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PubMed2005

Structure of a cellulose degrading bacterial community during anaerobic digestion.

It is widely accepted that cellulose is the rate-limiting substrate in the anaerobic digestion of organic solid wastes and that cellulose solubilisation is largely mediated by surface attached bacteria. However, little is known about the identity or the ecophysiology of cellulolytic microorganisms from landfills and anaerobic digesters. The aim of this study was to investigate an enriched cellulolytic microbial community from an anaerobic batch reactor. Chemical oxygen demand balancing was used to calculate the cellulose solubilisation rate and the degree of cellulose solubilisation. Fluorescence in situ hybridisation (FISH) was used to assess the relative abundance and physical location of three groups of bacteria belonging to the Clostridium lineage of the Firmicutes that have been implicated as the dominant cellulose degraders in this system. Quantitation of the relative abundance using FISH showed that there were changes in the microbial community structure throughout the digestion. However, comparison of these results to the process data reveals that these changes had no impact on the cellulose solubilisation in the reactor. The rate of cellulose solubilisation was approximately stable for much of the digestion despite changes in the cellulolytic population. The solubilisation rate appears to be most strongly affected by the rate of surface area colonisation and the biofilm architecture with the accepted model of first order kinetics due to surface area limitation applying only when the cellulose particles are fully covered with a thin layer of cells.

Anaerobiosis↗
PubMed1995

Differential regulation of surface immunoglobulin expression by various muramyl dipeptides in a murine pre-B cell line.

The murine pre-B cell line 70Z/3 responds to lipopolysaccharide (LPS), interleukin-1 (IL-1) or interferon-gamma (IGN gamma) by kappa gene transcription and expression of surface IgM (sIg). We found that muramyl dipeptide (MDP), a synthetic immunoadjuvant analog of a bacterial membrane structure, produced a weak increase in the number of sIg-positive 70Z/3 cells as measured by immunofluorescence staining. This number was significantly increased after exposure to MDP. Moreover, when MDP was used in combination with LPS, IL-1 or IFN gamma, an enhancement of sIg expression was observed showing an early influence of MDP in the presence of a second stimulant. Unexpectedly, two adjuvant-active analogs of MDP did not share its capacity to stimulate differentiation of the cell line when used alone or associated with other agents, indicating that adjuvanticity of MDP was not the only requirement. Two other products of bacterial origin, a Staphylococcus aureus cell extract (SAC) and the Toxic Shock Syndrome Toxin TSST-1 could neither enhance the kappa gene expression in 70Z/3 cells nor increase the MDP effect. The stimulating effect displayed by MDP could by related to NF-kappa B activation.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMedDate not supplied

[Uropathogenic Escherichia coli--ExPEC pathotype, the best characterized].

Uropathogenic E. coli strains are included in the recently defined pathotype ExPEC together with other E. coli isolates involved in various extraintestinal infections. These strains possess specific virulence factors such as adherence systems, toxins, iron uptake systems that distinguish them from the commensal strains. The expression of bacterial adherence structures requires a complex organization which involves a different number of proteins depending upon the assembly in fimbrial protrudings or afimbrial aggregates. However one could notice that their genetic determinants are usually similar. The toxins synthetized by ExPEC strains, mainly represented by hemolysin and cytotoxic necrotizing factor contribute to destruction of eukaryotic cells. Such siderophores as aerobactin confer the strains the advantage of capturing iron from the environment in which they must survive and replicate. Co-expression of virulence factors contributes to the host's defence system defeat and onset of infection. Quite often the codifying genes are localized in distinctive regions on the bacterial chromosome called pathogenicity-associated islands. The horizontal transfer of these DNA blocks among individuals belonging to the same species is one of the processes involved in the evolution of pathogenic strains.

