Distribution of alkB genes within n-alkane-degrading bacteria

Fifty-four bacterial strains belonging to 37 species were tested for their ability to assimilate short chain and/or medium chain liquid n-alkanes. A gene probe derived from the alkB gene of Pseudomonas oleovorans ATCC 29347 was utilized in hybridization experiments. Results of Southern hybridization of PCR-amplificates were compared with those of colony hybridization and dot blot hybridization. Strongest signals were received only from Gram-negative bacteria growing solely with short n-alkanes (C10). Hybridization results with soil isolates growing with n-alkanes of different chain lengths suggested as well that alkB genes seem to be widespread only in solely short-chain n-alkane-degrading pseudomonads. PCR products of Rhodococcus sp., Nocardioides sp., Gordona sp. and Sphingomonas sp. growing additionally or solely with medium-chain n-alkane as hexadecane had only few sequence identity with alkB though hybridizing with the gene probe. The derived amino acid sequence of the alkB-amplificate of Pseudomonas aureofaciens showed high homology (95%) with AlkB from Ps. oleovorans. alkB gene disruptants were not able to grow with decane.     

Initial reactions in the anaerobic oxidation of toluene and m-xylene by denitrifying bacteria.

Pseudomonas sp. strain T and Pseudomonas sp. strain K172 grow with toluene under denitrifying conditions. We demonstrated that anaerobic degradation of toluene was initiated by direct oxidation of the methyl group. Benzaldehyde and benzoate accumulated sequentially after toluene was added when cell suspensions were incubated at 5 degrees C. Strain T also grows anaerobically with m-xylene, and we demonstrated that degradation was initiated by oxidation of one methyl group. In cell suspensions incubated at 5 degrees C 3-methylbenzaldehyde and 3-methylbenzoate accumulated after m-xylene was added. Toluene- or m-xylene-grown strain T cells were induced to the same extent for oxidation of both hydrocarbons. In addition, the methyl group-oxidizing enzyme system of strain T also catalyzed the oxidation of each isomer of the chloro- and fluorotoluenes to the corresponding halogenated benzoate derivatives. In contrast, strain K172 only oxidized 4-fluorotoluene to 4-fluorobenzoate, probably because of the narrow substrate specificity of the methyl group-oxidizing enzymatic system. During anaerobic growth with toluene strains T and K172 produced two transformation products, benzylsuccinate and benzylfumarate. About 0.5% of the toluene carbon was converted to these products.     

Elucidation of the pathways of catabolic glutamate conversion in three thermophilic anaerobic bacteria

The glutamate catabolism of three thermophilic syntrophic anaerobes was compared based on the combined use of [(13)C] glutamate NMR measurements and enzyme activity determinations. In some cases the uptake of intermediates from different pathways was studied. The three organisms, Caloramator coolhaasii, Thermanaerovibrio acidaminovorans and strain TGO, had a different stoichiometry of glutamate conversion and were dependent on the presence of a hydrogen scavenger (Methanobacterium thermoautotrophicum Z245) to a different degree for their growth. C. coolhaasii formed acetate, CO(2), NH(4)(+) and H(2) from glutamate. Acetate was found to be formed through the beta-methylaspartate pathway in pure culture as well as in coculture. T. acidaminovorans converted glutamate to acetate, propionate, CO(2), NH(4)(+) and H(2). Most likely, this organism uses the beta-methylaspartate pathway for acetate formation. Propionate formation occurred through a direct oxidation of glutamate via succinyl-CoA and methylmalonyl-CoA. The metabolism of T. acidaminovorans shifted in favour of propionate formation when grown in coculture with the methanogen, but this did not lead to the use of a different glutamate degradation pathway. Strain TGO, an obligate syntrophic glutamate-degrading organism, formed propionate, traces of succinate, CO(2), NH(4)(+) and H(2). Glutamate was converted to propionate oxidatively via the intermediates succinyl-CoA and methylmalonyl-CoA. A minor part of the succinyl-CoA was converted to succinate and excreted.     

