The catalytic domain of the germination-specific lytic transglycosylase SleB from Bacillus anthracis displays a unique active site topology

Bacillus anthracis produces metabolically inactive spores. Germination of these spores requires germination‐specific lytic enzymes (GSLEs) that degrade the unique cortex peptidoglycan to permit resumption of metabolic activity and outgrowth. We report the first crystal structure of the catalytic domain of a GSLE, SleB. The structure revealed a transglycosylase fold with unique active site topology and permitted identification of the catalytic glutamate residue. Moreover, the structure provided insights into the molecular basis for the specificity of the enzyme for muramic‐δ‐lactam‐containing cortex peptidoglycan. The protein also contains a metal‐binding site that is positioned directly at the entrance of the substrate‐binding cleft. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

A widespread plant-fungal-bacterial symbiosis promotes plant biodiversity,plant nutrition and seedling recruitment

Highly diverse microbial assemblages colonize plant roots. It is still poorly understood whether different members of this root microbiome act synergistically by supplying different services (for example, different limiting nutrients) to plants and plant communities. In order to test this, we manipulated the presence of two widespread plant root symbionts, arbuscular mycorrhizal fungi and nitrogen-fixing rhizobia bacteria in model grassland communities established in axenic microcosms. Here, we demonstrate that both symbionts complement each other resulting in increased plant diversity, enhanced seedling recruitment and improved nutrient acquisition compared with a single symbiont situation. Legume seedlings obtained up to 15-fold higher productivity if they formed an association with both symbionts, opposed to productivity they reached with only one symbiont. Our results reveal the importance of functional diversity of symbionts and demonstrate that different members of the root microbiome can complement each other in acquiring different limiting nutrients and in driving important ecosystem functions.     

Cryobanking of viable biomaterials: implementation of new strategies for conservation purposes

Cryobanking, the freezing of biological specimens to maintain their integrity for a variety of anticipated and unanticipated uses, offers unique opportunities to advance the basic knowledge of biological systems and their evolution. Notably, cryobanking provides a crucial opportunity to support conservation efforts for endangered species. Historically, cryobanking has been developed mostly in response to human economic and medical needs — these needs must now be extended to biodiversity conservation. Reproduction technologies utilizing cryobanked gametes, embryos and somatic cells are already vital components of endangered species recovery efforts. Advances in modern biological research (e.g. stem cell research, genomics and proteomics) are already drawing heavily on cryobanked specimens, and future needs are anticipated to be immense. The challenges of developing and applying cryobanking for a broader diversity of species were addressed at an international conference held at Trier University (Germany) in June 2008. However, the magnitude of the potential benefits of cryobanking stood in stark contrast to the lack of substantial resources available for this area of strategic interest for biological science — and society at large. The meeting at Trier established a foundation for a strong global incentive to cryobank threatened species. The establishment of an Amphibian Ark cryobanking programme offers the first opportunity for global cooperation to achieve the cryobanking of the threatened species from an entire vertebrate class.     

ENaC, renal sodium excretion and extracellular ATP

Sodium balance determines the extracellular fluid volume and sets arterial blood pressure (BP). Chronically raised BP (hypertension) represents a major health risk in Western societies. The relationship between BP and renal sodium excretion (the pressure/natriuresis relationship) represents the key element in defining the BP homeostatic set point. The renin–angiotensin–aldosterone system (RAAS) makes major adjustments to the rates of renal sodium secretion, but this system works slowly over a period of hours to days. More rapid adjustments can be made by the sympathetic nervous system, although the kidney can function well without sympathetic nerves. Attention has now focussed on regulatory mechanisms within the kidney, including extracellular nucleotides and the P2 receptor system. Here, we discuss how extracellular ATP can control renal sodium excretion by altering the activity of epithelial sodium channels (ENaC) present in the apical membrane of principal cells. There remains considerable controversy over the molecular targets for released ATP, although the P2Y₂ receptor has received much attention. We review the available data and reflect on our own findings in which ATP-activated P2Y and P2X receptors make adjustments to ENaC activity and therefore sodium excretion.     