Adhesins, Escherichia coli↗
PubMed1993

Comparison of genome fingerprinting with conventional typing methods used on Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Two hundred Pseudomonas aeruginosa serial isolates from 61 cystic fibrosis patients were examined using restriction fragment length polymorphism in connection with pulsed field gel electrophoresis. A comparison was made of results obtained by genome fingerprinting and by conventional typing methods. It was possible to subdivide the majority of the genome types with the conventional typing methods, indicating the likelihood of bacterial phenotypes occurring in the lung of the cystic fibrosis patient. Two clusters of strains were observed among the 200 P. aeruginosa isolates from the 61 patients. Strains belonging to one cluster were present in 26 (42.6%) of the 61 patients. Strains belonging to the other cluster were present in 11 (18.0%) of the 61 patients. The occurrence of these clusters indicates that cross-infection has taken place among CF patients attending the Danish Cystic Fibrosis Centre. Conventional typing methods are based on the presence of specific bacterial surface structures. Therefore, conventional typing methods may sometimes lead to wrong classification of isolates from cystic fibrosis patients, especially if applied alone. A combination of two to six methods decreased the reproducibility of the typing results. The best combination was genome fingerprinting and phage typing, which yielded a reproducibility of 82.5%. Each time a method is added, the reproducibility decreases by an average of 14.4%.

Bacterial Typing Techniques↗
PubMed2001

Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing.

Quorum sensing is an example of community behavior prevalent among diverse bacterial species. The term "quorum sensing" describes the ability of a microorganism to perceive and respond to microbial population density, usually relying on the production and subsequent response to diffusible signal molecules. A significant number of gram-negative bacteria produce acylated homoserine lactones (acyl-HSLs) as signal molecules that function in quorum sensing. Bacteria that produce acyl-HSLs can respond to the local concentration of the signaling molecules, and high population densities foster the accumulation of inducing levels of acyl-HSLs. Depending upon the bacterial species, the physiological processes regulated by quorum sensing are extremely diverse, ranging from bioluminescence to swarming motility. Acyl-HSL quorum sensing has become a paradigm for intercellular signaling mechanisms. A flurry of research over the past decade has led to significant understanding of many aspects of quorum sensing including the synthesis of acyl-HSLs, the receptors that recognize the acyl-HSL signal and transduce this information to the level of gene expression, and the interaction of these receptors with the transcriptional machinery. Recent studies have begun to integrate acyl-HSL quorum sensing into global regulatory networks and establish its role in developing and maintaining the structure of bacterial communities.

4-Butyrolactone↗
PubMed2004

Anaerobic degradation of cis-1,2-dichloroethylene and vinyl chloride by Clostridium sp. strain DC1 isolated from landfill leachate sediment.

The bacterial community structure of anaerobic enrichment cultures that are capable of degrading both cis-1,2-dichloroethylene (cis-DCE) and vinyl chloride (VC) and isolation of the organism responsible for the degradation were investigated. Denaturing gradient gel electrophoresis (DGGE) of a PCR-amplified 16S rRNA gene from the cultures showed the possible predominance of Clostridium species. One isolate, designated strain DC1, was closely related to members of Clostridiaceae, based on 16S rRNA gene analysis, and the highest sequence similarity (98.9%) was obtained for Clostridium saccarobutylicum. In culture experiments, strain DC1 was shown to degrade cis-DCE and VC during the stationary phase of growth without accumulation of VC and/or ethene. The bacterial growth was not linked to the degradation of cis-DCE and VC. Stoichiometric analysis revealed that two moles of chloride ions as released from one mole of cis-DCE during the incubation period, indicating that cis-DCE was fully dechlorinated. The results appear consistent with the presence of a mechanism of oxidative dechlorination rather than respiratory reductive dechlorination; the latter is accompanied by transient formation of dechlorinated ethenes from cis-DCE and VC.

Journal Article↗
PubMed2006

Effect of chemical and physical parameters on a pulp mill biotreatment bacterial community.

The bacterial community composition in an activated sludge plant treatment from a bleached kraft pulp mill was monitored over a period of 209 days. Using DGGE and terminal-Restriction Fragment Length Polymorphism (t-RFLP) analysis we generated community DNA fingerprints over the time period. Both methods produce fingerprints that can be used to monitor stability in the system and generate fragments that can be associated with bacterial taxa. Chemical and physical parameters were also collected during that same time frame. We found a number of significant correlations with influent variables such as temperature, chemical oxygen demand (COD), Biochemical oxygen demand (BOD) and chloroform concentrations suggesting that these were the most likely parameters to influence the bacterial community structure. In addition several taxa correlated to important performance indicators such as COD/BOD removals and SVI. Multivariate analysis also confirmed the strong links between taxa variation and temperature, nutrient loads, chloroform and also one class of filaments. Establishing the identity of these taxa and their ecological preferences will greatly enhance our understanding and management of biological treatment systems.