Corrinoids in anaerobic bacteria

Abstract C₁-metabolizing bacteria were analyzed for their corrinoids. The autotrophic phototrophe Chloroflexus aurantiacus contains predominantly the light-sensitive coenzyme B₁₂. The corrinoid could be teh prostethic group of a methylmalonyl-CoA mutase, which is involved in the CO₂ fixing reaction sequence from proplonyl-CoA to succinyl CoA. Methanobacterium thermoautotrophicum and Sporomusa ovata contain only traces of light-sensitive corrinoids, indicating that the demethylation reaction is favored, if these corrinoids are involved in methyl transfer reactions. The chemical structure of the unique p -cresolyl cobamide is specific for the acetogenic bacterium S. ovata , rather than the corrinoid 'factor III' for methanogenic bacteria.     

Fermentative toluene degradation in anaerobic defined syntrophic cocultures

A syntrophic coculture of a new sulfate-reducing isolate, strain TRM1, with Wolinella succinogenes degraded toluene with either fumarate or NO3- as the terminal electron acceptor. Neither strain TRM1 nor W. succinogenes could metabolise toluene under these conditions in pure culture. Syntrophic degradation was 2-3 times slower than toluene utilisation by strain TRM1 in pure culture with sulfate as electron acceptor. The culture did not produce benzoate or fatty acids like acetate or propionate in detectable amounts. An increase in biomass of the syntrophic toluene-degrading culture was shown in a growth curve with nitrate as the terminal electron acceptor. Both partner organisms were detected microscopically at the end of the growth experiment. Syntrophic degradation of toluene with W. succinogenes and fumarate as the terminal electron acceptor was also demonstrated with the iron reducer Geobacter metallireducens. The results provide the first example of a fermentative oxidation of an aromatic hydrocarbon in a defined coculture.     

Unusual dehydrations in anaerobic bacteria

Abstract In amino acid fermenting anaerobic bacteria a set of unusual dehydratases is found which use 2-hydroxyacyl-CoA, 4-hydroxybutyryl-CoA or 5-hydroxyvaleryl-CoA as substrates. The extremely oxygen-sensitive 2-hydroxyacyl-CoA dehydratases catalysing the elimination of water from ( R )-lactyl-CoA to acryloyl-CoA or from ( R )-2-hydroxyglutaryl-CoA to glutaconyl-CoA contain iron-sulfur clusters as well as riboflavin and require additional activation by ATP. The dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA is catalysed by a moderately oxygen-sensitive enzyme also containing an iron-sulfur cluster and FAD. In all these reactions a non-activated C-H-bond at C₃ has to be cleaved by mechanisms not yet elucidated. The dehydration of 5-hydroxyvaleryl-CoA to 4-pentenoyl-CoA, however, has been characterised as a redox process mediated by enzyme-bound FAD. Finally, an iron-sulfur cluster-containing but pyridoxal-phosphate-independent l -serine dehydratase is described.     

Citrate metabolism in anaerobic bacteria

Abstract The regulation of anaerobic citrate metabolism is very diverse among different groups of bacteria. In organisms like Streptococcus lactis and Clostridium sporosphaeroides which lack citrate synthase, the activity of its antagonistic enzyme, citrate lyase, need not be regulated. Many anaerobes like Rhodocyclus gelatinosus and Clostridium sphenoides are able to synthesize their own l -glutamate and contain citrate synthase. In these bacteria the activity of citrate metabolizing enzymes which are involved in a cascade system are under strict control. In Rc. gelatinosus activation/inactivation of citrate lyase is controlled by acetylation/deacetylation which is catalyzed by its corresponding regulatory enzymes, citrate lyase ligase and citrate lyase deacetylase. In C. sphenoides inactivation of citrate lyase is accomplished by deacetylation as well as by changing in the enzyme conformation. Activation of citrate lyase is catalyzed by citrate lyase ligase whose activity in addition is modulated by phosphorylation/dephosphorylation. Further, electron transport process also seems to play a role in the inactivation of citrate metabolizing enzymes in enteric bacteria.     

