Structure of Putrescine Aminotransferase from Escherichia coli Provides Insights into the Substrate Specificity among Class III Aminotransferases

YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5′-phosphate (PLP) as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme''s substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 Å resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.     

Insights into the biology of Escherichia coli through structural proteomics

Escherichia coli has historically been an important organism for understanding a multitude of biological processes, and represents a model system as we attempt to simulate the workings of living cells. Many E. coli strains are also important human and animal pathogens for which new therapeutic strategies are required. For both reasons, a more complete and comprehensive understanding of the protein structure complement of E. coli is needed at the genome level. Here, we provide examples of insights into the mechanism and function of bacterial proteins that we have gained through the Bacterial Structural Genomics Initiative (BSGI), focused on medium-throughput structure determination of proteins from E. coli. We describe the structural characterization of several enzymes from the histidine biosynthetic pathway, the structures of three pseudouridine synthases, enzymes that synthesize one of the most abundant modified bases in RNA, as well as the combined use of protein structure and focused functional analysis to decipher functions for hypothetical proteins. Together, these results illustrate the power of structural genomics to contribute to a deeper biological understanding of bacterial processes.     

Molecular Taxonomy Provides New Insights into Anopheles Species of the Neotropical Arribalzagia Series

Phylogenetic analysis of partial mitochondrial cytochrome oxidase c subunit I (COI) and nuclear internal transcribed spacer 2 (ITS2) sequences were used to evaluate initial identification and to investigate phylogenetic relationships of seven Anopheles morphospecies of the Arribalzagia Series from Colombia. Phylogenetic trees recovered highly supported clades for An. punctimaculas.s., An. calderoni, An. malefactor s.l., An. neomaculipalpus, An. apicimacula s.l., An. mattogrossensis and An. peryassui. This study provides the first molecular confirmation of An. malefactorfrom Colombia and discovered conflicting patterns of divergence for the molecular markers among specimens from northeast and northern Colombia suggesting the presence of two previously unrecognized Molecular Operational Taxonomic Units (MOTUs). Furthermore, two highly differentiated An. apicimacula MOTUs previously found in Panama were detected. Overall, the combined molecular dataset facilitated the detection of known and new Colombian evolutionary lineages, and constitutes the baseline for future research on their bionomics, ecology and potential role as malaria vectors.     

Ancient DNA Provides New Insights into the Evolutionary History of New Zealand's Extinct Giant Eagle

Prior to human settlement 700 years ago New Zealand had no terrestrial mammals—apart from three species of bats—instead, approximately 250 avian species dominated the ecosystem. At the top of the food chain was the extinct Haast's eagle, Harpagornis moorei. H. moorei (10–15 kg; 2–3 m wingspan) was 30%–40% heavier than the largest extant eagle (the harpy eagle, Harpia harpyja), and hunted moa up to 15 times its weight. In a dramatic example of morphological plasticity and rapid size increase, we show that the H. moorei was very closely related to one of the world's smallest extant eagles, which is one-tenth its mass. This spectacular evolutionary change illustrates the potential speed of size alteration within lineages of vertebrates, especially in island ecosystems.     

Polysome Turnover During Amino Acid Starvation in Escherichia coli

The experiments presented in this paper support earlier evidence that ribosomes are released from polysomes when they encounter a codon for which no charged transfer ribonucleic acid is available. However, it is further shown that these ribosomes then reinitiate and resume translation. The size and the level of polysomes during deprival of an amino acid is a function of the frequency with which that particular amino acid appears in cellular proteins. Polysomes from starved cells are more stable than those from growing cells, and, moreover, polysomes from starved relaxed strains are more stable than those from starved stringent strains.     

Insights into the specificity of RNA cleavage by the Escherichia coli MazF toxin

The mazEF (chpA) toxin-antitoxin system of Escherichia coli is involved in the cell response to nutritional and antibiotic stresses as well as in bacterial-programmed cell death. Valuable information on the MazF toxin was derived from the determination of the crystal structure of the MazE/MazF complex and from in vivo data, suggesting that MazF promoted ribosome-dependent cleavage of messenger RNA. However, it was concluded from recent in vitro analyses using a MazF-(His6) fusion protein that MazF was an endoribonuclease that cleaved messenger RNA specifically at 5'-ACA-3' sites situated in single-stranded regions. In contrast, our work reported here shows that native MazF protein cleaves RNA at the 5' side of residue A in 5'-NAC-3' sequences (where N is preferentially U or A). MazF-dependent cleavage occurred at target sequences situated either in single- or double-stranded RNA regions. These activities were neutralized by a His6-MazE antitoxin. Although essentially consistent with previous in vivo reports on the substrate specificity of MazF, our results strongly suggest that the endoribonuclease activity of MazF may be modulated by additional factors to cleave messenger and other cellular RNAs.     

