Ancestral Genome Estimation Reveals the History of Ecological Diversification in Agrobacterium.

Horizontal gene transfer (HGT) is considered as a major source of innovation in bacteria, and as such is expected to drive adaptation to new ecological niches. However, among the many genes acquired through HGT along the diversification history of genomes, only a fraction may have actively contributed to sustained ecological adaptation. We used a phylogenetic approach accounting for the transfer of genes (or groups of genes) to estimate the history of genomes in Agrobacterium biovar 1, a diverse group of soil and plant-dwelling bacterial species. We identified clade-specific blocks of cotransferred genes encoding coherent biochemical pathways that may have contributed to the evolutionary success of key Agrobacterium clades. This pattern of gene coevolution rejects a neutral model of transfer, in which neighboring genes would be transferred independently of their function and rather suggests purifying selection on collectively coded acquired pathways. The acquisition of these synapomorphic blocks of cofunctioning genes probably drove the ecological diversification of Agrobacterium and defined features of ancestral ecological niches, which consistently hint at a strong selective role of host plant rhizospheres.

Improved curdlan production with discarded bottom parts of Asparagus spear.

BACKGROUND: This work evaluated the improvement of curdlan production of Agrobacterium sp. ATCC 31749 by using culture medium containing juice of discarded bottom part of green Asparagus spear (MJDA). Curdlan production was carried out using Agrobacterium sp. ATCC 31749 in flasks with different volumes of MJDA and its non-juice-adding control (CK) incubated in shaker at 30 °C, 200 rpm rotation for 168 h. RESULTS: All MJDA media increased Agrobacterium sp. ATCC 31749 cell mass and enhanced the cells' ability to utilise sucrose, the carbon source for curdlan biosynthesis, and thereby produced higher concentration of curdlan than CK which is used for commercial production of curdlan. After 168 h of fermentation, 10% MJDA produced 40.2 g/l of curdlan whiles CK produced 21.1 g/l. Curdlan production was increased by 90.4% higher in 10% MJDA than CK. Curdlan produced by 10% MJDA contains 1.2 and 1.5 µg/ml of Asparagus flavonoids and saponins respectively as additives which have wide range of health benefits. The mass of sucrose needed to produce 1.0 g curdlan by Agrobacterium sp. ATCC 31749 in CK is 1.7-fold more than in 10% MJDA. CONCLUSION: The results strongly revealed that 5-10% MJDA is a good curdlan fermentation media which increase curdlan production yield with cheaper cost of production and simultaneously reduce environmental waste resulting from the large scaled discarded bottom parts of green Asparagus spear during Asparagus production.

The essential features and modes of bacterial polar growth.

Polar growth represents a surprising departure from the canonical dispersed cell growth model. However, we know relatively little of the underlying mechanisms governing polar growth or the requisite suite of factors that direct polar growth. Underscoring how classic doctrine can be turned on its head, the peptidoglycan layer of polar-growing bacteria features unusual crosslinks and in some species the quintessential cell division proteins FtsA and FtsZ are recruited to the growing poles. Remarkably, numerous medically important pathogens utilize polar growth, accentuating the need for intensive research in this area. Here we review models of polar growth in bacteria based on recent research in the Actinomycetales and Rhizobiales, with emphasis on Mycobacterium and Agrobacterium species.

Improvement of degummed ramie fiber properties upon treatment with cellulase secreting immobilized A. larrymoorei A1.

Degummed ramie fiber is widely used in the textile industry. Cellulase enzyme can be effectively used for bio-polishing of the ramie fiber. We immobilized Agrobacterium larrymoorei A1, a potent extra-cellular cellulase producing bacteria, in Ca-alginate. The production of enzyme significantly increased with increasing alginate concentration and reached a maximum activity of 0.28 IU/ml at 20 g/l, which was 32% higher as compared to free cells. These immobilized cells were used on ramie fibers. Scanning electron micrograph (SEM) and differential interference contrast (DIC) studies showed increased smoothness and orientation of surface structure of the fibers after 19.5 h. The single fiber tenacity was almost same as compared to non-treated fiber and the initial modulus increased by 24.01%. The remarkable reusability of these immobilized cells provides a cost effective method for treatment of natural fibers containing cellulose.

A simple and fast protocol for the protein complex immunoprecipitation (Co-IP) of effector: host protein complexes.

