens to propose two independent sub-clades. Third, Agrobacterium tumefaciens demonstrates the genomic potential to synthesize the L configuration of fucose in its lipid polysaccharide, fostering its ability to colonize plant cells more effectively than Agrobacterium radiobacter.

The Synthesis and Role of ß-Alanine in Plants.

Most studies on amino acids are focused on the proteinogenic amino acids given their essential roles in protein synthesis among other pathways. In addition to 20 ubiquitous amino acids used in protein synthesis, plants synthesize over 250 non-proteinogenic amino acids that are involved in the synthesis of compounds that are anti-herbivory, anti-microbial, response to abiotic stresses, nitrogen storage, toxins against both vertebrates/invertebrates, and plant hormones among others. One such non-proteinogenic acid is ß-alanine, which is known mainly for studies on humans. ß-Alanine forms a part of pantothenate (vitamin B5), which is incorporated into the universal carbon shuttling compounds Coenzyme A and acyl carrier protein, in all organisms including plants. The focus of this review, however, is on the biosynthesis, metabolism, and the role of ß-alanine in plants. There are several functions of ß-alanine unique to plants. It is accumulated as a generic stress response molecule involved in protecting plants from temperature extremes, hypoxia, drought, heavy metal shock, and some biotic stresses. There is evidence of its participation in lignin biosynthesis and ethylene production in some species. It is further converted to the osmoprotective compound ß-alanine betaine in some species and converted to the antioxidant homoglutathione in others. The polyamines spermine/spermidine, propionate and uracil have been shown to be precursors of ß-alanine in plants. However, plants vary in terms of their biosynthetic pathways, and the primary metabolism of ß-alanine is far from settled.

Draft Genome Sequences of Five Proteobacteria Isolated from Lechuguilla Cave, New Mexico, USA, and Insights into Taxonomy and Quorum Sensing.

Genomic resources remain scarce for bacteria isolated from oligotrophic caves. We sequenced the genomes of five Proteobacteria isolated from Lechuguilla Cave in New Mexico. Genome-based phylogeny indicates that each strain belongs to a distinct genus. Two Rhizobiaceae isolates possess genomic potential for the biosynthesis of acyl-homoserine lactone.

Isolation, Whole-Genome Sequencing, and Annotation of Yimella sp. RIT 621, a Strain That Produces Antibiotic Compounds against Escherichia coli ATCC 25922 and Bacillus subtilis BGSC 168.

Here, we report the isolation, identification, whole-genome sequencing, and annotation of the bacterium Yimella sp. strain RIT 621. Concentrated spent medium extract treated with ethyl acetate was found to produce bactericidal compounds against the Gram-positive bacterium Bacillus subtilis BGSC 168 and the Gram-negative bacterium Escherichia coli ATCC 25922.