Insights into the mechanisms of the selectivity filter of Escherichia coli aquaporin Z

Aquaporin Z (AQPZ) is a tetrameric protein that forms water channels in the cell membrane of Escherichia coli. The histidine residue (residue 174) in the selectivity filter (SF) region plays an important role in the transport of water across the membrane. In this work, we perform equilibrium molecular dynamics (MD) simulations to illustrate the gating mechanism of the SF and the influences of residue 174 in two different protonation states: Hsd174 with the proton at Nδ, and Hse174 with the proton at Nε. We calculate the pore radii in the SF region versus the simulation time. We perform steered MD to compute the free-energy profile, i.e., the potential of mean force (PMF) of a water molecule through the SF region. We conduct a quantum mechanics calculation of the binding energy of one water molecule with the residues in the SF region. The hydrogen bonds formed between the side chain of Hsd174 and the side chain of residue 189 (Arg189) play important roles in the selectivity mechanism of AQPZ. The radii of the pores, the hydrogen-bond analysis, and the free energies show that it is easier for water molecules to permeate through the SF region of AQPZ with residue 174 in the Hse state than in the Hsd state.     

Ancient DNA Provides New Insights into the Evolutionary History of New Zealand's Extinct Giant Eagle

Prior to human settlement 700 years ago New Zealand had no terrestrial mammals—apart from three species of bats—instead, approximately 250 avian species dominated the ecosystem. At the top of the food chain was the extinct Haast's eagle, Harpagornis moorei. H. moorei (10–15 kg; 2–3 m wingspan) was 30%–40% heavier than the largest extant eagle (the harpy eagle, Harpia harpyja), and hunted moa up to 15 times its weight. In a dramatic example of morphological plasticity and rapid size increase, we show that the H. moorei was very closely related to one of the world's smallest extant eagles, which is one-tenth its mass. This spectacular evolutionary change illustrates the potential speed of size alteration within lineages of vertebrates, especially in island ecosystems.     

Analysis of Uromodulin Polymerization Provides New Insights into the Mechanisms Regulating ZP Domain-mediated Protein Assembly

Uromodulin is the most abundant protein secreted in urine, in which it is found as a high-molecular-weight polymer. Polymerization occurs via its zona pellucida (ZP) domain, a conserved module shared by many extracellular eukaryotic proteins that are able to assemble into matrices. In this work, we identified two motifs in uromodulin, mapping in the linker region of the ZP domain and in between protein cleavage and glycosylphosphatidylinositol (GPI)-anchoring sites, which regulate its polymerization. Indeed, mutations in either module led to premature intracellular polymerization of a soluble uromodulin isoform, demonstrating the inhibitory role of these motifs for ZP domain-mediated protein assembly. Proteolytic cleavage separating the external motif from the mature monomer is necessary to release the inhibitory function and allow protein polymerization. Moreover, we report absent or abnormal assembly into filaments of GPI-anchored uromodulin mutated in either the internal or the external motif. This effect is due to altered processing on the plasma membrane, demonstrating that the presence of the two modules has not only an inhibitory function but also can positively regulate protein polymerization. Our data expand previous knowledge on the control of ZP domain function and suggest a common mechanism regulating polymerization of ZP domain proteins.     