Bacteria↗
PubMed1999

Structural biology.

Protein crystallography has become a major technique for understanding cellular processes. This has come about through great advances in the technology of data collection and interpretation, particularly the use of synchrotron radiation. The ability to express eukaryotic genes in Escherichia coli is also important. Analysis of known structures shows that all proteins are built from about 1000 primeval folds. The collection of all primeval folds provides a basis for predicting structure from sequence. At present about 450 are known. Of the presently sequenced genomes only a fraction can be related to known proteins on the basis of sequence alone. Attempts are being made to determine all (or as many as possible) of the structures from some bacterial genomes in the expectation that structure will point to function more reliably than does sequence. Membrane proteins present a special problem. The next 20 years may see the experimental determination of another 40,000 protein structures. This will make considerable demands on synchrotron sources and will require many more biochemists than are currently available. The availability of massive structure databases will alter the way biochemistry is done.

Crystallography, X-Ray↗
PubMed2003

Nondigestible oligosaccharides enhance bacterial colonization resistance against Clostridium difficile in vitro.

Clostridium difficile is the principal etiologic agent of pseudomembranous colitis and is a major cause of nosocomial antibiotic-associated diarrhea. A limited degree of success in controlling C. difficile infection has been achieved by using probiotics; however, prebiotics can also be used to change bacterial community structure and metabolism in the large gut, although the effects of these carbohydrates on suppression of clostridial pathogens have not been well characterized. The aims of this study were to investigate the bifidogenicity of three nondigestible oligosaccharide (NDO) preparations in normal and antibiotic-treated fecal microbiotas in vitro and their abilities to increase barrier resistance against colonization by C. difficile by using cultural and molecular techniques. Fecal cultures from three healthy volunteers were challenged with a toxigenic strain of C. difficile, and molecular probes were used to monitor growth of the pathogen, together with growth of bifidobacterial and bacteroides populations, over a time course. Evidence of colonization resistance was assessed by determining viable bacterial counts, short-chain fatty acid formation, and cytotoxic activity. Chemostat studies were then performed to determine whether there was a direct correlation between bifidobacteria and C. difficile suppression. NDO were shown to stimulate bifidobacterial growth, and there were concomitant reductions in C. difficile populations. However, in the presence of clindamycin, activity against bifidobacteria was augmented in the presence of NDO, resulting in a further loss of colonization resistance. In the absence of clindamycin, NDO enhanced colonization resistance against C. difficile, although this could not be attributed to bifidobacterium-induced inhibitory phenomena.

Anti-Bacterial Agents↗
PubMed2005

Strategies for targeting the multidrug resistance-1 (MDR1)/P-gp transporter in human malignancies.

ATP-binding cassette (ABC) transporters are a super family of channel proteins that include multi-drug resistance 1 (MDR1/P-gp) and multi-drug resistance related proteins (MRPs) whose functions include the efflux of ions, nutrients, lipids, amino acids, peptides, proteins and drugs. The three-dimensional structures of bacterial and human ABC transporters demonstrate that these proteins are ATP-dependent molecular machines that scan the inner membrane leaflet for lipids/drugs and flip them to the outer membrane leaflet. In many human cancers, the level of expression of MDR1 is an important independent prognostic factor that determines response to combination chemotherapy. Intrinsic and acquired resistance to chemotherapy exposure are due to a high level of MDR1 expression that enhances drug efflux, with associated poor clinical outcome and lower complete remission (CR) rates. Recent clinical trials in hematological and solid malignancies have shown promise for a prolonged remission and improved overall survival when the MDR1 P-gp is inhibited when combined with chemotherapy. Structure-based homology modeling of these ABC transporters may help design novel drug candidates to both the membrane-spanning domain (MSD) and the nucleotide-binding domain (NBD) located within the cytoplasm. This review will highlight advances in the utilization of homology modeling in the drug discovery process and how this will impact on fundamental insights to the development of novel therapeutics that could alter and/or inhibit their functions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Gingival crevice microbiota from Chinese patients with gingivitis or necrotizing ulcerative gingivitis.

