Achromobacter

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 251 - 256
Advenella incenata gen . nov., sp . nov., a novel member of the Alcaligenaceae, isolated from various clinical samples; Coenye T et al.; A polyphasic taxonomic study of 14 isolates recovered from various human and veterinary clinical samples was performed . Phenotypically these isolates shared several characteristics with members of the Alcaligenaceae and related genera . Random amplified polymorphic DNA fingerprinting and whole-cell protein analysis suggested the presence of multiple genomic groups, which was confirmed by DNA-DNA hybridization experiments . 16S rRNA gene sequence analysis indicated that these isolates were related to the genera Pelistega, Taylorella, Oligella, Pigmentiphaga, Alcaligenes, Kerstersia, Achromobacter and Bordetella and belonged to the family Alcaligenaceae . Based on the results of the present study the organisms were classified in a novel genus, Advenella gen . nov . This genus comprises one named species, Advenella incenata sp . nov . (type strain LMG 22250(T)=CCUG 45225(T)) and five currently unnamed genomic species . The DNA G+C content of members of the novel genus Advenella is between 54.0 and 57.7 mol%.

Antimicrob Agents Chemother, 2005 Jan, 49(1), 104 - 10
Clonal relatedness and conserved integron structures in epidemiologically unrelated Pseudomonas aeruginosa strains producing the VIM-1 metallo-{beta}-lactamase from different Italian hospitals; Riccio ML et al.; Three epidemiologically independent Pseudomonas aeruginosa isolates, representative of the first VIM-1 metallo-beta-lactamase producers detected at three different hospitals in northern Italy, were investigated to determine their genomic relatedness and to compare the structures of the genetic supports for the VIM-1 determinants . The three isolates, all of serotype O11, appeared to be clonally related according to the results of genotyping by macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis and random amplification of polymorphic DNA . Investigation of the genetic support for the bla(VIM-1) determinant revealed that it was carried on identical or almost identical integrons (named In70.2 and In70.3) located within a conserved genomic context . The integrons were structurally related to In70 and In110, two plasmid-borne bla(VIM-1)-containing integrons from Achromobacter xylosoxidans and Pseudomonas putida isolates, respectively, from the same geographic area (northern Italy) and were found to be inserted close to the res site of a Tn5051-like transposon, different from any of those described previously, that was apparently carried on the bacterial chromosome . The present findings suggest that the three VIM-1-producing isolates are members of the same clonal complex which have been spreading in hospitals in northern Italy since the late 1990s and point to a common ancestry of their bla(VIM-1)-containing integrons.

Appl Environ Microbiol, 2004 Dec, 70(12), 7466 - 73
Cyclo(L-leucyl-L-prolyl) produced by Achromobacter xylosoxidans inhibits aflatoxin production by Aspergillus parasiticus; Yan PS et al.; Aflatoxins are potent carcinogenic and toxic substances that are produced primarily by Aspergillus flavus and Aspergillus parasiticus . We found that a bacterium remarkably inhibited production of norsolorinic acid, a precursor of aflatoxin, by A . parasiticus . This bacterium was identified as Achromobacter xylosoxidans based on its 16S ribosomal DNA sequence and was designated A . xylosoxidans NFRI-A1 . A . xylosoxidans strains commonly showed similar inhibition . The inhibitory substance(s) was excreted into the medium and was stable after heat, acid, or alkaline treatment . Although the bacterium appeared to produce several inhibitory substances, we finally succeeded in purifying a major inhibitory substance from the culture medium using Diaion HP20 column chromatography, thin-layer chromatography, and high-performance liquid chromatography . The purified inhibitory substance was identified as cyclo(L-leucyl-L-prolyl) based on physicochemical methods . The 50% inhibitory concentration for aflatoxin production by A . parasiticus SYS-4 (= NRRL2999) was 0.20 mg ml(-1), as determined by the tip culture method . High concentrations (more than 6.0 mg ml(-1)) of cyclo(L-leucyl-L-prolyl) further inhibited fungal growth . Similar inhibitory activities were observed with cyclo(D-leucyl-D-prolyl) and cyclo(L-valyl-L-prolyl), whereas cyclo(D-prolyl-L-leucyl) and cyclo(L-prolyl-D-leucyl) showed weaker activities . Reverse transcription-PCR analyses showed that cyclo(L-leucyl-L-prolyl) repressed transcription of the aflatoxin-related genes aflR, hexB, pksL1, and dmtA . This is the first report of a cyclodipeptide that affects aflatoxin production.

