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PubMed2000

Complete amino acid sequence of Japanese chestnut agglutinin.

The complete amino acid sequence of Japanese chestnut (Castanea crenata Sieb. et Zucc.) agglutinin (CCA) was determined. Analysis by SIMS of the acidic peptide obtained by pepsin digestion revealed that the N-terminal amino acid sequence should be Acetyl-Met-Glu-Glu. Prior to sequence analysis, redetermination of cysteine residues indicated the presence of one cysteine residue per subunit. The complete sequence was determined by endoproteinase Arg-C and Achromobacter protease I digestion, and CNBr cleavage. CCA consists of 309 amino acid residues with a high content of glycine (16.5 mol%) and one cysteine residue. The calculated molecular mass was 33, 387 Da including the N-terminal acetyl group. C-terminal sequence analysis of intact CCA gave only one sequence, HMEYF, indicating that no heterogeneous CCA formed by posttranslational cleavage at the C-terminal region, as occurs in some legume lectins. Analysis of the sequence of CCA itself revealed that CCA could be divided into two structural domains, the N-domain and the C-domain, almost at the center. These domains share about 35% identical residues, so CCA has a repeat sequence. Also, both domains show a homology to jacalin-related lectins with 27-38% identity. These results suggest that the structure of CCA resembles two molecules of jacalin-related lectin.

Agglutinins↗
PubMed1997

Characterization and distribution of O-glycosylated carbohydrates in the cell adhesion molecule, contact site A, from Dictyostelium discoideum.

This paper presents further investigation of the properties of carbohydrate II in the cell adhesion molecule, contact site A, from Dictyostelium discoideum. A purified contact site A was digested with Achromobacter protease I to produce a 31-kDa fragment to which carbohydrate II was mainly bound and a 21-kDa fragment containing the NH2 terminus of contact site A, which was identified as Ala-Pro-Thr-Ile-Thr-Ala. The NH2 terminus of the 31-kDa fragment was Thr-Glu-Ala-Thr-Thr-Ser. It was estimated from the cDNA sequence data of contact site A that more than 20 Ser/Thr residues exist as target sites for the O-linked oligosaccharides in the 31-kDa fragment, but not for the N-linked oligosaccharides. These results suggest that carbohydrate II exists as clustered O-linked oligosaccharides in the COOH terminus of contact site A. The results of two-dimensional electrophoresis confirm that oligosaccharides of contact site A contain sialic acids. Immunoelectron microscopy was carried out to define the organelle in which O-glycosylation by carbohydrate II occurs and how carbohydrate II antigens are distributed on the cell surface. The results show that O-glycosylation can occur in the Golgi apparatus in D. discoideum as observed in other cells, although this O-glycosylation was inhibited by tunicamycin. Furthermore, gold particles were densely concentrated in cell-cell contact regions but sparsely distributed in noncontact regions.

Animals↗
PubMed1996

High-resolution crystal structures of two polymorphs of cytochrome c' from the purple phototrophic bacterium rhodobacter capsulatus.

The structures of two polymorphs of cytochrome c' from Rhodobacter capsulatus (RCCP) strain M110 have been determined by the molecular replacement method. Iron anomalous scattering data were used to confirm the molecular replacement solution. The structures were refined at 1.72 angstrom and 2.0 angstrom resolution to R-values of 15.0% and 16.3%, respectively. The RCCP molecule is a dimer and each of the identical 129 residue subunits folds as a four-helical bundle with a covalently bound heme group in the center. This structural motif resembles that of cytochromes c' reported from Rhodospirillum molischianum (RMCP), Rhodospirillum rubrum (RRCP), Chromatium vinosum (CVCP), Achromobacter xyloseoxidans (AXCP) and Alcaligenes denitrificans (ADCP). However, the architecture of the RCCP dimer, that is, the mode of association of subunits, differs substantially from that of the other cytochromes c'. In the RCCP dimer, the subunits are roughly parallel with each other and only helix B of each subunit participates in formation of the dimer interface. Measurement of the solvent-accessible surface area indicates that the dimer interface is smaller in RCCP than in the other cytochromes c'. In RMCP, CVCP, RRCP, AXCP and ADCP the subunits cross each other to form an X shape, and two helices, A and B, of each subunit interact across the dimer interface. These results are consistent with hydrodynamic measurements, which show that there is an equilibrium between monomers and dimer in RCCP, whereas the dimer is the predominant form in the other cytochromes c' for which structures have been determined. Structural comparison of the six cytochromes c' reveal that they can be divided into two groups. In group 1 cytochromes c', CVCP and RCCP, the amino acid sequences and the folding of subunits are arranged in such a way as to allow the formation of a deep channel between helices B and C with direct solvent accessibility to the heme sixth ligand position. There is no such channel in group 2 cytochromes c', RMCP, RRCP, AXCP and ADCP. This may account, in part, for the differences in carbon monoxide binding.

