Showing papers on "Bacteria published in 1994"

Methods for general and molecular bacteriology

Abstract: Methodology for General and Molecular Microbiology Morphology Light microscopy Determinative and cytological light microscopy Electron microscopy Cell fractionation Antigen-antibody reactions Growth: Physicochemical factors in growth Nutrition and media Enrichment and isolation Solid, liquid/solid and semisolid culture Liquid culture Growth measurement Culture preservation Molecular Genetics: Gene mutation Gene transfer in Gram-negative bacteria Gene transfer in Gram-positive bacteria Plasmids Transposon mutagenesis Gene cloning and expression Polymerase chain reaction Nucleic acid analysis Metabolism: Physical analysis Chemical analysis Enzymatic activity Permeability and transport Systematics: Phenotypic characterization DNA sequence similarities Ribosomal RNA hybridization and gene sequencing Nucleic acid probes General Methods: Laboratory safety Photography Records and reports

Inactivation of antibiotics and the dissemination of resistance genes.

Abstract: The emergence of multidrug-resistant bacteria is a phenomenon of concern to the clinician and the pharmaceutical industry, as it is the major cause of failure in the treatment of infectious diseases. The most common mechanism of resistance in pathogenic bacteria to antibiotics of the aminoglycoside, beta-lactam (penicillins and cephalosporins), and chloramphenicol types involves the enzymic inactivation of the antibiotic by hydrolysis or by formation of inactive derivatives. Such resistance determinants most probably were acquired by pathogenic bacteria from a pool of resistance genes in other microbial genera, including antibiotic-producing organisms. The resistance gene sequences were subsequently integrated by site-specific recombination into several classes of naturally occurring gene expression cassettes (typically "integrons") and disseminated within the microbial population by a variety of gene transfer mechanisms. Although bacterial conjugation once was believed to be restricted in host range, it now appears that this mechanism of transfer permits genetic exchange between many different bacterial genera in nature.

Microbial ecology of dental plaque and its significance in health and disease.

Abstract: Dental plaque forms naturally on teeth and is of benefit to the host by helping to prevent colonization by exogenous species. The bacterial composition of plaque remains relatively stable despite regular exposure to minor environmental perturbations. This stability (microbial homeostasis) is due in part to a dynamic balance of both synergistic and antagonistic microbial interactions. However, homeostasis can break down, leading to shifts in the balance of the microflora, thereby predisposing sites to disease. For example, the frequent exposure of plaque to low pH leads to inhibition of acid-sensitive species and the selection of organisms with an aciduric physiology, such as mutans streptococci and lactobacilli. Similarly, plaque accumulation around the gingival margin leads to an inflammatory host response and an increased flow of gingival crevicular fluid. The subgingival microflora shifts from being mainly Gram-positive to being comprised of increased levels of obligately anaerobic, asaccharolytic Gram-negative organisms. It is proposed that disease can be prevented or treated not only by targeting the putative pathogens but also by interfering with the processes that drive the breakdown in homeostasis. Thus, the rate of acid production following sugar intake could be reduced by fluoride, alternative sweeteners, and low concentrations of antimicrobial agents, while oxygenating or redox agents could raise the Eh of periodontal pockets and prevent the growth and metabolism of obligately anaerobic species. These views have been incorporated into a modified hypothesis (the "ecological plaque hypothesis") to explain the relationship between the plaque microflora and the host in health and disease, and to identify new strategies for disease prevention.

PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid.

Abstract: A set of broad-range PCR primers for the 16S rRNA gene in bacteria were tested, along with three series of oligonucleotide probes to detect the PCR product. The first series of probes is broad in range and consists of a universal bacterial probe, a gram-positive probe, a Bacteroides-Flavobacterium probe, and two probes for other gram-negative species. The second series was designed to detect PCR products from seven major bacterial species or groups frequently causing meningitis: Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, Escherichia coli and other enteric bacteria, Listeria monocytogenes, and Staphylococcus aureus. The third series was designed for the detection of DNA from species or genera commonly considered potential contaminants of clinical samples, including cerebrospinal fluid (CSF): Bacillus, Corynebacterium, Propionibacterium, and coagulase-negative Staphylococcus spp. The primers amplified DNA from all 124 different species of bacteria tested. Southern hybridization testing of the broad-range probes with washes containing 3 M tetramethylammonium chloride indicated that this set of probes correctly identified all but two of the 102 bacterial species tested, the exceptions being Deinococcus radiopugnans and Gardnerella vaginalis. The gram-negative and gram-positive probes hybridized to isolates of two newly characterized bacteria, Alloiococcus otitis and Rochalimaea henselii, as predicted by Gram stain characteristics. The CSF pathogen and contaminant probe sequences were compared with available sequence information and with sequencing data for 32 different species. Testing of the CSF pathogen and contaminant probes against DNA from over 60 different strains indicated that, with the exception of the coagulase-negative Staphylococcus probes, these probes provided the correct identification of bacterial species known to be found in CSF.

