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PubMed2006

Structure and mechanism of a bacterial beta-glucosaminidase having O-GlcNAcase activity.

O-GlcNAc is an abundant post-translational modification of serine and threonine residues of nucleocytoplasmic proteins. This modification, found only within higher eukaryotes, is a dynamic modification that is often reciprocal to phosphorylation. In a manner analogous to phosphatases, a glycoside hydrolase termed O-GlcNAcase cleaves O-GlcNAc from modified proteins. Enzymes with high sequence similarity to human O-GlcNAcase are also found in human pathogens and symbionts. We report the three-dimensional structure of O-GlcNAcase from the human gut symbiont Bacteroides thetaiotaomicron both in its native form and in complex with a mimic of the reaction intermediate. Mutagenesis and kinetics studies show that the bacterial enzyme, very similarly to its human counterpart, operates via an unusual 'substrate-assisted' catalytic mechanism, which will inform the rational design of enzyme inhibitors.

Acetylglucosaminidase↗

PubMed1995

Crystal structure of the flavohemoglobin from Alcaligenes eutrophus at 1.75 A resolution.

The molecular structure of the flavohemoglobin from Alcaligenes eutrophus has been determined to a resolution of 1.75 A and refined to an R-factor of 19.6%. The protein comprises two fused modules: a heme binding module, which belongs to the globin family, and an FAD binding oxidoreductase module, which adopts a fold like ferredoxin reductase. The most striking deviation of the bacterial globin structure from those of other species is the movement of helix E in a way to provide more space in the vicinity of the distal heme binding site. A comparison with other members of the ferredoxin reductase family shows similar tertiary structures for the individual FAD and NAD binding domains but largely different interdomain orientations. The heme and FAD molecules approach each other to a minimal distance of 6.3 A and adopt an interplanar angle of 80 degrees. The electron transfer from FAD to heme occurs in a predominantly polar environment and may occur directly or be mediated by a water molecule.

Alcaligenes↗

PubMed2005

Application of 16S rDNA-PCR amplification and DGGE fingerprinting for detection of shift in microbial community diversity in Cu-, Zn-, and Cd-contaminated paddy soils.

Seven soils were sampled from farmland at different distances (0.01-5 km) from a copper and zinc smelter. The total contents of heavy metals in these soils ranged from 46 to 4895 mg Cu kg-1, 96 to 1133 mg Zn kg-1, and 6.9 to 28.8 mg Cd kg-1, respectively. The available fractions were highly correlated with total contents of the metals. In order to assess the impact of combined contamination of heavy metals on soil bacterial communities, denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplicons of 16S rDNA sequence of bacteria in soil was used. Bacterial community structure was affected to some extent by heavy metals. The number of DGGE bands in soils increased with increasing distance from the copper and zinc smelter. Clustering analysis of the DGGE profiles showed that bacteria in the seven soils belonged to three clusters. Bacterial communities in three soils sampled at 0.01-0.60 km from the smelter belonged to one cluster, and those in three soils sampled at 0.8-1.2 km from the smelter belong to another cluster. Bacterial community in soil farthest from the smelter belonged to a single cluster. This study demonstrated that heavy metal contamination decreased both biomass and diversity of bacterial community in the soil.

Cadmium↗

PubMed1997

Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis.

The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed. After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established. The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long). A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively. A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 34, 26, 17, 6, and 4%, respectively. The other 16 bacterial OTUs represent 13% of the clones. They were either affiliated with narrow phyla such as Planctomyces-Chlamydia, green nonsulfur bacteria, or Synergistes, or deeply branched on the phylogenetic tree. A large number of bacterial OTUs are not closely related to any other hitherto determined sequences. The most frequent bacterial OTUs represents less than 5% of the total bacterial SSU rDNA sequences. However, the 20 more frequent bacterial OTUs describe at least 50% of these sequences. Three of the six Archaea OTUs correspond to 95% of the Archaea population and are very similar to already known methanogenic species: Methanosarcina barkeri, Methanosarcina frisius, and Methanobacterium formicicum. In contrast, the three other Archaea OTUs are unusual and are related to thermophilic microorganisms such as Crenarchaea or Thermoplasma spp. Five percent of the sequences analyzed were chimeras and were removed from the analysis.