Amino Acid Sequence↗
PubMed1998

Prepro-leaders lacking N-linked glycosylation for secretory expression in the yeast Saccharomyces cerevisiae.

Synthetic prepro-leaders lacking consensus N-linked glycosylation sites confers secretion competence of correctly folded insulin precursor expressed in the yeast species Saccharomyces cerevisiae with a yield comparable to, or better than the alpha-factor prepro-leader. In contrast, the S. cerevisiae alpha-factor prepro-leader's three N-linked oligosaccharide chains are necessary for the ability to facilitate secretion of the insulin precursor from S. cerevisiae (T. Kjeldsen et al., Biotechnol. Appl. Biochem. 27, 109-115, 1998). Synthetic prepro-leader lacking both N-glycosylation and the dibasic Kex2 endoprotease processing site also efficiently facilitated secretion of a pro-leader/insulin precursor fusion protein in which the insulin precursor was correctly folded. The unprocessed pro-leader/insulin-precursor fusion protein was purified from culture medium and matured in vitro to desB30 insulin by Achromobacter lyticus lysyl-specific protease providing an alternative yeast expression system not dependent on the Kex2 endoprotease. The synthetic prepro-leader lacking N-linked glycosylation provides the opportunity for secretory expression in yeast utilizing either in vivo Kex2 endoprotease maturation of the fusion protein during secretion or in vitro maturation of the purified fusion protein with a suitable enzyme.

Alcaligenes↗
PubMed1995

Screening and characterization of microorganisms with glutaryl-7ADCA acylase activity.

A screening of microorganisms producing glutaryl-7ADCA acylase, an enzyme able to hydrolyse glutaric acid selectively from glutaryl-3-deacetoxy-7-aminocephalosporanic acid (glutaryl-7ADCA), has been carried out in soil samples. Five microorganisms expressing acylase activity were isolated and classified as Bacillus cereus, Achromobacter xylosooxidans, Bacillus sp., Pseudomonas sp. and Pseudomonas paucimobilis. The screening was carried out by preparing enrichment cultures containing glutaryl-7-ADCA or cephalosporin C as the selective carbon source. Four model compounds (adipoyl-, glutamyl- and glutaryl-p-nitroanilide and glutarylcoumarin), mimicking the glutaryl-7ADCA beta-lactam moiety, were synthesized as substrates suitable for the rapid screening of the microorganisms (2500) isolated from the enrichment cultures. A total of 300 strains were active on the model substrates and only 5 displayed acylase activity on glutaryl-7ADCA. The fermentation parameters, such as pH and inducer concentration, for the optimal acylase expression and acylase specificity towards the model substrates were different for each strain.

Amidohydrolases↗
PubMed1993

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd1-free background (NirS-) of Pseudomonas stutzeri.

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of "Achromobacter cycloclastes" nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd1 nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d1, indicating that both heterologous hosts synthesized a cytochrome cd1 without the d1-group.

Amino Acid Sequence↗
PubMed1983

In vitro activity of new cephalosporins against multi-resistant gram-negative bacteria.

The in vitro sensitivities of 47 multi-resistant gram-negative bacilli against six third generation cephalosporins were investigated. Overall, moxalactam was the most active compound and cefoperazone the least active. Ceftizoxime and ceftazidime were very active against Enterobacteriaceae. Ceftriaxone and ceftizoxime were active against Escherichia coli and Klebsiella spp., but were inactive against Pseudomonas spp.; Achromobacter sp. was resistant to all drugs, while Citrobacter freundii was sensitive to every antibiotic tested. These drugs may be clinically useful in infections with multi-resistant gram-negative bacteria.

Aeromonas↗
PubMedDate not supplied

Ochrobactrum anthropi bacteremia: report of four cases and short review.

