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PubMed1994

Crystal structure of the ferredoxin I from Desulfovibrio africanus at 2.3 A resolution.

The crystal structure of the ferredoxin I from the sulfate-reducing bacterium Desulfovibrio africanus (DaFdI) has been solved and refined by X-ray diffraction. The crystals are orthorhombic with a = 96.6 A, b = 58.1 A, and c = 20.7 A, space group P2(1)2(1)2, and two ferredoxin molecules per asymmetric unit. The initial electron density map has been obtained by combining phasing by molecular replacement methods, anomalous scattering, and noncrystallographic averaging. The final crystallographic R factor is 0.182 with 10-2.3 A resolution data. In parallel, the amino acid sequence was redetermined. This showed that DaFdI contains 64 residues (instead of 61) including one free cysteine, one histidine, and one tryptophan in the C-terminal part of the molecule. The current molecular model includes the two molecules of the asymmetric unit, 67 water molecules, and one sulfate ion. The DaFdI overall folding very closely resembles that of ferredoxins of known structure. Comparisons with the single cluster ferredoxins from Desulfovibrio gigas and Bacillus thermoproteolyticus show that the presence or the absence of a disulfide bridge does not significantly affect the folding of the other half of the molecule, including the characteristic alpha-helix of the single cluster ferreddoxins. Like other ferredoxins or analogs, the [4Fe-4S] iron--sulfur cluster presents, at 2.3 A resolution, a cubane-like geometry. By contrast, its immediate environment is different as it includes, besides the four cysteic sulfur ligands, the sulfur atom of the free cysteine. This sulfur atom, which is buried within the protein, is in van der Waals contact with one labile sulfur of the cluster and one liganded cysteic sulfur. The association of a [4Fe-4S] cluster with one free cysteic sulfur is similar to that previously found in both X-ray structures of Azotobacter vinelandii and Peptococcus aerogenes [Stout, C. D. (1989) J. Mol. Biol. 205, 545-555; Backes, G., et al. (1991) J. Am. Chem. Soc. 113, 2055-2064]. Chemical sequence analysis suggests that this characteristic [4Fe-4S] cluster sulfur environment is widely distributed among ferredoxins.

Amino Acid Sequence↗
PubMed1976

Oxidation-reduction properties of several low potential iron-sulfur proteins and of methylviologen.

Apparent oxidation-reduction potentials at pH 7.0 and 25 degrees C were determined using the H2-hydrogenase system with ferredoxins from the following sources: Clostridium pasteurianum, -403 mV; C tartarovorum, -424 mV; C. acidi-urici, -434 mV; Peptococcus aerogenes, -427 mV; Chromatium D, -482 mV (pH 8.0); B. polymyxa, Fd I, -377 mV, and Fd II, -422 mV; and spinach, -428 mV. The pH dependence of these values was variable, ranging from -2 to -24 mV/pH unit increase for different ferredoxins. Over the range of buffer concentrations between 0.05 and 0.2 M, the potentials did not vary significantly. The number of electrons transferred during reduction (as determined by integrations of EPR spectra and by dithionite titration) is 2 for the first five proteins, while potentiometric data for all the cases fit a Nernst equation for which n = 1. The E degrees' value for the redox indicator methylviologen at pH 7.4 was found to be -460 mV, according to both the H2-hydrogenase system and cyclic voltammetry, significantly different from the value previously reported at higher pH's. Additionally, the presence of C. pasteuranum ferredoxin appears to shift the E degrees value of methylviologen to even more negative values. An analysis of sources of error inherent with potential determinations with H2 and hydrogenase is presented. The electronic and EPR spectra of P. aerogenes ferredoxin, for which the x-ray structure has been published, are given here. It appears that the determination of potentials of ferredoxin and other low-potential porteins with the H2-hydrogenase system affords certain experimental advantages over alternative methods currently employed with these and similar substances.

Binding Sites↗
PubMed1975

Determination of the iron-sulfur distances in rubredoxin by x-ray absorption spectroscopy.