Appl Microbiol Biotechnol . 2004 Nov 12; {Epub ahead of print}
Phosphonium ionic liquids for degradation of phenol in a two-phase partitioning bioreactor; Baumann MD et al.; Six ionic liquids (ILs), which are organic salts that are liquid at room temperature, were tested for their biocompatibility with three xenobiotic-degrading bacteria, Pseudomonas putida, Achromobacter xylosoxidans, and Sphingomonas aromaticivorans . Of the 18 pairings, seven were found to demonstrate biocompatibility, with one IL (trihexyl(tetradecyl)phosphonium bis(trifluoromethylsulfonyl) amide) being biocompatible with all three organisms . This IL was then used in a two-phase partitioning bioreactor (TPPB), consisting of 1 l aqueous phase loaded with 1,580 mg phenol and 0.25 l IL, inoculated with the phenol degrader P . putida . This initially toxic aqueous level of phenol was substantially reduced by phenol partitioning into the IL phase, allowing the cells to utilize the reduced phenol concentration . The partitioning of phenol from the IL to the aqueous phase was driven by cellular demand and thermodynamic equilibrium . All of the phenol was consumed at a rate comparable to that of previously used organic-aqueous TPPB systems, demonstrating the first successful use of an IL with a cell-based system . A quantitative (31)P NMR spectroscopic assay for estimating the log P values of ILs is under development.

J Antimicrob Chemother, 2004 Dec, 54(6), 1057 - 61 Epub 2004 Oct 27.
Pitfalls of polymyxin antimicrobial susceptibility testing of Pseudomonas aeruginosa isolated from cystic fibrosis patients; Hogardt M et al.; Objectives and methods: With their potent activity against Gram-negative bacteria, the polymyxins are important alternative antibiotics for cystic fibrosis (CF) patients . A retrospective evaluation of polymyxin activity against 6001 Pseudomonas aeruginosa, 150 Achromobacter xylosoxidans and 506 Stenotrophomonas maltophilia CF isolates was initiated . In addition, we looked at how polymyxin susceptibility testing was affected by the testing method (agar dilution versus microdilution), the agent (polymyxin E versus polymyxin B), incubation time (24 h versus 48 h) and by different interpretative criteria (German DIN, French FSM, British BSAC) . RESULTS: Polymyxin B exhibited reasonable activity against P . aeruginosa (MIC(90)</=2 mg/L), whereas it was less active against A . xylosoxidans (MIC(90)</=16 mg/L) and S . maltophilia (MIC(90)</=16 mg/L) . During 2000-2002, polymyxin B resistance in P . aeruginosa, S . maltophilia and A . xylosoxidans was found to be 6.7%, 17.0% and 29.9% (corresponding to 12.4%, 20.7% and 35.4% of infected patients), respectively . When the agar dilution method was used, polymyxin E exhibited higher MICs than polymyxin B . The microdilution method produced lower polymyxin MICs than the agar dilution method . Therefore, the microdilution MICs after prolonged incubation (48 h) and the agar dilution MICs of polymyxin B correlated best (AUC of 0.93, r(2) of 0.44 and s of 0.83) . CONCLUSIONS: Polymyxin resistance among common CF pathogens is not rare, thus underlining the necessity of accurate susceptibility testing . When compared with the agar dilution method, it was found that the microdilution method is a valid, rapid and cost effective alternative for the determination of polymyxin activity . The performance of the microdilution method was most reliable after prolonged incubation (48 h) at a susceptibility breakpoint of </=4 mg/L according to the BSAC guidelines (specificity 91%, sensitivity 89%, 1.5% very major errors).

Toxicon, 2004 Dec 1, 44(7), 711 - 21
Amino acid sequence of a thrombin like enzyme, elegaxobin II, from the venom of Trimeresurus elegans (Sakishima-Habu); Oyama E et al.; The amino acid sequence of a thrombin like enzyme , named elegaxobin II, isolated from the venom of Trimeresurus elegans (Sakishima-habu) was determined by Edman sequencing of the peptides which was derived from digests with cyanogen bromide, achromobacter protease I, trypsin, endoproteinase Asp-N, and chymotrypsin . Elegaxobin II consisted of 233 amino acids and showed conservation of the catalytic amino acid residues (His(57), Asp(102), and Ser(195)) of chymotrypsin family serine protease in its amino acid sequence . The carboxyterminal amino acid, Leu, was determined using carboxypeptidase Y . This enzyme contains glucosamine and an N-linked glycosylation site . Elegaxobin II was 91% homologous in sequence to elegaxobin and protease I from the same snake venom, and it was 67, 75, 31 and 26% homologous in sequences to flavoxobin, KN-BJ 2, human kallikrein and bovine thrombin, respectively . Elegaxobin II lacked thrombin's ETW (146-148) loop, as well as its functionally important YPPW (60-insertion loop).

