Electrostatic effects in highly charged proteins: salt sensitivity of pKa values of histidines in staphylococcal nuclease

The pK(a) values of most histidines in small peptides and in myoglobin increase on average by 0.30 unit between 0.02 and 1.5 M NaCl [Kao et al. (2000) Biophys. J. 79, 1637]. The DeltapK(a) values reflect primarily the ionic strength dependence of the solvation energy; screening of Coulombic interactions contributes only in a minor way. This implies that Coulombic interactions are weak, or that attractive and repulsive contributions to the pK(a) values are balanced. To distinguish experimentally between these two possibilities, and to further characterize the magnitude and salt sensitivity of surface electrostatic interactions in proteins, the salt dependence of pK(a) values of histidines in staphylococcal nuclease was measured by (1)H NMR spectroscopy. Three of the four histidines titrated with significantly depressed pK(a) values, and the salt sensitivity of all histidine pK(a) values was substantial. In three cases, the pK(a) values increased by a full unit between 0.01 and 1.5 M KCl. Anion-specific effects were found; the pK(a) values measured under equivalent ionic strengths in SCN(-) and SO(4)(2-) were higher than in Cl(-); the order of the sensitivity of pK(a) values to anions was SCN(-) > Cl(-) > SO(4)(2-). Structure-based pK(a) calculations with continuum methods were performed to interpret the measured effects structurally and to test their ability to capture the experimental behavior. Calculations in which the protein interior was treated empirically with a dielectric constant of 20 reproduced the pK(a) values and their dependence on the concentration of Cl(-). According to the calculations, the pK(a) values are depressed because of unfavorable self-energies and repulsive Coulombic interactions. Their striking salt sensitivity reflects screening of weak, repulsive, Coulombic interactions among charges separated by more than 10 A. Long-range Coulombic interactions on the surfaces of proteins are weak, but they can add up to produce substantial electrostatic effects when positive and negative charges are not balanced.     

Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria

Multiple regulatory mechanisms for coping with stress co-exist in low G+C Gram-positive bacteria. Among these, the HrcA and CtsR repressors control distinct regulons in the model organism, Bacillus subtilis. We recently identified an orthologue of the CtsR regulator of stress response in the major pathogen, Staphylococcus aureus. Sequence analysis of the S. aureus genome revealed the presence of potential CtsR operator sites not only upstream from genes encoding subunits of the Clp ATP-dependent protease, as in B. subtilis, but also, unexpectedly, within the promoter regions of the dnaK and groESL operons known to be specifically controlled by HrcA. The tandem arrangement of the CtsR and HrcA operators suggests a novel mode of dual heat shock regulation by these two repressors. The S. aureus ctsR and hrcA genes were cloned under the control of the PxylA xylose-inducible promoter and used to demonstrate dual regulation of the dnaK and groESL operons by both CtsR and HrcA, using B. subtilis as a heterologous host. Direct binding by both repressors was shown in vitro by gel mobility shift and DNase I footprinting experiments using purified S. aureus CtsR and HrcA proteins. DeltactsR, DeltahrcA and DeltactsRDeltahrcA mutants of S. aureus were constructed, indicating that the two repressors are not redundant but, instead, act together synergistically to maintain low basal levels of expression of the dnaK and groESL operons in the absence of stress. This novel regulatory mode appears to be specific to Staphylococci.     

Behavioral syndrome in a native and an invasive hymenoptera species

Recent studies have focused on the role of behavior in biological invasions. Individuals may differ consistently in time for several behavioral traits (personality) which covary (behavioral syndrome) resulting in different behavioral types, some of them favoring invasion. Social hymenopterans have a strong potential to be invaders and their success depends primarily on the foundresses’ ability to found viable colonies. They are expected to be active, explorative and bold for optimally establishing their nest. In Europe, 2 hornet species coexist: the native Vespa crabro and the invasive Vespa velutina. These 2 species may compete for nesting sites and we suggest that the initial success of V. velutina has been favored by its behavior in outperforming V. crabro for the traits involved in nest initiation. Here, we (i) defined the personality of V. crabro and V. velutina, (ii) tested for the existence of behavioral syndrome in these species, and (iii) compared their performances using an open‐field test. Our results show that V. crabro foundresses behave consistently but not V. velutina; this lack of consistency being mainly due to reduced variance among individuals. This result questions the possibility of detecting consistent behavioral differences in species having recently undergone a strong bottleneck. Both species exhibit the same correlations between activity, boldness and exploration and V. velutina clearly outperforms V. crabro for all traits. Our results suggest that activity, boldness, and exploration are implicated in both hornet nest initiation and invasion process which contributed to explain why social hymenopterans are so successful at colonization.     

