Assembly and genetics of spore protective structures

The sporulation program in Bacillus subtilis ends in the formation of a highly resistant endospore that can withstand extremes of heat, mechanical disruption, ultraviolet irradiation, lytic enzymes and chemical attack. These properties are attributed mainly to the unique structure of spore coat and cortex, as well as to the physical state of the spore cytoplasm. The outermost layer of the spore, called the coat, has two morphologically distinct sublayers: an electron-dense outer coat and an electron-translucent inner coat. The coat is composed of more than 2 dozen proteins of varying size. Many coat genes and coat proteins have been isolated and characterized in detail, and studies of these have identified proteins with important roles in coat assembly, resistance and spore germination. We describe here characteristics of the coat proteins and propose a model for coat assembly based on recent work.     

Spore germination

Despite being relatively insensitive to environmental insult, the spore is responsive to low concentrations of chemical germinants, which induce germination. The process of bacterial spore germination involves membrane permeability changes, ion fluxes and the activation of enzymes that degrade the outer layers of the spore. A number of components in the spore that are required for the germination response have been identified, including a spore-specific family of receptor proteins (the GerA family), an ion transporter and cortex lytic enzymes. The germinant traverses the outer layers of the spore and interacts with its receptor in the inner membrane to initiate the cascade of germination events, but the molecular details of this signal transduction process remain to be identified.     

Overview: development in bacteria: spore formation in Bacillus subtilis

Like eukaryotes, bacteria possess complex developmental programs that drive environmental adaptation and morphological differentiation. In some species, these morphological changes are quite elaborate and result in major changes in cell appearance, including the formation of ornate appendages. The ease with which some bacteria can be manipulated makes them highly attractive model systems for developmental analysis. In this set of reviews, we tackle the best studied of these systems, spore formation in Bacillus subtilis. Construction of a spore initiates in response to starvation, takes each cell about 8 h and is directed by a tightly controlled genetic program. First, the cell creates an internal protoplast with its own copy of the chromosome. Over the next several hours, development continues as proteins synthesized within the protoplast as well as in the surrounding cell cytoplasm coalesce into the various complex structures that comprise the spore. The resulting cell is metabolically dormant and as close to indestructible as any cell found on earth. Nonetheless, the spore retains the ability to revive almost immediately when nutrient returns to the environment. Here, we review the genetic control of spore formation, the structure and assembly of several major spore components, the process of germination, and the environmental and disease implications of spores. As these reviews document, spore formation in B. subtilis has been among the most productive systems for understanding both the broad themes and the molecular basis of development. Not only does this system continue to add to our understanding of these questions, but it provides a particularly powerful means to address the cell biological dimension of development.     

Effect of plant lectins on Ustilago maydis in vitro

Ustilago maydis is an edible parasitic basidiomycete, which specifically infects corn (Zea mays) and teocintle (Z. diploperennis). To characterise the interaction between the basidiomycete and its host organism, we tested the effect of plant lectins with well-known sugar specificity on the growth and germination of U. maydis spores. Lectins specific for N-acetyl-D-galactosamine, such as those from Dolichos biflorus and Phaseolus lunatus, and the wheatgerm agglutinin specific for N-acetyl-D-glucosamine inhibited spore germination, but were ineffective in modifying U. maydis cell growth. The galactose-specific lectin from the corn coleoptyle inhibited both germination and cell growth, while the lectin concanavalin A (mannose/glucose specific) activated spore germination and growth. Our results suggest that specific saccharide-containing receptors participate in regulating the growth and maturation of U. maydis spores.     

Deciphering the BAR code of membrane modulators

The BAR domain is the eponymous domain of the “BAR-domain protein superfamily”, a large and diverse set of mostly multi-domain proteins that play eminent roles at the membrane cytoskeleton interface. BAR domain homodimers are the functional units that peripherally associate with lipid membranes and are involved in membrane sculpting activities. Differences in their intrinsic curvatures and lipid-binding properties account for a large variety in membrane modulating properties. Membrane activities of BAR domains are further modified and regulated by intramolecular or inter-subunit domains, by intermolecular protein interactions, and by posttranslational modifications. Rather than providing detailed cell biological information on single members of this superfamily, this review focuses on biochemical, biophysical, and structural aspects and on recent findings that paradigmatically promote our understanding of processes driven and modulated by BAR domains.     