Metabolic interactions between anaerobic bacteria in methanogenic environments.

Abstract: In methanogenic environments organic matter is degraded by associations of fermenting, acetogenic and methanogenic bacteria. Hydrogen and formate consumption, and to some extent also acetate consumption, by methanogens affects the metabolism of the other bacteria. Product formation of fermenting bacteria is shifted to more oxidized products, while acetogenic bacteria are only able to metabolize compounds when methanogens consume hydrogen and formate efficiently. These types of metabolic interaction between anaerobic bacteria is due to the fact that the oxidation of NADH and FADH2 coupled to proton or bicarbonate reduction in thermodynamically only feasible at low hydrogen and formate concentrations. Syntrophic relationships which depend on interspecies hydrogen or formate transfer were described for the degradation of e.g. fatty acids, amino acids and aromatic compounds.

Development of an rRNA-targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge.

Abstract: Enhanced biological phosphate removal in an anaerobic-aerobic activated sludge system has generally been ascribed to members of the genus Acinetobacter. A genus-specific 16S rRNA-targeted oligonucleotide probe was developed to investigate the role of Acinetobacter spp. in situ. Nonisotopic dot blot hybridization to 66 reference strains, including the seven described Acinetobacter spp., demonstrated the expected probe specificity. Fluorescent derivatives were used for in situ monitoring of Acinetobacter spp. in the anaerobic and aerobic compartments of a sewage treatment plant with enhanced biological phosphate removal. Microbial community structures were further analyzed with oligonucleotide probes specific for the alpha, beta, or gamma subclasses of the class Proteobacteria, for the Cytophaga-Flavobacterium cluster, for gram-positive bacteria with a high G + C DNA content, and for all bacteria. Total cell counts were determined by 49,6-diamidino-2-phenylindole staining. In both the anaerobic and the aerobic basins, the activated sludge samples were dominated by members of the class Proteobacteria belonging to the beta subclass and by gram-positive bacteria with a high G + C DNA content. Acinetobacter spp. constituted less than 10% of all bacteria. For both basins, the microbial community structures determined with molecular techniques were compared with the compositions of the heterotrophic saprophytic microbiota determined with agar plating techniques. Isolates on nutrient-rich medium were classified by whole-cell hybridization with rRNA-targeted probes and fatty acid analysis.(ABSTRACT TRUNCATED AT 250 WORDS) Images

Anaerobic oxidation of hydrocarbons in crude oil by new types of sulphate-reducing bacteria

Abstract: Many crude oil constituents are biodegradable in the presence of oxygen; however, a substantial anaerobic degradation has never been demonstrated. An unusually low content of n-alkanes in oils of certain deposits is commonly attributed to selective utilization of these hydrocarbons by aerobic microorganisms. On the other hand, oil wells and production fluids were shown to harbour anaerobic sulphate-reducing bacteria, but their actual electron donors and carbon sources were unknown. On the basis of nutritional properties of various bacterial isolates it was assumed that fatty acids and H2 are potential electron donors for sulphate reduction in situ. Here we demonstrate that hydrocarbons in crude oil are used directly by sulphate-reducing bacteria growing under strictly anoxic conditions. A moderately thermophilic pure culture selectively utilizes n-alkanes in oil for sulphate reduction to sulphide. In addition, a mesophilic sulphate-reducing enrichment culture is shown to oxidize alkylbenzenes in oil. Thus, sulphate-reducing bacteria utilizing aliphatic and aromatic hydrocarbons as electron donors may present a significant source of sulphide in oil deposits and oil production plants.

