Susana Castro-Sowinski

Ecological and agricultural significance of bacterial polyhydroxyalkanoates.

Abstract Polyhydroxyalkanoates (PHAs) are a group of carbon and energy storage compounds that are accumulated during suboptimal growth by many bacteria, and intracellularly deposited in the form of inclusion bodies. Accumulation of PHAs is thought to be used by bacteria to increase survival and stress tolerance in changing environments, and in competitive settings where carbon and energy sources may be limited, such as those encountered in the soil and the rhizosphere. Understanding the role that PHAs play as internal storage polymers is of fundamental importance in microbial ecology, and holds great potential for the improvement of bacterial inoculants for plants and soils. This review summarizes the current knowledge on the ecological function of PHAs, and their strategic role as survival factors in microorganisms under varying environmental stress is emphasized. It also explores the phylogeny of the PHA cycle enzymes, PHA synthase, and PHA depolymerase, suggesting that PHA accumulation was earlier acquired and maintained during evolution, thus contributing to microbial survival in the environment.

The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis

Azospirillum brasilense is a plant root-colonizing bacterium that exerts beneficial effects on the growth of many agricultural crops. Extracellular polysaccharides of the bacterium play an important role in its interactions with plant roots. The pRhico plasmid of A. brasilense Sp7, also named p90, carries several genes involved in synthesis and export of cell surface polysaccharides. We generated two Sp7 mutants impaired in two pRhico-located genes, noeJ and noeL, encoding mannose-6-phosphate isomerase and GDP-mannose 4,6-dehydratase, respectively. Our results demonstrate that in A. brasilense Sp7, noeJ and noeL are involved in lipopolysaccharide and exopolysaccharide synthesis. noeJ and noeL mutant strains were significantly altered in their outer membrane and cytoplasmic/periplasmic protein profiles relative to the wild-type strain. Moreover, both noeJ and noeL mutations significantly affected the bacterial responses to several stresses and antimicrobial compounds. Disruption of noeL, but not noeJ, affected the ability of the A. brasilense Sp7 to form biofilms. The pleiotropic alterations observed in the mutants could be due, at least partially, to their altered lipopolysaccharides and exopolysaccharides relative to the wild-type.

Natural Functions of Bacterial Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are energy- and intracellular carbon-storage compounds that can be mobilized and used when carbon is a limiting resource. Intracellular accumulation of PHA enhances the survival of several bacterial species under environmental stress conditions imposed in water and soil, such as UV irradiation, salinity, thermal and oxidative stress, desiccation, and osmotic shock. The ability to endure these stresses is linked to a cascade of events concomitant with PHA degradation and the expression of genes involved in protection against damaging agents. PHA synthesis involves enzymatic and transcriptional regulation, where the RpoS central stationary phase regulator sigma factor has been shown to be implicated. The energy generated during PHA degradation can also be used to drive various important energy-consuming pathways. In addition to its relevance for the plastic industry, PHA has important applications for agriculture, as those related to the production of reliable commercial inoculants, and in controlled release of insecticides when incorporated into degradable PHA granules.

Glycogen phosphorylase is involved in stress endurance and biofilm formation in Azospirillum brasilense Sp7

Here we report the identification of a glycogen phosphorylase (glgP) gene in the plant growth-promoting rhizobacterium Azospirillum brasilense, Sp7, and the characterization of a glgP marker exchange mutant of this strain. The glgP mutant showed a twofold reduction of glycogen phosphorylase activity and an increased glycogen accumulation as compared with wild-type Sp7, indicating that the identified gene indeed encodes a protein with glycogen phosphorylase activity. Interestingly, the glgP mutant had higher survival rates than the wild type after exposure to starvation, desiccation and osmotic pressure. The mutant was shown to be compromised in its biofilm formation ability. Analysis of the exopolysaccharide sugar composition of the glgP mutant revealed a decrease in the amount of glucose, accompanied by increases in rhamnose, fucose and ribose, as compared with the Sp7 exopolysaccharide. To the best of our knowledge, this is the first study that demonstrates GlgP activity in A. brasilense, and shows that glycogen accumulation may play an important role in the stress endurance of this bacterium.

Phenotypic variation in Azospirillum brasilense exposed to starvation

Bacteria have developed mechanisms that allow them maintaining cell viability during starvation and resuming growth when nutrients become available. Among these mechanisms are adaptive mutations and phase variation, which are often associated with DNA rearrangements. Azospirillum brasilense is a Gram-negative, nitrogen-fixing, plant growth-promoting rhizobacterium. Here we report phenotypic variants of A. brasilense that were collected after exposure to prolonged starvation or after re-isolation from maize roots. The variants differed in several features from the parental strains, including pigmentation, aggregation ability, EPS amount and composition and LPS structure. One of the phenotypic variants, overproducing EPS and showing an altered LPS structure, was further characterized and showed differential response to several stresses and antibiotics relative to its parental strain. Characterization of the variants by repetitive-PCR revealed that phenotypic variation was often associated with DNA rearrangements.

Cellular and biochemical response to Cr(VI) in Stenotrophomonas sp.

