Unique C-terminal extension and interactome of Mycobacterium tuberculosis GlmU impacts it's in vivo function and the survival of pathogen.

N-acetyl glucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme involved in the biosynthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a critical precursor for the synthesis of peptidoglycan and other cell wall components. The absence of a homolog in eukaryotes makes GlmU an attractive target for therapeutic intervention. Mycobacterium tuberculosis GlmU (GlmUMt) has features, such as a C-terminal extension, that are not present in GlmUorthologs from other bacteria. Here, we set out to determine the uniqueness of GlmUMt by performing in vivo complementation experiments using RvΔglmU mutant. We find that any deletion of the carboxy-terminal extension region of GlmUMt abolishes its ability to complement the function of GlmUMt. Results show orthologs of GlmU, including its closest ortholog, from Mycobacterium smegmatis, cannot complement the function of GlmUMt. Furthermore, the co-expression of GlmUMt domain deletion mutants with either acetyl or uridyltransferase activities failed to rescue the function. However, co-expression of GlmUMt point mutants with either acetyl or uridyltransferase activities successfully restored the biological function of GlmUMt, likely due to the formation of heterotrimers. Based on the interactome experiments, we speculate that GlmUMt participates in unique interactions essential for its in vivo function.

Transcriptional Comparison of Genes Associated with Photosynthesis, Photorespiration, and Photo-Assimilate Allocation and Metabolic Profiling of Rice Species.

The ever-increasing human population alongside environmental deterioration has presented a pressing demand for increased food production per unit area. As a consequence, considerable research effort is currently being expended in assessing approaches to enhance crop yields. One such approach is to harness the allelic variation lost in domestication. This is of particular importance since crop wild relatives often exhibit better tolerance to abiotic stresses. Here, we wanted to address the question as to why wild rice species have decreased grain production despite being characterized by enhanced rates of photosynthesis. In order to do so, we selected ten rice species on the basis of the presence of genome information, life span, the prominence of distribution, and habitat type and evaluated the expression of genes in photosynthesis, photorespiration, sucrose and starch synthesis, sucrose transport, and primary and secondary cell walls. We additionally measured the levels of a range of primary metabolites via gas chromatography-mass spectrometry. The results revealed that the wild rice species exhibited not only higher photosynthesis but also superior CO2 recovery by photorespiration; showed greater production of photosynthates such as soluble sugars and starch and quick transportation to the sink organs with a possibility of transporting forms such as RFOs, revealing the preferential consumption of soluble sugars to develop both primary and secondary cell walls; and, finally, displayed high glutamine/glutamic acid ratios, indicating that they likely exhibited high N-use efficiency. The findings from the current study thus identify directions for future rice improvement through breeding.

Next generation Glucose-1-phosphate thymidylyltransferase (RmlA) inhibitors: An extended SAR study to direct future design.

The monosaccharide l-Rhamnose is an important component of bacterial cell walls. The first step in the l-rhamnose biosynthetic pathway is catalysed by glucose-1-phosphate thymidylyltransferase (RmlA), which condenses glucose-1-phosphate (Glu-1-P) with deoxythymidine triphosphate (dTTP) to yield dTDP-d-glucose. In addition to the active site where catalysis of this reaction occurs, RmlA has an allosteric site that is important for its function. Building on previous reports, SAR studies have explored further the allosteric site, leading to the identification of very potent P. aeruginosa RmlA inhibitors. Modification at the C6-NH2 of the inhibitor's pyrimidinedione core structure was tolerated. X-ray crystallographic analysis of the complexes of P. aeruginosa RmlA with the novel analogues revealed that C6-aminoalkyl substituents can be used to position a modifiable amine just outside the allosteric pocket. This opens up the possibility of linking a siderophore to this class of inhibitor with the goal of enhancing bacterial cell wall permeability.

Combined GWAS and eQTL analysis uncovers a genetic regulatory network orchestrating the initiation of secondary cell wall development in cotton.