Bacteria, Anaerobic↗

PubMed2003

The Escherichia coli outer membrane cobalamin transporter BtuB: structural analysis of calcium and substrate binding, and identification of orthologous transporters by sequence/structure conservation.

Gram-negative bacteria possess specialized active transport systems that function to transport organometallic cofactors or carriers, such as cobalamins, siderophores, and porphyrins, across their outer membranes. The primary components of each transport system are an outer membrane transporter and the energy-coupling protein TonB. In Escherichiacoli, the TonB-dependent outer membrane transporter BtuB carries out active transport of cobalamin (Cbl) substrates across its outer membrane. Cobalamins bind to BtuB with nanomolar affinity. Previous studies implicated calcium in high-affinity binding of cyanocobalamin (CN-Cbl) to BtuB. We previously solved four structures of BtuB or BtuB complexes: an apo-structure of a methionine-substitution mutant (used to obtain experimental phases by selenomethionine single-wavelength anomalous diffraction studies); an apo-structure of wild-type BtuB; a binary complex of calcium and wild-type BtuB; and a ternary complex of calcium, CN-Cbl and wild-type BtuB. We present an analysis of the binding of calcium in the binary and ternary complexes, and show that calcium coordination changes upon substrate binding. High-affinity CN-Cbl binding and calcium coordination are coupled. We also analyze the binding mode of CN-Cbl to BtuB, and compare and contrast this binding to that observed in other proteins that bind Cbl. BtuB binds CN-Cbl in a manner very different from Cbl-utilizing enzymes and the periplasmic Cbl binding protein BtuF. Homology searches of bacterial genomes, structural annotation based on the presence of conserved Cbl-binding residues identified by analysis of our BtuB structure, and detection of homologs of the periplasmic Cbl-binding binding protein BtuF enable identification of putative BtuB orthologs in enteric and non-enteric bacterial species.

Amino Acid Motifs↗

PubMed1989

The structure of signal peptides from bacterial lipoproteins.

Statistical analysis of lipoprotein and non-lipoprotein signal peptides reveals that the two classes differ significantly only in the region close to the signal peptidase cleavage site. This region is apolar and has the consensus sequence LA(G,A) decreases C in the lipoproteins, but is polar and has small, uncharged residues in positions -3 and -1 in the non-lipoproteins. A simple search for matches to the lipoprotein consensus cleavage site suffices to discriminate between the two groups with close to 100% reliability.

Amino Acid Sequence↗

PubMed2005

Amyloid-like properties of bacterial inclusion bodies.

Bacterial inclusion bodies are major bottlenecks in protein production, narrowing the spectrum of relevant polypeptides obtained by recombinant DNA. While regarded as amorphous deposits formed by passive and rather unspecific precipitation of unfolded chains, we prove here that they are instead organized aggregates sharing important structural and biological features with amyloids. By using an Escherichia coli beta-galactosidase variant, we show that aggregation does not necessarily require unfolded polypeptide chains but rather depends on specific interactions between solvent-exposed hydrophobic stretches in partially structured species. In addition, purified inclusion bodies are efficient and highly selective nucleation seeds, promoting deposition of soluble homologous but not heterologous polypeptides in a dose-dependent manner. Finally, inclusion bodies bind amyloid-diagnostic dyes, which, jointly with Fourier transform infra red spectroscopy data, indicates a high level of organized intermolecular beta-sheet structure. The evidences of amyloid-like structure of bacterial inclusion bodies, irrespective of potential applications in bioprocess engineering, prompts the use of bacterial models to explore the molecular determinants of protein aggregation by means of simple biological systems.

Amyloid↗

PubMed2004

Induced resistance to infection of lobsters Homarus americanus by Aerococcus viridans (var.) homari, the bacterium causing gaffkemia.

