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PubMed2005

Design, synthesis, and activity of analogues of phosphinothricin as inhibitors of glutamine synthetase.

A new group of potent inhibitors of glutamine synthetase was designed and synthesized. The X-ray structure of bacterial glutamine synthetase complexed with phosphinothricin was used for computer-aided structure-based design of the inhibitors, in which the methyl group of phosphinothricin was chosen as the modification site. Amino and hydroxyl moieties were introduced into the phosphinic acid portion of the lead molecule to interact with ammonium binding site in the active cleft of the enzyme. Designed compounds were synthesized in enantiomerically pure form analogous to l-glutamic acid. In vitro kinetic studies with Escherichia coli glutamine synthetase confirmed the biological activity of the designed inhibitors, which with K(i) values in the micromolar range (K(i) = 0.59 microM for the most potent compound 2) appear to be slightly weaker inhibitors or equipotent to phosphinothricin.

Aminobutyrates↗
PubMed2004

Comparative genomic structure of prokaryotes.

Recent advances in DNA-sequencing technologies have made available an enormous resource of data for the study of bacterial genomes. The broad sample of complete genomes currently available allows us to look at variation in the gross features and characteristics of genomes while the detail of the sequences reveal some of the mechanisms by which these genomes evolve. This review aims to describe bacterial genome structures according to current knowledge and proposed hypotheses. We also describe examples where mechanisms of genome evolution have acted in the adaptation of bacterial species to particular niches.

Bacteria↗
PubMedDate not supplied

Effects of structural injure in the bile bacterial contamination after balloon transduodenal sphincteroplasty (papillary dilation) in dogs.

PURPOSE: To evaluate, in dogs, the biliary sphincter subjected to dilation by hydrostatic balloon by the point of view of structural alterations of the papilla and the biochemestry and bacterial contamination of the bile. METHODS: Twenty dogs were submitted to laparotomy, duodenotomy, and enlargement of the major duodenal papilla- GA(n=10) - with balloon of 8mm inflated with pressure of 0,5 atm, during 2 minutes or to the sham procedure - GB(n=10). Blood samples collected on times t(0 day), t(7 days) and t(28 days) were subjected to dosages of alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT) for cholestasis evaluation. The collected material from the gall bladder at the same times were registered and numbered to be submitted to culture in BHI, blood agar (rich, non-selective element) and Mac Conkey (selective element for Gram-negative bacillus. On the 28th day three fragments of the papilla were tranversally cut by the choledoc axis 3mm from the duodenal papilla and the cuts, stained with hematoxylin-eosin and Masson's tricome, were evaluated according to their inflammatory reaction. RESULTS: The GGT and ALP averages on the three periods in the groups A and B did not show significant differences, not being characterizes the cholestasis. The bacterian contamination was significantly higher in GA (2,19) than in GB (1,96); the contamination was lower in the initial time compared with 7 and 28 days (t0<t7 and t 28) in GA and GB (t0<t 28). The chronic inflammatory alteration of the mucosa occurred in 60% of the GA animals and in 80% of the GB animals. There was not mural inflammation. CONCLUSION: The duodenal papilla dilation or its simple manipulation leads to the bacterial contamination of the gall bladder, not associated with cholestasis. The morphologic lesions are more intense in the late phase, not associated with an eventual papilla esthenosis.

Alkaline Phosphatase↗
PubMed2001

Bacterial RNA polymerase.

The recently determined crystal structure of a bacterial core RNA polymerase (RNAP) provides the first glimpse of this family of evolutionarily conserved cellular RNAPs. Using the structure as a framework, a consistent picture of protein-nucleic acid interactions in transcription complexes has been accumulated from cross-linking experiments. The molecule can be viewed as a molecular machine, with distinct structural features hypothesized to perform specific functions. Comparison with the alpha-carbon backbone of a eukaryotic RNAP reveals close structural similarity.

Archaeal Proteins↗
PubMed2001

Percent G+C profiling accurately reveals diet-related differences in the gastrointestinal microbial community of broiler chickens.