The cotton fibre serves as a valuable experimental system to study cell wall synthesis in plants, but our understanding of the genetic regulation of this process during fibre development remains limited. We performed a genome-wide association study (GWAS) and identified 28 genetic loci associated with fibre quality in allotetraploid cotton. To investigate the regulatory roles of these loci, we sequenced fibre transcriptomes of 251 cotton accessions and identified 15 330 expression quantitative trait loci (eQTL). Analysis of local eQTL and GWAS data prioritised 13 likely causal genes for differential fibre quality in a transcriptome-wide association study (TWAS). Characterisation of distal eQTL revealed unequal genetic regulation patterns between two subgenomes, highlighted by an eQTL hotspot (Hot216) that established a genome-wide genetic network regulating the expression of 962 genes. The primary regulatory role of Hot216, and specifically the gene encoding a KIP-related protein, was found to be the transcriptional regulation of genes responsible for cell wall synthesis, which contributes to fibre length by modulating the developmental transition from rapid cell elongation to secondary cell wall synthesis. This study uncovered the genetic regulation of fibre-cell development and revealed the molecular basis of the temporal modulation of secondary cell wall synthesis during plant cell elongation.

Cytosolic invertases contribute to cellulose biosynthesis and influence carbon partitioning in seedlings of Arabidopsis thaliana.

In plants, UDP-glucose is the direct precursor for cellulose biosynthesis, and can be converted into other NDP-sugars required for the biosynthesis of wall matrix polysaccharides. UDP-glucose is generated from sucrose by two distinct metabolic pathways. The first pathway is the direct conversion of sucrose to UDP-glucose and fructose by sucrose synthase. The second pathway involves sucrose hydrolysis by cytosolic invertase (CINV), conversion of glucose to glucose-6-phosphate and glucose-1-phosphate, and UDP-glucose generation by UDP-glucose pyrophosphorylase (UGP). Previously, Barratt et al. (Proc. Natl Acad. Sci. USA, 106, 2009 and 13124) have found that an Arabidopsis double mutant lacking CINV1 and CINV2 displayed drastically reduced growth. Whether this reduced growth is due to deficient cell wall production caused by limited UDP-glucose supply, pleiotropic effects, or both, remained unresolved. Here, we present results indicating that the CINV/UGP pathway contributes to anisotropic growth and cellulose biosynthesis in Arabidopsis. Biochemical and imaging data demonstrate that cinv1 cinv2 seedlings are deficient in UDP-glucose production, exhibit abnormal cellulose biosynthesis and microtubule properties, and have altered cellulose organization without substantial changes to matrix polysaccharide composition, suggesting that the CINV/UGP pathway is a key metabolic route to UDP-glucose synthesis in Arabidopsis. Furthermore, differential responses of cinv1 cinv2 seedlings to exogenous sugar supplementation support a function of CINVs in influencing carbon partitioning in Arabidopsis. From these data and those of previous studies, we conclude that CINVs serve central roles in cellulose biosynthesis and carbon allocation in Arabidopsis.

Pathway-Directed Screen for Inhibitors of the Bacterial Cell Elongation Machinery.

New antibiotics are needed to combat the growing problem of resistant bacterial infections. An attractive avenue toward the discovery of such next-generation therapies is to identify novel inhibitors of clinically validated targets, like cell wall biogenesis. We have therefore developed a pathway-directed whole-cell screen for small molecules that block the activity of the Rod system of Escherichia coli This conserved multiprotein complex is required for cell elongation and the morphogenesis of rod-shaped bacteria. It is composed of cell wall synthases and membrane proteins of unknown function that are organized by filaments of the actin-like MreB protein. Our screen takes advantage of the conditional essentiality of the Rod system and the ability of the beta-lactam mecillinam (also known as amdinocillin) to cause a toxic malfunctioning of the machinery. Rod system inhibitors can therefore be identified as molecules that promote growth in the presence of mecillinam under conditions permissive for the growth of Rod- cells. A screen of ∼690,000 compounds identified 1,300 compounds that were active against E. coli Pathway-directed screening of a majority of this subset of compounds for Rod inhibitors successfully identified eight analogs of the MreB antagonist A22. Further characterization of the A22 analogs identified showed that their antibiotic activity under conditions where the Rod system is essential was strongly correlated with their ability to suppress mecillinam toxicity. This result combined with those from additional biological studies reinforce the notion that A22-like molecules are relatively specific for MreB and suggest that the lipoprotein transport factor LolA is unlikely to be a physiologically relevant target as previously proposed.