Contributions of Four Cortex Lytic Enzymes to Germination of Bacillus anthracis Spores

Bacterial spores remain dormant and highly resistant to environmental stress until they germinate. Completion of germination requires the degradation of spore cortex peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four GSLEs: CwlJ1, CwlJ2, SleB, and SleL. In this study, the cooperative action of all four GSLEs in vivo was investigated by combining in-frame deletion mutations to generate all possible double, triple, and quadruple GSLE mutant strains. Analyses of mutant strains during spore germination and outgrowth combined observations of optical density loss, colony-producing ability, and quantitative identification of spore cortex fragments. The lytic transglycosylase SleB alone can facilitate enough digestion to allow full spore viability and generates a variety of small and large cortex fragments. CwlJ1 is also sufficient to allow completion of nutrient-triggered germination independently and is a major factor in Ca²⁺-dipicolinic acid (DPA)-triggered germination, but its enzymatic activity remains unidentified because its products are large and not readily released from the spore''s integuments. CwlJ2 contributes the least to overall cortex digestion but plays a subsidiary role in Ca²⁺-DPA-induced germination. SleL is an N-acetylglucosaminidase that plays the major role in hydrolyzing the large products of other GSLEs into small, rapidly released muropeptides. As the roles of these enzymes in cortex degradation become clearer, they will be targets for methods to stimulate premature germination of B. anthracis spores, greatly simplifying decontamination measures.The Gram-positive bacterium Bacillus anthracis is the etiologic agent of cutaneous, gastrointestinal, and inhalational anthrax (24). An anthrax infection begins when the host is infected with highly resistant, quiescent B. anthracis spores (1, 24). Within the host, the spore''s sensory mechanism recognizes chemical signals, known as germinants, and triggers germination, which leads to the resumption of metabolism (36). Spores that have differentiated into vegetative cells produce a protective capsule and deadly toxins. These virulence factors allow the bacteria to evade the host''s immune system and establish an infection resulting in septicemia, toxemia, and frequently death (24). Although vegetative cells produce virulence factors that are potentially fatal, these cells cannot initiate infections and are much more susceptible to antimicrobial treatments than spores (24). Therefore, efficient triggering of spore germination may enhance current decontamination methods.Spores are highly resistant to many environmental insults because the spore core (cytoplasm) is dehydrated, dormant, and surrounded by multiple protective layers, including a modified layer of peptidoglycan (PG) known as the cortex (36). The cortex functions to maintain dormancy and heat resistance by preventing core rehydration (9). It is composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) sugars (Fig. ​(Fig.1).1). Peptide side chains on the NAM residues are either involved in interstrand cross-linking, cleaved to single l-alanine side chains, or fully removed with accompanying formation of muramic-δ-lactam (2, 31, 38). After germination is initiated by either nutrient or nonnutrient germinants, the cortex is depolymerized, resulting in complete core rehydration, resumption of metabolic activity, and outgrowth (33, 36).Open in a separate windowFIG. 1.Spore PG structure and hydrolysis. The central structure shows a representative spore PG strand with alternating NAG and NAM or muramic-δ-lactam (MδL) residues and with tetrapeptide or l-Ala side chains on the NAM residues. Forked arrows originate at sites of hydrolysis by the indicated enzymes and point to muropeptide products. The indicated “aG” muropeptide names are as previously published (7, 11). SleB lytic transglycosylase activity produces muropeptides terminating in anhydro-NAM. Cleavage at adjacent NAM residues produces the tetrasaccharide aG7a or aG7b, while cleavage further apart can produce octasaccharides or larger fragments. These can be further cleaved by muramidase treatment, resulting in the production of tetrasaccharide N, which terminates in NAM. The N-acetylglucosaminidase activity of SleL produces tetrasaccharides terminating in NAG, which can be further cleaved by muramidase to trisaccharides terminating in NAM.Cortex hydrolysis is driven by autolysins called germination-specific cortex lytic enzymes (GSLEs) that recognize the cortex-specific muramic-δ-lactam residues (2, 4, 21, 32). GSLEs fall into two classes: spore cortex lytic enzymes (SCLEs), which are thought to depolymerize intact cortical PG, and cortical fragment lytic enzymes (CFLEs), which further degrade partially hydrolyzed cortex (21). Both SCLEs and CFLEs have been identified in a variety of spore-forming species, including B. anthracis (11, 18, 19), Bacillus cereus (4, 20, 26), Bacillus megaterium (8, 34), Bacillus subtilis (13, 16, 25), Bacillus thuringiensis (12), and Clostridium perfringens (5, 23). Of the four GSLEs identified in B. anthracis, CwlJ1, CwlJ2, and SleB are predicted to be SCLEs (11), whereas SleL is thought to be a CFLE (18).Recently, independent studies showed that CwlJ1 and the lytic transglycosylase SleB (Fig. ​(Fig.1)1) play partially redundant roles and that either is sufficient for spore germination and outgrowth (10, 11). However, these same studies report conflicting results concerning the role of CwlJ2 during germination. Heffron et al. found no effect of CwlJ2 on the biochemistry of cortex hydrolysis or on colony-forming efficiency of spores (11). Giebel et al. reported that loss of CwlJ2 caused a minor defect in germination kinetics and that in the absence of SleB and CwlJ1, further loss of CwlJ2 had a major effect on colony forming efficiency (10). SleL in Bacillus anthracis is proposed to be an N-acetylglucosaminidase (Fig. ​(Fig.1)1) whose role is to further degrade cortex fragments resulting from SCLE hydrolysis (18). SleL is not essential for the completion of germination but does promote the release of small muropeptides to the spore''s surrounding environment (18).This study reports the effects of multiple deletion mutations affecting GSLEs on spore germination efficiency and kinetics of cortex hydrolysis. The data confirm the dominant roles played by CwlJ1 and SleB in the initiation of cortex hydrolysis and the major role of SleL in release of small cortex fragments. A minor role of CwlJ2 in nutrient-triggered germination and the contributions of CwlJ1 and CwlJ2 to Ca²⁺-dipicolinic acid (DPA)-triggered germination were revealed.     

