The crystal structure of DR2241 from Deinococcus radiodurans at 1.9 A resolution reveals a multi-domain protein with structural similarity to chelatases but also with two additional novel domains

A unique family of proteins have been identified in the Deinococcus genus with an N-terminal cobalamin (vitamin B(12)) chelatase domain denoted CbiX and an additional unique C-terminal domain with unknown function. Here we report the first crystal structure from this new family of proteins with the structure of Deinococcus radiodurans protein DR2241. The structure reveals a multi-domain protein where domains A (residues 1-132) has the same fold as the small CbiX (CbiX(S)), domains A and B (residues 1-272) follow the chelatase super-family fold and the two additional unique domains C and D have no structural homologues. Domain D harbours the sequence motifs CxxC and CxxxC, in which DR2241 gives the first evidence that these motifs bind a [4Fe-4S] iron-sulphur cluster. In solution there are indications of multimeric forms, and in the crystallographic asymmetric unit a tetramer is found where domains C and D are involved in stabilising the tetrameric assembly.     

A novel platform for accelerated pharmacodynamic profiling for lead optimization of anticancer drug candidates

Oncology drug discovery is, by definition, a target-rich enterprise. High-throughput screening (HTS) laboratories have supported a wide array of molecularly targeted and chemical genomic approaches for anticancer lead generation, and the number of hits emerging from such campaigns has increased dramatically. Although automation of HTS processes has eliminated primary screening as a bottleneck, the demands on secondary screening in appropriate cell-based assays have increased concomitantly with the numbers of hits delivered to therapeutic area laboratories. The authors describe herein the implementation of a novel platform using off-the-shelf solutions that have allowed them to efficiently characterize hundreds of HTS hits using a palette of Western blot-based pharmacodynamic assays. The platform employs a combination of a flatbed bufferless SDS-PAGE system, a dry ultra-rapid electroblotting apparatus, and a highly sensitive and quantitative infrared imaging system. Cumulatively, this platform has significantly reduced the cycle time for HTS hit evaluation. In addition, the routine use of this platform has resulted in higher quality data that have allowed the development of structure-activity databases that have tangibly improved lead optimization. The authors describe in detail the application of this platform, designated the Accelerated Pharmaco-Dynamic Profiler (APDP), to the annotation of inhibitors of 2 attractive oncology targets, BRAF kinase and Hsp90.     

Methanogen Colonisation Does Not Significantly Alter Acetogen Diversity in Lambs Isolated 17?h After Birth and Raised Aseptically

Reductive acetogenesis is not competitive with methanogenesis in adult ruminants, whereas acetogenic bacteria are the dominant hydrogenotrophs in the early rumen microbiota. The ecology of hydrogenotrophs in the developing rumen was investigated using young lambs, raised in sterile isolators, and conventional adult sheep. Two lambs were born naturally, left with their dams for 17?h and then placed into a sterile isolator and reared aseptically. They were inoculated with cellulolytic bacteria and later with Methanobrevibacter sp. 87.7 to investigate the effect of methanogen establishment on the rumen acetogen population since they lacked cultivable representatives of methanogens. Putative acetogens were investigated by acetyl-CoA synthase and formyltetrahydrofolate synthetase gene analysis and methanogens by methyl coenzyme reductase A gene analysis. Unexpectedly, a low abundant but diverse population of methanogens (predominantly Methanobrevibacter spp.) was identified in isolated lambs pre-inoculation with Mbb. sp 87.7, which was similar to the community structure in conventional sheep. In contrast, potential acetogen diversity in isolated lambs and conventional sheep was different. Potential acetogens affiliated between the Lachnospiraceae and Clostridiaceae in conventional sheep and with the Blautia genus and the Lachnospiraceae in isolated lambs. The establishment of Mbb. sp. 87.7 (1,000-fold increase in methanogens) did not substantially affect acetogen diversity.     