The objective of this study was to quantitatively compare the bacterial population structure in plaque from the gingival margin of two groups of 21 Chinese patients with gingivitis or necrotizing ulcerative gingivitis (NUG). Subjects were recruited in four dental clinics in Eastern China. Samples were quantitatively assessed by immunofluorescence and fluorescent in situ hybridization for taxa known to be associated with periodontal diseases. The analyses showed that the fusiform taxa (Fusobacterium nucleatum/Fusobacterium periodonticum, Leptotrichia buccalis, Tannerella forsythensis, and Capnocytophaga sp.), Campylobacter rectus, Prevotella intermedia, Prevotella nigrescens, Selenomonas sputigena, and treponemes were present in both groups with high prevalence. Porphyromonas gingivalis and Actinomyces gerencseriae were much more prevalent in the NUG group. Quantitatively, most taxa, including P. gingivalis, F. nucleatum and the treponemes, accounted, on average, for < 3% of the total bacterial cell number. Only P. intermedia/P. nigrescens, P. gingivalis, S. sputigena, A. gerencseriae, and the sum of all monitored suspected periodontal pathogens were significantly increased in the NUG group. The present study demonstrates for both groups a highly diverse plaque composition and suggests that, etiologically, the overall concentration and the concerted effects of the entire group of opportunistic pathogens thriving in NUG-associated plaque are of prime importance.

Adult↗
PubMed2004

Crystal structure and mechanism of a bacterial fluorinating enzyme.

Fluorine is the thirteenth most abundant element in the earth's crust, but fluoride concentrations in surface water are low and fluorinated metabolites are extremely rare. The fluoride ion is a potent nucleophile in its desolvated state, but is tightly hydrated in water and effectively inert. Low availability and a lack of chemical reactivity have largely excluded fluoride from biochemistry: in particular, fluorine's high redox potential precludes the haloperoxidase-type mechanism used in the metabolic incorporation of chloride and bromide ions. But fluorinated chemicals are growing in industrial importance, with applications in pharmaceuticals, agrochemicals and materials products. Reactive fluorination reagents requiring specialist process technologies are needed in industry and, although biological catalysts for these processes are highly sought after, only one enzyme that can convert fluoride to organic fluorine has been described. Streptomyces cattleya can form carbon-fluorine bonds and must therefore have evolved an enzyme able to overcome the chemical challenges of using aqueous fluoride. Here we report the sequence and three-dimensional structure of the first native fluorination enzyme, 5'-fluoro-5'-deoxyadenosine synthase, from this organism. Both substrate and products have been observed bound to the enzyme, enabling us to propose a nucleophilic substitution mechanism for this biological fluorination reaction.

Catalysis↗
PubMed1991

Lipoprotein lipase enhances the binding of chylomicrons to low density lipoprotein receptor-related protein.

Chylomicron catabolism is known to be initiated by the enzyme lipoprotein lipase (triacylglycero-protein acylhydrolase, EC 3.1.1.34). Chylomicron remnants, produced by lipolysis, are rapidly taken up by the liver via an apolipoprotein E (apoE)-mediated, receptor-dependent process. The low density lipoprotein (LDL) receptor-related protein (LRP) has been suggested as the potential apoE receptor. We have analyzed the binding of human chylomicrons to HepG2 cells in the absence and presence of lipoprotein lipase. Bovine and human lipoprotein lipases were able to increase the specific binding of the chylomicrons by up to 30-fold. This effect was not dependent on lipolysis but appeared to be due to the lipase protein itself. It was not found when a structurally unrelated, bacterial lipase was used. Using beta-migrating very low density lipoproteins (beta-VLDLs), known as a good ligand for LRP, binding studies were performed on LDL receptor-negative human fibroblasts. The binding was increased 40-fold by addition of lipoprotein lipase. Crosslinking experiments on cells with 125I-labeled apoE liposomes or lipoprotein lipase showed that both proteins were able to bind to LRP on the cell surface. The binding of apoE to LRP was highly increased by the addition of lipase. We conclude that lipoprotein lipase strongly enhances the binding of apoE-containing lipoproteins to LRP and therefore might play an important role in chylomicron catabolism not only because of its lipolytic activity but also because of its structural properties.

