Adaptation of Pseudomonas fluorescens to the plant rhizosphere

Saprophytic Pseudomonas are common root-colonizing bacteria that can improve plant health. Efficient exploitation of these bacteria in agriculture requires knowledge of traits that enhance ecological performance in the rhizosphere. Here, I describe the development and application of a promoter-trapping technology (IVET) that enables the isolation of Pseudomonas fluorescens genes that show elevated levels of expression in the rhizosphere. Using IVET, 20 P. fluorescens genes were identified that are induced during rhizosphere colonization, and their patterns of expression were analysed in laboratory media and in the rhizosphere. Fourteen genes showed significant homology to sequences in GenBank that are involved in nutrient acquisition, stress response, or secretion; six showed no homology. Seven of the rhizosphere-induced ( rhi ) genes have homology to known non- Pseudomonas genes. One of the rhi genes ( hrcC ) is a component of a type III secretion pathway, not previously known in non-parasitic bacteria. Together, these genes provide a view of the rhizosphere environment as perceived by a rhizosphere colonist, and suggest that the nature of the association between P. fluorescens and the plant root may be more complex and intimate than previously thought.     

Gamma-proteobacteria Aquicella lusitana gen. nov., sp. nov., and Aquicella siphonis sp. nov. infect protozoa and require activated charcoal for growth in laboratory media

Several isolates, belonging to two new species of the same novel genus of gamma-proteobacteria, were recovered from drilled well (borehole) and spa water at S?o Gemil in central Portugal. These organisms are phylogenetically most closely related to the strictly intracellular uncultured species of the genus Rickettsiella, which cause disease in arthropods, and to the facultatively intracellular species of the genus Legionella, some of which cause Legionnaires' disease and Pontiac fever. The S?o Gemil strains grew only on media containing charcoal, as is also true of the species of the genus LEGIONELLA: Unlike the vast majority of Legionella isolates, the new isolates did not require L-cysteine or ferric pyrophosphate for growth but like the legionellae had an absolute requirement for alpha-ketoglutarate. Strains SGT-39(T) and SGT-56 grew consistently between 30 and 43 degrees C, while strains SGT-108(T) and SGT-109 grew between 30 and 40 degrees C. The pH ranges for growth of these organisms were surprisingly narrow: strains SGT-39(T) and SGT-56 grew between pH 6.3 and 7.3, while strains SGT-108(T) and SGT-109 grew between pH 6.3 and 7.0. Both organisms proliferated in the amoeba Hartmannella vermiformis but did not grow in U937 human cells. Based on 16S rRNA gene sequence analysis and physiological, biochemical, and chemical analysis we describe two new species of one novel genus; one species is represented by strain SGT-39(T), for which we propose the name Aquicella lusitana, while strain SGT-108(T) represents a second species of the same genus, for which we propose the name Aquicella siphonis.     

Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose

The wrinkly spreader (WS) genotype of Pseudomonas fluorescens SBW25 colonizes the air-liquid interface of spatially structured microcosms resulting in formation of a thick biofilm. Its ability to colonize this niche is largely due to overproduction of a cellulosic polymer, the product of the wss operon. Chemical analysis of the biofilm matrix shows that the cellulosic polymer is partially acetylated cellulose, which is consistent with predictions of gene function based on in silico analysis of wss. Both polar and non-polar mutations in the sixth gene of the wss operon (wssF ) or adjacent downstream genes (wssGHIJ ) generated mutants that overproduce non-acetylated cellulose, thus implicating WssFGHIJ in acetylation of cellulose. WssGHI are homologues of AlgFIJ from P. aeruginosa, which together are necessary and sufficient to acetylate alginate polymer. WssF belongs to a newly established Pfam family and is predicted to provide acyl groups to WssGHI. The role of WssJ is unclear, but its similarity to MinD-like proteins suggests a role in polar localization of the acetylation complex. Fluorescent microscopy of Calcofluor-stained biofilms revealed a matrix structure composed of networks of cellulose fibres, sheets and clumped material. Quantitative analyses of biofilm structure showed that acetylation of cellulose is important for effective colonization of the air-liquid interface: mutants identical to WS, but defective in enzymes required for acetylation produced biofilms with altered physical properties. In addition, mutants producing non-acetylated cellulose were unable to spread rapidly across solid surfaces. Inclusion in these assays of a WS mutant with a defect in the GGDEF regulator (WspR) confirmed the requirement for this protein in expression of both acetylated cellulose polymer and bacterial attachment. These results suggest a model in which WspR regulation of cellulose expression and attachment plays a role in the co-ordination of surface colonization.     