Lethal effects of heat on bacterial physiology and structure

High temperatures have profound effects on the structural and physiological properties of sporulating and non-sporulating bacteria, with membranes, RNA, DNA, ribosomes, protein and enzymes all affected. Nevertheless, it is apparent that no one single event is responsible for cell death. The induction of intracellular heat-shock proteins and the activation of extracellular alarmones in vegetative cells exposed to mildly lethal temperatures are important cell responses. In bacterial spores, several factors contribute to the overall resistance to moist (wet) and dry heat; the latter, but not the former, induces mutations. Heat resistance develops during sporulation, when spore-specific heat-shock proteins are also produced. Heat sensitivity is regained during germination of spores.     

E-cadherin and plakoglobin recruit plakophilin3 to the cell border to initiate desmosome assembly

A decrease in the levels of the desmosomal plaque protein, plakophilin3 (PKP3), leads to a decrease in desmosome size and cell-cell adhesion. To test the hypothesis that PKP3 is required for desmosome formation, the recruitment of desmosomal components to the cell surface was studied in the PKP3 knockdown clones. The PKP3 knockdown clones showed decreased cell border staining for multiple desmosomal proteins, when compared to vector controls, and did not form desmosomes in a calcium switch assay. Further analysis demonstrated that PKP3, plakoglobin (PG) and E-cadherin are present at the cell border at low concentrations of calcium. Loss of either PG or E-cadherin led to a decrease in the levels of PKP3 and other desmosomal proteins at the cell border. The results reported here are consistent with the model that PG and E-cadherin recruit PKP3 to the cell border to initiate desmosome formation.     

The β6/β7 region of the Hsp70 substrate-binding domain mediates heat-shock response and prion propagation

Hsp70 is a highly conserved chaperone that in addition to providing essential cellular functions and aiding in cell survival following exposure to a variety of stresses is also a key modulator of prion propagation. Hsp70 is composed of a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). The key functions of Hsp70 are tightly regulated through an allosteric communication network that coordinates ATPase activity with substrate-binding activity. How Hsp70 conformational changes relate to functional change that results in heat shock and prion-related phenotypes is poorly understood. Here, we utilised the yeast [PSI ⁺] system, coupled with SBD-targeted mutagenesis, to investigate how allosteric changes within key structural regions of the Hsp70 SBD result in functional changes in the protein that translate to phenotypic defects in prion propagation and ability to grow at elevated temperatures. We find that variants mutated within the β6 and β7 region of the SBD are defective in prion propagation and heat-shock phenotypes, due to conformational changes within the SBD. Structural analysis of the mutants identifies a potential NBD:SBD interface and key residues that may play important roles in signal transduction between domains. As a consequence of disrupting the β6/β7 region and the SBD overall, Hsp70 exhibits a variety of functional changes including dysregulation of ATPase activity, reduction in ability to refold proteins and changes to interaction affinity with specific co-chaperones and protein substrates. Our findings relate specific structural changes in Hsp70 to specific changes in functional properties that underpin important phenotypic changes in vivo. A thorough understanding of the molecular mechanisms of Hsp70 regulation and how specific modifications result in phenotypic change is essential for the development of new drugs targeting Hsp70 for therapeutic purposes.     

Volatile metabolites of aspergilli in relation to spore germination of some keratinophilic fungi

Summary The effect of volatile metabolites produced by 8 Aspergilli (i.e.,Aspergillus candidus, A. chevalieri, A. flavus, A. fumigatus, A. nidulans, A. niger, A. ochraceous andA. tamarii) on spore germination were tested againstAuxarthron conjugatum, Chrysosporium pannicola, Keratinomyces ajelloi andMicrosporum gypseum. The volatile metabolites inhibited the spore germination of all the test fungi.Acknowledgment. The authors thank Professor G.P. Mishra for the use of the facilities and S.K.D. acknowledges the fellowship award by I.C.M.R. New Delhi.     

