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PubMed1988

Alteration of bacterial DNA structure, gene expression, and plasmid encoded antibiotic resistance following exposure to enoxacin.

Enoxacin inhibits growth of Escherichia coli K12 strains primarily by binding to the GyrA subunit of DNA gyrase (topoisomerase II); strains with gyrA, but not gyrB, mutations are less susceptible to the bactericidal effects of this agent. In sensitive strains, enoxacin completely inhibits DNA synthesis within 5 min and produces drug-gyrase-DNA complexes at numerous sites throughout the E. coli chromosome, as shown by the formation of linear DNA molecules after detergent treatment. Enoxacin, even at subminimal inhibitory concentrations, induces the bacterial SOS system, even in partially resistant gyrA strains. This drug also inhibits the induced expression of the lacZ encoded beta-galactosidase, regardless of whether this gene is located on the chromosome, a low copy number F' plasmid or high copy number Col E1 related plasmids. This inhibition of gene expression at subminimal inhibitory concentrations is likely to be a factor, in addition to gyrase inhibition, in the elimination of Col E1 plasmids and to the reduction in R plasmid conjugal transfer. Enoxacin enhances the bactericidal effects of kanamycin in both in-vitro and in-vivo models, suggesting that this quinolone may be effective in the treatment of infections due to strains resistant to antibacterials as a consequence of plasmid encoded resistance determinants.

Anti-Bacterial Agents↗
PubMed2000

Bacterial community structure associated with a dimethylsulfoniopropionate-producing North Atlantic algal bloom.

The bacteria associated with oceanic algal blooms are acknowledged to play important roles in carbon, nitrogen, and sulfur cycling, yet little information is available on their identities or phylogenetic affiliations. Three culture-independent methods were used to characterize bacteria from a dimethylsulfoniopropionate (DMSP)-producing algal bloom in the North Atlantic. Group-specific 16S rRNA-targeted oligonucleotides, 16S ribosomal DNA (rDNA) clone libraries, and terminal restriction fragment length polymorphism analysis all indicated that the marine Roseobacter lineage was numerically important in the heterotrophic bacterial community, averaging >20% of the 16S rDNA sampled. Two other groups of heterotrophic bacteria, the SAR86 and SAR11 clades, were also shown by the three 16S rRNA-based methods to be abundant in the bloom community. In surface waters, the Roseobacter, SAR86, and SAR11 lineages together accounted for over 50% of the bacterial rDNA and showed little spatial variability in abundance despite variations in the dominant algal species. Depth profiles indicated that Roseobacter phylotype abundance decreased with depth and was positively correlated with chlorophyll a, DMSP, and total organic sulfur (dimethyl sulfide plus DMSP plus dimethyl sulfoxide) concentrations. Based on these data and previous physiological studies of cultured Roseobacter strains, we hypothesize that this lineage plays a role in cycling organic sulfur compounds produced within the bloom. Three other abundant bacterial phylotypes (representing a cyanobacterium and two members of the alpha Proteobacteria) were primarily associated with chlorophyll-rich surface waters of the bloom (0 to 50 m), while two others (representing Cytophagales and delta Proteobacteria) were primarily found in deeper waters (200 to 500 m).

Alphaproteobacteria↗
PubMed2002

The structure of bacterial outer membrane proteins.

Integral membrane proteins come in two types, alpha-helical and beta-barrel proteins. In both types, all hydrogen bonding donors and acceptors of the polypeptide backbone are completely compensated and buried while nonpolar side chains point to the membrane. The alpha-helical type is more abundant and occurs in cytoplasmic (or inner) membranes, whereas the beta-barrels are known from outer membranes of bacteria. The beta-barrel construction is described by the number of strands and the shear number, which is a measure for the inclination angle of the beta-strands against the barrel axis. The common right-handed beta-twist requires shear numbers slightly larger than the number of strands. Membrane protein beta-barrels contain between 8 and 22 beta-strands and have a simple topology that is probably enforced by the folding process. The smallest barrels form inverse micelles and work as enzymes or they bind to other macromolecules. The medium-range barrels form more or less specific pores for nutrient uptake, whereas the largest barrels occur in active Fe(2+) transporters. The beta-barrels are suitable objects for channel engineering, because the structures are simple and because many of these proteins can be produced into inclusion bodies and recovered therefrom in the exact native conformation.

Bacterial Outer Membrane Proteins↗
PubMed1994

Isolation, characterization and structure of bacterial flagellar motors containing the switch complex.