Two rate-limiting steps in the kinetic mechanism of the serine/threonine specific protein kinase ERK2: a case of fast phosphorylation followed by fast product release

Extracellular regulated protein kinase 2 (ERK2) is a eukaryotic protein kinase whose activity is regulated by mitogenic stimuli. To gain insight into the catalytic properties of ERK2 and to complement structure-function studies, we undertook a pre-steady state kinetic analysis of the enzyme. To do this, ERK2 was quantitatively activated by MAPKK1 in vitro by monitoring the stoichiometry and site specificity of phosphorylation using a combination of protein mass spectrometry, tryptic peptide analysis, and (32)P radiolabeling. Using a quench-flow apparatus, MgATP(2-) was rapidly mixed (<1 ms) with both ERK2 and the protein substrate EtsDelta138 in the presence of a saturating total concentration (20 mM) of magnesium ion at 27 degrees C and pH 7.5. An exponential burst of product was observed over the first few milliseconds that followed mixing. This burst had an amplitude alpha of 0.44 and was followed by a slower linear phase. The pre-steady state burst is consistent with two partially rate-limiting enzymatic steps, which have the following rate constants: k(2) = 109 +/- 9 s(-1) and k(3) = 56 +/- 4 s(-1). These are attributed to rapid phosphorylation of EtsDelta138 and the process of product release, respectively. Single-turnover experiments provided an independent determination of k(2) (106 +/- 25 s(-1)). The observed catalytic constant (k(cat)(obs)) was found to be sensitive to the concentration of ERK2. The data fit a model in which ERK2 monomers form dimers and suggest that both the monomeric and dimeric forms of ERK2 are active with catalytic constants (k(cat)) of 25 and 37 s(-1), respectively. In addition, the model suggests that in the presence of saturating concentrations of both magnesium and substrates ERK2 subunits dissociate with a dissociation constant (K(d)) of 32 +/- 16 nM.     

Antagonistic coevolution between a bacterium and a bacteriophage

Antagonistic coevolution between hosts and parasites is believed to play a pivotal role in host and parasite population dynamics, the evolutionary maintenance of sex and the evolution of parasite virulence. Furthermore, antagonistic coevolution is believed to be responsible for rapid differentiation of both hosts and parasites between geographically structured populations. Yet empirical evidence for host-parasite antagonistic coevolution, and its impact on between-population genetic divergence, is limited. Here we demonstrate a long-term arms race between the infectivity of a viral parasite (bacteriophage; phage) and the resistance of its bacterial host. Coevolution was largely driven by directional selection, with hosts becoming resistant to a wider range of parasite genotypes and parasites infective to a wider range of host genotypes. Coevolution followed divergent trajectories between replicate communities despite establishment with isogenic bacteria and phage, and resulted in bacteria adapted to their own, compared with other, phage populations.     