J Biol Chem, 2004 Dec 24, 279(52), 54161 - 72 Epub 2004 Oct 15.
Protein sequence analysis, cloning, and expression of flammutoxin, a pore-forming cytolysin from Flammulina velutipes . Maturation of dimeric precursor to monomeric active form by carboxyl-terminal truncation; Tomita T et al.; Flammutoxin (FTX), a 31-kDa pore-forming cytolysin from Flammulina velutipes, is specifically expressed during the fruiting body formation . We cloned and expressed the cDNA encoding a 272-residue protein with an identical N-terminal sequence with that of FTX but failed to obtain hemolytically active protein . This, together with the presence of multiple FTX family proteins in the mushroom, prompted us to determine the complete primary structure of FTX by protein sequence analysis . The N-terminal 72 and C-terminal 107 residues were sequenced by Edman degradation of the fragments generated from the alkylated FTX by enzymatic digestions with Achromobacter protease I or Staphylococcus aureus V8 protease and by chemical cleavages with CNBr, hydroxylamine, or 1% formic acid . The central part of FTX was sequenced with a surface-adhesive 7-kDa fragment, which was generated by a tryptic digestion of FTX and recovered by rinsing the wall of a test tube with 6 M guanidine HCl . The 7-kDa peptide was cleaved with 12 M HCl, thermolysin, or S . aureus V8 protease to produce smaller peptides for sequence analysis . As a result, FTX consisted of 251 residues, and protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein . Recombinant FTX (rFTX) with or without the C-terminal 20 residues (rFTX271 or rFTX251, respectively) was prepared to study the maturation process of FTX . Like natural FTX, rFTX251 existed as a monomer in solution and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes, causing hemolysis . In contrast, rFTX271, existing as a dimer in solution, bound to the cells but failed to form pore complex . The dimeric rFTX271 was converted to hemolytically active monomers upon the cleavage between Lys(251) and Met(252) by trypsin.

J Biol Chem, 2004 Dec 17, 279(51), 53374 - 8 Epub 2004 Oct 07.
Structure-based engineering of Alcaligenes xylosoxidans copper-containing nitrite reductase enhances intermolecular electron transfer reaction with pseudoazurin; Kataoka K et al.; The intermolecular electron transfer from Achromobacter cycloclastes pseudoazurin (AcPAZ) to wild-type and mutant Alcaligenes xylosoxidans nitrite reductases (AxNIRs) was investigated using steady-state kinetics and electrochemical methods . The affinity and the electron transfer reaction constant (k(ET)) are considerably lower between AcPAZ and AxNIR (K(m) = 1.34 mM and k(ET) = 0.87 x 10(5) M(-1) s(-1)) than between AcPAZ and its cognate nitrite reductase (AcNIR) (K(m) = 20 microM and k(ET) = 7.3 x 10(5) M(-1) s(-1)) . A negatively charged hydrophobic patch, comprising seven acidic residues around the type 1 copper site in AcNIR, is the site of protein-protein interaction with a positively charged hydrophobic patch on AcPAZ . In AxNIR, four of the negatively charged residues (Glu-112, Glu-133, Glu-195, and Asp-199) are conserved at the corresponding positions of AcNIR, whereas the other three residues are not acidic amino acids but neutral amino acids (Ala-83, Ala-191, and Gly-198) . Seven mutant AxNIRs with additional negatively charged residues surrounding the hydrophobic patch of AxNIR (A83D, A191E, G198E, A83D/A191E, A93D/G198E, A191E/G198E, and A83D/A191E/G198E) were prepared to enhance the specificity of the electron transport reaction between AcPAZ and AxNIR . The k(ET) values of these mutants become progressively larger as the number of mutated residues increases . The K(m) and k(ET) values of A83D/A191E/G198E (K(m) = 88 microM and k(ET) = 4.1 x 10(5) M(-1) s(-1)) are 15-fold smaller and 4.7-fold larger than those of wild-type AxNIR, respectively . These results suggest that the introduction of negatively charged residues into the docking surface of AxNIR facilitates both the formation of electron transport complex and the electron transfer reaction.