Broiler chickens from eight commercial farms in Southern Finland were analyzed for the structure of their gastrointestinal microbial community by a nonselective DNA-based method, percent G+C-based profiling. The bacteriological impact of the feed source and in-farm whole-wheat amendment of the diet was assessed by percent G+C profiling. Also, a phylogenetic 16S rRNA gene (rDNA)-based study was carried out to aid in interpretation of the percent G+C profiles. This survey showed that most of the 16S rDNA sequences found could not be assigned to any previously known bacterial genus or they represented an unknown species of one of the taxonomically heterogeneous genera, such as Ruminococcus or Clostridium. The data from bacterial community profiling were analyzed by t-test, multiple linear regression, and principal-component statistical approaches. The percent G+C profiling method with appropriate statistical analyses detected microbial community differences smaller than 10% within each 5% increment of the percent G+C profiles. Diet turned out to be the strongest determinant of the cecal bacterial community structure. Both the source of feed and local feed amendment changed the bacteriological profile significantly, whereas profiles of individual farms with identical feed regimens hardly differed from each other. This suggests that the management of typical Finnish farms is relatively uniform or that hygiene on the farm, in fact, has little impact on the structure of the cecal bacterial community. Therefore, feed compounders should have a significant role in the modulation of gut microflora and consequently in prevention of gastrointestinal disorders in farm animals.

Animal Feed↗
PubMed2002

Directed molecular evolution of ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase catalyzes a rate-limiting reaction in prokaryotic glycogen and plant starch biosynthesis. Despite sharing similar molecular size and catalytic and allosteric regulatory properties, the prokaryotic and higher plant enzymes differ in higher-order protein structure. The bacterial enzyme is encoded by a single gene whose product of ca. 50,000 Da assembles into a homotetrameric structure. Although the higher plant enzyme has a similar molecular size, it is made up of a pair of large subunits and a pair of small subunits, encoded by different genes. To identify the basis for the evolution of AGPase function and quaternary structure, a potato small subunit homotetrameric mutant, TG-15, was subjected to iterations of DNA shuffling and screened for enzyme variants with up-regulated catalytic and/or regulatory properties. A glycogen selection/screening regimen of buoyant density gradient centrifugation and iodine vapor colony staining on glucose-containing media was used to increase the stringency of selection. This approach led to the isolation of a population of AGPase small subunit homotetramer enzymes with enhanced affinity toward ATP and increased sensitivity to activator and/or greater resistance to inhibition than TG-15. Several enzymes displayed a shift in effector preference from 3-phosphoglycerate to fructose-6 phosphate or fructose-1,6-bis-phosphate, effectors used by specific bacterial AGPases. Our results suggest that evolution of AGPase, with regard to quaternary structure, allosteric effector selectivity, and effector sensitivity, can occur through the introduction of a few point mutations alone with low-level recombination hastening the process.

Directed Molecular Evolution↗
PubMed2002

Crystal structure of porcine mitochondrial NADP+-dependent isocitrate dehydrogenase complexed with Mn2+ and isocitrate. Insights into the enzyme mechanism.

The crystal structure of porcine heart mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH) complexed with Mn2+ and isocitrate was solved to a resolution of 1.85 A. The enzyme was expressed in Escherichia coli, purified as a fusion protein with maltose binding protein, and cleaved with thrombin to yield homogeneous enzyme. The structure was determined by multiwavelength anomalous diffraction phasing using selenium substitution in the form of selenomethionine as the anomalous scatterer. The porcine NADP+-IDH enzyme is structurally compared with the previously solved structures of IDH from E. coli and Bacillus subtilis that share 16 and 17% identity, respectively, with the mammalian enzyme. The porcine enzyme has a protein fold similar to the bacterial IDH structures with each monomer folding into two domains. However, considerable differences exist between the bacterial and mammalian forms of IDH in regions connecting core secondary structure. Based on the alignment of sequence and structure among the porcine, E. coli, and B. subtilis IDH, a putative phosphorylation site has been identified for the mammalian enzyme. The active site, including the bound Mn2+-isocitrate complex, is highly ordered and, therefore, mechanistically informative. The consensus IDH mechanism predicts that the Mn2+-bound hydroxyl of isocitrate is deprotonated prior to its NADP+-dependent oxidation. The present crystal structure has an active site water that is well positioned to accept the proton and ultimately transfer the proton to solvent through an additional bound water.