GPI-anchored SKS proteins regulate root development through controlling cell polar expansion and cell wall synthesis.

Glycosylphosphatidylinositol-anchored proteins were reported to be involved in many developmental progresses in Arabidopsis. Here I report that, a group of homologous glycosylphosphatidylinositol-anchored proteins from SKU5-Similar family regulate seedling root development of Arabidopsis through controlling cell polar expansion and cell wall synthesis. Due to the irregular expansion of root cells and the defective synthesis of cell walls, their knockout mutants generated shorter roots with irregularly shaped root cells, and thicker cell walls.

Beyond force generation: Why is a dynamic ring of FtsZ polymers essential for bacterial cytokinesis?

We propose that the essential function of the most highly conserved protein in bacterial cytokinesis, FtsZ, is not to generate a mechanical force to drive cell division. Rather, we suggest that FtsZ acts as a signal-processing hub to coordinate cell wall synthesis at the division septum with a diverse array of cellular processes, ensuring that the cell divides smoothly at the correct time and place, and with the correct septum morphology. Here, we explore how the polymerization properties of FtsZ, which have been widely attributed to force generation, can also be advantageous in this signal processing role. We suggest mechanisms by which FtsZ senses and integrates both mechanical and biochemical signals, and conclude by proposing experiments to investigate how FtsZ contributes to the remarkable spatial and temporal precision of bacterial cytokinesis.

Defining the rate-limiting processes of bacterial cytokinesis.

Bacterial cytokinesis is accomplished by the essential 'divisome' machinery. The most widely conserved divisome component, FtsZ, is a tubulin homolog that polymerizes into the 'FtsZ-ring' ('Z-ring'). Previous in vitro studies suggest that Z-ring contraction serves as a major constrictive force generator to limit the progression of cytokinesis. Here, we applied quantitative superresolution imaging to examine whether and how Z-ring contraction limits the rate of septum closure during cytokinesis in Escherichia coli cells. Surprisingly, septum closure rate was robust to substantial changes in all Z-ring properties proposed to be coupled to force generation: FtsZ's GTPase activity, Z-ring density, and the timing of Z-ring assembly and disassembly. Instead, the rate was limited by the activity of an essential cell wall synthesis enzyme and further modulated by a physical divisome-chromosome coupling. These results challenge a Z-ring-centric view of bacterial cytokinesis and identify cell wall synthesis and chromosome segregation as limiting processes of cytokinesis.

Genetic Determinants of Penicillin Tolerance in Vibrio cholerae.

Many bacteria are resistant to killing (tolerant) by typically bactericidal antibiotics due to their ability to counteract drug-induced cell damage. Vibrio cholerae, the cholera agent, displays an unusually high tolerance to diverse inhibitors of cell wall synthesis. Exposure to these agents, which in other bacteria leads to lysis and death, results in a breakdown of the cell wall and subsequent sphere formation in V. cholerae Spheres readily recover to rod-shaped cells upon antibiotic removal, but the mechanisms mediating the recovery process are not well characterized. Here, we found that the mechanisms of recovery are dependent on environmental conditions. Interestingly, on agarose pads, spheres undergo characteristic stages during the restoration of rod shape. Drug inhibition and microscopy experiments suggest that class A penicillin binding proteins (aPBPs) play a more active role than the Rod system, especially early in sphere recovery. Transposon insertion sequencing (TnSeq) analyses revealed that lipopolysaccharide (LPS) and cell wall biogenesis genes, as well as the sigma E cell envelope stress response, were particularly critical for recovery. LPS core and O-antigen appear to be more critical for sphere formation/integrity and viability than lipid A modifications. Overall, our findings demonstrate that the outer membrane is a key contributor to beta lactam tolerance and suggest a role for aPBPs in cell wall biogenesis in the absence of rod-shape cues. Factors required for postantibiotic recovery could serve as targets for antibiotic adjuvants that enhance the efficacy of antibiotics that inhibit cell wall biogenesis.