A putative complex of the three switch proteins, FliG, FliM and FliN appears to be directly involved in torque generation and control of direction of rotation. We have developed a preparative procedure for flagellar motors that retains these proteins as evidenced by Western blots using anti-FliG, anti-FliM and anti-FliN antibodies. Immunogold labeling with these three antibodies shows that the three switch proteins are localized to the motor. Electron micrographs of frozen-hydrated preparations reveal a large, new component we have termed the "C ring complex" attached to the cytoplasmic face of the M ring. In a three-dimensional reconstruction of the cylindrically averaged structure, the M-S ring complex appears thicker and wider by the addition of extra material to the cytoplasmic surface of the M ring. In addition, extending into the cytoplasm from the thickened M ring is the C ring complex, a thin-walled cylinder having a length of 170 A and an outer diameter of 450 A compared to the 290 A diameter of the M ring. We provide evidence that the thickened M ring contains FliG and that the C ring complex may contain FliM and FliN. The large diameter of the C ring complex may permit interaction with the M ring and with the circlet of studs thought to be the MotA/MotB complex.

Bacterial Proteins↗
PubMed2004

The first structure of a bacterial class B Acid phosphatase reveals further structural heterogeneity among phosphatases of the haloacid dehalogenase fold.

AphA is a periplasmic acid phosphatase of Escherichia coli belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases. The crystal structure of AphA has been determined at 2.2A and its resolution extended to 1.7A on an AuCl(3) derivative. This represents the first crystal structure of a class B bacterial phosphatase. Despite the lack of sequence homology, the AphA structure reveals a haloacid dehalogenase-like fold. This finding suggests that this fold could be conserved among members of the DDDD superfamily of phosphohydrolases. The active enzyme is a homotetramer built by using an extended N-terminal arm intertwining the four monomers. The active site of the native enzyme, as prepared, hosts a magnesium ion, which can be replaced by other metal ions. The structure explains the non-specific behaviour of AphA towards substrates, while a structure-based alignment with other phosphatases provides clues about the catalytic mechanism.

Acid Phosphatase↗
PubMedDate not supplied

[Antigenic bacterial polysaccharides. Structure of O-specific polysaccharides from Pseudomonas cepacia serogroups C, I, O1, and O4].

Like some Pseudomonas cepacia serogroups studied earlier, serogroups C, I (Nakamura), O1 and O4 (Heidt) are characterized by the presence of at least two structurally different O-antigenic polysaccharide chains in cell-wall lipopolysaccharides. On the basis of acid hydrolysis, methylation, 1H- and 13C-NMR spectroscopy, including computer-assisted 13C-NMR-based analysis, the complete structures of the predominant polysaccharides of serogroups I (I), C and O4 (III) and the minor polysaccharides of serogroups I (II) and O1 (V) were established, and the structure of the predominant polysaccharide of serogroup O1 (IV) established earlier (Cox A. D., Wilkinson S. G.@Carbohydr. Res. 1990. V. 195. No 2. P. 295-301) was confirmed.

Burkholderia cepacia↗
PubMed1996

Pit structure on bacterial cell surface.

The yellow-pigmented bacterium isolated from a ditch was a gram negative rod with a G+C content of 63 mol%, and was classified in the genus Sphingomonas. Electron microscopy revealed that the bacterial cell surface was covered with many large plaits. When grown in a medium containing a polysaccharide as an essential nutrient, a pit of 0.02-0.1 micrometers in diameter was formed on the cell surface, and a thin section showed the rearrangement of the plaits and the presence of a region where the cell membrane sinks into the cytosol. The dependence of the pit formation on the presence of macromolecule may predict the existence of a direct uptake mechanism for macromolecules through a mouth-like pit, possibly in endocytosis fashion. The confirmation of the pit structure is the first such finding in the history of microbiology and may provide a new insight into the cell morphology and biochemistry of macromolecule transport in microbial cell system.

Alginates↗
PubMed1998

Quasi- and nonequivalence in the structure of bacterial flagellar filament.

In supercoiled forms of flagellar filaments, which are thought to be produced by combinations of two distinct subunit lattices, the lattices are elastically deformed in 11 different ways, depending on their azimuthal positions on the circumference of a tube with 11 protofilaments. Those two interactions are nonequivalent as opposed to quasiequivalent ones in elastically deformed lattices of otherwise identical interactions. The term nonequivalence is defined to represent different bonding interactions, and quasiequivalent is used to describe deformed but conserved bonding interactions. By using two distinct lattices that were accurately determined by x-ray fiber diffraction, 10 possible supercoiled forms of flagellar filaments were simulated, based on a bistable-subunit packing model. Comparison to the observed forms showed good agreement, indicating that the model and determined lattice parameters effectively represent realistic features of the structure. The simulated quasiequivalent lattices have been compared to the two nonequivalent lattices, revealing an interesting feature: the maximum deviation in the intersubunit distance by elastic deformation is almost three-quarters of the difference between the two distinct lattices, demonstrating a balanced coexistence of a well-defined conformational distinction and extensive adaptability in the molecular structure of flagellin and its packing interactions.