Appl Microbiol Biotechnol, 2004 Oct, 65(5), 620 - 6 Epub 2004 Jul 28.
Mesophilic aerobic degradation of a metal lubricant by a biological consortium; Iwashita S et al.; The metal-forming industries require the use of greases to lubricate metal surfaces during manufacturing operations, and the residues of these lubricants must be removed prior to finishing processes to protect and improve the appearance of the final product . An aqueous, biological metal-cleaning process operating under mild conditions (pH 9, 42 degrees C) eliminates the use of environmentally unfriendly cleaning materials such as chlorinated solvents by employing microorganisms to degrade greases and oils naturally . This process was characterized in terms of initial degradation rates of a representative metal lubricant and by phylogenetic identification of the active bacteria . The metal lubricant in a surfactant solution was degraded by a bacterial consortium, and its concentration was determined by a novel gas chromatography assay . The maximum degradation rate Vmax and the apparent Km were obtained as 45 mg/(day mg protein) and 24 g/l on cellular basis, and 4.6 g/(day l) and 33 g/l on a volumetric basis, respectively . Mineralization of the metal lubricant was shown by analyzing the evolved CO2 and Cl-, and the bacterial consortium utilized the metal lubricant as a sole carbon and energy source (micro=0.05+/-0.01 h(-1) at 0.5 vol% lubricant concentration) . The active bacteria in the biological metal-cleaning process were identified as Bacillus licheniformis for the higher lubricant concentrations (3, 5, and 7.5 vol%), Bacillus cereus at 1 vol%, and Pseudomonas aeruginosa, Rhizobiaceae strain M100, and Achromobacter sp . LMG 5431 at 0.3 vol%.

Chemosphere, 2004 Nov, 57(5), 401 - 12
Bacterial communities and enzyme activities of PAHs polluted soils; Andreoni V et al.; Three soils (i.e . a Belgian soil, B-BT, a German soil, G, and an Italian agricultural soil, I-BT) with different properties and hydrocarbon-pollution history with regard to their potential to degrade phenanthrene were investigated . A chemical and microbiological evaluation of soils was done using measurements of routine chemical properties, bacterial counts and several enzyme activities . The three soils showed different levels of polycyclic aromatic hydrocarbons (PAHs), being their contamination strictly associated to their pollution history . High values of enzyme activities and culturable heterotrophic bacteria were detected in the soil with no or negligible presence of organic pollutants . Genetic diversity of soil samples and enrichment cultures was measured as bands on denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences from the soil and enrichment community DNAs . When analysed by Shannon index (H'), the highest genetic biodiversity (H'=2.87) was found in the Belgian soil B-BT with a medium-term exposition to PAHs and the poorest biodiversity (H'=0.85) in the German soil with a long-term exposition to alkanes and PAHs and where absence, or lower levels of enzyme activities were measured . For the Italian agricultural soil I-BT, containing negligible amounts of organic pollutants but the highest Cu content, a Shannon index=2.13 was found . The enrichment of four mixed cultures capable of degrading solid phenanthrene in batch liquid systems was also studied . Phenanthrene degradation rates in batch systems were culture-dependent, and simple (one-slope) and complex (two-slope) kinetic behaviours were observed . The presence of common bands of microbial species in the cultures and in the native soil DNA indicated that those strains could be potential in situ phenanthrene degraders . Consistent with this assumption are the decrease of PAH and phenanthrene contents of Belgian soil B-BT and the isolation of phenanthrene-degrading bacteria . From the fastest phenanthrene-degrading culture C(B-BT), representative strains were identified as Achromobacter xylosoxidans (100%), Methylobacterium sp . (99%), Rhizobium galegae (99%), Rhodococcus aetherovorans (100%), Stenotrophomonas acidaminiphila (100%), Alcaligenes sp . (99%) and Aquamicrobium defluvium (100%) . DGGE-profiles of culture C(B-BT) showed bands attributable to Rhodococcus, Achromobacter, Methylobacterium rhizobium, Alcaligenes and Aquamicrobium . The isolation of Rhodococcus aetherovorans and Methylobacterium sp . can be consistent with the hypothesis that different phenanthrene-degrading strategies, cell surface properties, or the presence of xenobiotic-specific membrane carriers could play a role in the uptake/degradation of solid phenanthrene.