Amino Acid Sequence↗
PubMed1979

Macromolecular structural transitions in Pf1 filamentous bacterial virus.

The filamentous bacterial virus Pf1 is a simple model for biological filaments. We have studied the structure of the virion and report here that the helix parameters of Pf1 change sharply with temperature at about 8 degrees C. Local interactions between protein subunits change by only a few tenths of an angstrom, but the changes are amplified between one end and the other of the virion to a rotation of 15 turns and a translation of 1,000 A. The limited nature of the phase transition is probably due to the constraints of 'knobs-into-holes' interaction between side chains of adjacent alpha-helical protein subunits. Treatment of the virion with ether causes a rearrangement of protein subunits into sheets, with the alpha-helices normal to the plane of the sheet. This phase transition suggests a model for virion assembly in the bacterial membrane.

Bacteriophages↗
PubMed1991

Structure and function of a bacterial mRNA stabilizer: analysis of the 5' untranslated region of ompA mRNA.

The 5' untranslated region (UTR) of the Escherichia coli ompA transcript functions in vivo as a growth rate-regulated mRNA stabilizer. The secondary structure of this mRNA segment has been determined by a combination of three methods: phylogenetic analysis, in vitro probing with a structure-specific RNase, and methylation by dimethylsulfate in vivo and in vitro. These studies reveal that despite extensive sequence differences, the 5' UTRs of the ompA transcripts of E. coli, Serratia marcescens, and Enterobacter aerogenes can fold in a remarkably similar fashion. Furthermore, the Serratia and Enterobacter ompA 5' UTRs function as effective mRNA stabilizers in E. coli. Stabilization of mRNA by the Serratia ompA 5' UTR is growth rate dependent. These findings indicate that the features of the ompA 5' UTR responsible for its ability to stabilize mRNA in a growth rate-regulated manner are to be found among the structural similarities shared by these diverse evolutionary variants.

Bacterial Outer Membrane Proteins↗
PubMed2002

Sugar specificity of bacterial CMP kinases as revealed by crystal structures and mutagenesis of Escherichia coli enzyme.

Bacterial cytidine monophosphate (CMP) kinases are characterised by an insert enlarging their CMP binding domain, and by their particular substrate specificity. Thus, both CMP and 2'-deoxy-CMP (dCMP) are good phosphate acceptors for the CMP kinase from Escherichia coli (E. coli CMPK), whereas eukaryotic UMP/CMP kinases phosphorylate the deoxynucleotides with very low efficiency. Four crystal structures of E. coli CMPK complexed with nucleoside monophosphates differing in their sugar moiety were solved. Both structures with CMP or dCMP show interactions with the pentose that were not described so far. These interactions are lost with the poorer substrates AraCMP and 2',3'-dideoxy-CMP. Comparison of all four structures shows that the pentose hydroxyls are involved in ligand-induced movements of enzyme domains. It also gives a structural basis of the mechanism by which either ribose or deoxyribose can be accommodated. In parallel, for the four nucleotides the kinetic results of the wild-type enzyme and of three structure-based variants are presented. The phosphorylation rate is significantly decreased when either of the two pentose interacting residues is mutated. One of these is an arginine that is highly conserved in all known nucleoside monophosphate kinases. In contrast, the other residue, Asp185, is typical of bacterial CMP kinases. It interacts with Ser101, the only residue conserved in all CMP binding domain inserts. Mutating Ser101 reduces CMP phosphorylation only moderately, but dramatically reduces dCMP phosphorylation. This is the first experimental evidence of a catalytic role involving the characteristic insert of bacterial CMP kinases. Furthermore, this role concerns only dCMP phosphorylation, a feature of this family of enzymes.

Amino Acid Sequence↗
PubMed1992

Poly(A) RNA in Escherichia coli: nucleotide sequence at the junction of the lpp transcript and the polyadenylate moiety.

