<Emphasis Type=Italic>Pseudomonas</Emphasis> siderophores in the sputum of patients with cystic fibrosis

The lungs of patients with cystic fibrosis become chronically infected with the bacterium Pseudomonas aeruginosa, which heralds progressive lung damage and a decline in health. Iron is a crucial micronutrient for bacteria and its acquisition is a key factor in infection. P. aeruginosa can acquire this element by secreting pyoverdine and pyochelin, iron-chelating compounds (siderophores) that scavenge iron and deliver it to the bacteria. Siderophore-mediated iron uptake is generally considered a key factor in the ability of P. aeruginosa to cause infection. We have investigated the amounts of pyoverdine in 148 sputum samples from 36 cystic fibrosis patients (30 infected with P. aeruginosa and 6 as negative controls). Pyoverdine was present in 93 samples in concentrations between 0.30 and 51 μM (median 4.6 μM) and there was a strong association between the amount of pyoverdine and the number of P. aeruginosa present. However, pyoverdine was not present, or below the limits of detection (~0.3 μM), in 21 sputum samples that contained P. aeruginosa. Pyochelin was also absent, or below the limits of detection (~1 μM), in samples from P. aeruginosa-infected patients with little or no detectable pyoverdine. Our data show that pyoverdine is an important iron-scavenging molecule for P. aeruginosa in many cystic fibrosis patients, but other P. aeruginosa iron-uptake systems must be active in some patients to satisfy the bacterial need for iron.     

Processing of intermediates in recombination and DNA repair: identification of a new endonuclease that specifically cleaves Holliday junctions.

The formation and subsequent resolution of Holliday junctions are critical stages in recombination. We describe a new Escherichia coli endonuclease that resolves Holliday intermediates by junction cleavage. The 14 kDa Rus protein binds DNA containing a synthetic four-way junction (X-DNA) and introduces symmetrical cuts in two strands to give nicked duplex products. Rus also processes Holliday intermediates made by RecA into products that are characteristic of junction resolution. The cleavage activity on X-DNA is remarkably similar to that of RuvC. Both proteins preferentially cut the same two strands at the same location. Increased expression of Rus suppresses the DNA repair and recombination defects of ruvA, ruvB and ruvC mutants. We conclude that all ruv strains are defective in junction cleavage, and discuss pathways for Holliday junction resolution by RuvAB, RuvC, RecG and Rus.     

Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination

Summary Recombinant plasmids carrying ruvA, ruvB, or both were constructed and used to investigate the genetic defects in a collection of UV-sensitive ruv mutants. The results revealed that efficient survival of UV-irradiated cells depends on both ruvA and ruvB, and on a third gene, ruvC, located upstream of the ruvAB operon. Southern blotting analysis was used to locate insertions in ruv and to examine putative deletion mutants. Two Tn10 insertions were located to the region encoding ruvA. Since these insertions caused a deficiency in the activities of both ruvA and ruvB, we concluded that they must exert a polar effect on ruvB. Two putative ruv deletion mutants were shown to be the result of deletion-inversion events mediated during imprecise excision of Tn10. The relevant inversion breakpoints in these mutants were located to ruvA and ruvC.     

Modulation of glycogen and trehalose levels in Micromonospora echinospora (ATCC 15837)

The growth of Micromonospora echinospora was studied in high and low C/N ratio medium using both batch and continuous culture. Asparagine was consumed rapidly in batch cultures where it served as both a nitrogen and carbon source. Glucose consumption was low suggesting that asparagine functions as the major carbon source under these conditions. The effect of nutrient limitation on the accumulation of storage carbohydrate in batch culture revealed an intimate association between nitrogen limitation and the accumulation of carbonaceous reserves. This study revealed that glycogen constituted the major carbohydrate reserve associated with the onset of sporulation. Intracellular trehalose levels were found to be relatively low and may have been affected by the availability of carbon. Continuous culture studies revealed a correlation between glycogen accumulation and increasing growth rate. It was also found that elevated cellular ATP levels correlated with the increase in glycogen, and reduced glycolytic activity. At the higher growth rates cellular ATP levels were elevated and coincided with reduced activity of the key glycolytic enzyme, phosphofructokinase, suggesting that glycogen can act as a convenient energy reservoir when excess carbon flux dictates.     