Acta Crystallogr D Biol Crystallogr, 1996 Sep, 52(Pt 5), 1027 - 9
Crystallization and preliminary X-ray diffraction analysis of two lysinal derivatives of Achromobacter protease I; Oda Y; Two crystal forms of lysinal derivatives of Achromobacter protease I have been obtained . The first, modified by benzyloxycarbonyl-Val-lysinal crystallizes in the monoclinic space group P2(1) with unit-cell dimensions of a = 39.6, b = 71.2, c = 45.6 A and beta = 98.4 degrees . The second, modified by benzyloxycarbonyl-Leu-Leu-lysinal crystallizes in the orthorhombic space group I222 (or I2(1)2(1)2(1)) with unit-cell dimensions of a = 98.7, b = 102.2 and c = 55.8 A . The space groups and the unit-cell dimensions of the present two lysinal derivatives are different to those of the protease and TLCK- modified one . The space group of the protease is P1 with cell dimensions a = 39.53, b = 40.34, c = 43.92 A, alpha = 114.81, beta = 113.75 and gamma = 74.00 degrees and that of the TLCK-modified one is also P1 with cell dimensions of a = 37.30, b = 42.74, c = 48.02 A, alpha = 120.10, beta = 112.81 and gamma = 68.54 degrees . Diffraction to 1.9 A resolution for the Val-lysinal modified crystal and to 2.2 A resolution for the Leu-Leu-lysinal modified crystal has been observed using a rotating-anode X-ray generator . Full structure determinations of these lysinal-modified protease crystals may lead to an understanding of the molecular basis of enzyme-substrate interactions in the catalytic process of this protease.

Appl Microbiol Biotechnol . 2004 Jul 28; {Epub ahead of print}
Mesophilic aerobic degradation of a metal lubricant by a biological consortium; Iwashita S et al.; The metal-forming industries require the use of greases to lubricate metal surfaces during manufacturing operations, and the residues of these lubricants must be removed prior to finishing processes to protect and improve the appearance of the final product . An aqueous, biological metal-cleaning process operating under mild conditions (pH 9, 42show $132# degrees show $132#C) eliminates the use of environmentally unfriendly cleaning materials such as chlorinated solvents by employing microorganisms to degrade greases and oils naturally . This process was characterized in terms of initial degradation rates of a representative metal lubricant and by phylogenetic identification of the active bacteria . The metal lubricant in a surfactant solution was degraded by a bacterial consortium, and its concentration was determined by a novel gas chromatography assay . The maximum degradation rate V(max) and the apparent K(m) were obtained as 45 mg/(day mg protein) and 24 g/l on cellular basis, and 4.6 g/(day l) and 33 g/l on a volumetric basis, respectively . Mineralization of the metal lubricant was shown by analyzing the evolved CO(2) and Cl(-), and the bacterial consortium utilized the metal lubricant as a sole carbon and energy source (micro=0.05+/-0.01 h(-1) at 0.5 vol% lubricant concentration) . The active bacteria in the biological metal-cleaning process were identified as Bacillus licheniformis for the higher lubricant concentrations (3, 5, and 7.5 vol%), Bacillus cereus at 1 vol%, and Pseudomonas aeruginosa, Rhizobiaceae strain M100, and Achromobacter sp . LMG 5431 at 0.3 vol%.

Plant Physiol Biochem, 2004 Jun, 42(6), 565 - 72
Plant growth-promoting bacteria confer resistance in tomato plants to salt stress; Mayak S et al.; The object of the work is to evaluate whether rhizobacteria populating dry salty environments can increase resistance in tomato to salt stress . Seven strains of plant growth-promoting bacteria that have 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity were isolated from soil samples taken from the Arava region of southern Israel . Following growth of these seedlings in the presence of 43 mM NaCl for 7 weeks, the bacterium that promoted growth to the greatest extent was selected for further study . DNA analysis of the 16S RNA indicated that the selected bacterium was Achromobacter piechaudii . This bacterium significantly increased the fresh and dry weights of tomato seedlings grown in the presence of up to 172 mM NaCl salt . The bacterium reduced the production of ethylene by tomato seedlings, which was otherwise stimulated when seedlings were challenged with increasing salt concentrations, but did not reduce the content of sodium . However, it slightly increased the uptake of phosphorous and potassium, which may contribute in part to activation of processes involved in the alleviation of the effect of salt . In the presence of salt the bacterium increased the water use efficiency (WUE) . This may suggest that the bacterium act to alleviate the salt suppression of photosynthesis . However, the detailed mechanism was not elucidated . The work described in this report is a first step in the development of productive agricultural systems in saline environments.