A vaccine composed of steam sterilized (autoclaved) cells of a virulent strain of Aerococcus viridans (var.) homari was effective in protecting lobsters Homarus americanus against gaffkemia. At 15 degrees C the heat-killed vaccines (HKV) at concentrations between 1 and 5 x 10(7) particles kg(-1) lobster body wt induced maximal protection in induction periods ranging from 7 to 11 d. Protection was substantial over the course of a 30 d post-induction trial period. Spring-caught lobsters (i.e. those more fully rehabilitated following ecdysis) gained more protection (LD50 = 1.9 x 10(4)) from the vaccination than did those caught in the late fall-early winter period (lobsters that were not yet fully recovered from ecdysis) (LD50 = 3.2 x 10(3)). The protection offered by the HK vaccine was comparable to that induced by a vaccine produced by incubating the pathogen with low concentrations (2 pg ml(-1)) of the antibiotic vancomycin. The bacterins produced by both methods exhibited similar new properties: (1) agglutination at low titres by lobster hemolymph serum, suggesting an impaired capsule layer, and (2) increased permeability to the large Alcian Blue molecule. With both vaccines, the protection may be a direct result of increased exposure to intact bacterial cell structures by the lobster defences, an exposure which otherwise would be prevented by an intact capsule.

Agglutination Tests↗

PubMed1994

Amoebapores, a family of membranolytic peptides from cytoplasmic granules of Entamoeba histolytica: isolation, primary structure, and pore formation in bacterial cytoplasmic membranes.

Three peptides with pore-forming activity were isolated from the cytoplasmic granules of pathogenic Entamoeba histolytica by acidic extraction, gel filtration and reversed-phase high-performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic alpha-helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore-forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram-positive bacteria. The amoebapores represent a distinct family of membrane-active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity on the parasite.

Amino Acid Sequence↗

PubMed2022

A graph-based approach for the visualisation and analysis of bacterial pangenomes.

BACKGROUND: The advent of low cost, high throughput DNA sequencing has led to the availability of thousands of complete genome sequences for a wide variety of bacterial species. Examining and interpreting genetic variation on this scale represents a significant challenge to existing methods of data analysis and visualisation. RESULTS: Starting with the output of standard pangenome analysis tools, we describe the generation and analysis of interactive, 3D network graphs to explore the structure of bacterial populations, the distribution of genes across a population, and the syntenic order in which those genes occur, in the new open-source network analysis platform, Graphia. Both the analysis and the visualisation are scalable to datasets of thousands of genome sequences. CONCLUSIONS: We anticipate that the approaches presented here will be of great utility to the microbial research community, allowing faster, more intuitive, and flexible interaction with pangenome datasets, thereby enhancing interpretation of these complex data.

Bacteria↗

PubMed2002

Detecting structural and functional differences in activated sludge bacterial communities originating from laboratory treatment of elementally and totally chlorine-free bleaching effluents.

The ability to differentiate functional and structural diversity of bacterial communities present in activated sludges adapted to elementally (ECF) and totally (TCF) chlorine-free bleaching effluents was evaluated. Community function was evaluated through substrate utilization patterns in BiologGN microplates, and taxonomic structure was evaluated by fluorescent in situ hybridization using probes targeting the Eubacteria; the alpha, beta, and gamma subclasses of the Proteobacteria; and gram-positive bacteria with high GC content. Over 6-week sampling periods, ECF-and TCF-adapted sludge bacterial communities presented reproducible substrate utilization patterns that through principal components (PCs) analysis, separated the ECF samples from the TCF samples. Application of the fluorescent in situ hybridization technique was complicated by the intense autofluorescence of the bleaching effluent sludge samples that interfered with detection of specific hybridization signals. The most notable difference in community structure detected using the chosen set of probes was the relatively greater proportion of cells of the alpha subclass in TCF sludge (27%) than in ECF sludge (6%). Nonspecific hybridization with beta and gamma probes was relatively high, but both sludges appeared to have similar proportions of cells of the beta (20-22%) and gamma (11-12%) subclasses. The two sludges presented relatively few gram-positive cells with high GC content (<0.2% of eubacterial counts). Differences in both metabolic potential and taxonomic structure of the microbial communities in the ECF- and TCF-activated sludges were detected. The kinetics of the development of these differences in treatment plants and their relationships with treatment efficiency and production process conditions should now be evaluated.