Apolipoproteins E↗
PubMed2005

The selection of mixed microbial inocula in environmental biotechnology: example using petroleum contaminated tropical soils.

The impact of inorganic N and P additions on a tropical soil contaminated with petroleum hydrocarbons was investigated using molecular and culture techniques. Microcosms were incubated for 42 days and sampled at 0, 1, 7, 28 and 42 days. Changes in bacterial community structure were determined using denaturing gradient gel electrophoresis (DGGE) of the rRNA following reverse transcription PCR using primers specific to the V3 region of the 16S rRNA gene. To identify which components of the microbial community were changing during incubation, PCR amplicons were resolved using DGGE and the banding patterns analyzed using stepwise discriminant function analysis (SDA). SDA showed that the number of bands needed to recover the differences between samples over time could be reduced from the initial 11 bands for the 16S rRNA transcript to 3 bands. Sequences originating from the rRNA gels (16S rRNA transcripts) were recovered in clades containing known cultured isolates of Bacillus marisflavi, Microbacterium oxydans and Pseudomonas oleovorans. Isolation studies on these soils using lubricant oil as a carbon source yielded 317 bacterial isolates, 3 of which showed high sequence similarity (>96%) with the 16S rRNA transcripts identified using SDA as being important in differentiating between bacterial communities over time. These isolates were then tested singly and in combination for their ability to degrade lubricant oil. These analyses demonstrated that the consortium selected using the combined molecular-SDA approach was more effective at degrading the lubricant in both liquid media and in contaminated sand than the single isolates.

Bacteria↗
PubMed2006

Molecular analysis of the bacterial community in a continental high-temperature and water-flooded petroleum reservoir.

Water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China was analysed for its bacterial community and diversity. The bacteria were characterized by their 16S rRNA genes. A 16S rRNA gene clone library was constructed from the community DNA, and using restriction fragment length polymorphism analysis, 337 randomly selected clones were clustered with 74 operational taxonomic units. Sequencing and phylogenetic analyses showed that the screened clones were affiliated with Gammaproteobacteria (85.7%), Thermotogales (6.8%), Epsilonproteobacteria (2.4%), low-G+C Gram-positive (2.1%), high-G+C Gram-positive, Betaproteobacteria and Nitrospira (each <1.0%). Thermopilic bacteria were found in the high-temperature water from the flooded petroleum reservoir, as well as mesophilic bacteria such as Pseudomonas-like clones. The mesophilic bacteria were probably introduced into the reservoir as it was being exploited. This work provides significant information on the structure of bacterial communities in high-temperature, long-term water-flooded petroleum reservoirs.

Bacteria↗
PubMed1980

[Lysozyme in sow's milk and its importance to bacterial population of the gastrointestinal tract in suckling piglets].

The lysozyme level was found to reach its highest point on the second day from parturition, followed by strong decline to a much lower value which then remained constant up to the 30th day of lactation. The rise recorded from the period between farrowing and the second day after birth as well as the decline up to the fourth day and the constant level up to the 30th day from parturition were statistically secured. Lysozyme levels in the milk of sows with no previous lactation record was found to be lower with significance than those recordable from sows with something between six and nine previous lactations. The conclusion has been that lysozyme levels are strongly affected by both lactation time and number of lactations. Lysozyme concentrations in sow milk on the first and second days from parturition were betweeen 6.8 and 11.0 microgram/ml, depending on the number of previous lactations. Such high lysozyme levels in sow foremilk over the first three days of age as well as all findings so far gained on the structure of bacterial cell walls are likely to suggest that the above enzyme affects bacterial population of the gastro-intestinal tract of suckling.

Animals↗
PubMed2003

Effects of 2-hydroxy-4-(methylthio) butanoic acid (HMB) on microbial growth in continuous culture.