The substrate determines the rate and pattern of neutral lipid synthesized by isolated human sebaceous glands

Lipogenesis from different substrates was determined in isolated human sebaceous glands after 17-20 h in culture. Rates of total lipogenesis were 1003 +/- 141, 842 +/- 90, 481 +/- 57 pmol.h-1 gland-1 +/- SE from acetate, lactate and glucose, respectively, when present as sole substrates: the rate from glucose was significantly lower (P less than 0.01). Squalene synthesis was greatest from acetate at 479 +/- 44 pmol.h-1.gland-1; significantly higher than from lactate (281 +/- 45 pmol.h-1.gland-1) or glucose at 119 +/- 18 pmol.h-1.gland-1. Wax ester plus cholesterol ester synthesis showed similar dependence on substrate but triglyceride synthesis was unaffected. We conclude that the added substrate determines both the rate and pattern of non-polar lipid synthesized by isolated human sebaceous glands.     

DNA sequence of a plasmid-encoded dihydrofolate reductase

Summary The sequence of the methotrexate-resistant dihydrofolate reductase (DHFR) gene borne by the plasmid R-388 was determined. The gene was subcloned and mapped by an in vitro mutagenesis method involving insertion of synthetic oligonucleotide decamers encoding the BamHI recognition site. Sites of insertion that destroyed the methotrexate resistance fell in two regions separated by 300 bp within a 1.2 kb fragment. One of these regions encodes a 78 amino acid polypeptide homologous to another drug-resistant DHFR. The second region essential for DHFR expression appears to be the promoter of the DHFR gene.     

Functional implications of structure-based sequence alignment of proteins in the extracellular pectate lyase superfamily.

Pectate lyases are plant virulence factors that degrade the pectate component of the plant cell wall. The enzymes share considerable sequence homology with plant pollen and style proteins, suggesting a shared structural topology and possibly functional relationships as well. The three-dimensional structures of two Erwinia chrysanthemi pectate lyases, C and E, have been superimposed and the structurally conserved amino acids have been identified. There are 232 amino acids that superimpose with a root-mean-square deviation of 3 A or less. These amino acids have been used to correct the primary sequence alignment derived from evolution-based techniques. Subsequently, multiple alignment techniques have allowed the realignment of other extracellular pectate lyases as well as all sequence homologs, including pectin lyases and the plant pollen and style proteins. The new multiple sequence alignment reveals amino acids likely to participate in the parallel beta helix motif, those involved in binding Ca2+, and those invariant amino acids with potential catalytic properties. The latter amino acids cluster in two well-separated regions on the pectate lyase structures, suggesting two distinct enzymatic functions for extracellular pectate lyases and their sequence homologs.