Bacteria↗
PubMed2000

Slowing of proton transport processes in the structure of bacterial reaction centers and bacteriorhodopsin in the presence of dipyridamole.

Dipyridamole, 2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido(5, 4-d)pyrimidine, is employed in clinical practice as a vasodilator. It can also inhibit a specific membrane protein (glycoprotein P) which pumps anticancer drugs out of tumor cells. Dipyridamole (10-4 M) markedly slows down the kinetics of the electrogenic phase of the photoelectric response in Rhodobacter sphaeroides chromatophores. This phase is due to proton transfer from the external medium to the secondary quinone acceptor in the reaction center. In purple membranes of bacterium Halobacterium salinarium containing bacteriorhodopsin dipyridamole (in its charged state) significantly slowed the kinetics of proton transfer from the primary donor, Asp-96 (in membranes from bacteria of wild type), or from the external medium (in D96N mutant) to the Schiff base. It is suggested that dipyridamole can influence the structural-dynamic state of membrane proteins including modification of the structure of their hydrogen bonds involved in proton-transport processes.

Bacteriorhodopsins↗
PubMed2003

Solid-state 13C and 1H spin diffusion NMR analyses of the microfibril structure for bacterial cellulose.

To obtain further information about the cause for the rather large splitting of the C4 resonance line into the downfield (C4D) and upfield (C4U) lines in CP/MAS 13C NMR spectra for native cellulose, 13C and 1H spin diffusion measurements have been conducted by using different types of bacterial cellulose samples. In 13C spin diffusion measurements, the C4D resonance line is selectively inverted by the Dante pi pulse sequence and the 13C spin diffusion is allowed to proceed from the C4D carbons to other carbons including the C4U carbons with use of the 13C4-enriched bacterial cellulose sample. The analysis based on the simple spin diffusion theory for the process experimentally observed reveals that the C4U carbons may be located at distances less than about 1 nm from the C4D carbons. In 1H spin diffusion measurements, poly(vinyl alcohol) (PVA) films in which ribbon assemblies of bacterial cellulose are dispersed are employed and the 1H spin diffusion process is examined from the water-swollen PVA continuous phase to the dispersed ribbon assemblies by the 13C detection through the 1H-13C CP technique. As a result, it is found that the C4D and C4U carbons are almost equally subjected to the 1H spin diffusion from the PVA phase, indicating that the C4U carbons are not localized in some limited area, e.g. in the surfacial region, but are distributed in the whole area in the microfibrils. These experimental results suggest that the C4U carbons may exist as structural defects probably due to conformational irregularity associated with disordered hydrogen bonding of the CH(2)OH groups in the microfibrils.

Acetobacter↗
PubMed1991

Structure and bacterial receptor activity of a human salivary proline-rich glycoprotein.

Using an overlay technique, we previously showed that the Gram-negative periodontal pathogen Fusobacterium nucleatum binds to a glycoprotein of Mr 89,000 (Prakobphol, A., Murray, P., and Fischer, S.J. (1987) Anal. Biochem. 164, 5-11) in the parotid saliva of some individuals. We now show that deglycosylation of the purified glycoprotein results in loss of receptor activity. Amino acid analysis of the protein core showed predominantly proline, glycine, and glutamic acid/glutamine, a characteristic of proline-rich glycoproteins (PRG). The amino terminus contained repeating sequences of Ser-Gln-Gly-Pro-Pro-Pro-Arg-Pro-Gly-Lys-Pro-Glu-Gly-Pro-Pro-Pro- Gln-Gly that had significant compositional and sequence homology to that encoded by exon 3 of the PRB3 gene. We analyzed the PRG oligosaccharides by a combination of mass spectrometry techniques and nuclear magnetic resonance spectroscopy. Twenty-seven highly fucosylated structures were identified. The most abundant was as follows (where Fuc is fucose). (formula; see text) To understand the structural basis of F. nucleatum binding, we screened glycolipids and neoglycolipids carrying carbohydrate structures related to those of the PRG for receptor activity; components with unsubstituted terminal lactosamine residues best supported adherence. Neoglycolipids constructed from PRG oligosaccharides were also receptors. Treatment with beta-galactosidase, but not alpha-fucosidase, abolished binding, suggesting that unsubstituted lactosamine units, including the 6-antenna of the major oligosaccharide, mediate F. nucleatum adherence.