An ‘open’ structure of the RecOR complex supports ssDNA binding within the core of the complex

Efficient DNA repair is critical for cell survival and the maintenance of genome integrity. The homologous recombination pathway is responsible for the repair of DNA double-strand breaks within cells. Initiation of this pathway in bacteria can be carried out by either the RecBCD or the RecFOR proteins. An important regulatory player within the RecFOR pathway is the RecOR complex that facilitates RecA loading onto DNA. Here we report new data regarding the assembly of Deinococcus radiodurans RecOR and its interaction with DNA, providing novel mechanistic insight into the mode of action of RecOR in homologous recombination. We present a higher resolution crystal structure of RecOR in an ‘open’ conformation in which the tetrameric RecR ring flanked by two RecO molecules is accessible for DNA binding. We show using small-angle neutron scattering and mutagenesis studies that DNA binding does indeed occur within the RecR ring. Binding of single-stranded DNA occurs without any major conformational changes of the RecOR complex while structural rearrangements are observed on double-stranded DNA binding. Finally, our molecular dynamics simulations, supported by our biochemical data, provide a detailed picture of the DNA binding motif of RecOR and reveal that single-stranded DNA is sandwiched between the two facing oligonucleotide binding domains of RecO within the RecR ring.     

The diversity of Escherichia coli serotypes and biotypes in cattle faeces

AIM: To study the diversity of commensal Escherichia coli populations shed in faeces of cattle fed on different diets. METHODS AND RESULTS: Thirty Brahman-cross steers were initially fed a high grain (80%) diet and then randomly allocated into three dietary treatment groups, fed 80% grain, roughage, or roughage + 50% molasses. Up to eight different E. coli isolates were selected from primary isolation plates of faecal samples from each animal. Fifty-two distinct serotypes, including nine different VTEC strains, were identified from a total of 474 E. coli isolates. Cattle fed a roughage + molasses diet had greater serotype diversity (30 serotypes identified) than cattle fed roughage or grain (21 and 17 serotypes identified respectively). Cluster analysis showed that serotypes isolated from cattle fed roughage and roughage + molasses diets were more closely associated than serotypes isolated from cattle fed grain. Resistance to one or more of 11 antimicrobial agents was detected among isolates from 20 different serotypes. Whilst only 2.3% of E. coli isolates produced enterohaemolysin, 25% were found to produce alpha-haemolysin. CONCLUSIONS: Diverse non-VTEC populations of E. coli serotypes are shed in cattle faeces and diet may affect population diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information on the serotype diversity and phenotypic traits of predominant E. coli populations in cattle faeces, which could be sources of environmental contamination.     

The reaction mechanism of phospholipase D from Streptomyces sp. strain PMF. Snapshots along the reaction pathway reveal a pentacoordinate reaction intermediate and an unexpected final product

Almost all enzyme-catalysed phosphohydrolytic or phosphoryl transfer reactions proceed through a five-coordinated phosphorus transition state. This is also true for the phospholipase D superfamily of enzymes, where the active site usually is made up of two identical sequence repeats of an HKD motif, positioned around an approximate 2-fold axis, where the histidine and lysine residues are essential for catalysis. An almost complete reaction pathway has been elucidated by a series of experiments where crystals of phospholipase D from Streptomyces sp. strain PMF (PLD(PMF)) were soaked for different times with (i) a soluble poor, short-chained phospholipid substrate and (ii) with a product. The various crystal structures were determined to a resolution of 1.35-1.75 A for the different time-steps. Both substrate and product-structures were determined in order to identify the different reaction states and to examine if the reaction actually terminated on formation of phosphatidic acid (the true product of phospholipase D action) or could proceed even further. The results presented support the theory that the phospholipase D superfamily shares a common reaction mechanism, although different family members have very different substrate preferences and perform different catalytic reactions. Results also show that the reaction proceeds via a phosphohistidine intermediate and provide unambiguous identification of a catalytic water molecule, ideally positioned for apical attack on the phosphorus and consistent with an associative in-line phosphoryl transfer reaction. In one of the experiments an apparent five-coordinate phosphorus transition state is observed.     