2-Hydroxy-4-(methylthio) butanoic acid (HMB) positively affects milk composition and yield, potentially through ruminal actions. Four continuous culture fermenters were used to determine the optimal concentration of HMB for digestibility of organic matter (OM), neutral detergent fiber (NDF), acid detergent fiber (ADF), and hemicellulose and synthesis of microbial N. A highly degradable mix of hay and grain was used as a basal diet to simulate a typical lactation diet. Three concentrations of HMB (0, 0.055, and 0.110%) and one concentration of dl-Met (0.097%) were infused into the fermenters according to a 4 x 4 Latin square design. Digesta samples were collected during the last 3 d of each of the four 10-d experimental periods. Digestibility of OM, hemicellulose, and NDF was largely insensitive to treatment. Digestibility of ADF showed a quadratic effect to supplementation of HMB, with 0.055% having lower digestibility than 0 or 0.110%. Total production of VFA was not influenced by HMB supplementation, but differences in concentration and production of individual VFA were seen. Isobutyrate increased linearly with increasing HMB supplementation. Propionate concentration decreased linearly with increased HMB supplementation, but propionate production showed a quadratic trend (P = 0.13). A higher concentration of acetate was detected for dl-Met compared with the highest HMB concentration. There were trends (P < 0.15) for dl-Met to decrease the production of isobutyrate and to lower the concentration of butyrate when compared with HMB. Microbial efficiency was not different among treatments. The proportion of bacterial N produced from NH3-N decreased linearly with increasing HMB, and bacteria receiving dl-Met synthesized more N from NH3-N than those receiving HMB. These data suggest that supplementation of HMB may have a sparing effect on branched chain volatile fatty acids because the fatty acids are not needed to provide carbon for synthesis of valine, isoleucine and leucine with ammonia. Comparisons of bacterial community structure in the fermenter effluent samples using PCR amplicons containing the ribosomal intergenic spacer region and its flanking partial 16S ribosomal RNA gene showed no distinct banding patterns, though treatments tended to group together. Both Met and HMB affect the rumen microbial population, but Met supplied as dl-Met does not act identically to that supplied as HMB.

Ammonia↗
PubMed1994

Genetic and biochemical analysis of the fission yeast ribonucleoprotein particle containing a homolog of Srp54p.

Mammalian signal recognition particle (SRP), a complex of six polypeptides and one 7SL RNA molecule, is required for targeting nascent presecretory proteins to the endoplasmic reticulum (ER). Earlier work identified a Schizosaccharomyces pombe homolog of human SRP RNA and showed that it is a component of a particle similar in size and biochemical properties to mammalian SRP. The recent cloning of the gene encoding a fission yeast protein homologous to Srp54p has made possible further characterization of the subunit structure, subcellular distribution, and assembly of fission yeast SRP. S. pombe SRP RNA and Srp54p co-sediment on a sucrose velocity gradient and coimmunoprecipitate, indicating that they reside in the same complex. In vitro assays demonstrate that fission yeast Srp54p binds under stringent conditions to E. coli SRP RNA, which consists essentially of domain IV, but not to the full-length cognate RNA nor to an RNA in which domain III has been deleted in an effort to mirror the structure of bacterial homologs. Moreover, the association of S. pombe Srp54p with SRP RNA in vivo is disrupted by conditional mutations not only in domain IV, which contains its binding site, but in domains I and III, suggesting that the particle may assemble cooperatively. The growth defects conferred by mutations throughout SRP RNA can be suppressed by overexpression of Srp54p, and the degree to which growth is restored correlates inversely with the severity of the reduction in protein binding. Conditional mutations in SRP RNA also reduce its sedimentation with the ribosome/membrane pellet during cell fractionation. Finally, immunoprecipitation under native conditions of an SRP-enriched fraction from [35S]-labeled fission yeast cells suggests that five additional polypeptides are complexed with Srp54p; each of these proteins is similar in size to a constituent of mammalian SRP, implying that the subunit structure of this ribonucleoprotein is conserved over vast evolutionary distances.

Base Sequence↗