Ochrobactrum anthropi, formerly "Achromobacter" CDC group Vd, is a nonfermentative, nonfastidious gram-negative bacillus, that only recently has been given attention as a potential human pathogen. Over a 2-year period, we observed four patients with multiple blood cultures that were positive for the organism. The patients had acute leukemia as underlying disease, and presented with clinical and microbiologic features consistent with catheter-related bacteremia. In three of the patients the infection initially appeared to be unrelated to chemotherapy-associated profound neutropenia and occurred early after, or was the reason for, hospital admission. The antimicrobial susceptibility of the isolates varied: unlike previously reported cases, resistance in some of our isolates included aminoglycosides, newer fluoroquinolones, and trimethoprim-sulfamethoxazole. Despite in vitro susceptibility to imipenem in initial isolates, treatment of two patients with this agent obviously failed to eradicate the organism, and the patients either relapsed with bacteremia shortly after discontinuation of treatment or remained persistently febrile and bacteremic. O. anthropi appears to be increasingly recognized as a human opportunist pathogen associated with intravascular catheters and unpredictable multiple antibiotic resistance.

Adult↗
PubMed1975

Bacterial degradation of polychlorinated biphenyls II. Rate studies.

Polychlorinated biphenyls (Aroclor 1221, 1242 and 1254) at concentrations up to 0.1% in glucose did not inhibit the growth of lake water bacteria. The bacteria used Aroclor 1221 and 1242 but not 1254 as sole carbon and energy sources for growth. Less than 1% of lake water bacteria, however, possess this ability. Seven bacterial isolates from Aroclor agar plates were identified; five belonged to Achromobacter sp. and two were Pseudomonas sp. The metabolic breaksown of Aroclor 1221 was followed. THE MIXTURE WAS COMPLETELY DEGRADED INTO SEVERAL LOW MOLECULAR WEIGHT COMPOUNDS AFTER ONE MONTH INCUBATION. Unchlorinated biphenyl was degraded at a faster rate than 2-chlorobiphenyl and 4-chlorobiphenyl isomers.

Bacteria↗
PubMed1978

Bacterial utilization of cyclo(glycl-L-prolyl).

3 strains of soil bacteria (2 Achromobacter, 1 Flavobacterium) were isolated by growth on cyclo(Gly-L-Pro) as carbon/nitrogen source. Good growth required yeast extract supplements. Utilization of cyclo(Gly-L-Pro) was inducible. Many efforts failed to obtain active cell-free preparations. Injected radioactive cyclo(Gly-L-Pro) was excreted intact by the albino rat; in contrast, injected radioactive H-Gly-L-Pro-OH was extensively metabolized.

Dipeptides↗
PubMed1990

Inhibitory effect of halocyamine, an antimicrobial substance from ascidian hemocytes, on the growth of fish viruses and marine bacteria.

Halocyamine A, an antimicrobial substance isolated from hemocytes of the solitary ascidian Halocynthia roretzi, inhibited in vitro the growth of fish RNA viruses (infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus). Pretreatment of RNA virus with halocyamine A reduced the infectivity of the virus toward host cells. The growth of marine bacteria, Achromobacter aquamarinus and Pseudomonas perfectomarinus, was also inhibited by halocyamine A but that of Alteromonas putrefaciens and Vibrio anguillarum was not. These results suggest that halocyamine may have a role in the defense mechanisms of H. roretzi against marine viruses and bacteria.

Animals↗
PubMed1988

Comparative in vitro activity of the new oral cephalosporin cefixime.

Cefixime was 8 to 10 times more active than cefaclor and augmentin against isolates of Escherichia coli, Klebsiella pneumoniae and Salmonella typhi, MIC90 values ranging from 0.06 to 0.25 micrograms/ml. However, none of these three drugs was particularly active against isolates of more resistant gram-negative bacilli such as Enterobacter, Serratia, Citrobacter, Acinetobacter, Providencia and Achromobacter spp. The lowest MIC values for gram-negative bacilli were seen with ciprofloxacin, except for isolates of Acinetobacter, where cotrimoxazole was the most active of the five drugs studied. Augmentin and ciprofloxacin exhibited the lowest MICs for isolates of streptococci and corynebacteria. Although cefixime may be among the most active oral beta-lactam drugs, it does not appear to be useful for treatment of infections caused by more resistant gram-negative bacilli.

Amoxicillin↗
PubMedDate not supplied

Isolation and partial characterization of a 46-kd allergen of Bermuda grass pollen.