Chlorine↗

PubMedDate not supplied

[Study of extracellular structures of Agrobacterium involved in bacterial and plant interactions].

Agrobacterial cells produced straight microfibrils not only when in contact with wheat seedling roots, but also when in contact with each other. After 2 h of incubation, agrobacterial cells were found to form aggregates, in which the cells were in contact either directly or through thick straight microfibrils (bridges) of an unknown composition. The majority of the microfibrils were susceptible to attack by cellulase, although some of them showed resistance to this enzyme. Like the wild-type flagellated agrobacteria, their bald mutants produced long straight microfibrils. The cells surface structures of agrobacteria were examined by labeling them immunocytochemically with colloidal gold conjugated antibodies against O-specific lipopolysaccharides, Vir proteins, and cellulase. Agrobacterial cells treated with acetosyringone and brought into contact were found to contain subpolar and polar cell surface structures. Antibodies against the VirB2 protein were able to interact with a tuft of thin microfibrils located on one pole of the agrobacterial cell, whose vir genes were induced by acetosyringone, but were unable to interact with the surface structures of the agrobacterial cells aggregated in liquid medium in the absence of wheat seedlings.

Bacterial Outer Membrane Proteins↗

PubMed1990

Protein H1: a role for chromatin structure in the regulation of bacterial gene expression and virulence?

There has been a recent revival of interest in one of the most abundant Escherichia coli proteins, H1 (also called H-NS). This protein was first identified many years ago as a major component of the bacterial nucleoid, and has been characterized biochemically by several groups. However, no clear function for the protein emerged from these studies. Our thinking has been transformed by recent findings which complement the biochemistry with genetic data. Several mutations, selected over many years by virtue of their diverse effects on gene expression, have turned out to be allelic and to fall within the structural gene for H1. Bringing together the genetics and the biochemistry has demonstrated that the whole is worth more than the sum of the parts! These findings have far-reaching implications for the mechanisms by which gene expression is regulated and also, perhaps, for the control of bacterial virulence.

Amino Acid Sequence↗

PubMed2004

Molecular modeling of mammalian cytochrome P450s.

The cytochrome P450 enzymes collectively metabolize a wide range of xenobiotic and endogenous compounds. The broad substrate specificity of this superfamily derives from the multiplicity of P450s whose unique substrate specificity profiles reflect underlying differences in primary sequence. Experimental structures of P450s, where available, have provided great insight into the basis of substrate recognition. However, for those mammalian P450s whose structures have not been determined, homology modeling has become an increasingly important tool for understanding substrate specificity and mechanism. P450 modeling is often a challenging task, owing to the rather low sequence identity between target and template proteins. Although mammalian P450 models have previously been based on bacterial P450 structures, the recent advent of mammalian P450 structures holds great potential for generating more accurate homology models. Consequently, the substrate recognition properties of several mammalian P450s have been rationalized using the predicted substrate binding site of recently developed models. This review summarizes the major concepts and current approaches of molecular modeling of P450s.

Algorithms↗

PubMed1976

Isolation and analysis of the lipid A backbone. Lipid A structure of lipopolysaccharides from various bacterial groups.

A degradation procedure of lipopolysaccharides was worked out which allows the isolation of the reduced backbone of lipid A in a total yield of between 20 and 30%. This procedure was applied to lipopolysaccharides of S forms (Salmonella minnesota, Shigella flexneri 5b, Escherichia coli 086, E. coli 0111, Xanthomonas sinensis, Rhodopseudomonas gelatinosa) and R mutants (Salmonella minnesota, Shigella flexneri, 5b, E. coli BB9 and E. coli EH 100). Chemical analysis, reaction with beta-N-acetyl-glucosaminidase and application of methylation analysis revealed that the lipid A backbone of all strains contains beta 1', 6-linked glucosamine disaccharides carrying two phosphate groups, one in glycosidic and one in ester linkage, a structure, identified previously in the Salmonella minnesota Re mutant.