Peroxisome Size Provides Insights into the Function of Autophagy-related Proteins

Autophagy is a major pathway of intracellular degradation mediated by formation of autophagosomes. Recently, autophagy was implicated in the degradation of intracellular bacteria, whose size often exceeds the capacity of normal autophagosomes. However, the adaptations of the autophagic machinery for sequestration of large cargos were unknown. Here we developed a yeast model system to study the effect of cargo size on the requirement of autophagy-related (Atg) proteins. We controlled the size of peroxisomes before their turnover by pexophagy, the selective autophagy of peroxisomes, and found that peroxisome size determines the requirement of Atg11 and Atg26. Small peroxisomes can be degraded without these proteins. However, Atg26 becomes essential for degradation of medium peroxisomes. Additionally, the pexophagy-specific phagophore assembly site, organized by the dual interaction of Atg30 with functionally active Atg11 and Atg17, becomes essential for degradation of large peroxisomes. In contrast, Atg28 is partially required for all autophagy-related pathways independent of cargo size, suggesting it is a component of the core autophagic machinery. As a rule, the larger the cargo, the more cargo-specific Atg proteins become essential for its sequestration.     

Changes in the Plasma Membrane of Escherichia coli During Magnesium Starvation

The effect of Mg(++) starvation on the structure of the Escherichia coli cell membrane was studied with the freeze-etch technique. Special attention was paid to changes within the plane of the membrane, which in normal exponentially growing cells has a netlike arrangement of particles 2 to 6 nm in diameter. During Mg(++) starvation, a paracrystalline particle pattern appeared on the plasma membrane, and large areas devoid of particles were seen. Although these changes are reproducibly associated with Mg(++) starvation of the bacteria, no decrease in the Mg(++) content of the cell envelope per se was detected, even after 24 hr of Mg(++) deprivation. The structural changes caused by Mg(++) deprivation appeared to involve specific and permanent alterations in membrane development. The absence of other nutrients or divalent cations did not induce similar alterations.     

Phenomenon of transient repression in Escherichia coli

Paigen, Kenneth (Roswell Park Memorial Institute, Buffalo, N.Y.). Phenomenon of transient repression in Escherichia coli. J. Bacteriol. 91:1201-1209. 1966.-A family of mutants has been obtained in Escherichia coli K-12 in which beta-galactosidase is not inducible for approximately one cell generation after the cells are transferred to glucose from other carbon sources. After that period; the enzyme can be induced at the level appropriate to glucose-grown cultures of the parent cells. Among a wide variety of carbon sources, the only one capable of eliciting a state of transient repression is glucose. Conversely, transient repression occurs when cells are transferred to glucose from any of a variety of other carbon sources. The only exceptions to this so far discovered are lactose, gluconate, and xylose. Susceptibility to transient repression in mutants can also be induced in glucose-grown cells by a period of starvation. Mutant cells which have become susceptible to transient repression lose susceptibility in the presence of glucose only when they are under conditions which permit active protein synthesis. The presence of an inducer of beta-galactosidase is not required during this time, nor does pre-induction for beta-galactosidase diminish the susceptibility of mutants. At least two other catabolite repression-sensitive enzymes (galactokinase and tryptophanase) are also sensitive to transient repression, and the two phenomena are probably related. The absolute specificity of glucose and the pattern of response seen after growth in different carbon sources suggest that the endogenous metabolite which produces these repressions is far more readily derived from glucose in metabolism than it is from any other exogenous carbon source.     

Pathway Analysis of GWAS Provides New Insights into Genetic Susceptibility to 3 Inflammatory Diseases

Although the introduction of genome-wide association studies (GWAS) have greatly increased the number of genes associated with common diseases, only a small proportion of the predicted genetic contribution has so far been elucidated. Studying the cumulative variation of polymorphisms in multiple genes acting in functional pathways may provide a complementary approach to the more common single SNP association approach in understanding genetic determinants of common disease. We developed a novel pathway-based method to assess the combined contribution of multiple genetic variants acting within canonical biological pathways and applied it to data from 14,000 UK individuals with 7 common diseases. We tested inflammatory pathways for association with Crohn''s disease (CD), rheumatoid arthritis (RA) and type 1 diabetes (T1D) with 4 non-inflammatory diseases as controls. Using a variable selection algorithm, we identified variants responsible for the pathway association and evaluated their use for disease prediction using a 10 fold cross-validation framework in order to calculate out-of-sample area under the Receiver Operating Curve (AUC). The generalisability of these predictive models was tested on an independent birth cohort from Northern Finland. Multiple canonical inflammatory pathways showed highly significant associations (p 10⁻³–10⁻²⁰) with CD, T1D and RA. Variable selection identified on average a set of 205 SNPs (149 genes) for T1D, 350 SNPs (189 genes) for RA and 493 SNPs (277 genes) for CD. The pattern of polymorphisms at these SNPS were found to be highly predictive of T1D (91% AUC) and RA (85% AUC), and weakly predictive of CD (60% AUC). The predictive ability of the T1D model (without any parameter refitting) had good predictive ability (79% AUC) in the Finnish cohort. Our analysis suggests that genetic contribution to common inflammatory diseases operates through multiple genes interacting in functional pathways.     

