Multiple Copies of flhDC in Paraburkholderia unamae Regulate Flagellar Gene Expression, Motility, and Biofilm Formation.

FlhDC is a heterohexameric complex that acts as a master regulator of flagellar biosynthesis genes in numerous bacteria. Previous studies have identified a single flhDC operon encoding this complex. However, we found that two flhDC loci are present throughout Paraburkholderia, and two additional flhC copies are also present in Paraburkholderia unamae. Systematic deletion analysis in P. unamae of the different flhDC copies showed that one of the operons, flhDC1, plays the predominant role, with deletion of its genes resulting in a severe inhibition of motility and biofilm formation. Expression analysis using promoter-lacZ fusions and real-time quantitative PCR support the primary role of flhDC1 in flagellar gene regulation, with flhDC2 a secondary contributor. Phylogenetic analysis shows the presence of the flhDC1 and flhDC2 operons throughout Paraburkholderia. In contrast, Burkholderia and other bacteria only carry the copy syntenous with flhDC2. The variations in impact each copy of flhDC has on downstream processes indicate that regulation of FlhDC in P. unamae, and likely other Paraburkholderia species, is regulated at least in part by the presence of multiple copies of these genes. IMPORTANCE Motility is important in the colonization of plant roots by beneficial and pathogenic bacteria, with flagella playing essential roles in host cell adhesion, entrance, and biofilm formation. Flagellar biosynthesis is energetically expensive. Its complex regulation by the FlhDC master regulator is well studied in peritrichous flagella expressing enterics. We report the unique presence throughout Paraburkholderia of multiple copies of flhDC. In P. unamae, the flhDC1 copy showed higher expression and a greater effect on swim motility, flagellar development, and regulation of downstream genes, than the flhDC2 copy that is syntenous to flhDC in Escherichia coli and pathogenic Burkholderia spp. The flhDC genes have evolved differently in these plant-growth-promoting bacteria, giving an additional layer of complexity in gene regulation by FlhDC.

A reliable method for the selection and confirmation of transconjugants of plant growth-promoting bacteria especially plant-associated Burkholderia spp.

Selectable markers, e.g., antibiotic resistance, for conjugation experiments are not always effective for slow-growing plant growth promoting bacteria such as Burkholderia. We used PCAT medium containing Congo Red for selecting Burkholderia transconjugants. This method allows for the reliable selection of transconjugants of these novel plant growth-promoting bacteria.

DNA-Based Authentication of Botanicals and Plant-Derived Dietary Supplements: Where Have We Been and Where Are We Going?

Herbal medicines and botanicals have long been used as sole or additional medical aids worldwide. Currently, billions of dollars are spent on botanicals and related products, but minimal regulation exists regarding their purity, integrity, and efficacy. Cases of adulteration and contamination have led to severe illness and even death in some cases. Identifying the plant material in botanicals and phytomedicines using organoleptic means or through microscopic observation of plant parts is not trivial, and plants are often misidentified. Recently, DNA-based methods have been applied to these products because DNA is not changed by growth conditions unlike the chemical constituents of many active pharmaceutical agents. In recent years, DNA barcoding methods, which are used to identify species diversity in the Tree of Life, have been also applied to botanicals and plant-derived dietary supplements. In this review, we recount the history of DNA-based methods for identification of botanicals and discuss some of the difficulties in defining a specific bar code or codes to use. In addition, we describe how next generation sequencing technologies have enabled new techniques that can be applied to identifying these products with greater authority and resolution. Lastly, we present case histories where dietary supplements, decoctions, and other products have been shown to contain materials other than the main ingredient stipulated on the label. We conclude that there is a fundamental need for greater quality control in this industry, which if not self-imposed, that may result from legislation.

Complete Genome Sequence of Micromonospora Strain L5, a Potential Plant-Growth-Regulating Actinomycete, Originally Isolated from Casuarina equisetifolia Root Nodules.

Micromonospora species live in diverse environments and exhibit a broad range of functions, including antibiotic production, biocontrol, and degradation of complex polysaccharides. To learn more about these versatile actinomycetes, we sequenced the genome of strain L5, originally isolated from root nodules of an actinorhizal plant growing in Mexico.

Rhizobium common nod genes are required for biofilm formation.