Acetylglucosamine↗

PubMed1997

Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy.

BACKGROUND: Carboxypeptidase G enzymes hydrolyze the C-terminal glutamate moiety from folic acid and its analogues, such as methotrexate. The enzyme studied here, carboxypeptidase G2 (CPG2), is a dimeric zinc-dependent exopeptidase produced by Pseudomonas sp. strain RS-16. CPG2 has applications in cancer therapy: following its administration as an immunoconjugate, in which CPG2 is linked to an antibody to a tumour-specific antigen, it can enzymatically convert subsequently administered inactive prodrugs to cytotoxic drugs selectively at the tumour site. CPG2 has no significant amino acid sequence homology with proteins of known structure. Hence, structure determination of CPG2 was undertaken to identify active-site residues, which may in turn provide ideas for protein and/or substrate modification with a view to improving its therapeutic usefulness. RESULTS: We have determined the crystal structure of CPG2 at 2.5 A resolution using multiple isomorphous replacement methods and non-crystallographic symmetry averaging. Each subunit of the molecular dimer consists of a larger catalytic domain containing two zinc ions at the active site, and a separate smaller domain that forms the dimer interface. The two active sites in the dimer are more than 60 A apart and are presumed to be independent; each contains a symmetric distribution of carboxylate and histidine ligands around two zinc ions which are 3.3 A apart. This distance is bridged by two shared zinc ligands, an aspartic acid residue and a hydroxyl ion. CONCLUSIONS: We find that the CPG2 catalytic domain has structural homology with other zinc-dependent exopeptidases, both those with a single zinc ion and those with a pair of zinc ions in the active site. The closest structural homology is with the aminopeptidase from Aeromonas proteolytica, where the similarity includes superposable zinc ligands but does not extend to the rest of the active-site residues, consistent with the different substrate specificities. The mechanism of peptide cleavage is likely to be very similar in these two enzymes and may involve the bridging hydroxyl ion ligand acting as a primary nucleophile.

Aminopeptidases↗

PubMed2000

Structure elucidation of glycoprotein glycans and of polysaccharides by NMR spectroscopy.

The applicability of 1H-NMR spectroscopy for the determination of the primary and tertiary structure of carbohydrate-containing molecules is demonstrated. For classes of known compounds the characterization can be based on chemical shifts observed in 1D NMR spectra with or without the aid of a computer database. For more complex structure determinations 2D NMR techniques are required. Here the application of 2D NMR is demonstrated for the primary structure determination of two bacterial exopolysaccharides, for the spatial structure determination of a disaccharide and a glycoprotein hormone.

Bacterial Proteins↗

PubMed2002

Molecular phylogenetic analyses of reverse-transcribed bacterial rRNA obtained from deep-sea cold seep sediments.

A depth profile of naturally occurring bacterial community structures associated with the deep-sea cold seep push-core sediment in the Japan Trench at a depth of 5343 m were evaluated using molecular phylogenetic analyses of RNA reverse transcription-PCR (RT-PCR) amplified 16S crDNA fragments. A total of 137 clones of bacterial crDNA (complimentary rDNA) phylotypes (phylogenetic types) obtained at three different depths (2-4, 8-10 and 14-16 cm) were identified in partial crDNA sequencings. crDNA phylotypes from the cold seep sediment were dominantly composed of delta- and epsilon-Proteobacteria (36% and 42% respectively). Phylotype analysis of crDNA clone libraries and terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the majority of bacterial components shifted from delta- Proteobacteria to epsilon-Proteobacteria with increasing depth. Among the delta-proteobacterial crDNA clones, the sequences related to the genus Desulfosarcina were dominant. Almost all sequences of crDNA belonging to epsilon-Proteobacteria were affiliated with the same cluster (epsilon-CSG: epsilon-proteobacterial cold seep group), and were closely related with rDNA sequences from deep-sea hydrothermal vent environments.

Base Sequence↗