The type I macrophage scavenger receptor binds to gram-positive bacteria and recognizes lipoteichoic acid.

Abstract: Macrophage scavenger receptors exhibit unusually broad binding specificity for polyanionic ligands and have been implicated in atherosclerosis and various host defense functions. Using a radiolabeled, secreted form of the type I bovine macrophage scavenger receptor in an in vitro binding assay, we have found that this receptor binds to intact Gram-positive bacteria, including Streptococcus pyogenes, Streptococcus agalactiae, Staphylococcus aureus, Enterococcus hirae, and Listeria monocytogenes. Competition binding studies using purified lipoteichoic acid, an anionic polymer expressed on the surface of most Gram-positive bacteria, show that lipoteichoic acids are scavenger receptor ligands and probably mediate binding of the receptor to Gram-positive bacteria. Lipoteichoic acids, for which no host cell receptors have previously been identified, are implicated in the pathogenesis of septic shock due to Gram-positive bacteria. Scavenger receptors may participate in host defense by clearing lipoteichoic acid and/or intact bacteria from tissues and the circulation during Gram-positive sepsis. Since scavenger receptors have been previously shown to bind to and facilitate bloodstream clearance of Gram-negative bacterial endotoxin (lipopolysaccharide), these receptors may provide a general mechanism for macrophage recognition and internalization of pathogens and their cell surface components.

Biological activities of two fungistatic antibiotics produced by Bacillus cereus UW85.

Abstract: Cultures and culture filtrates of Bacillus cereus UW85 suppress damping-off of alfalfa caused by Phytophthora medicaginis. We studied the role in disease suppression of two antibiotics from culture filtrates of UW85 that reversibly inhibited growth of P. medicaginis. We purified the two antibiotics by cation-exchange chromatography and high-voltage paper electrophoresis and showed that one of them, designated zwittermicin A, was an aminopolyol of 396 Da that was cationic at pH 7.0; the second, designated antibiotic B, appeared to be an aminoglycoside containing a disaccharide. Both antibiotics prevented disease of alfalfa seedlings caused by P. medicaginis. Purified zwittermicin A reversibly reduced elongation of germ tubes derived from cysts of P. medicaginis, and antibiotic B caused swelling of the germ tubes. Mutants generated with Tn917 or mitomycin C treatment were screened either for antibiotic accumulation in an agar plate diffusion assay or for the ability to suppress damping-off disease of alfalfa. Of 2,682 mutants screened for antibiotic accumulation, 5 mutants were substantially reduced in antibiotic accumulation and disease-suppressive activity. Of the 1,700 mutants screened for disease-suppressive activity, 3 mutants had reduced activity and they accumulated less of both antibiotics than did the parent strain. The amount of antibiotic accumulated by the mutants was significantly correlated with the level of disease suppression. Addition of either zwittermicin A or antibiotic B to alfalfa plants inoculated with a culture of a nonsuppressive mutant resulted in disease suppression. These results demonstrate that B. cereus UW85 produces two fungistatic antibiotics that contribute to suppression of damping-off disease of alfalfa.

Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion.

Abstract: Legionella pneumophila is considered to be a facultative intracellular parasite. Therefore, the ability of these bacteria to enter, i.e., invade, eukaryotic cells is expected to be a key pathogenic determinant. We compared the invasive ability of bacteria grown under standard laboratory conditions with that of bacteria grown in Acanthamoeba castellanii, one of the protozoan species that serves as a natural host for L. pneumophila in the environment. Amoeba-grown L. pneumophila cells were found to be at least 100-fold more invasive for epithelial cells and 10-fold more invasive for macrophages and A. castellanii than were L. pneumophila cells grown on agar. Comparison of agar- and amoeba-grown L. pneumophila cells by light and electron microscopy demonstrated dramatic differences in the morphology and structure of the bacteria. Analyses of protein expression in the two strains of bacteria suggest that these phenotypic differences may be due to the expression of new proteins in amoeba-grown L. pneumophila cells. In addition, the amoeba-grown bacteria were found to enter macrophages via coiling phagocytosis at a higher frequency than agar-grown bacteria did. Replication of L. pneumophila in protozoans present in domestic water supplies may be necessary to produce bacteria that are competent to enter mammalian cells and produce human disease.