Important marine habitat off east Antarctica revealed by two decades of multi‐species predator tracking

Satellite telemetry data are a key source of animal distribution information for marine ecosystem management and conservation activities. We used two decades of telemetry data from the East Antarctic sector of the Southern Ocean. Habitat utilization models for the spring/summer period were developed for six highly abundant, wide‐ranging meso‐ and top‐predator species: Adélie Pygoscelis adeliae and emperor Aptenodytes forsteri penguins, light‐mantled albatross Phoebetria palpebrata, Antarctic fur seals Arctocephalus gazella, southern elephant seals Mirounga leonina, and Weddell seals Leptonychotes weddellii. The regional predictions from these models were combined to identify areas utilized by multiple species, and therefore likely to be of particular ecological significance. These areas were distributed across the longitudinal breadth of the East Antarctic sector, and were characterized by proximity to breeding colonies, both on the Antarctic continent and on subantarctic islands to the north, and by sea‐ice dynamics, particularly locations of winter polynyas. These areas of important habitat were also congruent with many of the areas reported to be showing the strongest regional trends in sea ice seasonality. The results emphasize the importance of on‐shore and sea‐ice processes to Antarctic marine ecosystems. Our study provides ocean‐basin‐scale predictions of predator habitat utilization, an assessment of contemporary habitat use against which future changes can be assessed, and is of direct relevance to current conservation planning and spatial management efforts.     

RusA proteins from the extreme thermophile Aquifex aeolicus and lactococcal phage r1t resolve Holliday junctions

The RusA protein of Escherichia coli is a DNA structure-specific endonuclease that resolves Holliday junction intermediates formed during DNA replication, recombination and repair by introducing symmetrically paired incisions 5' to CC dinucleotides. It is encoded by the defective prophage DLP12, which raises the possibility that it may be of bacteriophage origin. We show that rusA-like sequences are indeed often associated with prophage sequences in the genomes of several bacterial species. They are also found in many bacteriophages, including Lactococcus lactis phage r1t. However, rusA is also present in the chromosome of the hyperthermophilic bacterium Aquifex aeolicus. In this case, there is no obvious association of rusA with prophage-like sequences. Given the ancient lineage of Aquifex aeolicus, this observation provides the first indication that RusA may be of bacterial origin. The RusA proteins of A. aeolicus and bacteriophage r1t were purified and shown to resolve Holliday junctions. The r1t enzyme also promotes DNA repair in strains lacking the RuvABC resolvase. Both enzymes cleave junctions in a sequence-dependent manner, but the A. aeolicus RusA shows a different sequence preference (3' to TG) from the E. coli protein (5' to CC), and the r1t RusA has relaxed sequence dependence, requiring only a single cytosine.     

Relative orientation between the beta-ionone ring and the polyene chain for the chromophore of rhodopsin in native membranes

Rotational resonance solid state nuclear magnetic resonance has been used to determine the relative orientation of the beta-ionone ring and the polyene chain of the chromophore 11-Z-retinylidene of rhodopsin in rod outer segment membranes from bovine retina. The bleached protein was regenerated with either 11-Z-[8,18-(13)C(2)]retinal or 11-Z-[8,16/17(13)C(2)]retinal, the latter having only one (13)C label at either of the chemically equivalent positions 16 and 17. Observation of (13)C selectively enriched in the ring methyl groups, C16/17, revealed alternative conformational states for the ring. Minor spectral components comprised around 26% of the chromophore. The major conformation (approximately 74%) has the chemical shift resolution required for measuring internuclear distances to (13)C in the retinal chain (C8) separately from each of these methyl groups. The resulting distance constraints, C8 to C16 and C17 (4.05 +/- 0.25 A) and from C8 to C18 (2.95 +/- 0.15 A), show that the major portion of retinylidene in rhodopsin has a twisted 6-s-cis conformation. The more precise distance measurement made here between C8 and C18 (2.95 A) predicts that the chain is twisted out-of-plane with respect to the ring by a modest amount (C5-C6-C7-C8 torsion angle = -28 +/- 7 degrees ).     

Mechanism of arsenate resistance in the ericoid mycorrhizal fungus Hymenoscyphus ericae

Arsenate resistance is exhibited by the ericoid mycorrhizal fungus Hymenoscyphus ericae collected from As-contaminated mine soils. To investigate the mechanism of arsenate resistance, uptake kinetics for arsenate (H(2)AsO(4)(-)), arsenite (H(3)AsO(3)), and phosphate (H(2)PO(4)(-)) were determined in both arsenate-resistant and -non-resistant H. ericae. The uptake kinetics of H(2)AsO(4)(-), H(3)AsO(3), and H(2)PO(4)(-) in both resistant and non-resistant isolates were similar. The presence of 5.0 microM H(2)PO(4)(-) repressed uptake of H(2)AsO(4)(-) and exposure to 0.75 mM H(2)AsO(4)(-) repressed H(2)PO(4)(-) uptake in both H. ericae. Mine site H. ericae demonstrated an enhanced As efflux mechanism in comparison with non-resistant H. ericae and lost approximately 90% of preloaded cellular As (1-h uptake of 0.22 micromol g(-1) dry weight h(-1) H(2)AsO(4)(-)) over a 5-h period in comparison with non-resistant H. ericae, which lost 40% of their total absorbed H(2)AsO(4)(-). As lost from the fungal tissue was in the form of H(3)AsO(3). The results of the present study demonstrate an enhanced H(3)AsO(3) efflux system operating in mine site H. ericae as a mechanism for H(2)AsO(4)(-) resistance. The ecological significance of this mechanism of arsenate resistance is discussed.