Plant pathogens are responsible for enormous damage in natural and cultured ecosystems. One strategy most pathogenic organisms follow is the secretion of effector proteins that manipulate the host immune system to suppress defense responses. There is considerable interest in finding host targets of pathogen effectors as this helps to shape our understanding of how those proteins work in planta. The presented protocol describes a protein complex immunoprecipitation method aimed at verifying protein-protein interactions derived from protein complementation assays like Yeast-two-Hybrid.

Design and construction of multigenic constructs for plant biotechnology using the GoldenBraid cloning strategy.

GoldenBraid (GB) is an iterative and standardized DNA assembling system specially designed for Multigene Engineering in Plant Synthetic Biology. GB is based on restriction-ligation reactions using type IIS restriction enzymes. GB comprises a collection of standard DNA pieces named "GB parts" and a set of destination plasmids (pDGBs) that incorporate the multipartite assembly of standardized DNA parts. GB reactions are extremely efficient: two transcriptional units (TUs) can be assembled from several basic GBparts in one T-DNA less than 24 h. Moreover, larger assemblies comprising 4-5 TUs are routinely built in less than 2 working weeks. Here we provide a detailed view of the GB methodology. As a practical example, a Bimolecular Fluorescence Complementation construct comprising four TUs in a 12 kb DNA fragment is presented.

Carboxymethyl cellulose coating decreases toxicity and oxidizing capacity of nanoscale zerovalent iron.

Nanoscale zerovalent iron (NZVI) with modified surface via coating with organic stabilizers has been documented with enhanced colloidal stability and dispersity. Therefore, the expanded application potential and accompanying intrinsic exposure of such nanoparticle can be anticipated. In our study, carboxymethyl cellulose (CMC)-stabilized NZVI (CNZVI) exerted minimized oxidative stress response and slower disruption of cell membrane integrity, resulting in mitigated cytotoxicity towards bacteria Agrobacterium sp. PH-08 as compared with the uncoated counterpart. The corrosive oxidation of both nanoparticles in oxygenic water provided a better understanding of coating effect. The decreased oxidative degradation of probe 4-chlorophenol with CNZVI than NZVI implicated a weaker oxidizing capacity, which might overweight massive adhesion-mediated redox damage and explain the different exposure outcome. However, enhanced evolution of iron oxide as well as the promoted production of hydrogen peroxide adversely demonstrated CMC-coating facilitated iron corrosion by oxygen, suggesting CMC was most likely to act as a radical scavenger and compete with organics or bacteria for oxidants. Moreover, XRD, XPS and TEM results showed that the spherical NZVI was oxidized to form needle-shaped iron oxide-hydroxide (γFeOOH) with no detectable oxidative stress for PH-08, alleviating worries regarding exotoxicological impact of iron nanotechnology.

Agrobacterium-mediated genetic transformation of Pogostemon cablin (Blanco) Benth. Using leaf explants: bactericidal effect of leaf extracts and counteracting strategies.

An optimized protocol for Agrobacterium tumefaciens-mediated transformation of patchouli using leaf disk explants is reported. In vitro antibacterial activity of leaf extracts of the plants revealed Agrobacterium sensitivity to the extracts. Fluorometric assay of bacterial cell viability indicated dose-dependent cytotoxic activity of callus extract against Agrobacterium cells. Addition of 0.1% Tween 20 and 2 g/l L-glutamine to Agrobacterium infection medium counteracted the bactericidal effect and significantly increased the T-DNA delivery to explants. A short preculture of explants for 2 days followed by infection with Agrobacterium in medium containing 150 µM of acetosyringone were found essential for efficient T-DNA delivery. Cocultivation for 3 days at 22 °C in conjunction with other optimized factors resulted in maximum T-DNA delivery. The Agrobacterium-mediated transformation of leaf disk explants were found significantly related to physiological age of the explants, age and origin of the of the donor plant. Leaf explants from second node of the 3-month-old in vivo plants showed highest transformation efficiency (94.3%) revealed by transient GUS expression assay. Plants selected on medium containing 20 mg/l kanamycin showed stable GUS expression in leaves and stem. The elongated shoots readily developed roots on kanamycin-free rooting medium and on transfer to soil, plants were successfully established. Polymerase chain reaction (PCR) and reverse-transcriptase PCR analysis in putative plants confirmed their transgenic nature. The established transformation method should provide new opportunities for the genetic improvement of patchouli for desirable trait.

Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant.

VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.