Chromium (Cr)-resistant bacteria isolated from a soil with 6 g kg(-1) of Cr were identified based on 16S rRNA gene sequence analysis as a Stenotrophomonas, and designated as JD1. Growth of JD1 was accompanied by transformation of Cr(VI) to Cr(III) in liquid medium initially containing 300 mg L(-1) Cr(VI), the maximum concentration allowing growth. JD1 produced the highest levels of a Cr(VI)-binding exopolysaccharide when grown in medium with 100 mg L(-1) Cr(VI). The relative exopolysaccharide monosaccharide composition was analysed by HPLC, which showed that rhamnose+galactose was the major component, and that its relative level increased when cells were grown with Cr(VI). JD1 grew as a biofilm on various inert surfaces. Biofilm macromolecular composition analysis indicated that the relative levels of exopolysaccharide and protein were more abundant in biofilms grown in 100 mg L(-1) Cr(VI), whereas relative uronic acid levels remained constant. Biofilm cells exposed to Cr(VI) were elongated, grouped in clusters and exopolysaccharide obtained from the biofilm extracellular matrix had an enhanced capacity to bind Cr(VI). Exopolysaccharide production and composition, and biofilm growth are discussed as a mechanism of protection that allows survival during Cr(VI) stress.

Legume Crops, Importance and Use of Bacterial Inoculation to Increase Production

Legumes are flowering plants that produce seedpods. They have colonized several ecosystems (from rain forests and arctic/alpine regions to deserts; Schrire et al., 2005), and have been found in most of the archaeological record of plants. Early in 37 B.C. Varro said “Legumes should be planted in light soils, not so much for their own crop as for the good they do to subsequent crops” (Graham & Vance, 2003), recognizing the importance of multiple cropping and intercropping production.

A Thioredoxin of Sinorhizobium meliloti CE52G Is Required for Melanin Production and Symbiotic Nitrogen Fixation

A miniTn5-induced mutant of a melanin-producing strain of Sinorhizobium meliloti (CE52G) that does not produce melanin was mapped to a gene identified as a probable thioredoxin gene. It was proved that the thiol-reducing activity of the mutant was affected. Addition to the growth medium of substrates that induce the production of melanin (L-tyrosine, guaiacol, orcinol) increased the thioredoxin-like (trxL) mRNA level in the wild-type strain. The mutant strain was affected in the response to paraquat-induced oxidative stress, symbiotic nitrogen fixation, and both laccase and tyrosinase activities. The importance of thioredoxin in melanin production in bacteria, through the regulation of laccase or tyrosinase activities, or both, by the redox state of structural or catalytic SH groups, is discussed.

Removal of bacteria and Cryptosporidium from water by micelle–montmorillonite complexes

AbstractThis study aimed at testing the application of nano-composites of micelle–montmorillonite for removal of bacteria and parasites from water. The suitability of these complexes for efficient adsorption of microorganisms was expected on account of their large surface areas, large excess of positive charge, and existence of large hydrophobic domains. Tests included removal from water of bacteria: Gram negative (Escherichia coli K-12), Gram positive (Bacillus megaterium), and a protozoan parasite, Cryptosporidium parvum. Micelles of the organic cations Benzyldimethylhexadecylammonium (BDMHDA), or Octadecyltrimethylammonium complexed with the clay-mineral montmorillonite were shown to reduce by 3–6 orders of magnitude the numbers of microorganisms in water as tested in suspension and by filters (20 cm in length) packed with the complexes mixed with excess sand (100:1, w/w). Respiration tests demonstrated that E. coli K-12 cells adsorbed to micelle (BDMHDA)–montmorillonite complex lost their viability. K...

The Pattern of Secreted Molecules During the Co-Inoculation of Alfalfa Plants With Sinorhizobium meliloti and Delftia sp. strain JD2: An Interaction That Improves Plant Yield.

Delftia sp. strain JD2 is a plant-growth-promoting bacterium that enhances legume nodulation and growth, acting as nodule-assisting bacterium during the co-inoculation of plants with rhizobial strains. In this work, we evaluate how the co-inoculation of alfalfa with Sinorhizobium meliloti U143 and JD2 increases plant yield under greenhouse conditions and we analyze the pattern of secreted bioactive compounds which may be involved in the microbe-plant communication. The chemical composition of extracellular cultures (EC) produced in hydroponic conditions (collected 4, 7, and 14 days after bacterial treatment) were characterized using different chromatographic and elucidation techniques. In addition, we assessed the effect that plant irrigation with cell-free EC, produced during co-inoculation experiments, would have on plant yield. Results showed increased alfalfa shoot and root matter, suggesting that U143-JD2 co-inoculation might be a beneficial agricultural practice. The pattern of secreted secondary metabolites among treatments showed important differences. Qualitative and quantitative changes in phenolic compounds (including flavonoids), organic acids, and volatile compounds were detected during the early microbe-plant interaction, suggesting that the production of some molecules positively affects the microbe-plant association. Finally, the irrigation of co-inoculated plants with cell-free EC under greenhouse conditions increased plant yield over agronomic expectations. This effect might be attributed to the bioactive secondary metabolites incorporated during the irrigation.