The esterase B from Sphingobium sp. SM42 has the new de-arenethiolase activity against cephalosporin antibiotics.

The esterase B (EstB) from Sphingobium sp. SM42, which was previously reported to be active towards dibutyl phthalate, can cleave some small aromatic ring side chains from cephalosporin derivatives. A new name, de-arenethiolase, has been proposed to represent this activity. We present the in vitro characterization of the activity of purified EstB toward cephalosporin substrates. Interestingly, EstB was highly active against cefoperazone and cefazolin resulting in 83 and 67% decreases in killing zone diameter, respectively. EstB also demonstrated a moderate activity towards ceftriaxone (18%) and cefotaxime (16%) while exhibiting no activity against cephalosporin C and cefixime. HPLC analysis indicated that EstB catalyzed the cleavage of the C-S bond found in cephalosporin derivatives to release the corresponding free aromatic ring side chains.

Experimental measurement of endocytosis in fungal hyphae.

The present study examines the notion that polarized exocytosis in the tips of growing hyphae creates an excess of plasma membrane and thus the need for its removal by endocytosis. To measure endocytosis experimentally, we developed a photobleaching (FRAP) procedure to count endocytic events in hyphae of Neurospora crassa carrying a fluorescent tag on the actin-binding protein fimbrin (FIM-1-GFP). Given 40 nm as the average diameter of endocytic vesicles, we calculated that about 12.5% of the plasma membrane discharged in the apex becomes endocytosed in the subapex. According to our calculations, the GFP-tagged hyphae of N. crassa, measured under the constrained conditions of confocal microscopic examination, needed about 8800 vesicles/min to extend their plasma membrane or about 9800/min, if we include predicted demands for cell wall growth and extracellular secretion. Our findings support the notion that exocytosis and endocytosis operate in tandem with the latter serving as a compensatory process to remove any excess of plasma membrane generated by the intense exocytosis in the hyphal tips. Presumably, this tandem arrangement evolved to support the hallmark features of fungi namely rapid cell extension and abundant secretion of hydrolytic enzymes.

Engineering cell wall synthesis mechanism for enhanced PHB accumulation in E. coli.

The rigidity of bacterial cell walls synthesized by a complicated pathway limit the cell shapes as coccus, bar or ellipse or even fibers. A less rigid bacterium could be beneficial for intracellular accumulation of poly-3-hydroxybutyrate (PHB) as granular inclusion bodies. To understand how cell rigidity affects PHB accumulation, E. coli cell wall synthesis pathway was reinforced and weakened, respectively. Cell rigidity was achieved by thickening the cell walls via insertion of a constitutive gltA (encoding citrate synthase) promoter in front of a series of cell wall synthesis genes on the chromosome of several E. coli derivatives, resulting in 1.32-1.60 folds increase of Young's modulus in mechanical strength for longer E. coli cells over-expressing fission ring FtsZ protein inhibiting gene sulA. Cell rigidity was weakened by down regulating expressions of ten genes in the cell wall synthesis pathway using CRISPRi, leading to elastic cells with more spaces for PHB accumulation. The regulation on cell wall synthesis changes the cell rigidity: E. coli with thickened cell walls accumulated only 25% PHB while cell wall weakened E. coli produced 93% PHB. Manipulation on cell wall synthesis mechanism adds another possibility to morphology engineering of microorganisms.

Coarse-grained simulations of bacterial cell wall growth reveal that local coordination alone can be sufficient to maintain rod shape.