Biochemistry (Mosc), 2004 May, 69(5), 501 - 5
Structural investigations and identification of the extracellular bacteriolytic endopeptidase L1 from Lysobacter sp . XL1; Muranova TA et al.; The N-terminal amino acid sequence (23 amino acid residues) and the amino acid composition of the extracellular bacteriolytic enzyme L1 of 21 kD from the bacterium Lysobacter sp . XL1 have been determined . The enzyme was hydrolyzed by trypsin, the resulting peptides were isolated, and their primary structures were determined . A high extent of homology (92%) of the N-terminal amino acid sequence and the primary structure of isolated peptides of the enzyme L1 (62 amino acid residues or 31% of protein sequence) to the corresponding sites of alpha-lytic proteinases (EC 3.4.21.12) of Lysobacter enzymogenes and Achromobacter lyticus was found . These data allowed identification of the endopeptidase L1 of Lysobacter sp . XL1 as alpha-lytic proteinase EC 3.4.21.12.

Int Microbiol, 2004 Mar, 7(1), 27 - 34
Nucleotide sequence and expression of the ncr nickel and cobalt resistance in Hafnia alvei 5-5; Park JE et al.; The structural genes for the nickel and cobalt resistance of the conjugative plasmid pEJH 501 of Hafnia alvei 5-5, contained on a SalI-EcoRI fragment of 4.8 kb, were cloned and sequenced . The DNA sequence included five genes in the following order: ncrA, ncrB, ncrC, ncrY, and ncrX . The predicted amino acid sequences of ncrA were homologous to the amino acid sequences of nreB of Achromobacter xylosoxidans 31A . Expression of ncr with the T7 RNA polymerase-promoter system allowed Escherichia coli BL21 (DE3) to overexpress NcrA, NcrB, and NcrC but not NcrY, and NcrX . The apparent molecular masses of NcrA, NcrB, and NcrC were 30, 33, and 17 kDa, respectively . Primer-extension analysis showed that ncr mRNA started at nucleotide position 23 upstream from ncrA . The promoter region of the ncr operon possessed a strong, putative -35 element of sigma(32)-type promoter sequence, and transcriptional 'lacZ fusion studies indicated that the -35 element influenced sigma(32)-specific transcription.

Methods Mol Biol, 2004, 268, 465 - 70
Determination of esterolytic and lipolytic activities of lactic acid bacteria; Medina RB et al.; Lactic acid bacteria (LAB) are considered weakly lipolytic compared with many other groups of bacteria (e.g., Pseudomonas, Bacillus, and Achromobacter) . The esterolytic and lipolytic systems of dairy LAB remain poorly characterized . Esterases from lactic acid bacteria, yeasts, and Pseudomonas organisms may be involved in the development of fruity flavors in foods, and pregastric lipase and esterases are essential for the development of typical flavor in Italian cheese . Microbial lipases and esterases may improve quality or accelerate the maturation of cheeses, cured bacon, and fermented sausages.Lipases are defined as glycerol ester hydrolases (EC 3.1.1.3) that hydrolyze tri-, di-, and monoglycerides present at an oil-water interface . Esterases (EC 3.1.1.6) hydrolyze esters in solution and may also hydrolyze tri- and especially di- and monoglycerides containing short-chain fatty acids.Some probiotic strains of LAB can hydrolyze the triglycerides, releasing most short and medium chain, and essential fatty acids, which are valuable to today's health-conscious consumer . Medium chain fatty acids (C6-C14), in particular, have become accepted treatment for patients with malabsorption symptoms, a variety of metabolic disorders, cholesterol problems, and infant malnutrition . These probiotic bacteria could alleviate lipase deficiency in the digestive tract during digestion (steatorrhea).In this chapter, we describe different methods routinely used in our laboratory to determine the esterolytic and lipolytic activity of LAB . These techniques include the use of alpha- and beta-naphthyl derivatives of fatty acids (chromogenic method), the p-nitrophenyl (pNP) derivative of fatty acids (chromogenic method), and triglycerides (agar-well assay technique and titrimetric test) as substrates.