The high intensity x-ray flux from the synchrotron radiation at the Stanford Synchroton Radiation Project has been used to study the extended x-ray absorption fine structure (EXAFS) of the iron-sulfur protein Peptococcus aerogenes rubredoxin. Absorption measurements were made from 7080 eV, which is below the K-edge of iron, to about 650 eV above the edge and structure was obtained over the entire region. By means of a model iron-sulfur compound for evaluating the phase shifts, the variation of the absorption above the edge of lyophilized, oxidized rubredoxin was converted to iron-sulfur distances. The data were fitted with a least squares program to a model in which three distances R3 were kept equal and the fourth R1 was allowed to differ. The mean square error was constant over a region of this parameter space, becoming twice as large at R3 = 2.217, R1 = 2.389 and R3 = 2.268, R1 = 2.108 A. These values, which are the extreme differences allowed by the present data, are definitely closer to being equal than those found by the determination of the x-ray diffraction crystal structure of the similar protein from Clostridium pasteurianum. However, the average distance from our experiment is in excellent agreement with the average distance from the crystal structure determination. Preliminary EXAFS measurements were also made on the oxidized rubredoxin in solution at pH 7.0. The spectra were unchanged, indicating that the average iron-sulfur distance change is less than 0.02 A. Upon reduction the average iron-sulfur bond length increased by about 0.05 A. Since the EXAFS measurements can give accurate determinations of distances in proteins both in crystals and solution, the technique should be widely applicable.

Binding Sites↗
PubMed1988

Structure of ferredoxin I from Azotobacter vinelandii.

The structure of Azotobacter vinelandii ferredoxin I (Av FdI, 106 amino acids) has been redetermined, based on x-ray diffraction data from tetragonal crystals of the native protein and two heavy atom derivatives. The current model differs greatly from the one previously reported and is in agreement with arguments based on various spectroscopic and other methods. The unit cell parameters are a = b = 55.62 A and c = 95.51 A, whereas the space group was found to be P4(1)2(1)2 instead of P4(3)2(1)2. The sequence of the first half of Av FdI is closely homologous with ferredoxin from Peptococcus aerogenes (Pa Fd, 54 amino acids) and the fold of the corresponding chain is almost identical. The ligands of the 3Fe complex are Cys-8, -16, and -49, corresponding to three of the four ligands in complex I of Pa Fd; the ligands of the 4Fe complex are Cys-20, -39, -42, and -45, corresponding to the four ligands in complex II of Pa Fd.

Azotobacter↗
PubMed1996

Site-specific mutagenesis of Rhodobacter capsulatus ferredoxin I, FdxN, that functions in nitrogen fixation. Role of extra residues.

One of the two [4Fe-4S]-type clusters of the Rhodobacter capsulatus ferredoxin I, FdxN, was modified through site-specific mutagenesis of the distinctive features of the second cluster-binding motif, Cys38-X2-Cys41-X8-Cys50-X3-Cys54-X4-Cys59. First, various mutagenized products were tested to learn whether they could rescue the decreased capacity of an fdxN-null strain MSA1 to fix nitrogen: the phenotype of MSA1 was reassessed to Nifs (slow growth by nitrogen fixation) from our previous description of Nif- (Saeki, K., Suetsugu, Y., Tokuda, K., Miyatake, Y., Young, D. A., Marrs, B. L. and Matsubara, H. (1991) J. Biol. Chem. 266, 12889-12895). Substitution of Cys59 to Ser yielded an almost fully active product, while that of Cys54 did not. Gradual deletions and deletion-substitution of the 8 residues between Cys41 and Cys50 also yielded active products. Second, three of the modified FdxN proteins were subjected to purification. Only the GA protein, whose 8 residues between positions 42 and 49 were replaced by the Gly-Ala sequence, was purified. The GA protein and the authentic FdxN showed similar optical properties. The two clusters in the former had Em values of -490 and -430 mV, while those in the latter had an identical value of -490 mV, when determined by EPR analysis. It was concluded that: 1) Cys59 is not a ligand to [4Fe-4S] clusters but is important for structural integrity, 2) the residues between positions 42 and 49 may form a "loop-out" from a structure analogous to the Peptococcus aerogenes ferredoxin, and 3) the loop-out region does not have functional significance in nitrogen fixation but may be responsible for maintaining the highly negative redox potential of one of the two clusters.