Although it has been known for some time that bacterial mRNA molecules carry polyadenylate moieties at their 3' ends, nothing is known about the molecular structure of bacterial poly(A) RNA. To define the polyadenylylation site of a specific bacterial mRNA, we took advantage of the presence of elevated levels of poly(A) RNA in cells of Escherichia coli deficient in exoribonucleases and synthesized DNA complementary to polyadenylylated lipoprotein mRNA, encoded by the lpp gene, by using avian myeloblastosis virus reverse transcriptase and an oligo(dT)-containing primer. The 5'-terminal portion of the cDNA was amplified by the polymerase chain reaction and appropriate oligonucleotide primers, and the amplified DNA was cloned in pUC18 and subjected to nucleotide sequence analysis. Four clones were found to contain the entire 3'-terminal coding region of lpp mRNA, with poly(A) attached to either of two sites in the downstream untranslated region of the transcript. In one type of clone, the polyadenylate moiety was attached at the putative transcription termination site of lpp mRNA, whereas other clones lacked the stem-loop structure of the rho-independent transcription terminator and the polyadenylate moiety was attached to the residue just preceding the terminal stem-loop of the primary transcript. A model for the polyadenylylation of bacterial mRNA is proposed in which poly(A) polymerase and exonucleases compete for the 3' ends of mRNA molecules.

Amino Acid Sequence↗
PubMed1979

An X-ray study of the cytoplasmic membranes of two gram-positive bacteria.

X-ray diffraction diagrams of partially disordered one-dimensional lattices of isolated bacterial cytoplasmic membranes are described and they provide a basis for suggesting possible molecular structures of bacterial membranes. Biochemical and electron microscope evidence points towards a lipid bilayer with a high degree of fluidity. The protein molecules are in a disordered configuration in the membrane.

Bacillus↗
PubMed2006

Structure and reactivity of a thermostable prokaryotic nitric-oxide synthase that forms a long-lived oxy-heme complex.

In an effort to generate more stable reaction intermediates involved in substrate oxidation by nitric-oxide synthases (NOSs), we have cloned, expressed, and characterized a thermostable NOS homolog from the thermophilic bacterium Geobacillus stearothermophilus (gsNOS). As expected, gsNOS forms nitric oxide (NO) from l-arginine via the stable intermediate N-hydroxy l-arginine (NOHA). The addition of oxygen to ferrous gsNOS results in long-lived heme-oxy complexes in the presence (Soret peak 427 nm) and absence (Soret peak 413 nm) of substrates l-arginine and NOHA. The substrate-induced red shift correlates with hydrogen bonding between substrate and heme-bound oxygen resulting in conversion to a ferric heme-superoxy species. In single turnover experiments with NOHA, NO forms only in the presence of H(4)B. The crystal structure of gsNOS at 3.2 AA of resolution reveals great similarity to other known bacterial NOS structures, with the exception of differences in the distal heme pocket, close to the oxygen binding site. In particular, a Lys-356 (Bacillus subtilis NOS) to Arg-365 (gsNOS) substitution alters the conformation of a conserved Asp carboxylate, resulting in movement of an Ile residue toward the heme. Thus, a more constrained heme pocket may slow ligand dissociation and increase the lifetime of heme-bound oxygen to seconds at 4 degrees C. Similarly, the ferric-heme NO complex is also stabilized in gsNOS. The slow kinetics of gsNOS offer promise for studying downstream intermediates involved in substrate oxidation.

Bacillaceae↗
PubMed2006

Fluorescent in situ hybridization (FISH) for the detection of bacterial community in activated sludge from textile factories.

Conventional methods used to study the bacterial community structure in activated sludge are not sufficient enough to determine the compositions of the bacterial populations responsible for biodegradation. Activated sludge samples from 3 textile factories were analyzed by fluorescent in situ hybridization (FISH) using rRNA probes and by phase-contrast microscopy. In Factory-I, the predominant groups were the beta-subclass of Proteobacteria and the cytophaga-flavobacterium (CF) cluster (33.3% and 31.0%) followed by gamma-subclass (17.1%), high G+C DNA (HGC) gram-positive (15.4%) and alpha-subclass (3.2%). Factory-II showed a similar pattern (32.7%, 31.8%, 17.5%, 16.4%, 1.6%) but with lower concentrations, while Factory-III showed predominant alpha- and beta-subclasses (25.2%, 25.0%) and CF cluster (24.8%) followed by the gamma-subclass (13.6%) and the HGC (11.4%) at much lower concentrations. The floc characteristic for factory-I and -II was normal, however factory-III had diffuse and atypical flocs. In conclusion, the FISH technique provided comprehensive information on the bacterial consortia of activated sludge samples. The compositions of the bacterial community and their concentrations together with the floc characteristics might be some of the reasons that affect the operational efficiencies among the 3 textile factories.