Cyn d Bd46K, a 46-kD component of Bermuda grass (Cynodon dactylon) pollen, had been identified as an allergenic constituent. In the present study two-dimensional (2D) gel electrophoresis illustrated the presence of five acidic isoforms in Cyn d Bd46K, and this molecule was purified by monoclonal antibody (MAb) affinity chromatography for further characterization. Using a digoxigenin-labeled lectin-binding assay, the elucidating protein was disclosed to be a glycoprotein with terminal mannose. The involvement of a carbohydrate moiety in the allergenicity and antigenicity of the elucidated molecule was demonstrated with sodium-periodate-treated Cyn d Bd46K, which reduced binding to its specific MAb and human IgE. We were unable to identify the N-terminal amino acid sequences of Cyn d Bd46K, but some internal amino acid sequences were disclosed by microsequencing some fragments cleaved by Achromobacter protease I and fractionated by reversed-phase column chromatography. The amino acid sequences of 4 identified Cyn d Bd46K internal peptide fragments were found to be 25-71% identical with that of cytochrome c oxidase III from corn grass pollen. The present study provided important information for future experiments on the molecular cloning of the elucidated allergen.

Allergens↗
PubMed2003

Antibiotic treatment of multidrug-resistant organisms in cystic fibrosis.

Respiratory tract infection with eventual respiratory failure is the major cause of morbidity and mortality in cystic fibrosis (CF). Infective exacerbations need to be treated promptly and effectively to minimize potentially accelerated attrition of lung function. The choice of antibiotic depends on in vitro sensitivity patterns. However, physicians treating patients with CF are increasingly faced with infection with multidrug-resistant isolates of Pseudomonas aeruginosa. In addition, innately resistant organisms such as Burkholderia cepacia complex, Stenotrophomonas maltophilia and Achromobacter (Alcaligenes) xylosoxidans are becoming more prevalent. Infection with methicillin-resistant Staphylococcus aureus (MRSA) is also a problem. These changing patterns probably result from greater patient longevity and increased antibiotic use for acute exacerbations and maintenance care. Multidrug-resistant P. aeruginosa infection may be treated successfully by using two antibiotics with different mechanisms of action. In practice antibiotic choices have usually been made on a best-guess basis, but recent research suggests that more directed therapy can be achieved through the application of multiple-combination bactericidal testing (MCBT). Aerosol delivery of tobramycin for inhalation solution achieves high endobronchial concentrations that may overcome bacterial resistance as defined by standard laboratory protocols. Resistance to colistin is rare and this antibiotic should be seen as a valuable second-line drug to be reserved for multidrug-resistant P. aeruginosa. The efficacy of new antibiotic groups such as the macrolides needs to be evaluated.CF units should adopt strict segregation policies to interrupt person-to-person spread of B. cepacia complex. Treatment of panresistant strains is difficult and often arbitrary. Combination antibiotic therapy is recommended, usually tobramycin and high-dose meropenem and/or ceftazidime, but the choice of treatment regimen should always be guided by the clinical response.The clinical significance of S. maltophilia, A. xylosoxidans and MRSA infection in CF lung disease remains uncertain. If patients show clinical decline and are chronically colonized/infected with either of the former two pathogens, treatment is recommended but efficacy data are lacking. There are defined microbiological reasons for attempting eradication of MRSA but there are no proven deleterious effects of this infection on lung function in patients with CF. Various treatment protocols exist but none has been subject to a randomized, controlled trial. Multidrug-resistant microorganisms are an important and growing issue in the care of patients with CF. Each patient infected with such strains should be assessed individually and antibiotic treatment planned according to in vitro sensitivity, patient drug tolerance, and results of in vitro studies which may direct the physician to antibiotic combinations most likely to succeed.

Anti-Bacterial Agents↗
PubMed2006

Biotransformation of isoeugenol to vanillin by Pseudomonas putida IE27 cells.

The ability to produce vanillin and/or vanillic acid from isoeugenol was screened using resting cells of various bacteria. The vanillin- and/or vanillic-acid-producing activities were observed in strains belonging to the genera Achromobacter, Aeromonas, Agrobacerium, Alcaligenes, Arthrobacter, Bacillus, Micrococcus, Pseudomonas, Rhodobacter, and Rhodococcus. Strain IE27, a soil isolate showing the highest vanillin-producing activity, was identified as Pseudomonas putida. We optimized the culture and reaction conditions for vanillin production from isoeugenol using P. putida IE27 cells. The vanillin-producing activity was induced by adding isoeugenol to the culture medium but not vanillin or eugenol. Under the optimized reaction conditions, P. putida IE27 cells produced 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20 degrees C after a 24-h incubation in the presence of 10% (v/v) dimethyl sulfoxide.