Amino Acid Sequence↗
PubMed1997

Y13C Azotobacter vinelandii ferredoxin I. A designed [Fe-S] ligand motif contains a cysteine persulfide.

Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S]2+/+ in the [3Fe-4S]+/0 location of native (7Fe) Azotobacter vinelandii ferredoxin I (AvFdI) by providing the correct three-dimensional orientation of cysteine ligands without introducing a CysXXCysXXCys motif. Tyr13 of AvFdI occupies the position of the fourth ligating cysteine in the homologous and structurally characterized 8Fe ferredoxin from Peptococcus aerogenes and a Y13C variant of AvFdI could be easily modeled as an 8Fe protein. However, characterization of purified Y13C FdI by UV-visible spectra, circular dichroism, electron paramagnetic resonance spectroscopies, and by x-ray crystallography revealed that the protein failed to use the introduced cysteine as a ligand and retained its [3Fe-4S]+/0 cluster. Further, electrochemical characterization showed that the redox potential and pH behavior of the cluster were unaffected by the substitution of Tyr by Cys. Although Y13C FdI is functional in vivo it does differ significantly from native FdI in that it is extremely unstable in the reduced state possibly due to increased solvent exposure of the [3Fe-4S]0 cluster. Surprisingly, the x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified. Thus, if NifS is involved in modifying the introduced cysteine there must be specificity to the reaction.

Amino Acid Sequence↗
PubMed1978

A reliable test for differentiation and presumptive identification of certain clinically significant anaerobes.

A modification of the slide catalase test was evaluated to determine its reliability for the presumptive identification of certain anaerobic gram-negative and gram-positive organisms isolated from clinical specimens. A total of 650 fresh isolates was evaluated. Growth from a trypticase blood agar plate or Schaedler's agar with laked blood was transferred to a slide and a drop of 15% H2O2 with Tween 80 added. Veillonella alcalescens, Proprionibacterium species, Peptococcus species, and Bacteroides fragilis, B. thetaiotaomicron, B. distasonis, B. ovatus, and B. vulgatus were positive by this method. Comparative slide and tube catalase tests were performed with the above-mentioned bacteroides species by growing each isolate on trypticase soy agar, Brucella agar, brain-heart infusion agar, each with and without added hemin, and on Schaedler's agar. Four solutions of H2O2 were used, 3 and 15%, and 3 and 15% with Tween 80. The addition of Tween 80 to H2O2 increased the sensitivity of the reaction with both slide and tube methods. The 15% H2O2 with Tween 80 consistently gave the most rapid and persistent positive reaction. A slide catalase test in combination with a Gram stain should be performed on each anaerobic isolate; this would significantly decrease the time required for presumptive identification of many anaerobic organisms.

Bacteria↗
PubMedDate not supplied

The role of Bacteroides melaninogenicus and other anaerobes in periodontal infections.

Recent taxonomic and anatomical studies of dental plaque associated with periodontal health and disease have demonstrated that differences in the microbial populations in plaque may be responsible for the initiation and progression of disease. The consistent isolation of large numbers of anaerobic and capnophilic bacteria from the depths of periodontal lesions has suggested an important role for these organisms. Bacteria that have been isolated include Capnocytophaga (Bacteroides ochraceus), other species of Bacteroides, Fusobacterium, Selenomonas, spirochetes, Campylobacter, Veillonella, Actinomyces, Propionibacterium, Peptococcus, and other genera. The periodontopathic potential of oral strains of Bacteroides melaninogenicus has been explored in a number of investigations because these organisms are consistently isolated from periodontal lesions. Studies of B. melaninogenicus have included purification of a capsular substance, characterization of the lipopolysaccharide and a variety of toxic substances and lytic enzymes, and ecologic aspects of its colonization. Understanding of the nature and pathogenic mechanisms of the oral microbiota may lead to control of this pandemic infection.

Bacteroides Infections↗
PubMedDate not supplied

Tubo-ovarian abscess: contemporary approach to management.