Biofilm-growing intestinal anaerobic bacteria

Sessile growth of anaerobic bacteria from the human intestinal tract has been poorly investigated, so far. We recently reported data on the close association existing between biliary stent clogging and polymicrobial biofilm development in its lumen. By exploiting the explanted stents as a rich source of anaerobic bacterial strains belonging to the genera Bacteroides, Clostridium, Fusobacterium, Finegoldia, Prevotella, and Veillonella, the present study focused on their ability to adhere, to grow in sessile mode and to form in vitro mono- or dual-species biofilms. Experiments on dual-species biofilm formation were planned on the basis of the anaerobic strains isolated from each clogged biliary stent, by selecting those in which a couple of anaerobic strains belonging to different species contributed to the polymicrobial biofilm development. Then, strains were investigated by field emission scanning electron microscopy and confocal laser scanning microscopy to reveal if they are able to grow as mono- and/or dual-species biofilms. As far as we know, this is the first report on the ability to adhere and form mono/dual-species biofilms exhibited by strains belonging to the species Bacteroides oralis, Clostridium difficile, Clostridium baratii, Clostridium fallax, Clostridium bifermentans, Finegoldia magna, and Fusobacterium necrophorum.     

Studies on anaerobic bacteria

Summary A new chromogenic anaerobe, Clostridium roseum nov. spec., has been found. It is characterized by: red-orange pigment, turning purplish on oxidation; gelatin liquefaction and other evidence of proteolysis; nitrate reduction; fermentation of various carbohydrates including pectin; close resemblance to Cl. acetobutylicum in corn mash fermentation, with the same neutral products, acetone, ethyl alcohol and butyl alcohol, in nearly the same ratios; agglutinative specificity and separation from Cl. acetobutylicum and Cl. felsineum, as well as several less nearly physiologically related butyric anaerobes.     

Halophilic anaerobic fermentative bacteria

In hypersaline environments bacteria are exposed to a high osmotic pressure caused by the surrounding high salt concentrations. Halophilic microorganisms have specific strategies for balancing the osmotic pressure and surviving in these extreme conditions. Halophilic fermentative bacteria form taxonomically and phylogenetically a coherent group mainly belonging to the order Halanaerobiales. In this review, halophilic anaerobic fermentative bacteria in terms of taxonomy and phylogeny, special characteristics, survival strategies, and potential for biotechnological applications in a wide variety of branches, such as production of hydrogen, are discussed.     

Effect of carbon starvation on toluene degradation activity by toluene monooxygenase-expressing bacteria

Subsurface bacteria commonly exist in a starvation state with only periodic exposure to utilizable sources of carbon and energy. In this study, the effect of carbon starvation on aerobic toluene degradation was quantitatively evaluated with a selection of bacteria representing all the known toluene oxygenase enzyme pathways. For all the investigated strains, the rate of toluene biodegradation decreased exponentially with starvation time. First-order deactivation rate constants for TMO-expressing bacteria were approximately an order of magnitude greater than those for other oxygenase-expressing bacteria. When growth conditions (the type of growth substrate and the type and concentration of toluene oxygenase inducer) were varied in the cultures prior to the deactivation experiments, the rate of deactivation was not significantly affected, suggesting that the rate of deactivation is independent of previous substrate/inducer conditions. Because TMO-expressing bacteria are known to efficiently detoxify TCE in subsurface environments, these findings have significant implications for in situ TCE bioremediation, specifically for environments experiencing variable growth-substrate exposure conditions.     

Activity-dependent fluorescent labeling of bacteria that degrade toluene via toluene 2,3-dioxygenase

Alternative substrates for the toluene 2,3-dioxygenase pathway of several pseudomonads served as enzyme-activity-dependent fluorescent probes for the bacteria. Phenylacetylene and cinnamonitrile were transformed to fluorescent and brightly colored products by Pseudomonas putida F1, Pseudomonas fluorescens CFS215, and Burkholderia (Pseudomonas) strain JS150. Active bacteria transformed phenylacetylene, producing bright yellow solutions containing the putative product 2-hydroxy-6-oxo-7-octyn-2,4-dienoate. Transformation of cinnamonitrile resulted in bright orange solutions due to accumulation of the putative product 2-hydroxy-6-oxo-8-cyanoocta-2,4,7-trienoate. Chemical and physical properties of the products supported their identification, which indicated that the first three enzymes of the pathway catalyzed product formation. Phenylacetylene labeled bacteria with green fluorescence emission; bacteria were concentrated on black 0.2-μm-pore-size polycarbonate filters containing polyvinylpyrrolidone (PVP) as a wetting agent. Bacteria labeled with cinnamonitrile were fluorescent orange; labeling was effective with bacteria trapped on PVP-free polycarbonate filters. Production of the enzymes involved in labeling of P. putida F1 and P. fluorescens CFS215 was induced by growth (on arginine) in the presence of toluene; cells grown on arginine without toluene were not labeled. Labeling of P. putida F1 by phenylacetylene was inhibited by toluene, indicating that the same enzymatic pathway was required for transformations of both substrates. Bacteria expressing other toluene-degrading enzymatic pathways were not fluorescently labeled with phenylacetylene. Received: 30 July 1997 / Received revision: 1 November 1997 / Accepted: 21 November 1997     