Bacteria are surrounded by a peptidoglycan (PG) cell wall that must be remodeled to allow cell growth. While many structural details and properties of PG and the individual enzymes involved are known, how the process is coordinated to maintain cell integrity and rod shape is not understood. We have developed a coarse-grained method to simulate how individual transglycosylases, transpeptidases, and endopeptidases could introduce new material into an existing unilayer PG network. We find that a simple model with no enzyme coordination fails to maintain cell wall integrity and rod shape. We then iteratively analyze failure modes and explore different mechanistic hypotheses about how each problem might be overcome by the macromolecules involved. In contrast to a current theory, which posits that long MreB filaments are needed to coordinate PG insertion sites, we find that local coordination of enzyme activities in individual complexes can be sufficient to maintain cell integrity and rod shape. We also present possible molecular explanations for the existence of monofunctional transpeptidases and glycosidases (glycoside hydrolases), trimeric peptide crosslinks, cell twisting during growth, and synthesis of new strands in pairs.

Nitrogen Source Dependent Changes in Central Sugar Metabolism Maintain Cell Wall Assembly in Mitochondrial Complex I-Defective frostbite1 and Secondarily Affect Programmed Cell Death.

For optimal plant growth, carbon and nitrogen availability needs to be tightly coordinated. Mitochondrial perturbations related to a defect in complex I in the Arabidopsis thalianafrostbite1 (fro1) mutant, carrying a point mutation in the 8-kD Fe-S subunit of NDUFS4 protein, alter aspects of fundamental carbon metabolism, which is manifested as stunted growth. During nitrate nutrition, fro1 plants showed a dominant sugar flux toward nitrogen assimilation and energy production, whereas cellulose integration in the cell wall was restricted. However, when cultured on NH4⁺ as the sole nitrogen source, which typically induces developmental disorders in plants (i.e., the ammonium toxicity syndrome), fro1 showed improved growth as compared to NO3- nourishing. Higher energy availability in fro1 plants was correlated with restored cell wall assembly during NH4⁺ growth. To determine the relationship between mitochondrial complex I disassembly and cell wall-related processes, aspects of cell wall integrity and sugar and reactive oxygen species signaling were analyzed in fro1 plants. The responses of fro1 plants to NH4⁺ treatment were consistent with the inhibition of a form of programmed cell death. Resistance of fro1 plants to NH4⁺ toxicity coincided with an absence of necrotic lesion in plant leaves.

Nitrogen fertilizer application affects lodging resistance by altering secondary cell wall synthesis in japonica rice (Oryza sativa).

Stem mechanical strength is an important agricultural quantitative trait that is closely related to lodging resistance in rice, which is known to be reduced by fertilizer with higher levels of nitrogen. To understand the mechanism that regulates stem mechanical strength in response to nitrogen, we analysed stem morphology, anatomy, mechanical properties, cell wall components, and expression of cell wall-related genes, in two varieties of japonica rice, namely, Wuyunjing23 (lodging-resistant variety) and W3668 (lodging-susceptible variety). The results showed that higher nitrogen fertilizer increased the lodging index in both varieties due to a reduction in breaking strength and bending stress, and these changes were larger in W3668. Cellulose content decreased slightly under higher nitrogen fertilizer, whereas lignin content reduced remarkably. Histochemical staining revealed that high nitrogen application decreased lignin deposition in the secondary cell wall of the sclerenchyma cells and vascular bundle cells compared with the low nitrogen treatments, while it did not alter the pattern of cellulose deposition in these cells in both Wuyunjing23 and W3668. In addition, the expression of the genes involved in lignin biosynthesis, OsPAL, OsCoMT, Os4CL3, OsCCR, OsCAD2, OsCAD7, OsCesA4, and OsCesA7, were also down-regulated under higher nitrogen conditions at the early stage of culm growth. These results suggest that the genes involved in lignin biosynthesis are down-regulated by higher nitrogen fertilizer, which causes lignin deficiency in the secondary cell walls and the weakening of mechanical tissue structure. Subsequently, this results in these internodes with reduced mechanical strength and poor lodging resistance.

Mutations in MurE, the essential UDP-N-acetylmuramoylalanyl-D-glutamate 2,6-diaminopimelate ligase of Corynebacterium glutamicum: effect on L-lysine formation and analysis of systemic consequences.