Amino Acid Sequence↗
PubMed2002

Influence of drying-rewetting frequency on soil bacterial community structure.

Soil drying and rewetting represents a common physiological stress for the microbial communities residing in surface soils. A drying-rewetting cycle may induce lysis in a significant proportion of the microbial biomass and, for a number of reasons, may directly or indirectly influence microbial community composition. Few studies have explicitly examined the role of drying-rewetting frequency in shaping soil microbial community structure. In this experiment, we manipulated soil water stress in the laboratory by exposing two different soil types to 0, 1, 2, 4, 6, 9, or 15 drying-rewetting cycles over a 2-month period. The two soils used for the experiment were both collected from the Sedgwick Ranch Natural Reserve in Santa Ynez, CA, one from an annual grassland, the other from underneath an oak canopy. The average soil moisture content over the course of the incubation was the same for all samples, compensating for the number of drying-rewetting cycles. At the end of the 2-month incubation we extracted DNA from soil samples and characterized the soil bacterial communities using the terminal restriction fragment length polymorphism (T-RFLP) method. We found that drying-rewetting regimes can influence bacterial community composition in oak but not in grass soils. The two soils have inherently different bacterial communities; only the bacteria residing in the oak soil, which are less frequently exposed to moisture stress in their natural environment, were significantly affected by drying-rewetting cycles. The community indices of taxonomic diversity and richness were relatively insensitive to drying-rewetting frequency. We hypothesize that drying-rewetting induced shifts in bacterial community composition may partly explain the changes in C mineralization rates that are commonly observed following exposure to numerous drying-rewetting cycles. Microbial community composition may influence soil processes, particularly in soils exposed to a significant level of environmental stress.

Classification↗
PubMed2006

In vivo DNase I sensitivity of the Streptomyces coelicolor chromosome correlates with gene expression: implications for bacterial chromosome structure.

For a bacterium, Streptomyces coelicolor A3(2) contains a relatively large genome (8.7 Mb) with a complex and adaptive pattern of gene regulation. We discovered a correlation between the physical structure of the S.coelicolor genome and the transcriptional activity of the genes therein. Twelve genes were surveyed throughout 72 h of growth for both in vivo sensitivity to DNase I digestion and levels of transcription. DNase I-sensitivity correlated positively with transcript levels, implying that it was predictive of gene expression, and indicating increased accessibility of transcribed DNA. The genome was fractionated based on the sensitivity to DNase I digestion, with the low molecular weight (frequently cut) fraction highly enriched for actively transcribed sequences when compared to the infrequently cut fraction, which was representative of the entire genome. This approach will allow comparison of nucleoid proteins, and any modifications thereof, associated with transcriptionally active and inactive regions of the bacterial genome.

Chromosomes, Fungal↗
PubMed2003

Bacterial community structure and physiological state in a biofilm reactor degrading 4-chloroaniline.

Degradation of 4-chloroaniline in the presence of aniline by a microbial community in a laboratory-scale biofilm reactor was evaluated. The starter inoculum was isolated and reconstructed from a percolating column enrichment of Indonesian agricultural soil. The capacity to mineralise and detoxify 4-chloroaniline in the presence of aniline was demonstrated by the biofilm reactor when operated at high hydraulic retention time (HRT; 0.87 h). At low HRT (0.23 h and 0.39 h) 4-chlorocatechol accumulated in the effluent, accompanied by a decrease in dechlorination and detoxification. When returned to high HRT (2.14 h), the accumulation of 4-chlorocatechol stopped and the extent of dechlorination and detoxification increased. Bacteria other than the original inoculum appeared in the reactor when the operating mode was switched from closed cycle to open cycle. One of these bacteria, identified as Pseudomonas putida R1 by partial 16S rDNA sequencing, subsequently dominated the reactor at every HRT imposed. PCR-based single-strand conformational polymorphism of 16 s rDNA and traditional cultivation procedures indicated that the bacterial composition in the reactor shifted in response to applied HRT. The relationship between the bacterial abundance and the degradation capacity of the reactor is discussed.

Aniline Compounds↗
PubMed1999

Gram-positive sepsis. Mechanisms and differences from gram-negative sepsis.