Two hundred and thirty-two patients with tubo-ovarian abscesses (TOAs) were evaluated. Ruptured TOAs were documented in seven (3%) of the patients. One hundred and seventy-five patients with TOAs were treated with antibiotics alone; for 15 of these patients, TOAs were confirmed by laparoscopy. The remaining 57 patients required surgical intervention: drainage (five patients), unilateral salpingo-oophorectomy (19) and total abdominal hysterectomy and bilateral salpingo-oophorectomy (33). A unilateral TOA was present in 163 patients (70%). Seventy-six patients with TOAs used intrauterine contraceptive devices, and in this group, 54 (71%) patients had unilateral TOAs. The most common microorganisms that were recovered from these TOAs were Escherichia coli, Bacteroides fragilis, Bacteroides species, Peptostreptococcus, Peptococcus, and aerobic streptococci. Sixty-eight percent of the patients treated with an antimicrobial regimen that included clindamycin had a decrease in the size of the TOA, while only 36.5% of those receiving antimicrobial regimens without clindamycin had a decrease in the size of the TOA (P less than .01). Long-term follow-up information (two to 10 years) was available for 58 of the patients treated with antibiotics alone. Eighteen (31%) required subsequent surgery; 12 had persistent TOAs; and six, chronic salpingo-oophoritis. Intrauterine pregnancy was documented in eight (13.8%) patients. Of the 19 patients treated with unilateral adnexectomy, two ultimately required hysterectomy and contralateral adnexectomy, while three patients in this group subsequently became pregnant (one ectopic and two intrauterine).

Abscess↗
PubMedDate not supplied

Cefoperazone therapy for obstetric and gynecologic infections.

The safety and clinical efficacy of cefoperazone therapy for obstetric and gynecologic infections have been evaluated in clinical trials involving 107 women hospitalized at nine medical centers. The indications for cefoperazone therapy have included post-partum and postoperative uterine infections, pelvic inflammatory disease, postoperative pelvic infections, and miscellaneous pelvic infections. A variety of aerobic and anaerobic bacteria, including members of the genera Staphylococcus, Streptococcus, Escherichia, Klebsiella, Proteus, Neisseria, Bacteroides, Peptococcus, and Peptostreptococcus, were isolated from these patients. More than 80% of these isolates were inhibited in vitro by cefoperazone concentrations less than or equal to 2.0 micrograms/ml. Symptomatic cures were achieved in 91% of patients, and 96% of isolated pathogens were eradicated during therapy. Adverse drug-related reactions occurred in only 4% of patients. Follow-up laboratory evaluations demonstrated only minor abnormalities that affected a small number of patients. These results indicate that cefoperazone offers safe and effective therapy for common obstetric and gynecologic infections.

Adolescent↗
PubMedDate not supplied

Anaerobic oral and dental infection.

Anaerobes make up a significant part of the oral and dental indigenous and pathogenic flora. Their role in periodontal disease, root canal infections, infections of the hard and soft oral tissue, as well as their importance as foci for disseminated infectious disease is well established. Despite the ubiquitous involvement of bacteria, significant progress in our understanding of specific microbial etiologies has occurred only in the past decade. Estimates of the number of species recovered from samples of subgingival plaque range from 250 to 400, a large portion made up by anaerobes. Common anaerobic isolates include Fusobacterium, Bacteroides, Actinomyces, Peptococcus, Peptostreptococcus, Selenomonas, Eubacterium, Propionibacterium, and Treponema. Recently, several significant advances in our knowledge have set the stage for future research. First, circulating levels of hormones in pregnant women were shown to be stimulatory to Bacteroides species, which were associated with increased levels of gingival infection. Second, bacterial invasion of the soft and hard periodontal tissues has been documented in gingivitis, advanced periodontitis, and localized juvenile periodontitis. The frequency and identity of invading bacteria will determine the implications for diagnosis and treatment. Third, antibacterial "probes" aimed at anaerobic (and capnophilic) bacteria have had promising results in controlling and arresting oral, dental, and peridontal anaerobic infections.

Anti-Bacterial Agents↗
PubMedDate not supplied

Role of anaerobic bacteria in bite-wound infections.