OBJECTIVES: To explore systemic effects of mutations in the UDP-N-acetylmuramoylalanyl-D-glutamate 2,6-diaminopimelate ligase (MurE) of Corynebacterium glutamicum, that leads to extracellular L-lysine accumulation by this bacterium. RESULTS: The analysis of a mutant cohort of C. glutamicum strains carrying all possible 20 amino acids at position 81 of MurE revealed unexpected effects on cellular properties. With increasing L-lysine accumulation the growth rate of the producing strain is reduced. A dynamic flux balance analysis including the flux over MurE fully supports this finding and suggests that further reductions at this flux control point would enhance L-lysine accumulation even further. The strain carrying the best MurE variant MurE-G81K produces 37 mM L-lysine with a yield of 0.17 g/g (L-lysine·HCl/glucose·H2O), bearing no other genetic modification. Interestingly, among the strains with high L-lysine titers, strain variants occur which, despite possessing the desired amino acid substitutions in MurE, have regained close to normal growth and correspondingly lower L-lysine accumulation. Genome analyses of such variants revealed the transposition of mobile genetic elements which apparently annulled the favorable consequences of the MurE mutations on L-lysine formation. CONCLUSION: MurE is an attractive target to achieve high L-lysine accumulation, and product formation is inversely related to the specific growth rate. Moreover, single point mutations leading to elevated L-lysine titers may cause systemic effects on different levels comprising also major genome modifications. The latter caused by the activity of mobile genetic elements, most likely due to the stress conditions being characteristic for microbial metabolite producers.

Economy, efficiency, and the evolution of pollen tube growth rates.

PREMISE: Pollen tube growth rate (PTGR) is an important aspect of male gametophyte performance because of its central role in the fertilization process. Theory suggests that under intense competition, PTGRs should evolve to be faster, especially if PTGR accurately reflects gametophyte quality. Oddly, we know remarkably little about how effectively the work of tube construction is translated to elongation (growth and growth rate). Here we test the prediction that pollen tubes grow equally efficiently by comparing the scaling of wall production rate (WPR) to PTGR in three water lilies that flower concurrently: Nymphaea odorata, Nuphar advena and Brasenia schreberi. METHODS: Single-donor pollinations on flower or carpel pairs were fixed just after pollen germination (time A) and 45 min later (time B). Mean PTGR was calculated as the average increase in tube length over that growth period. Tube circumferences (C) and wall thicknesses (W) were measured at time B. For each donor, WPR = mean (C × W) × mean PTGR. KEY RESULTS: Within species, pollen tubes maintained a constant WPR to PTGR ratio, but species had significantly different ratios. N. odorata and N. advena had similar PTGRs, but for any given PTGR, they had the lowest and highest WPRs, respectively. CONCLUSIONS: We showed that growth rate efficiencies evolved by changes in the volume of wall material used for growth and in how that material was partitioned between lateral and length dimensions. The economics of pollen tube growth are determined by tube design, which is consequent on trade-offs between efficient growth and other pollen tube functions.

Characterizing visible and invisible cell wall mutant phenotypes.

About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with 'invisible' phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basis of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall.

Crystal structures of d-alanine-d-alanine ligase from Xanthomonas oryzae pv. oryzae alone and in complex with nucleotides.

D-Alanine-D-alanine ligase (DDL) catalyzes the biosynthesis of d-alanyl-d-alanine, an essential bacterial peptidoglycan precursor, and is an important drug target for the development of antibacterials. We determined four different crystal structures of DDL from Xanthomonas oryzae pv. oryzae (Xoo) causing Bacteria Blight (BB), which include apo, ADP-bound, ATP-bound, and AMPPNP-bound structures at the resolution between 2.3 and 2.0 Å. Similarly with other DDLs, the active site of XoDDL is formed by three loops from three domains at the center of enzyme. Compared with d-alanyl-d-alanine and ATP-bound TtDDL structure, the γ-phosphate of ATP in XoDDL structure was shifted outside toward solution. We swapped the ω-loop (loop3) of XoDDL with those of Escherichia coli and Helicobacter pylori DDLs, and measured the enzymatic kinetics of wild-type XoDDL and two mutant XoDDLs with the swapped ω-loops. Results showed that the direct interactions between ω-loop and other two loops are essential for the active ATP conformation for D-ala-phosphate formation.