Antimicrobial Potential of Lactic Acid Bacteria

Abstract: The preservative effect of lactic acid bacteria during the manufacture and subsequent storage of fermented foods is mainly due to the acidic conditions that they create in the food during their development. This souring effect is primarily due to the fermentative conversion of carbohydrates to organic acids (lactic and acetic acid) with a concomitant lowering of the pH of the food, an important characteristic that leads to an increased shelf-life and safety of the final product. In recent decades, it has become clear that the overall inhibitory action of lactic acid bacteria is due to more complex antagonistic systems produced by the starter cultures. Lactic acid bacteria are capable of producing and excreting inhibitory substances other than lactic and acetic acid. These substances are antagonistic to a wide spectrum of microorganisms, and thus can make significant contributions to their preservative action. They are produced in much smaller amounts than lactic acid and acetic acid, and include formic acid, free fatty acids, ammonia, ethanol, hydrogen peroxide, diacetyl, acetoin, 2,3-butanediol, acetaldehyde, benzoate, bacteriolytic enzymes, bacteriocins and antibiotics, as well as several less well-defined or completely unidentified inhibitory substances (Klaenhammer, 1988; Daeschel, 1989; Lind-gren & Dobrogosz, 1990; Schillinger, 1990; Piard & Desmazeaud, 1991, 1992; Vandenbergh, 1993). Some of these substances display antagonistic activity towards many food spoilage and foodborne pathogenic microorganisms, including psychrotrophic lactobacilli and leuconostocs, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Listeria monocytogenes, Staphyloc-occus aureus, etc. The competitive removal of essential substrates, the accumulation of D-amino acids, a lowering of oxidation-reduction potential and coaggregation may further restrict undesirable microorganisms. Unfortunately, in some instances the antibiosis will be detrimental by inhibition of other desirable lactic strains composing the mixed starter culture.

Resistance to arsenic compounds in microorganisms

Abstract: Arsenic ions, frequently present as environmental pollutants, are very toxic for most microorganisms. Some microbial strains possess genetic determinants that confer resistance. In bacteria, these determinants are often found on plasmids, which has facilitated their study at the molecular level. Bacterial plasmids conferring arsenic resistance encode specific efflux pumps able to extrude arsenic from the cell cytoplasm thus lowering the intracellular concentration of the toxic ions. In Gram-negative bacteria, the efflux pump consists of a two-component ATPase complex. ArsA is the ATPase subunit and is associated with an integral membrane subunit, ArsB. Arsenate is enzymatically reduced to arsenite (the substrate of ArsB and the activator of ArsA) by the small cytoplasmic ArsC polypeptide. In Gram-positive bacteria, comparable arsB and arsC genes (and proteins) are found, but arsA is missing. In addition to the wide spread plasmid arsenic resistance determinant, a few bacteria confer resistance to arsenite with a separate determinant for enzymatic oxidation of more-toxic arsenite to less-toxic arsenate. In contrast to the detailed information on the mechanisms of arsenic resistance in bacteria, little work has been reported on this subject in algae and fungi.

Some rumen ciliates have endosymbiotic methanogens

Abstract: Most of the small ciliate protozoa, including Dasytricha ruminantium and Entodinium spp. living in the rumen of sheep, were found to have intracellular bacteria. These bacteria were not present in digestive vacuoles. They showed characteristic coenzyme F420 autofluorescence and they were detected with a rhodamine-labelled Archaea-specific oligonucleotide probe. The measured volume percent of autofluorescing bacteria (1%) was close to the total volume of intracellular bacteria estimated from TEM stereology. Thus it is likely that all of the bacteria living in the cytoplasm of these ciliates were endosymbiotic methanogens, using H2 evolved by the host ciliate to form methane. Intracellular methanogens appear to be much more numerous than those attached to the external cell surface of ciliates.

Respiratory chains and bioenergetics of acetic acid bacteria.