Topographic prominence as a method for cluster identification in single‐molecule localisation data

Single‐molecule localisation based super‐resolution fluorescence imaging produces maps of the coordinates of fluorescent molecules in a region of interest. Cluster analysis algorithms provide information concerning the clustering characteristics of these molecules, often through the generation of cluster heat maps based on local molecular density. The goal of this study was to generate a new cluster analysis method based on a topographic approach. In particular, a topographic map of the level of clustering across a region is generated based on Getis' variant of Ripley's K‐function. By using the relative heights (topographic prominence, TP) of the peaks in the map, cluster characteristics can be identified more accurately than by using previously demonstrated height thresholds. Analogous to geological TP, the concepts of wet and dry TP and topographic isolation are introduced to generate binary maps. The algorithm is validated using simulated and experimental data and found to significantly outperform previous cluster identification methods.

Illustration of the topographic prominence based cluster analysis algorithm.     

Studying sources of incongruence in arthropod molecular phylogenies: Sea spiders (Pycnogonida) as a case study

In this report, we analyze the phylogeny of Pycnogonida using the three nuclear and three mitochondrial markers currently sequenced for studying inter- and intrafamilial relationships within Arthropoda: 18S and 28S rRNA genes, Histone H3, cytochrome c oxidase subunit 1 (CO1), 12S and 16S rRNA genes. We identify several problems in previous studies, due to the use of inappropriate sequences (taxonomic misidentification, DNA contamination, sequencing errors, missing data) or taxa (outgroup choice). Our analyses show that most markers are not powerful to study the phylogeny of sea spiders. The results suggest however a recent diversification of the group (Mesozoic rather than Paleozoic) and the early divergence of Austrodecidae, followed by Colossendeidae, Pycnogonidae and Rhynchothoracidae. Except Ammotheidae and Callipallenidae, all other families were recovered as monophyletic. Analyses of synonymous sites in CO1 sequences reveal an extreme heterogeneity of nucleotide composition within sea spiders, as six unrelated species show a reverse strand-specific bias. We therefore suggest that several independent reversals of asymmetric mutational constraints occurred during the evolution of Pycnogonida, as a consequence of genomic inversions involving either the control region or a fragment containing the CO1 gene. These hypotheses are supported by the comparison of two complete mitochondrial genomes of sea spiders (Achelia bituberculata and Nymphon gracile) with that of Limulus.     

Determinants of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Activation by Soluble CD4 and Monoclonal Antibodies

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1β, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.     

Links between archaeal community structure, vegetation type and methanogenic pathway in Alaskan peatlands

Although northern peatlands contribute significantly to natural methane emissions, recent studies of the importance and type of methanogenesis in these systems have provided conflicting results. Mechanisms controlling methanogenesis in northern peatlands remain poorly understood, despite the importance of methane as a greenhouse gas. We used 16S rRNA gene retrieval and denaturing gradient gel electrophoresis (DGGE) to analyse archaeal communities in 15 high-latitude peatland sites in Alaska and three mid-latitude peatland sites in Massachusetts. Archaeal community composition was analysed in the context of environmental, vegetation and biogeochemical factors characterized in a parallel study. Phylogenetic analysis revealed that Alaskan sites were dominated by a cluster of uncultivated crenarchaeotes and members of the families Methanomicrobiaceae and Methanobacteriaceae, which are not acetoclastic. Members of the acetoclastic family Methanosarcinaceae were not detected, whereas those of the family Methanosaetaceae were either not detected or were minor. These results are consistent with biogeochemical evidence that acetoclastic methanogenesis is not a predominant terminal decomposition pathway in most of the sites analysed. Ordination analyses indicated a link between vegetation type and archaeal community composition, suggesting that plants (and/or the environmental conditions that control their distribution) influence both archaeal community activity and dynamics.     

The blood contains multiple distinct progenitor populations with clonogenic B and T lineage potential

The thymus is seeded by bone marrow-derived progenitors that circulate in the blood. Multiple cell types can be found in the thymus early after i.v. administration or in steady state, but most fail to satisfy the known characteristics of true T progenitors. Cells that do conform to classical definitions retain multilineage potential, but surprisingly, cannot make B cells. Because acquisition of the T lineage fate among noncommitted progenitors is a lengthy process, the absence of B cell potential in early thymocytes suggests that B and T lineages diverge prethymically. To test this suggestion, we screened numerous presumptive progenitor populations for T cell growth and differentiation potential, as well as for clonogenic T or B cell development. We find that blood and marrow each contain multiple distinct subsets that display growth and differentiation potential consistent with being canonical T progenitors. Assessment of clonogenic potential further shows that although all blood and marrow populations have high T cell cloning potential, no T/non-B cells are apparent. These data suggest that either true thymic reconstitution potential derives from a small T/non-B cell subset of one of these populations, or that most of the cells defined as canonical progenitors within the thymus do not, in fact, reside in the mainstream of T progenitor differentiation.     

Crystal structure of bacteriophage T4 5' nuclease in complex with a branched DNA reveals how flap endonuclease-1 family nucleases bind their substrates

Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5' nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5' arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5' arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5' arm, lies above a magnesium binding site. The less ordered 3' arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5' arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.