Animals↗
PubMed2006

Contribution of alginate and levan production to biofilm formation by Pseudomonas syringae.

Exopolysaccharides (EPSs) play important roles in the attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional, complex structure of bacterial biofilms. To elucidate the spatial distribution and function of the EPSs levan and alginate during biofilm formation, biofilms of Pseudomonas syringae strains with different EPS patterns were compared. The mucoid strain PG4180.muc, which produces levan and alginate, and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow chambers. Confocal laser scanning microscopy with fluorescently labelled lectins was applied to investigate the spatial distribution of levan and an additional as yet unknown EPS in flow-chamber biofilms. Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centres of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose as confirmed by an enzyme-linked lectin-sorbent assay using peroxidase-linked ConA. Time-course studies revealed that expression of the levan-forming enzyme, levansucrase, occurred mainly during early exponential growth of both planktonic and sessile cells. Thus, accumulation of levan in biofilm voids hints to a function as a nutrient storage source for later stages of biofilm development. The presence of a third EPS besides levan and alginate was indicated by binding of the lectin from Naja mossambica to a fibrous structure in biofilms of all P. syringae derivatives. Production of the as yet uncharacterized additional EPS might be more important for biofilm formation than the syntheses of levan and alginate.

Alginates↗
PubMed1975

Influence of molecular structure on the tolerogenicity of bacterial dextrans. I. The alpha1--6-linked epitope of dextran B512.

Native dextran B512 is a near-linear glucose polymer with 96 per cent alpha1--6 and 4 per cent alpha1--3 linkages and a molecular weight (mol. wt) of 8 X 10(7). Sheep RBC sensitized with its O-stearoyl derivative (prepared by a modified method) have been used satisfactorily in direct PFC assays. B512 immunizes BALB/c mice optimally with doses of 1--10 mug and produces B-cell tolerance with 1 mg upwards. The specificity of the response determined by PFC inhibition analysis, is directed towards an alpha1--6-linked epitope. High dose tolerance is not preceded by immunity and is stable on cell transfer to irradiated recipients in which responsiveness becomes perceptible after 4--6 weeks. Progressive depolymerization of this polysaccharide reduces immunogenicity and tolerogenicity, both of which are extinguished when the mol. wt falls to 2 X 10(4). Optimal immunization with B512 is succeeded by partial tolerance (previously characterized by analogous levan experiments as a B-cell exhaustion process). The tolerance threshold dose of B512 is reduced 1000-fold during immunosuppression with cyclophosphamide. PFC inhibition studies supported the contention that tolerogenicity of polysaccharides is influenced by their overall binding capacities. A direct relationship between inhibitory and tolerogenic activities was found both with B512 fractions of varying mol. wt and with heterologous dextrans. The similarities between B512 and levan argue against the association of a highly branched structure with greater tolerogenicity. The effect of reducing the percentage of alpha1--6 linkages in dextrans suggests, however, that epitope density probably plays a contributory role in determining the outcome of interaction between polysaccharides and B cells.

Animals↗
PubMed2003

Ring-like pore structures of SecA: implication for bacterial protein-conducting channels.

SecA, an essential component of the general protein secretion pathway of bacteria, is present in Escherichia coli as soluble and membrane-integral forms. Here we show by electron microscopy that SecA assumes two characteristic forms in the presence of phospholipid monolayers: dumbbell-shaped elongated structures and ring-like pore structures. The ring-like pore structures with diameters of 8 nm and holes of 2 nm are found only in the presence of anionic phospholipids. These ring-like pore structures with larger 3- to 6-nm holes (without staining) were also observed by atomic force microscopic examination. They do not form in solution or in the presence of uncharged phosphatidylcholine. These ring-like phospholipid-induced pore-structures may form the core of bacterial protein-conducting channels through bacterial membranes.

Adenosine Triphosphatases↗