Abstract: Publisher Summary The chapter discusses the respiratory chains and bioenergetics of acetic acid bacteria. Acetic acid bacteria are obligate aerobes and well known as “vinegar producers.” They produce acetic acid from ethanol by two sequential catalytic reactions of membrane-bound alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Besides alcohols and aldehydes, acetic acid bacteria are able to oxidize various sugars and sugar alcohols such as D-glucose, glycerol, and D-sorbitol. Such oxidation reactions are called “oxidative fermentations”, because they involve incomplete oxidation of alcohols or sugars accompanied by accumulation of the corresponding oxidation products in huge amounts in the growth medium. Bacteria capable of effecting oxidative fermentations are called “oxidative bacteria,” of which the most prominent are acetic acid bacteria. Acetic acid bacteria are also important for the fermentation industries to produce biomaterials such as vinegar and L-sorbose. Acetic acid bacteria are classified into two genera, Gluconobacter and Acetobacter of the family Acetobacteraceae. Gluconobacter species catalyze highly active oxidation reactions on ethanol or D-glucose— including also oxidative reactions on sugars such as D-gluconic acid, D-sorbitol, and glycerol. By contrast, Acetobacter species have a highly active ethanoloxidizing system but not enzymes for sugar oxidation. The respiratory chain in Acetobacter spp. has ubiquinone, cytochrome b , cytochrome c , and a terminal ubiquinol oxidase, which is either cytochrome a 1 or cytochrome o .

Mechanism of electrical enhancement of efficacy of antibiotics in killing biofilm bacteria.

Abstract: The bioelectric effect, in which electric fields are used to enhance the efficacy of biocides and antibiotics in killing biofilm bacteria, has been shown to reduce the very high concentrations of these antibacterial agents needed to kill biofilm bacteria to levels very close to those needed to kill planktonic (floating) bacteria of the same species. In this report, we show that biofilm bacteria are readily killed by an antibiotic on all areas of the active electrodes and on the surfaces of conductive elements that lie within the electric field but do not themselves function as electrodes. Considerations of electrode geometry indicate that very low (< 100 microA/cm2) current densities may be effective in this electrical enhancement of antibiotic efficacy against biofilm bacteria, and flow experiments indicate that this bioelectric effect does not appear to depend entirely on the possible local electrochemical generation of antibacterial molecules or ions. These data are expected to facilitate the use of the bioelectric effect in the prevention and treatment of device-related bacterial infections that are caused by bacteria that grow in biofilms and thereby frustrate antibiotic chemotherapy.

Evolution of cytochrome oxidase, an enzyme older than atmospheric oxygen.

Abstract: Cytochrome oxidase is a key enzyme in aerobic metabolism. All the recorded eubacterial (domain Bacteria) and archaebacterial (Archaea) sequences of subunits 1 and 2 of this protein complex have been used for a comprehensive evolutionary analysis. The phylogenetic trees reveal several processes of gene duplication. Some of these are ancient, having occurred in the common ancestor of Bacteria and Archaea, whereas others have occurred in specific lines of Bacteria. We show that eubacterial quinol oxidase was derived from cytochrome c oxidase in Gram-positive bacteria and that archaebacterial quinol oxidase has an independent origin. A considerable amount of evidence suggests that Proteobacteria (Purple bacteria) acquired quinol oxidase through a lateral gene transfer from Gram-positive bacteria. The prevalent hypothesis that aerobic metabolism arose several times in evolution after oxygenic photosynthesis, is not sustained by two aspects of the molecular data. First, cytochrome oxidase was present in the common ancestor of Archaea and Bacteria whereas oxygenic photosynthesis appeared in Bacteria. Second, an extant cytochrome oxidase in nitrogen-fixing bacteria shows that aerobic metabolism is possible in an environment with a very low level of oxygen, such as the root nodules of leguminous plants. Therefore, we propose that aerobic metabolism in organisms with cytochrome oxidase has a monophyletic and ancient origin, prior to the appearance of eubacterial oxygenic photosynthetic organisms.

Use of the Escherichia coli beta-glucuronidase (gusA) gene as a reporter gene for analyzing promoters in lactic acid bacteria.