Modulation of protein biophysical properties by chemical glycosylation: biochemical insights and biomedical implications

Glycosylation constitutes one of the most important posttranslational modifications employed by biological systems to modulate protein biophysical properties. Due to the direct biochemical and biomedical implications of achieving control over protein stability and function by chemical means, there has been great interest in recent years towards the development of chemical strategies for protein glycosylation. Since current knowledge about glycoprotein biophysics has been mainly derived from the study of naturally glycosylated proteins, chemical glycosylation provides novel insights into its mechanistic understanding by affording control over glycosylation parameters. This review presents a survey of the effects that natural and chemical glycosylation have on the fundamental biophysical properties of proteins (structure, dynamics, stability, and function). This is complemented by a mechanistic discussion of how glycans achieve such effects and discussion of the implications of employing chemical glycosylation as a tool to exert control over protein biophysical properties within biochemical and biomedical applications. Received 15 December 2006; received after revision 28 March 2007; accepted 25 April 2007     

Phylogenetic relationship of organisms obtained by ribosomal protein comparison

The evolutionary relationships of ribosomal proteins from eubacteria, archaea, eukaryotes, chloroplasts and mitochondria were examined by their degree of conservation, their structural and functional properties and by the occurrence of conserved structural elements. The structural domains formed by the different protein families were studied. The occurrence of monophyletic groups was investigated for each protein family within the archaea. Phylogenetic trees were constructed between these organisms and together with the homologous sequences of the other phylogenetic domains. All organisms belonging to the archaea clearly formed a monophyletic group. The conserved sequence motifs were checked for the potential to form similar secondary structural elements. Received 24 October 1996; accepted 30 October 1996     

Protein synthesis during germination of heterothallic yeast ascospores

Protein synthesis during ascospore germination of the heterothallicSaccharomyces cerevisiae strain AP-3 was investigated. Protein synthesis in the germinating ascospores appeared to begin approximately 20 min following glucose initiation. Since RNA synthesis did not start until approximately 70 min after the onset of germination, strain AP-3 ascospores must contain RNA which is ready for immediate translation. Both trehalase and glyceraldehyde-3-phosphate dehydrogenase activities were found to be affected by the onset of germination. Trehalase activity was found to increase severalfold following 60 min of spore germination but remained relatively constant over the subsequent 120 min examined. Dehydrogenase activity was not detectable in AP-3 ascospores but was measurable in germinating ascospores.     

Protein synthesis during germination of heterothallic yeast ascospores.

Protein synthesis during ascospore germination of the heterothallic Saccharomyces cerevisiae strain AP-3 was investigated. Protein synthesis in the germinating ascospores appeared to begin approximately 20 min following glucose initiation. Since RNA synthesis did not start until approximately 70 min after the onset of germination, strain AP-3 ascospores must contain RNA which is ready for immediate translation. Both trehalase and glyceraldehyde-3-phosphate dehydrogenase activities were found to be affected by the onset of germination. Trehalase activity was found to increase severalfold following 60 min of spore germination but remained relatively constant over the subsequent 120 min examined. Dehydrogenase activity was not detectable in AP-3 ascospores but was measurable in germinating ascospores.     

Membrane shaping by the Bin/amphiphysin/Rvs (BAR) domain protein superfamily

BAR domain superfamily proteins have emerged as central regulators of dynamic membrane remodeling, thereby playing important roles in a wide variety of cellular processes, such as organelle biogenesis, cell division, cell migration, secretion, and endocytosis. Here, we review the mechanistic and structural basis for the membrane curvature-sensing and deforming properties of BAR domain superfamily proteins. Moreover, we summarize the present state of knowledge with respect to their regulation by autoinhibitory mechanisms or posttranslational modifications, and their interactions with other proteins, in particular with GTPases, and with membrane lipids. We postulate that BAR superfamily proteins act as membrane-deforming scaffolds that spatiotemporally orchestrate membrane remodeling.     