In legume nitrogen-fixing symbioses, rhizobial nod genes are obligatory for initiating infection thread formation and root nodule development. Here we show that the common nod genes, nodD1ABC, whose products synthesize core Nod factor, a chitin-like oligomer, are also required for the establishment of the three-dimensional architecture of the biofilm of Sinorhizobium meliloti. Common nod gene mutants form a biofilm that is a monolayer. Moreover, adding Nod Factor antibody to S. meliloti cells inhibits biofilm formation, while chitinase treatment disrupts pre-formed biofilms. These results attest to the involvement of core Nod factor in rhizobial biofilm establishment. However, luteolin, the plant-derived inducer of S. meliloti's nod genes, is not required for mature biofilm formation, although biofilm establishment is enhanced in the presence of this flavonoid inducer. Because biofilm formation is plant-inducer-independent and because all nodulating rhizobia, both alpha- and beta-proteobacteria have common nod genes, the role of core Nod factor in biofilm formation is likely to be an ancestral and evolutionarily conserved function of these genes.

ENOD40 Gene Expression and Cytokinin Responses in the Nonnodulating, Nonmycorrhizal (NodMyc) Mutant, Masym3, of Melilotus alba Desr.

Several nonnodulating, nonmycorrhizal (Nod(-)Myc(-)) mutants of Melilotus alba Desr. (white sweetclover) have been described. However, the details of their responses to Sinorhizobium meliloti have not been fully elucidated. We investigated rhizobial entry and colonization using Confocal Scanning Laser Microscopy on the Masym1-5 mutants and isolated an early nodulin (ENOD40) gene from wild-type M. alba. We focused on Masym3, the least responsive of the mutants to S. meliloti and VA-fungi, to determine its response to cytokinin. Cytokinin appears to be a downstream signal in the nodule developmental pathway based not only on our previous observations whereby Nod(-)Myc(-) alfalfa roots treated with cytokinin accumulated several ENOD gene transcripts, but also on recent reports showing the importance of cytokinin receptors for nodulation. Here we show that applying 10(-6) M 6-benzylaminopurine to uninoculated Masym3 roots elicited ENOD40 transcript accumulation. In addition, Masym3 root hairs inoculated with either wild-type S. meliloti or Nod(-)S. meliloti expressing the trans-zeatin synthase gene of Agrobacterium tumefaciens exhibited tip swelling, suggesting that cytokinin mediated this response. However, Masym3 root hair tips swelled following inoculation with Nod(-)S. meliloti or after mock-inoculation, a response resembling the phenotype of root hairs, after handling, of the Medicago truncatula mutant, dmi2. Mtdmi2 is Nod(-)Myc(-) due to a defect in a gene encoding a Nodule Receptor Kinase (NORK). Like Mtdmi2, the root hair swelling response appears in part to be mediated by touch because Masym3 root hairs not contacted by either bacteria or drops of water or buffer remain elongated and do not exhibit tip swelling.

Identification of botanicals and potential contaminants through RFLP and sequencing.

Botanical supplements for health enhancement are being increasingly used in the United States, but no safeguards are formally in place to ensure that they are not contaminated with non-efficacious or potentially harmful plant material. A molecular approach, which allows the authentication of botanical ingredients and detection of contaminating plant material by analyzing the ITS-1 region by PCR-RFLP and subsequent sequencing, is described. When using starting material from which DNA can be obtained, this method has the potential for identifying both primary and contaminating plant material in botanical dietary supplements.

Investigation of Four Classes of Non-nodulating White Sweetclover (Melilotus alba annua Desr.) Mutants and Their Responses to Arbuscular-Mycorrhizal Fungi.

The nitrogen-fixing symbiosis between Rhizobiaceae and legumes is one of the best-studied interactions established between prokaryotes and eukaryotes. The plant develops root nodules in which the bacteria are housed, and atmospheric nitrogen is fixed into ammonia by the rhizobia and made available to the plant in exchange for carbon compounds. It has been hypothesized that this symbiosis evolved from the more ancient arbuscular mycorrhizal (AM) symbiosis, in which the fungus associates with roots and aids the plant in the absorption of mineral nutrients, particularly phosphate. Support comes from several fronts: 1) legume mutants where Nod(-) and Myc(-) co-segregate, and 2) the fact that various early nodulin (ENOD) genes are expressed in legume AM. Both strongly argue for the idea that the signal transduction pathways between the two symbioses are conserved. We have analyzed the responses of four classes of non-nodulating Melilotus alba (white sweetclover) mutants to Glomus intraradices (the mycorrhizal symbiont) to investigate how Nod(-) mutations affect the establishment of this symbiosis. We also re-examined the root hair responses of the non-nodulating mutants to Sinorhizobium meliloti (the nitrogen-fixing symbiont). Of the four classes, several sweetclover sym mutants are both Nod(-) and Myc(-). In an attempt to decipher the relationship between nodulation and mycorrhiza formation, we also performed co-inoculation experiments with mutant rhizobia and Glomus intraradices on Medicago sativa, a close relative of M. alba. Even though sulfated Nod factor was supplied by some of the bacterial mutants, the fungus did not complement symbiotically defective rhizobia for nodulation.