Extinguished operant responding shows stimulus specificity

The experiment tested for stimulus specificity in extinguished operant responding. Eight pigeons pecked keys for food reinforcers delivered by a variable interval (VI) 60-s schedule. The key was illuminated with red light during some sessions and white light during others. Then, responding was placed on extinction. During some sessions of extinction, the color of the key light remained constant throughout the session (red or white). During other sessions the color changed at 30 min into the session (red to white or white to red). Response rate increased after the change of key color in extinction. If it is assumed that key color is part of the stimulus to which subjects habituate, then these results are consistent with McSweeney and Swindell's [J. Gen. Psychol. 129 (2002) 364] suggestion that responding declines in extinction partly because subjects habituate to the stimuli that support conditioned responding. Habituation is relatively specific to the exact nature of the stimulus presented. Therefore, changes in the stimulus violate stimulus specificity and restore habituated responding. The results are also consistent with other theories that attribute extinction to a reduction of stimulus control [e.g., Psychol. Bull. 114 (1993) 80; J. Exp. Psychol.: Anim. Behav. Process. 16 (1990) 235], but considerations such as parsimony and testability favor the habituation hypothesis over these theories.     

Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen

C.S. MCSWEENEY, B. PALMER, R. BUNCH AND D.O. KRAUSE. 2001 .
Aims: To determine the effect of condensed tannins in Calliandra calothyrsus (calliandra) on rumen microbial function.
Methods and Results: Microbial populations, ruminal protein synthesis and fermentation end-products were measured in sheep fed roughage hay supplemented with calliandra (30%), with and without inclusions of polyethylene glycol (PEG) to counteract the effect of tannin. Molecular and conventional enumeration techniques were used to quantify rumen bacteria, fungi and protozoa, and protein synthesis was predicted from estimates of urinary purine excretion. The total number of cellulolytic bacteria, including populations of Fibrobacter succinogenes and Ruminococcus spp., was significantly lower in sheep supplemented with calliandra and these populations increased when animals were treated with PEG. By contrast, protozoa and fungi and the microbial group containing Bacteroides - Porphyromonas - Prevotella bacteria appeared to be less affected. The efficiency of microbial protein synthesis in the rumen was not altered significantly.
Conclusions: Calliandra caused significant shifts in rumen microbial populations without changing the efficiency of protein synthesis.
Significance and Impact of the Study: The effect of calliandra tannins on rumen digestion may result more from complexing with nutrients than direct inhibition of micro-organisms.     

A comparison of reproductive patterns and adult dispersal in sympatric introduced and native marine crabs: implications for species characteristics of invaders

The introduction and translocation of nonindigenous marine species is widespread and can pose severe threats to biodiversity and ecosystem functioning. Predicting which species are potential invaders is of particular interest to ecologists. One approach is to identify characteristics that predispose a species to becoming a successful invader. Since its introduction in the 1980’s, the invasive Asian shore crab, Hemigrapsus sanguineus, has shown a remarkable ability to colonize rocky intertidal habitats along the east coast of the United States. In Long Island Sound H. sanguineus occurs sympatrically with the functionally equivalent, but non-invasive, native Atlantic mud crab, Panopeus herbstii. The presence of both species at the same site allowed us to make a detailed, simultaneous assessment of life history traits and adult dispersibility of co-occurring invading and native crab species. We investigated fecundity and maturation rates, length of breeding season and brood production for both species, and conducted field experiments using mark–recapture techniques to determine mobility patterns. Our results show that the nonindigenous Asian crab has a greater reproductive potential than the native mud crab as evidenced by a longer breeding season, multiple brood production and higher fecundity rates. Field experiments confirmed previous studies indicating H. sanguineus is a highly mobile crab, and further demonstrated that adult Asian crabs are more likely than mud crabs to disperse from their shelter/refuge sites. Recovery rates for native mud crabs were significantly higher than those for Asian crabs in three experimental trials, across sites and years. This work provides new information about life history characteristics of both species and supports the hypothesis that high reproductive potential combined with high adult dispersal ability may be important factors associated with the invasion/establishment success of the Asian shore crab. More study is needed, however, to determine the applicability of these findings to other highly successful marine invaders.