Bromodeoxyuridine labeling and flow cytometric identification of replicating Saccharomyces cerevisiae cells: lengths of cell cycle phases and population variability at specific cell cycle positions.

An immunofluorescent staining procedure has been developed to identify, with flow cytometry, replicating cells of Saccharomyces cerevisiae after incorporation of bromodeoxyuridine (BrdUrd) into the DNA. Incorporation of BrdUrd is made possible by using yeast strains with a cloned thymidine kinase gene from the herpes simplex virus. An exposure time of 4 min to BrdUrd results in detectable labeling of the DNA. The BrdUrd/DNA double staining procedure has been optimized and the flow cytometry measurements yield histograms comparable to data typically obtained for mammalian cells. On the basis of the accurate assessment of cell fractions in individual cell cycle phases of the asynchronously growing cell population, the average duration of the cell cycle phases has been evaluated. For a population doubling time of 100 min it was found that cells spend in average 41 min in the replicating phase and 24 min in the G2+M cell cycle period. Assuming that mother cells immediately reenter the S phase after cell division, daughter cells spend 65 min in the G1 cell cycle phase. Together with the single cell fluorescence parameters, the forward-angle light scattering intensity (FALS) has been determined as an indicator of cell size. Comparing different temporal positions within the cell cycle, the determined FALS distributions show the lowest variability at the beginning of the S phase. The developed procedure in combination with multiparameter flow cytometry should be useful for studying the kinetics and regulation of the budding yeast cell cycle.     

The secretion signal of YopN, a regulatory protein of the Yersinia enterocolitica type III secretion pathway

The type III secretion signal of Yersinia enterocolitica YopN was mapped using a gene fusion approach. yopN codons 1 to 12 were identified as critical for signal function. Several synonymous mutations that abolish secretion of hybrid proteins without altering the codon specificity of yopN mRNA were identified.     

Isolation of microorganisms for biological detoxification of lignocellulosic hydrolysates

Acid pretreatment of lignocellulosic biomass releases furan and phenolic compounds, which are toxic to microorganisms used for subsequent fermentation. In this study, we isolated new microorganisms for depletion of inhibitors in lignocellulosic acid hydrolysates. A sequential enrichment strategy was used to isolate microorganisms from soil. Selection was carried out in a defined mineral medium containing a mixture of ferulic acid (5 mM), 5-hydroxymethylfurfural (5-HMF, 15 mM), and furfural (20 mM) as the carbon and energy sources, followed by an additional transfer into a corn stover hydrolysate (CSH) prepared using dilute acid. Subsequently, based on stable growth on these substrates, six isolates—including five bacteria related to Methylobacterium extorquens, Pseudomonas sp, Flavobacterium indologenes, Acinetobacter sp., Arthrobacter aurescens, and one fungus, Coniochaeta ligniaria—were chosen. All six isolates depleted toxic compounds from defined medium, but only C. ligniaria C8 (NRRL 30616) was effective at eliminating furfural and 5-HMF from CSH. C. ligniaria NRRL 30616 may be useful in developing a bioprocess for inhibitor abatement in the conversion of lignocellulosic biomass to fuels and chemicals.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable     

Flux analysis uncovers key role of functional redundancy in formaldehyde metabolism

Genome-scale analysis of predicted metabolic pathways has revealed the common occurrence of apparent redundancy for specific functional units, or metabolic modules. In many cases, mutation analysis does not resolve function, and instead, direct experimental analysis of metabolic flux under changing conditions is necessary. In order to use genome sequences to build models of cellular function, it is important to define function for such apparently redundant systems. Here we describe direct flux measurements to determine the role of redundancy in three modules involved in formaldehyde assimilation and dissimilation in a bacterium growing on methanol. A combination of deuterium and ¹⁴C labeling was used to measure the flux through each of the branches of metabolism for growth on methanol during transitions into and out of methylotrophy. The cells were found to differentially partition formaldehyde among the three modules depending on the flux of methanol into the cell. A dynamic mathematical model demonstrated that the kinetic constants of the enzymes involved are sufficient to account for this phenomenon. We demonstrate the role of redundancy in formaldehyde metabolism and have uncovered a new paradigm for coping with toxic, high-flux metabolic intermediates: a dynamic, interconnected metabolic loop.     

Cell cycle regulation of the Escherichia coli nrd operon: requirement for a cis-acting upstream AT-rich sequence.