Emerg Infect Dis, 2004 Mar, 10(3), 470 - 7
Amoebae-resisting bacteria isolated from human nasal swabs by amoebal coculture; Greub G et al.; Amoebae feed on bacteria, and few bacteria can resist their microbicidal ability . Amoebal coculture could therefore be used to selectively grow these amoebae-resisting bacteria (ARB), which may be human pathogens . To isolate new ARB, we performed amoebal coculture from 444 nasal samples . We recovered 7 (1.6%) ARB from 444 nasal swabs, including 4 new species provisionally named Candidatus Roseomonas massiliae, C . Rhizobium massiliae, C . Chryseobacterium massiliae, and C . Amoebinatus massiliae . The remaining isolates were closely related to Methylobacterium extorquens, Bosea vestrii, and Achromobacter xylosoxidans . Thus, amoebal coculture allows the recovery of new bacterial species from heavily contaminated samples and might be a valuable approach for the recovery of as-yet unrecognized emerging pathogens from clinical specimens.

J Korean Med Sci, 2004 Apr, 19(2), 291 - 3
Pacemaker lead endocarditis caused by Achromobacter xylosoxidans; Ahn Y et al.; We report the case of a 35-yr-old patient who presented with high fever and chills . He had undergone a patch closure of the ventricular septal defect 18 yr before . One year later, a VVI pacemaker was implanted via the right subclavian vein because of complete heart block . Nine years after that, a new VVI pacemaker with another right ventricular electrode was inserted controlaterally and the old pacing lead was abandoned . Trans-thoracic and trans-esophageal echocardiogram identified the pacemaker lead in the right ventricle (RV) attaching hyperechoic materials and also a fluttering round hyperechoic mass with a stalk in the RV outflow tract . Cultures in blood and pus from pacemaker lead grew Achromobacter xylosoxidans . A diagnosis of pacemaker lead endocarditis due to Achromobacter xylosoxidans was made . In this regards, the best treatment is an immediate removal of the entire pacing system and antimicrobial therapy.

Eur J Clin Microbiol Infect Dis, 2004 Apr, 23(4), 336 - 9 Epub 2004 Mar 13.
Persistent colonization of nine cystic fibrosis patients with an Achromobacter (Alcaligenes) xylosoxidans clone; Kanellopoulou M et al.; The present study was conducted to investigate the increasing incidence of Achromobacter (previously Alcaligenes) xylosoxidans isolates being recovered from sputum samples of cystic fibrosis patients at a cystic fibrosis department for adults in Athens, Greece . During the 1-year study period, a total of 34 isolates were detected persistently in 9 of 71 cystic fibrosis patients . The isolates exhibited resistance to multiple antimicrobial agents . Isolates that were recovered repeatedly from each patient exhibited identical macrorestriction profiles with pulsed-field gel electrophoresis, indicating that the same strain persisted in the lungs of these patients . Isolates from five of the patients were genetically related, suggesting a common-source outbreak of Achromobacter xylosoxidans colonization or infection.

J Appl Microbiol, 2004, 96(4), 844 - 52
Degradation of 2,4,6-tribromophenol by bacterial cells attached to chalk collected from a contaminated aquifer; Nejidat A et al.; AIM: To investigate the factors governing the adhesion and activity of the 2,4,6-tribromophenol (TBP) degrading bacterium Achromobacter piechaudii TBPZ-N61 on chalk from a contaminated aquifer . METHODS AND RESULTS: Adhesion kinetics of TBPZ-N61 to grey and white chalk from a polluted fractured chalk aquifer was tested in a batch system . Both grey and white chalk contain ca 80% CaCO3, while grey chalk contains more organic matter (2.4%) than the white chalk (0.3%) and also contains Dolmite and Clinoptilolite . Adhesion of the bacterial cells to the chalk particles (<0.2 mm) occurred rapidly (96% of the cells within 15 min) . Langmuir-fitted adhesion isotherms suggest that cells in the stationary phase, which are more hydrophobic, adhere to both grey and white chalk more efficiently than cells in the logarithmic growth phase . Increasing the pH (from 6.7 to 8.1) caused a significant reduction in cell adhesion to the chalk . Activity of attached cells was evaluated in both batch and column experiments . Logarithmic cells adhering to white and grey chalk were more active in TBP degradation than cells in suspension . In column experiments, significant TBP degradation was retained up to 30 days after a single injection of TBPZ cells . Thereafter, activity was fully recovered by amendment of yeast extract . Chalk surfaces that were incubated in situ in contaminated groundwater for 20 days still allowed the adhesion and activity of TBPZ cells . CONCLUSIONS: Taken together, our results show that bacteria adhere efficiently to specific sites on the chalk surfaces, and that sustained bacterial activity of the attached cells can be achieved by adding a carbon source such as yeast extract which also overcome toxic constituents that may occur in some chalk types . SIGNIFICANCE AND IMPACT OF THE STUDY: Bioremediation of TBP-contaminated chalk aquifers is made possible by the injection of bacterial cultures.