Oxygen tolerance in anaerobic pathogenic bacteria

A prerequisite for successful identification of anaerobic pathogenic bacteria from samples of clinical material is the method of cultivation. Currently, several methods of cultivation in anaerobic environment are used: cultivation in anaerobic box, anaerobic jar, and in nonrecurring cultivation system. Here, we determined the suitability of the above methods of cultivation using the estimation of the growth (diameters of colony size) of commonly isolated anaerobic pathogens (Bacteroides fragilis, Clostridium difficile, and Clostridium perfringens). The tested bacterial strains were exposed to atmospheric oxygen for various time periods and then they were cultivated using different anaerobic cultivation systems. Maximum growth differed, depending on the type of cultivation and the strain used. Thus, largest zone diameters, in the majority of measurements, were achieved in the anaerobic box. However, nonrecurring cultivation system seemed better in several cases; this applied to the cultivation of C. perfringens after 15, 30, and 60 min exposure to atmospheric oxygen as well as the cultivation of B. fragilis after 30 and 60 min of oxygen exposure. The cultivation in anaerobic box was the most convenient method for growth of C. difficile. In almost all cases, higher growth was observed in nonrecurring cultivation system than in the system of anaerobic jar. On the other hand, no significant differences were observed among these anaerobic cultivation systems which confirmed their applicability (taking into account some individual features concerning the optimization of cultivations) for identification of pathogenic anaerobes.     

Radical-mediated dehydration reactions in anaerobic bacteria

Most dehydratases catalyse the elimination of water from beta-hydroxy ketones, beta-hydroxy carboxylic acids or beta-hydroxyacyl-CoA. The electron-withdrawing carbonyl functionalities acidify the alpha-hydrogens to enable their removal by basic amino acid side chains. Anaerobic bacteria, however, ferment amino acids via alpha- or gamma-hydroxyacyl-CoA, dehydrations of which involve the abstraction of a beta-hydrogen, which is ostensibly non-acidic (pK ca. 40). Evidence is accumulating that beta-hydrogens are acidified via transient conversion of the CoA derivatives to enoxy radicals by one-electron transfers, which decrease the pK to 14. The dehydrations of (R)-2-hydroxyacyl-CoA to (E)-2-enoyl-CoA are catalysed by heterodimeric [4Fe-4S]-containing dehydratases, which require reductive activation by an ATP-dependent one-electron transfer mediated by a homodimeric protein with a [4Fe-4S] cluster between the two subunits. The electron is further transferred to the substrate, yielding a ketyl radical anion, which expels the hydroxyl group and forms an enoxy radical. The dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA involves a similar mechanism, in which the ketyl radical anion is generated by one-electron oxidation. The structure of the FAD- and [4Fe-4S]-containing homotetrameric dehydratase is related to that of acyl-CoA dehydrogenases, suggesting a radical-based mechanism for both flavoproteins.     

Energy conservation in chemotrophic anaerobic bacteria

[This corrects the article on p. 100 in vol. 41.].     