Resculpting the binding pocket of APC superfamily LeuT-fold amino acid transporters

Amino acid transporters are essential components of prokaryote and eukaryote cells, possess distinct physiological functions, and differ markedly in substrate specificity. Amino acid transporters can be both drug targets and drug transporters (bioavailability, targeting) with many monogenic disorders resulting from dysfunctional membrane transport. The largest collection of amino acid transporters (including the mammalian SLC6, SLC7, SLC32, SLC36, and SLC38 families), across all kingdoms of life, is within the Amino acid-Polyamine-organoCation (APC) superfamily. The LeuT-fold is a paradigm structure for APC superfamily amino acid transporters and carriers of sugars, neurotransmitters, electrolytes, osmolytes, vitamins, micronutrients, signalling molecules, and organic and fatty acids. Each transporter is specific for a unique sub-set of solutes, specificity being determined by how well a substrate fits into each binding pocket. However, the molecular basis of substrate selectivity remains, by and large, elusive. Using an integrated computational and experimental approach, we demonstrate that a single position within the LeuT-fold can play a crucial role in determining substrate specificity in mammalian and arthropod amino acid transporters within the APC superfamily. Systematic mutation of the amino acid residue occupying the equivalent position to LeuT V104 titrates binding pocket space resulting in dramatic changes in substrate selectivity in exemplar APC amino acid transporters including PAT2 (SLC36A2) and SNAT5 (SLC38A5). Our work demonstrates how a single residue/site within an archetypal structural motif can alter substrate affinity and selectivity within this important superfamily of diverse membrane transporters.     

Structure, mechanism and catalytic duality of thiamine-dependent enzymes

Thiamine is an essential cofactor that is required for processes of general metabolism amongst all organisms, and it is likely to have played a role in the earliest stages of the evolution of life. Here, we review from a structural perspective the enzymatic mechanisms that involve this cofactor. We explore asymmetry within homodimeric thiamine diphosphate (ThDP)-dependent enzyme structures and discuss how this may be correlated with the kinetic properties of half-of-the-sites reactivity, and negative cooperativity. It is likely these structural and kinetic hallmarks may arise through reciprocal coupling of active sites. This mode of communication between distant active sites is not unique to ThDP-dependent enzymes, but is widespread in other classes of oligomeric enzyme. Thus, it appears likely to be a general phenomenon reflecting a powerful mechanism of accelerating the rate of a chemical pathway. Finally, we speculate on the early evolutionary history of the cofactor and its ancient association with protein and RNA.     

Structural and biochemical aspects of keratan sulphate in the cornea

Keratan sulphate (KS) is the predominant glycosaminoglycan (GAG) in the cornea of the eye, where it exists in proteoglycan (PG) form. KS-PGs have long been thought to play a pivotal role in the establishment and maintenance of the array of regularly-spaced and uniformly-thin collagen fibrils which make up the corneal stroma. This characteristic arrangement of fibrils allows light to pass through the cornea. Indeed, perturbations to the synthesis of KS-PG core proteins in genetically altered mice lead to structural matrix alterations and corneal opacification. Similarly, mutations in enzymes responsible for the sulphation of KS-GAG chains are causative for the inherited human disease, macular corneal dystrophy, which is manifested clinically by progressive corneal cloudiness starting in young adulthood.     

Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins)

The DD-peptidase enzymes (penicillin-binding proteins) catalyze the final transpeptidation reaction of bacterial cell wall (peptidoglycan) biosynthesis. Although there is now much structural information available about these enzymes, studies of their activity as enzymes lag. It is now established that representatives of two low-molecular-mass classes of DD-peptidases recognize elements of peptidoglycan structure and rapidly react with substrates and inhibitors incorporating these elements. No members of other DD-peptidase classes, including the high-molecular-mass enzymes, essential for bacterial growth, appear to interact strongly with any particular elements of peptidoglycan structure. Rational design of inhibitors for these enzymes is therefore challenging.     

Host recognition by toxigenic plant pathogens

Certain fungal pathogens release host-selective (or host-specific) toxins (HST) as a host recognition factor during spore germination at the infection site on plants. Prior to penetration of the pathogen into its host, the released toxin specifically binds to a putative receptor on the host cells and initiates signaling mechanisms leading to pleiotropic effects on cells. Of these, the crucial one negates the general and inducible defense reactions of the cells. This is accomplished by a signal from the HST, which is transduced through a path way at or near the step of plasma membrane modulation, which is directly or indirectly triggered by the HST. This mechanism operates even though the toxin may affect mitochondria or chloroplasts as the primary target organelle. The fungal spore is able to penetrate the so-called 'narcotized cell' and completes the initial colonization of the host. The host recognition process may take place without necessitating host cell death, even in the case of perthophytic parasites. At the molecular level, HST-mediated recognition of the host by a pathogen requires strict stereochemical precision like a lock and key.