Benzaldehydes↗
PubMed1997

D-lysine production from L-lysine by successive chemical racemization and microbial asymmetric degradation.

In order to develop a practical process for D-lysine production from L-lysine, successive chemical racemization and microbial asymmetric degradation were investigated. The racemization of L-lysine proceeded quantitatively at elevated temperatures. A sample of 1000 strains of bacteria, fungi, yeast and actinomyces were screened for the ability to degrade L-lysine asymmetrically. Microorganisms belonging to the Achromobacter, Agrobacterium, Candida, Comamonas, Flavobacterium, Proteus, Providencia, Pseudomonas and Yarrowia genera exhibited a high L-lysine-degrading activity. Comamonas testosteroni IAM 1048 was determined to be the best strain and used as a biocatalyst for eliminating the L isomer. The degradation rate of L-lysine with C. testosteroni IAM 1048 was influenced by pH, temperature and agitation speed. Under the optimal conditions, the L isomer in a 100-g/l mixture of racemic lysine was completely degraded within 72 h, with 47 g D-lysine/l left in the reaction mixture. Crystalline D-lysine, with a chemical purity greater than 99% and optical purity of 99.9% enantiomeric excess, was obtained at a yield of 38% from the reaction mixture by simple purification. An engineering analysis of L-lysine racemization and microbial degradation was carried out to establish the basis of process design for D-lysine production.

Biodegradation, Environmental↗
PubMed2002

Metal-ligand interactions in perturbed blue copper sites: a paramagnetic (1)H NMR study of Co(II)-pseudoazurin.

Pseudoazurin is an electron transfer copper protein, a member of the cupredoxin family. The protein is frequently found in denitrifying bacteria, where it is the electron donor of nitrite reductase. The copper at the active site is coordinated to His40, Cys78, His81 and Met86 in a distorted tetragonal geometry. We have recorded and assigned the (1)H NMR spectra of Co(II)-substituted pseudoazurin from Achromobacter cycloclastes. The (1)H NMR spectrum of Co(II)-pseudoazurin closely resembles that of Co(II)-rusticyanin, reflecting an altered conformation for the Met-Co(II)-Cys moiety in both proteins, compared to Co(II)-azurin, amicyanin and stellacyanin. The electron spin density onto the Sgamma(Cys) is larger in Co(II)-pseudoazurin compared to Co(II)-rusticyanin. Instead, the Co(II)-Met interaction is similar in both derivatives. Hence, the different metal-ligand interactions might be independently modulated by the protein structure. The present work also shows that the electron spin density onto the Co(II)-S(cys) bond is sensibly smaller than the Cu(II)-S(cys). Notwithstanding, NMR data on Co(II)-substituted blue copper proteins can be safely extrapolated to native Cu(II) proteins.

Alcaligenes↗
PubMed2006

Analysis of the composition of bacterial communities in oil reservoirs from a southern offshore Brazilian basin.

The aim of this study was to characterize and compare the bacterial community structure of two distinct oil samples from a petroleum field in Brazil by using both molecular, based on the construction of 16S rRNA gene libraries, and cultivation methods. Statistical comparisons of libraries based on Amplified Ribosomal DNA Restriction Analysis (ARDRA) data revealed no significant differences between the communities recovered in the non-biodegraded (NBD) and highly biodegraded oils (HBD). BlastN analysis of the 16S rRNA gene sequences representative of distinct ribotypes from both oils showed the presence of nine different bacterial genera in these samples, encompassing members of the genera Arcobacter, Halanaerobium, Marinobacter, Propionibacterium, Streptomyces, Leuconostoc, Acinetobacter, Bacillus and Streptococcus. Enrichments obtained using oil as inoculum and sole carbon source yielded bacterial isolates showing high 16S rRNA gene sequence similarity with Achromobacter xylosoxidans, Bacillus subtilis, Brevibacillus sp., Dietzia sp. and Methylobacterium sp. Comparison between the data obtained using cultivation-independent and enrichment cultures suggests that different selection of community members may occur when using distinct approaches. All the organisms found, except for Leuconostoc sp. and Streptococus sp., have been previously reported in the literature as hydrocarbon degraders and/or associated to oil field environments.

Bacteria↗