Adenylate Energy Charge in Escherichia coli During Growth and Starvation

The value of the adenylate energy charge, [(adenosine triphosphate) + (1/2) (adenosine diphosphate)]/[(adenosine triphosphate) + (adenosine diphosphate) + (adenosine monophosphate)], in Escherichia coli cells during growth is about 0.8. During the stationary phase after cessation of growth, or during starvation in carbon-limited cultures, the energy charge declines slowly to a value of about 0.5, and then falls more rapidly. During the slow decline in energy charge, all the cells are capable of forming colonies, but a rapid fall in viability coincides with the steep drop in energy charge. These results suggest that growth can occur only at energy charge values above about 0.8, that viability is maintained at values between 0.8 and 0.5, and that cells die at values below 0.5. Tabulation of adenylate concentrations previously reported for various organisms and tissues supports the prediction, based on enzyme kinetic observations in vitro, that the energy charge is stabilized near 0.85 in intact metabolizing cells of a wide variety of types.     

A Proteomic Approach Provides New Insights into the Control of Soil-Borne Plant Pathogens by Bacillus Species

Beneficial microorganisms (also known as biopesticides) are considered to be one of the most promising methods for more rational and safe crop management practices. We used Bacillus strains EU07, QST713 and FZB24, and investigated their inhibitory effect on Fusarium. Bacterial cell cultures, cell-free supernatants and volatiles displayed varying degrees of suppressive effect. Proteomic analysis of secreted proteins from EU07 and FZB24 revealed the presence of lytic enzymes, cellulases, proteases, 1,4-β-glucanase and hydrolases, all of which contribute to degradation of the pathogen cell wall. Further proteomic investigations showed that proteins involved in metabolism, protein folding, protein degradation, translation, recognition and signal transduction cascade play an important role in the control of Fusarium oxysporum. Our findings provide new knowledge on the mechanism of action of Bacillus species and insight into biocontrol mechanisms.     

Comparative Genome Analysis Provides Insights into the Pathogenicity of Flavobacterium psychrophilum

Flavobacterium psychrophilum is a fish pathogen in salmonid aquaculture worldwide that causes cold water disease (CWD) and rainbow trout fry syndrome (RTFS). Comparative genome analyses of 11 F. psychrophilum isolates representing temporally and geographically distant populations were used to describe the F. psychrophilum pan-genome and to examine virulence factors, prophages, CRISPR arrays, and genomic islands present in the genomes. Analysis of the genomic DNA sequences were complemented with selected phenotypic characteristics of the strains. The pan genome analysis showed that F. psychrophilum could hold at least 3373 genes, while the core genome contained 1743 genes. On average, 67 new genes were detected for every new genome added to the analysis, indicating that F. psychrophilum possesses an open pan genome. The putative virulence factors were equally distributed among isolates, independent of geographic location, year of isolation and source of isolates. Only one prophage-related sequence was found which corresponded to the previously described prophage 6H, and appeared in 5 out of 11 isolates. CRISPR array analysis revealed two different loci with dissimilar spacer content, which only matched one sequence in the database, the temperate bacteriophage 6H. Genomic Islands (GIs) were identified in F. psychrophilum isolates 950106-1/1 and CSF 259–93, associated with toxins and antibiotic resistance. Finally, phenotypic characterization revealed a high degree of similarity among the strains with respect to biofilm formation and secretion of extracellular enzymes. Global scale dispersion of virulence factors in the genomes and the abilities for biofilm formation, hemolytic activity and secretion of extracellular enzymes among the strains suggested that F. psychrophilum isolates have a similar mode of action on adhesion, colonization and destruction of fish tissues across large spatial and temporal scales of occurrence. Overall, the genomic characterization and phenotypic properties may provide new insights to the mechanisms of pathogenicity in F. psychrophilum.