A statistically derived parameterization for the collagen triple-helix

The triple-helix is a unique secondary structural motif found primarily within the collagens. In collagen, it is a homo- or hetero-tripeptide with a repeating primary sequence of (Gly-X-Y)(n), displaying characteristic peptide backbone dihedral angles. Studies of bulk collagen fibrils indicate that the triple-helix must be a highly repetitive secondary structure, with very specific constraints. Primary sequence analysis shows that most collagen molecules are primarily triple-helical; however, no high-resolution structure of any entire protein is yet available. Given the drastic morphological differences in self-assembled collagen structures with subtle changes in assembly conditions, a detailed knowledge of the relative locations of charged and sterically bulky residues in collagen is desirable. Its repetitive primary sequence and highly conserved secondary structure make collagen, and the triple-helix in general, an ideal candidate for a general parameterization for prediction of residue locations and for the use of a helical wheel in the prediction of residue orientation. Herein, a statistical analysis of the currently available high-resolution X-ray crystal structures of model triple-helical peptides is performed to produce an experimentally based parameter set for predicting peptide backbone and C(beta) atom locations for the triple-helix. Unlike existing homology models, this allows easy prediction of an entire triple-helix structure based on all existing high-resolution triple-helix structures, rather than only on a single structure or on idealized parameters. Furthermore, regional differences based on the helical propensity of residues may be readily incorporated. The parameter set is validated in terms of the predicted bond lengths, backbone dihedral angles, and interchain hydrogen bonding.     

Psychromonas antarcticus gen. nov., sp. nov., a new aerotolerant anaerobic, halophilic psychrophile isolated from pond sediment of the McMurdo Ice Shelf, Antarctica

A gram-negative, rod- to oval-shaped, aerotolerant anaerobic bacterium was isolated from an anaerobic enrichment inoculated with sediment taken from below the cyanobacterial mat of a high-salinity pond near Bratina Island on the McMurdo Ice Shelf, Antarctica. The organism was positive for terminal oxidase and catalase and was motile by means of a polar flagellum. Optimal growth of anaerobic cultures occurred at 12° C, at pH 6.5, and at an NaCl concentration of 3% (w/v). Of a variety of polysaccharides tested, only starch and glycogen supported growth. No growth was observed on cellulosic substrates and xylan, and the organism was unable to attack esculin. Monosaccharides and disaccharides, including the cyanobacterial cell-wall constituent N-acetyl glucosamine, were fermented. Per 100 mol of hexose, the following products (in mol) were formed: acetate, 60; formate, 130; ethanol, 56; lactate, 73; CO₂, 15; and butyrate, 2. Propionate, ethanol, n-propanol, n-butanol and succinate were not detectable in the culture medium (< 1 mol per 100 mol of monomer). Hydrogen was not detected in the head space (detection limit < 10^–5 atm). Growth yields in aerobic static liquid cultures were slightly higher than those in anaerobic culture, and fermentation favoured acetate at the expense of electron sink products. Growth was inhibited in aerobic shaking cultures, and the organism did not utilize nitrate or sulfate as electron acceptors. The G+C content of the DNA from the bacterium was 42.8 mol%. A phylogenetic analysis indicated that the organism is a member of the γ-subgroup of Proteobacteria, but that it is distinct from other members of this group based on the sequence of its 16S rRNA gene, mol% G+C, morphology, and physiological and biochemical characteristics. It is designated as a new genus and species; the type strain is star-1 (DSM 10704). Received: 17 June 1996 / Accepted: 13 October 1997     

Strategies for dealing with conformational sampling in structural calculations of flexible or kinked transmembrane peptides.

Peptides corresponding to transmembrane (TM) segments from membrane proteins provide a potential route for the determination of membrane protein structure. We have determined that 2 functionally critical TM segments from the mammalian Na+/H+ exchanger display well converged structure in regions separated by break points. The flexibility of these break points results in conformational sampling in solution. A brief review of available NMR structures of helical membrane proteins demonstrates that there are a number of published structures showing similar properties. Such flexibility is likely indicative of kinks in the full-length protein. This minireview focuses on methods and protocols for NMR structure calculation and analysis of peptide structures under conditions of conformational sampling. The methods outlined allow the identification and analysis of structured peptides containing break points owing to conformational sampling and the differentiation between oligomerization and ensemble-averaged observation of multiple peptide conformations.