Abstract: A transcriptional fusion vector, designated pNZ272, based on the promoterless beta-glucuronidase gene (gusA) of Escherichia coli as a reporter gene, has been constructed for lactic acid bacteria. The replicon of pNZ272 was derived from the Lactococcus lactis plasmid pSH71, allowing replication in a wide range of gram-positive bacteria and E. coli. The applicability of pNZ272 and the expression of the gusA gene in L. lactis was demonstrated in shotgun cloning experiments with lactococcal chromosomal and bacteriophage DNA. In addition, three defined lactococcal promoters were inserted in pNZ272: the plasmid-derived lacA promoter, the chromosomal usp45 promoter, and a promoter from bacteriophage phi SK11G. The three resulting plasmids showed beta-glucuronidase activity in a gusA-deficient E. coli strain and in four species of lactic acid bacteria belonging to the genera Lactobacillus, Lactococcus, and Leuconostoc. The copy numbers of the gusA-expressing plasmids were similar within a single species of lactic acid bacteria. However, the specific beta-glucuronidase activity and the gusA mRNA levels varied considerably both within a single species and among different species of lactic acid bacteria. The transcriptional start site of all three promoters was determined and found to be identical in the different species. The results of this comparative promoter analysis indicate that the requirements for efficient transcription initiation differ among the lactic acid bacteria studied.

Perception of Rhizobium nodulation factors by tomato cells and inactivation by root chitinases.

Abstract: The bacterial genera Rhizobium and Bradyrhizobium, nitrogen-fixing symbionts of legumes, secrete specific lipo-chitooligosaccharides that induce the formation of nodules on their host plants. When preparations of such nodulation-inducing factors (Nod factors) were added to suspension-cultured tomato cells, a rapid and transient alkalinization of the culture medium occurred. Lipo-oligosaccharide preparations from Rhizobium or Bradyrhizobium treated with flavonoids, known inducers of Nod factor synthesis, were up to 100 times more potent in inducing alkalinization than the ones from untreated bacteria. The activity was absent from preparations of the mutant strain Rhizobium sp. NGR234 delta nodABC, unable to produce any Nod factors. Preparations of Nod factors from various bacteria as well as individual, highly purified Nod factors from Rhizobium sp. NGR(pA28) induced alkalinization in the tomato cell cultures at nanomolar concentrations. This demonstrates that Nod factors can be perceived by tomato, a nonhost of rhizobia. Using the alkalinization response as a sensitive bioassay, Nod factors were found to be inactivated by plant chitinases. Root chitinases purified from different legumes differed in their potential to inactivate differently substituted Nod factors produced by Rhizobium sp. NGR(pA28). This indicates that the specificity of the bacterium-host plant interaction may be due, at least in part, to differential inactivation of Nod factors by root chitinases.

The proteolytic system of lactic acid bacteria

Abstract: Although there are pronounced differences in proteolytic capacity between the different species of lactic acid bacteria, numerous strains are known to contain proteolytic systems that allow them to grow on protein-rich substrates such as meat, vegetables and milk. There are two characteristics that differentiate these lactic acid bacteria from many other proteolytic microorganisms. First, lactic acid bacteria are fastidious organisms with multiple amino acid auxotrophies and as a consequence their growth is critically dependent on efficient systems for the degradation of proteins and the transport of amino acids and small peptides. Second, several lactic acid bacteria contain a proteolytic system that is highly specific and results in the production of unique peptides. In retrospect, it is likely that these characteristic proteolytic properties, together with the capacity to produce lactic acid from sugars, are the primary factors that have allowed the selection of certain strains of lactic acid bacteria as starter cultures for industrial fermentations. This certainly applies to the lactic acid bacteria that initiate fermentations in milk, a medium with a high content of lactose and αsl-and β-casein, the major milk proteins.

Metabolism of sulfate-reducing prokaryotes

Abstract: Dissimilatory sulfate reduction is carried out by a heterogeneous group of bacteria and archaea that occur in environments with temperatures up to 105 degrees C. As a group together they have the capacity to metabolize a wide variety of compounds ranging from hydrogen via typical organic fermentation products to hexadecane, toluene, and several types of substituted aromatics. Without exception all sulfate reducers activate sulfate to APS; the natural electron donor(s) for the ensuing APS reductase reaction is not known. The same is true for the reduction of the product bisulfite; in addition there is still some uncertainty as to whether the pathway to sulfide is a direct six-electron reduction of bisulfite or whether it involves trithionate and thiosulfate as intermediates. The study of the degradation pathways of organic substrates by sulfate-reducing prokaryotes has led to the discovery of novel non-cyclic pathways for the oxidation of the acetyl moiety of acetyl-CoA to CO2. The most detailed knowledge is available on the metabolism of Desulfovibrio strains, both on the pathways and enzymes involved in substrate degradation and on electron transfer components and terminal reductases. Problems encountered in elucidating the flow of reducing equivalents and energy transduction are the cytoplasmic localization of the terminal reductases and uncertainties about the electron donors for the reactions catalyzed by these enzymes. New developments in the study of the metabolism of sulfate-reducing bacteria and archaea are reviewed.