The expression of the nrd operon encoding ribonucleotide reductase in Escherichia coli has been shown to be cell cycle regulated. To identify the cis-acting elements required for the cell cycle regulation of the nrd promoter, different 5' deletions as well as site-directed mutations were translationally fused to a lacZ reporter gene. The expression of beta-galactosidase from these nrd-lacZ fusions in single-copy plasmids was determined with synchronously growing cultures obtained by repeated phosphate starvation as well as with exponentially growing cultures by flow cytometry analysis. Although Fis and DnaA, two regulatory proteins that bind at multiple sites on the E. coli chromosome, have been found to regulate the nrd promoter, the results in this study demonstrated that neither Fis nor DnaA was required for nrd cell cycle regulation. A cis-acting upstream AT-rich sequence was found to be required for the cell cycle regulation. This sequence could be replaced by a different sequence that maintained the AT richness. A flow cytometry analysis that combined specific immunofluorescent staining of beta-galactosidase with a DNA-specific stain was developed and employed to study the nrd promoter activity in cells at specific cell cycle positions. The results of the flow cytometry analysis confirmed the results obtained from studies with synchronized cells.     

Ethanol production from SPORL-pretreated lodgepole pine: preliminary evaluation of mass balance and process energy efficiency

Lodgepole pine from forest thinnings is a potential feedstock for ethanol production. In this study, lodgepole pine was converted to ethanol with a yield of 276 L per metric ton of wood or 72% of theoretical yield. The lodgepole pine chips were directly subjected to sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) pretreatment and then disk-milled; the recovered cellulose substrate was quais-simultaneously saccharified enzymatically and fermented to ethanol using commercial cellulases and Saccharomyces cerevisiae D5A. The liquor stream from the pretreatment containing hydrolyzed sugars mainly from hemicelluloses was fermented by the same yeast strain after detoxification using an XAD resin column. The SPORL pretreatment was conducted at 180°C for a period of 25 min with a liquor-to-wood ratio of 3:1 (v/w) in a laboratory digester. Three levels of sulfuric acid charge (0.0%, 1.4%, and 2.2% on an oven dried wood basis in w/w) and three levels of sodium bisulfite charge (0.0%, 4.0%, and 8.0% in w/w) were applied. Mechanical and thermal energy consumption for milling and pretreatment were determined. These data were used to determine the efficiency of sugar recoveries and net ethanol energy production values and to formulate a preliminary mass and energy balance.     

Acyl Enzyme Intermediates in Sortase-Catalyzed Pilus Morphogenesis in Gram-Positive Bacteria