The etiologic agents usually involved in wound infections due to human or animal bites are the aerobic skin flora of the victim, e.g., Staphylococcus aureus, and/or the aerobic oral flora of the biter, e.g., Pasteurella multocida. While anaerobic bacteria are predominant in the normal oral flora of humans and animals, their importance in the pathogenesis of bite-wound infections has not been stressed. Most investigators in this field have either not cultured these wounds for anaerobic bacteria or not utilized optimal culture techniques. In a series of studies on human and animal bite wounds, methods that are optimal for recovery of anaerobic bacteria were used. Anaerobes were found in significant quantities in 39% of animal bite wounds, 50% of human bite wounds, and 56% of clenched-fist injuries. Several species of anaerobes usually were present in the wounds and always were present in mixed culture with aerobic oral flora. The anaerobes most commonly isolated included Bacteroides asaccharolyticus, Bacteroides bivius, Bacteroides disiens, Bacteroides melaninogenicus, Bacteroides oralis, Bacteroides ruminicola, Bacteroides pneumosintes, Bacteroides ureolyticus, Fusobacterium nucleatum, Fusobacterium russii, Peptococcus species, Peptostreptococcus species, and Veillonella species. Initial, empiric antimicrobial therapy for bite wounds should be directed against potential anaerobic as well as aerobic pathogens.

Animals↗
PubMedDate not supplied

Anaerobes as normal oral flora.

The human mouth provides a suitable habitat for numerous bacterial genera. Anaerobic genera or genera that include anaerobic members found in the oral cavity are Actinomyces, Arachnia, Bacteroides, Bifidobacterium, Eubacterium, Fusobacterium, Lactobacillus, Leptotrichia, Peptococcus, Peptostreptococcus, Propionibacterium, Selenomonas, Treponema, and Veillonella. The incidence of anaerobes varies with age of the individual and with specific sites sampled. In edentulous infants, the incidence of anaerobes is relatively low. In adults, anaerobes are invariably present but are more prevalent in samples from the gingival sulcus than they are in samples from the gingival margin, tooth surfaces, buccal mucosa, tongue, or saliva. In samples from the healthy gingival sulcus, anaerobic, gram-positive bacilli are found in the range of 5%-14%; gram-negative bacilli in the range of 13%-29%; Veillonella in the range of 2%-8%; and gram-positive cocci in the range of 1%-15% of the cultivable flora. From marginal plaque and plaque from the tooth surface, gram-positive bacilli, gram-positive cocci, and Veillonella appear to be the predominant anaerobes. In saliva, Veillonella are the most numerous anaerobes.

Actinomyces↗
PubMedDate not supplied

Cefonicid in a once-daily regimen for treatment of osteomyelitis in an ambulatory setting.

Fifteen patients with bone joint infections were treated with 1.0 g of cefonicid administered intravenously or intramuscularly once daily. Single organisms isolated included Staphylococcus aureus (from six patients), Staphylococcus epidermidis (three), and Peptococcus species (one). For four patients infection was polymicrobial, and for one patient no organism was isolated. The mean duration of therapy was 40.4 days, only 10.9 days of which were spent in the hospital. The remainder of therapy was administered intramuscularly in an ambulatory setting. Therapy was successful in all 12 assessable patients. No clinical or bacteriologic relapse occurred in the follow-up period of three to 13 months. The occurrence of adverse effects prompted discontinuation of cefonicid therapy in three patients. Minimum savings in hospital-bed costs alone were $64,350, with 390 hospital days avoided. The minimum savings in work income were $10,010, with 182 days of absenteeism avoided. These data are preliminary but suggest efficacy of cefonicid in a mode of therapy that could have profound cost benefits.

Adult↗
PubMedDate not supplied

Efficacy of sulbactam plus ampicillin in gynecologic infections.