Fate of Listeria monocytogenes in murine macrophages: evidence for simultaneous killing and survival of intracellular bacteria.

Abstract: The intracellular survival of the ubiquitous pathogen Listeria monocytogenes was studied in primary cultures of bone marrow-derived mouse macrophages. Bacteria were able to grow rapidly in these cells, with an apparent multiplication rate of about 40 min. Electron microscopy demonstrated that intracellular bacterial replication was the consequence of simultaneous intracellular killing and replication of bacteria in the same cells. Within the first hour following phagocytosis, most bacteria were destroyed in the phagosomal compartment to which they were confined. This was due to early transfer of hydrolytic enzymes to phagosomes, undoubtedly via phagosome-lysosome (P-L) fusion, as demonstrated by a quantitative analysis after staining for a lysosomal marker, acid phosphatase. One hour after infection, about 14% of the bacteria were free in the cytoplasm, in which they multiplied and induced actin polymerization and spreading to adjacent macrophages, as in epithelial cells. By using the 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine staining procedure, direct evidence is presented that all phagosomes were acidified immediately after phagocytosis, thus indicating that intraphagosomal bacteria were exposed to an acidic environment that might favor vacuolar lysis by listeriolysin O. Intracellular growth in macrophages, therefore, appears to be the result of a competition between the expression of the hydrolytic activity of these cells following P-L fusion and the capacity of L. monocytogenes to escape from the acidified phagosomal compartment before P-L fusion has occurred. The finding that concomitant intracellular killing and survival of L. monocytogenes occurs in the same macrophages might explain the high immunogenicity observed in vivo with live bacteria, as opposed to killed bacteria.

Characterization of a new thermophilic sulfate-reducing bacterium Thermodesulfovibrio yellowstonii, gen. nov. and sp. nov.: its phylogenetic relationship to Thermodesulfobacterium commune and their origins deep within the bacterial domain.

Abstract: A thermophilic sulfate-reducing vibrio isolated from thermal vent water in Yellowstone Lake, Wyoming, USA is described. The gram-negative, curved rod-shaped cells averaged 0.3 micrometer wide and 1.5 micrometers long. They were motile by means of a single polar flagellum. Growth was observed between 40 degrees and 70 degrees C with optimal growth at 65 degrees C. Cultures remained viable for one year at 27 degrees C although spore-formation was not observed. Sulfate, thiosulfate and sulfite were used as electron acceptors. Sulfur, fumarate and nitrate were not reduced. In the presence of sulfate, growth was observed only with lactate, pyruvate, hydrogen plus acetate, or formate plus acetate. Pyruvate was the only compound observed to support fermentative growth. Pyruvate and lactate were oxidized to acetate. Desulfofuscidin and c-type cytochromes were present. The G + C content was 29.5 mol%. The divergence in the 16 S ribosomal RNA sequences between the new isolate and Thermodesulfobacterium commune suggests that these two thermophilic sulfate-reducing bacteria represent different genera. These two bacteria depict a lineage that branches deeply within the Bacteria domain and which is clearly distinct from previously defined phylogenetic lines of sulfate-reducing bacteria. Strain YP87 is described as the type strain of the new genus and species Thermodesulfovibrio yellowstonii.

Pseudomonas aeruginosa invades corneal epithelial cells during experimental infection.