In gram-positive bacteria, covalently linked pilus polymers are assembled by a specific transpeptidase enzyme called pilus-specific sortase. This sortase is postulated to cleave the LPXTG motif of a pilin precursor between threonine and glycine and to form an acyl enzyme intermediate with the substrate. Pilus polymerization is believed to occur through the resolution of this intermediate upon specific nucleophilic attack by the conserved lysine located within the pilin motif of another pilin monomer, which joins two pilins with an isopeptide bond formed between threonine and lysine. Here, we present evidence for sortase reaction intermediates in Corynebacterium diphtheriae. We show that truncated SrtA mutants that are loosely bound to the cytoplasmic membrane form high-molecular-weight complexes with SpaA polymers secreted into the extracellular milieu. These complexes are not formed with SpaA pilin mutants that have alanine substitutions in place of threonine in the LPXTG motif or lysine in the pilin motif. The same phenotype is observed with alanine substitutions of either the conserved cysteine or histidine residue of SrtA known to be required for catalysis. Remarkably, the assembly of SpaA pili, or the formation of intermediates, is abolished with a SrtA mutant missing the membrane-anchoring domain. We infer that pilus polymerization involves the formation of covalent pilin-sortase intermediates, which occurs within a molecular platform on the exoplasmic face of the cytoplasmic membrane that brings together both sortase and its cognate substrates in close proximity to each other, likely surrounding a secretion apparatus. We present electron microscopic data in support of this picture.Adherence to specific host tissue is a key step in bacterial colonization and the establishment of a successful infection by bacterial pathogens. Bacteria express a variety of adhesive cell surface molecules to bind host cells or other substrates in their natural habitat. The proteinaceous filaments known as pili or fimbriae are a clinically important class of these molecules. Both gram-negative and gram-positive bacteria express pili (6, 8). The gram-positive bacterial pili are unique in three respects (12, 25, 31). First, they represent heterodimeric or heterotrimeric protein polymers in which individual pilin subunits are covalently joined to each other (2, 9, 32). Second, the polymer itself is covalently attached to the cell wall (3, 31). Third, unlike pilus assembly in gram-negative bacteria, many of which require chaperones (26), the polymerization of the gram-positive pili and their cell wall attachment require specific transpeptidase enzymes called sortases (31).Mazmanian and colleagues discovered the sortase SrtA as an enzyme that linked the surface protein A of Staphylococcus aureus to its cell wall (15). Genome sequences revealed that sortases are ubiquitously expressed in gram-positive bacteria, including significant pathogens, such as Actinomyces naeslundii, Bacillus cereus, Corynebacterium diphtheriae, Enterococcus faecalis, Streptococcus agalactiae, and Streptococcus pneumoniae (4, 5, 28). Sortases are classified according to their functions and phylogenic relationships (4, 5). The class that closely matches SrtA of S. aureus in structure and function is now called a housekeeping sortase. Its function is to attach numerous surface proteins to the cell wall (16). Common to each of these cell surface proteins is a cell wall sorting signal with an LPXTG motif that is absolutely necessary for cell wall anchoring (18). Elegant genetic, biochemical, and structural work by the Schneewind laboratory illuminated the universal reaction mechanism of protein sorting in the gram-positive cell wall (14). Cell wall anchoring of surface proteins is catalyzed in two steps. In the first step, SrtA cleaves the TG peptide bond of the LPXTG motif of protein A and forms an acyl enzyme intermediate involving the threonine of protein A and the catalytic cysteine of sortase (22, 27, 29). In the second step, the cleaved protein A is transferred to the cell wall when a nucleophile amine from the lipid II precursor attacks and resolves the acyl enzyme intermediate (20, 21, 30). This seminal work formed the basis of our current model of pilus assembly catalyzed by pilus-specific sortases (12).We have used C. diphtheriae as a model for studies of the mechanism of pilus biogenesis. The corynebacterial genome encodes six different sortases (32). We now know that while five of these sortases (SrtA to -E) are devoted to pilus assembly, even the housekeeping sortase, SrtF, is required for efficient attachment of pili to the cell wall (23). Corynebacteria produce three distinct types of heterotrimeric pili, which are encoded by three pilus islands, each encoding three pilins (namely, SpaABC, SpaDEF, and SpaGHI) plus one or two cognate sortases essential for the assembly of the respective pilus (7, 24, 32). In each case, the prototype pilus represents a shaft structure made of a specific major pilin (namely, SpaA, SpaD, and SpaH) (12). Each type of pilus also contains a minor pilin at the tip (SpaC, SpaF, and SpaG) and another minor pilin dispersed along the shaft, as well as at the base of the pilus (SpaB, SpaE, and SpaI) (12). How are these polymers assembled, and how are they attached to the cell wall? All pilin proteins are predicted to contain in their amino termini a hydrophobic signal sequence necessary for export to the exoplasm by the Sec machinery. In addition, like the cell wall-anchored protein A of S. aureus, a cell wall sorting signal including the LPXTG motif is also present at the carboxy terminus of each of the Spa proteins of corynebacteria and other pilus proteins found in different gram-positive organisms (17). It is thus logical to imagine that the pilus-specific sortase utilizes the LPXTG motif for pilus polymerization, its cell wall anchoring, or both. Substantial genetic, biochemical, and ultrastructural analyses have proved this prediction. Consequently, Ton-That and Schneewind proposed a model of pilus assembly which posited that the basic mechanism of catalysis is conserved between cell wall sorting of surface proteins and the assembly of the pilus (31).According to our current working model (Fig. ​(Fig.1A),1A), the prototype SpaA pilus is assembled as follows. SrtA, which is essential and also specific for SpaA pilus formation, captures and cleaves cognate pilins at the LPXTG motif and forms an acyl enzyme intermediate. To form a dimer of SpaA and SpaC, the proposed tip entity, a conserved lysine in the SpaA pilin motif attacks the Cys-Thr bond of the SpaC-SrtA acyl enzyme intermediate. Shaft formation ensues by the cyclic addition of SpaA to the SpaC-SpaA dimer and the SpaC-SpaA_n oligomer formed in the preceding reaction. When a SpaB is attached to the growing pilus terminus by a similar mechanism involving a critical lysine of SpaB, it acts as a switch, terminating pilus polymerization in favor of cell wall anchoring (11). This happens by the classic resolution reaction mentioned above, which involves the lipid II precursor (28), followed by its linkage to the cell wall (11). Alternatively, the SpaB-containing pilus can elongate further by adding a SpaA subunit to SpaB (11). This model explains all the available genetic and biochemical data we have obtained so far in the corynebacterial system, as well as other systems reported by various investigators.Open in a separate windowFIG. 1.(A) Working model of pilus assembly in C. diphtheriae. Spa pilins are synthesized in the cytoplasm and transported across the cytoplasmic membrane by the Sec machinery. The membrane-bound pilus-specific sortase SrtA cleaves the Spa pilins at the LPXTG motif and forms an acyl enzyme intermediate with the substrates. Pilus polymerization occurs when this intermediate is resolved by a nucleophilic attack by the lysine residue within the pilin motif of an adjacent intermediate. Cell wall anchoring terminates pilus polymerization when SpaB is incorporated into the pilus base by the housekeeping sortase, SrtF (see the text for details). (B) Membrane localization of the pilus-specific sortase SrtA. Corynebacteria grown to mid-log phase were separated from the culture medium (M) by centrifugation. The cell wall (W) was removed from its protoplast by muramidase treatment of the cells. The protoplasts were lysed, and membrane (P*) and cytoplasmic (C) compartments were obtained by ultracentrifugation. Protein samples were separated on 4 to 12% Tris-glycine gradient gels and detected by immunoblotting them with the specific antisera α-SrtA, α-SecA, and α-SpaA. The positions of molecular mass markers (kDa) are indicated. WT, wild type.Significantly, there has been no report demonstrating the proposed intermediates of pilus assembly, to our knowledge. The present study was initiated to explore this key element of our model of pilus assembly, as well as the localization of the sortase in the membrane and its organization in the exoplasmic membrane in relation to the cognate pilins and the general secretion machinery.     