The role of anaerobic bacteria in bacteremia

Anaerobic bacteria remain an important cause of bloodstream infections and account for 1–17% of positive blood cultures. This review summarizes the epidemiology, microbiology, predisposing conditions, and treatment of anaerobic bacteremia (AB) in newborns, children, adults and in patients undergoing dental procedures. The majority of AB are due to Gram-negative bacilli, mostly Bacteroides fragilis group. The other species causing AB include Peptostreptococcus, Clostridium spp., and Fusobacterium spp. Many of these infections are polymicrobial. AB in newborns is associated with prolonged labor, premature rupture of membranes, maternal amnionitis, prematurity, fetal distress, and respiratory difficulty. The predisposing conditions in children include: chronic debilitating disorders such as malignant neoplasm, hematologic abnormalities, immunodeficiencies, chronic renal insufficiency, or decubitus ulcers and carried a poor prognosis. Predisposing factors to AB in adults include malignant neoplasms, hematologic disorders, transplantation of organs, recent gastrointestinal or obstetric gynecologic surgery, intestinal obstruction, diabetes mellitus, post-splenectomy, use of cytotoxic agents or corticosteroids, and an undrained abscess. Early recognition and appropriate treatment of these infections are of great clinical importance.     

The role of anaerobic bacteria in sinusitis

The normal oropharyngeal flora contained aerobic and anaerobic bacteria that can cause respiratory infections including sinusitis. Some of these bacteria can interfere with the growth of potential pathogens and may play a role in preventing infections. Anaerobic bacteria emerge as pathogens as the infection becomes chronic. This may be the result of the selective pressure of antimicrobial agents that enable resistant anaerobic organisms to survive, and from the development over time of conditions appropriate for anaerobic growth, which include the reduction in oxygen tension and an increase in acidity within the sinus cavity. Anaerobes were isolated in acute maxillary sinusitis of odontogenic origin and in over half of the patients with chronic sinusitis whenever proper techniques for their cultivation were employed. These organisms were also recovered in acute sinusitis that was associated with dental infections. The predominant isolates were pigmented Prevotella and Porphyromonas, Fusobacterium and Peptostreptococcus spp.     

Antimicrobial susceptibility of anaerobic bacteria in Bulgaria

ObjectivesThe antimicrobial susceptibility of anaerobic bacteria isolated from clinical specimens in the referent for Bulgaria anaerobic laboratory was studied in a period of 25 years/1983–2007/.MethodsNCCLS – recommended agar dilution methods were used. β-lactamase activity was determined with nitrocefin discs.ResultsThe 29 antimicrobial agents included in the study were divided according to their in vitro activity against the anaerobic isolates into 4 main groups for guiding empirical treatment: 1st group of metronidazole, chloramphenicol, meropenem, imipenem and combinations of β-lactam antibiotics with sulbactam – with high activity and drugs of choice for treatment; 2nd group – clindamycin, cefoxitin, carbenicillin/and azlocillin, piperacillin/ – with a good activity and low percent of resistant strains; 3rd group – of tetracycline and erythromycin with higher percent of resistant strains including the new macrolides as josamycin, clarithromycin, roxithromycin and azithromycin; 4th group – penicillins/ampicillin, amoxicillin, penicillin/and cephalosporins/cefamandole, cefazolin, cefotaxime and cefoperazone/ – not suitable for treatment of infections including Bacteroides fragilis group strains, with a very high percent of resistant strains, probably due to β-lactamase activity in most of the strains.ConclusionA continued updating and a follow-up in the changes of antibiotic susceptibility are necessary in every country as resistance patterns vary not only between geographical regions but also even among medical centers and hospitals which may be connected with differences in antibiotic usage in man and animals.     

Interactions between amino-acid-degrading bacteria and methanogenic bacteria in anaerobic digestion

The degradation of amino acids in anaerobic digestion was examined in terms of the interactions between amino-acid-degrading bacteria and methanogenic bacteria. Certain amino acids were degraded oxidatively by dehydrogenation, with methanogenic bacteria acting as H(2) acceptors. The inhibition of methanogenesis by chloroform also inhibited the degradation of these amino acids and/or caused variations in the composition of volatile acids produced from them. The presence of glycine reduced the inhibitory effect caused by chloroform, probably because glycine acted as an H(2) acceptor in place of methanogenic bacteria. This fact suggested that the coupled oxidation-reduction reactions between two amino acids-one acting as the H(2) donor and the other acting as the H(2) acceptor-may occur in the anaerobic digestion of proteins or amino-acid mixtures. The conversion of some proteins to volatile acids was not affected when methanogenesis was inhibited by chloroform. This suggested that the component amino acids of proteins may be degraded by the coupled oxidation-reduction reactions and that the degradation of proteins may not be dependent on the activity of methanogenic bacteria as H(2) acceptors.