Abstract: Pseudomonas aeruginosa is considered an extracellular pathogen. Using assays to determine intracellular survival in the presence of gentamicin, we have demonstrated that some strains of P. aeruginosa are able to invade corneal cells during experimental bacterial keratitis in mice. Although intracellular bacteria were detectable 15 min after inoculation, the number of intracellular bacteria increased in a time-dependent manner over a 24-h period. Levels of invasion were similar when bacteria were grown as a biofilm on solid medium and when they were grown in suspension. Intracellular bacteria survived in vitro for at least 24 h, although only minimal bacterial multiplication within cells was observed. P. aeruginosa PAK and Escherichia coli HB101 did not cause disease in this model and were not isolated from corneas after 24 h even when an inoculum of 10(8) CFU was applied. Transmission electron microscopy of corneal epithelium from eyes infected for 8 h revealed that intracellular bacteria were present within membrane-bound vacuoles, which suggests that bacterial entry was an endocytic process. At 24 h, the observation of many bacteria free in the cytoplasm indicated that P. aeruginosa was able to escape the endocytic vacuole. The ability of some P. aeruginosa strains to invade corneal epithelial cells may contribute to the pathogenesis or to the progression of disease, since intracellular bacteria can evade host immune effectors and antibiotics commonly used to treat infection.

Bacteria as Risk Markers for Periodontitis.

Abstract: Specific microbial species have been closely associated with periodontitis Through longitudinal studies, some of these microbial species have been implicated in the etiology of progressive periodontal disease Although putative periodontal pathogens are often isolated from individuals with severe periodontitis, they also frequently inhabit the subgingival environment and are not always associated with advanced disease In this respect, it is becoming increasingly apparent that there is no single etiology of the various periodontal diseases Destructive periodontal diseases are the result of environmental, host, and bacterial factors Microorganisms, however, are essential components of any model for progressive periodontitis This paper selectively reviews bacteria as risk markers for periodontitis Attention focuses on bacteria in conjunction with behavioral patterns (oral hygiene habits and smoking) and host response (gingival crevicular fluid substances) as risk markers for periodontitis Prospective

The sourdough microflora. Interactions between lactic acid bacteria and yeasts: metabolism of amino acids.

Abstract: Co-culture of Lactobacillus brevis subsp. lindneri or L. plantarum with Saccharomyces cerevisiae or S. exiguus from sourdough did not modify the yield of the yeasts but gave higher growth rates and final yields of both lactic acid bacteria (LAB) than in their respective mono-cultures. Co-cultures of L. brevis subsp. lindneri with S. cerevisiae or S. exiguus in a medium without valine or leucine, which are essential for growth of the LAB, led to growth of the LAB due to excretion of these amino acids by the yeasts.

Identification of 4-hydroxyhexanoic acid as a new constituent of biosynthetic polyhydroxyalkanoic acids from bacteria

Abstract: Twenty-four different strains of aerobic Gram-negative bacteria, mainly belonging to the genera Alcaligenes, Paracoccus, Pseudomonas and Methylobacterium, were examined with respect to their ability to utilize 4-hydroxyvaleric acid (4HV), 4-valerolactone (4VL) and 3-hydroxypropionic acid (3HP) as carbon sources for growth and for accumulation of polyhydroxyalkanoic acid (PHA). A gas chromatographic (GC) method for the detection of 3-hydroxyalkanoic acid methyl esters has been extended for the detection of derivatives obtained from the methanolysis of 4-hydroxybutyric acid (4HB) and 4HV. Most of the Alcaligenes species and P. oxalaticus Ox1 accumulated a terpolyester consisting of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV) and 4HV as constituents from 4HV or 4VL as sole carbon sources in batch, fed-batch or two-stage fed-batch cultures. Poly(3HB-co-3HV-co-4HV) accumulated from 4HV by A. eutrophus strain NCIB 11599 amounted to approximately 50% of the cell dry matter and was composed of 42.0 mol % 3HB, 52.2 mol % 3HV and 5.6 mol % 4HV, respectively. Pseudomonads, which belong to the rRNA homology group I, were not able to incorporate 4HV. With 3HP as carbon source, the GC analysis provided evidence for the presence of 3HP in the PHA of many bacteria. Nuclear magnetic resonance spectroscopic analysis confirmed that, for example, A. eutrophus strain TF93 accumulated poly(3HB-co-3HP) with 98 mol % 3HB and 2 mol % 3HP if the cells were cultivated in the presence of 0.5% (w/v) 3HP.