Assembly of distinct pilus structures on the surface of Corynebacterium diphtheriae

Different surface organelles contribute to specific interactions of a pathogen with host tissues or infectious partners. Multiple pilus gene clusters potentially encoding different surface structures have been identified in several gram-positive bacterial genomes sequenced to date, including actinomycetales, clostridia, corynebacteria, and streptococci. Corynebacterium diphtheriae has been shown to assemble a pilus structure, with sortase SrtA essential for the assembly of a major subunit SpaA and two minor proteins, SpaB and SpaC. We report here the characterization of a second pilus consisting of SpaD, SpaE, and SpaF, of which SpaD and SpaE form the pilus shaft and SpaF may be located at the pilus tip. The structure of the SpaDEF pilus contains no SpaABC pilins as detected by immunoelectron microscopy. Neither deletion of spaA nor sortase srtA abolishes SpaDEF pilus formation. The assembly of the SpaDEF pilus requires specific sortases located within the SpaDEF pilus gene cluster. Although either sortase SrtB or SrtC is sufficient to polymerize SpaDF, the incorporation of SpaE into the SpaD pili requires sortase SrtB. In addition, an alanine in place of the lysine of the SpaD pilin motif abrogates pilus polymerization. Thus, SpaD, SpaE, and SpaF constitute a different pilus structure that is independently assembled and morphologically distinct from the SpaABC pili and possibly other pili of C. diphtheriae.     

Desulfohaplosamate, a new phosphate-containing steroid from Dasychalina sp., is a selective cannabinoid CB2 receptor ligand

From the polar organic extract of the Indonesian sponge Dasychalina sp. we have isolated haplosamate A (1), a unique C(28) sterol containing a sulfate group at C-3 and a methyl phosphate at C-15, along with its new desulfo analogue 2, whose structure has been secured by detailed NMR investigation. Compounds 1 and 2, as well as their semi-synthetic analogues 3-5, have been evaluated for interaction with CB(1) and CB(2) receptors through a binding test. Desulfohaplosamate (2) showed a selective affinity for CB(2) receptors in the low μM range, while a semi-synthetic derivative with cleaved ring B showed a complete loss of affinity for both receptors, highlighting the importance of an intact steroid nucleus. To our knowledge, haplosamate derivatives represent the first CB receptor ligands belonging to the class of steroids.