The efficacy of sulbactam plus ampicillin in the treatment of various gynecologic infections was evaluated in 24 women (median age, 35 years). Ten women had pelvic cellulitis plus vaginal cuff abscess; six, pyeloperitonitis; three, vaginal cuff abscess; three, surgical wound sepsis; one, tubo-ovarian abscess; and one, endometritis. Surgical procedures preceding infection included abdominal hysterectomy, ovarian cyst removal, ectopic pregnancy, correction of cystocele, and uterine dilatation and curettage. Twenty patients received 1 g of sulbactam plus 1 g of ampicillin per dose; four received 0.5 g of sulbactam plus 1 g of ampicillin per dose. The combination was given iv every 6 hr for three to four days and then im every 8 hr for three to five days (mean treatment duration, seven days). Pus cultures yielded Enterobacteriaceae (21 cases), enterococci (two), Bacteroides fragilis (12), other Bacteroides species (five), Peptococcus species (nine), Peptostreptococcus species (seven), and other anaerobes (five). Six infections were purely anaerobic; 18 were mixed. All but two infections were cured by both clinical and bacteriologic criteria, with no adverse reactions. Parenteral sulbactam/ampicillin seems safe and effective in the treatment of gynecologic infections of moderate severity.

Adult↗
PubMed1977

Quantitative bacteriology of the vaginal flora.

Quantitative bacteriology was performed on vaginal secretions from healthy adult women. The analysis included a single sample from 17 college students and 35 samples from five volunteers collected at intervals of three to five days throughout the menstrual cycle. Mean concentrations in all 52 specimens were 10(8.1) aerobic bacteria/g and 10(9.1) anaerobic bacteria/g. The rank of predominant organisms, according to rates of recovery in concentrations of greater than 10(5) colony-forming units/g, was anaerobic and facultative Lacrobacillus species, Peptococcus species, Bacteroides species, Staphylococcus epidermidis, Corynebacterium species, Peotostreptococcus species, and Eubacterium species. Sequential samples collected throughout the menstrual cycle showed relatively consistent mean levels of anaerobes and a significant decrease in concentrations of aerobes in premenstrual specimens compared with those in the specimens collected in the week following onset of menses. Analysis of sequential specimens from each of the five individuals showed considerable variation in species recovered. These data indicate that the vaginal flora in healthy adult women is a dynamic ecosystem in which anaerobes are usually the numerically dominant bacteria.

Adolescent↗
PubMed1981

Bacterial interference between Clostridium difficile and normal fecal flora.

Clostridium difficile has been shown to be the cause of virtually all cases of pseudomembranous colitis related to the administration of antimicrobial agents. It is possible that some antimicrobial agents alter the normal bacterial flora of the gastrointestinal tract so as to permit colonization and/or proliferation by C. difficile. The inhibitory activity of representative fecal bacteria from 23 anaerobic and aerobic genera against C. difficile was examined using two in vitro procedures. Strains of bacteria in six of the genera inhibited the multiplication of C. difficile, with Lactobacillus organisms and group D enterococci displaying the most antagonistic activity. C. difficile was examined for its ability to inhibit the multiplication of several fecal strains of anaerobic and aerobic bacteria. All eight strains of C. difficile tested inhibited the growth of particular strains of bacteria in the genera Bacteroides, Peptococcus, and Peptostreptococcus.

Clostridium↗
PubMed1984

Clinical and microbiological characterization of patients with nonspecific vaginosis associated with motile, curved anaerobic rods.

The vaginal secretions of 20 normal control subjects and 21 patients with motile, curved anaerobic rods were cultured for aerobic and anaerobic bacteria, Chlamydia trachomatis, herpes simplex virus, and Trichomonas vaginalis. Extensive histories and physical examinations of the patients and microscopic appearance and gas-liquid chromatography patterns of vaginal secretions were compared between the two groups. The patients who had motile rods in their vaginal secretions more frequently presented with a history of complaints about foul-smelling discharge (18 [86%] of 21); discharge noted during physical examination at their introitus (15 [71%] of 21); a vaginal pH greater than 4.5 (21 [100%] of 21); and a highly specific microscopic appearance of their secretions. The secretions were characterized by the absence of lactobacilli, the presence of highly motile, curved bacilli, and an increased number of background bacteria when compared with normal patients. Patients had more frequent anaerobic isolates than did controls (P less than .001), with increased numbers of Peptococcus, Peptostreptococcus, Propionibacterium, and Bacteroides species. All patients with motile bacteria in their secretions met the criteria of the syndrome of nonspecific vaginosis that has been previously described.

Adult↗