This article has reviewed the mechanisms by which gram-positive bacteria lead to septic shock, with regard to bacterial structure and toxicology and the host responses elicited both in animal models and in the clinical setting. Gram-positive organisms are better suited to invade host tissues and elicit, in general, a brisker phagocytic response than gram-negative organisms. The lack of endotoxin in the outer cell wall is compensated for by the presence of exposed peptidoglycan and a range of other toxic secreted products. It appears that cell wall components of gram-positive bacteria may signal via the same receptor as gram-negative endotoxin, although the type of signal and coreceptor may differ. Both animal and clinical data suggest that, unlike endotoxin-mediated shock, gram-positive infection produces a modest TNF response only and does not respond well to anti-TNF therapies. This leads one to conclude that the mechanisms leading to shock in gram-positive infection may be multifactorial and perhaps more difficult to treat. A thorough review of gram-positive mechanisms of sepsis is hampered by a lack of basic research in this field. Understanding of gram-negative bacterial structure and the regulation of virulence genes is at an advanced stage, yet the molecular tools to analyse virulence factors in the gram-positive genome have only recently become available. There is a paucity of good animal models of gram-positive infection and a lack of microbiologic data from some of the major trials in sepsis that might have given greater insight into the mechanisms leading to shock in various infections.

Animals↗
PubMed2003

Assessment of bacterial community structure in the deep sub-seafloor biosphere by 16S rDNA-based techniques: a cautionary tale.

Investigations into the deep marine environment have demonstrated the presence of a significant microbial biomass buried deep within sediments on a global scale. It is now believed that this deep biosphere plays a major role in the global cycling of elements and contains a large reservoir of organic carbon. This paper reports the development of a DNA extraction protocol that addresses the particular problems faced in applying molecular ecological techniques to samples containing very low biomass. Sediment samples were collected from different geographical locations within the Pacific Ocean and include the Ocean Drilling Program (ODP) Leg 190, Nankai Trough Accretionary Prism. Seven DNA extraction protocols were tested and a commercially available DNA extraction kit with modifications was shown to produce higher yields of polymerase chain reaction (PCR)-amplifiable DNA than standard laboratory methods. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene diversity revealed that template DNA from these extremely low biomass sediment samples was susceptible to PCR bias and random amplification. We propose that it is essential to screen 16S rRNA gene products for bacterial diversity by DGGE or other rapid fingerprinting methods, prior to their use in establishing a representative clone library of deep sub-seafloor bacteria. This represents a cautionary approach to analysis of microbial diversity in such sub-seafloor ecosystems.

Bacteria↗
PubMed2003

Denaturing gradient gel electrophoresis analysis to study bacterial community structure in pockets of periodontitis patients.

Bacteria are involved in the onset and progression of periodontitis. A promising molecular technique, denaturing gradient gel electrophoresis (DGGE), to study microbial population dynamics in the subgingival pocket is presented. Twenty-three samples were taken from the subgingival pockets of nine patients and six healthy family members. From four periodontitis patients, 12 samples were evaluated before, 1 day after and 3 months after treatment. Part of the 16S rRNA gene of all bacteria was amplified by PCR and separated by DGGE, creating banding patterns representative of the community structure. Shifts in composition and diversity of the microbial population could be determined semiquantitatively, and this showed that treatment resulted in a decrease in the diversity of the population. After 3 months a microbial population 33-47% different from the population before treatment had re-established. Intense bands representing Exiguobacterium aurantiacum were present in 13 out of 25 samples, indicating that this species may play a role in periodontal disease.

Actinomyces↗
PubMed2007

Bacterial community structure associated with the Antarctic soft coral, Alcyonium antarcticum.

The structure and composition of microbial communities inhabiting the soft coral Alcyonium antarcticum were investigated across three differentially contaminated sites within McMurdo Sound, Antarctica. Diverse microbial communities were revealed at all sites using culture-based analysis, denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene clone-library analysis, and FISH. Phylogenetic analysis of isolates and retrieved sequences demonstrated close affiliation with known psychrophiles from the Antarctic environment and high similarity to Gammaproteobacteria clades of sponge-associated microorganisms. The majority of bacteria detected with all techniques reside within the Gammaproteobacteria, although other phylogenetic groups including Alpha- and Betaproteobacteria, Bacteroidetes, Firmicutes, Actinomycetales, Planctomycetes, and Chlorobi and bacteria from the functional group of sulfate-reducing bacteria were also present. Multivariate (nMDS) analysis of DGGE banding patterns and principal component analysis of quantitative FISH data revealed no distinct differences in community composition between differentially contaminated sites. Rather, conserved coral-associated bacterial groups were observed within and between sites, providing evidence to support specific coral-microbial interactions. This is the first investigation of microbial communities associated with Antarctic soft corals, and the results suggest that spatially stable microbial associations exist across an environmental impact gradient.

Animals↗