Biochem Biophys Res Commun, 2004 Mar 26, 316(1), 107 - 13
pH-profile crystal structure studies of C-terminal despentapeptide nitrite reductase from Achromobacter cycloclastes; Li HT et al.; Crystal structures of C-terminal despentapeptide nitrite reductase (NiRc-5) from Achromobacter cycloclastes were determined from 1.9 to 2.3A at pH 5.0, 5.4, and 6.2 . NiRc-5, that has lost about 30% activity, is found to possess quite similar trimeric structures as the native enzyme . Electron density and copper content measurements indicate that the activity loss is not caused by the release of type 2 copper (T2Cu) . pH-profile structural comparisons with native enzyme reveal that the T2Cu active center in NiRc-5 is perturbed, accounting for the partial loss of enzyme activity . This perturbation likely results from the less constrained conformations of two catalytic residues, Asp98 and His255 . Hydrogen bonding analysis shows that the deletion of five residues causes a loss of more than half the intersubunit hydrogen bonds mediated by C-terminal tail . This study shows that the C-terminal tail plays an important role in controlling the conformations around the T2Cu site at the subunit interface, and helps keep the optimum microenvironment of active center for the full enzyme activity of AcNiR.

Protein Expr Purif, 2003 Dec, 32(2), 288 - 92
Cytoplasmic expression of the Achromobacter xylosoxidans blue copper nitrite reductase in Escherichia coli and characterisation of the recombinant protein; Ho WH et al.; The gene of the Achromobacter xylosoxidans (DSM 2402) blue copper-containing nitrite reductase was amplified using the polymerase chain reaction . DNA sequence analysis reveals that the amino acid sequence is identical to those of the GIFU1051 and the NCIMB11015 A . xylosoxidans nitrite reductases . The gene encoding the mature coding region for DSM 2402 nitrite reductase was cloned into a pET-vector, overexpressed in the cytoplasm of Escherichia coli BL21(DE3), and the expressed holoprotein was purified to apparent homogeneity by cation-exchange chromatography . The recombinant blue copper-containing nitrite reductase was obtained in high yields of 70mgL(-1) of culture . The specific catalytic activity as well as the electronic absorption and electron paramagnetic resonance spectra agree with corresponding data for the native protein . Mass spectroscopic analysis of the recombinant nitrite reductase gave a molecular weight of 36659.1Da for the apo-protein monomer, in agreement with the expected molecular mass based on the amino acid sequence.

Am J Respir Med, 2003, 2(4), 321 - 32
Antibiotic treatment of multidrug-resistant organisms in cystic fibrosis; Conway SP et al.; 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.

J Paediatr Child Health, 2003 Dec, 39(9), 665 - 7
The microbiology of glue ear in Australian Aboriginal children; Stuart J et al.; OBJECTIVE: To study the bacterial cultures of middle ear aspirates from 27 Aboriginal children with otitis media with effusion . METHODS: Standard bacteriological techniques were used to analyse the middle-ear aspirates collected during surgery to insert grommets in 27 Aboriginal children . Swabs of the tympanic membrane were taken for comparison . RESULTS: Forty-five aspirates were collected from 59 myringotomies . Positive cultures were obtained from 19 of these (13 children) with potentially pathogenic organisms identified in 11 children including Staphylococcus, Pseudomonas, Haemophilus influenzae, Moraxella, Achromobacter, Enterobacter and Corynebacterium . CONCLUSION: This is only the second study to look at the bacteria in middle ear effusions in Aboriginal children . Streptococcus pneumoniae was notable in its absence as was found in a previous study.

Am J Respir Crit Care Med, 2003 Oct 15, 168(8), 918 - 51
Pathophysiology and management of pulmonary infections in cystic fibrosis; Gibson RL et al.; This comprehensive State of the Art review summarizes the current published knowledge base regarding the pathophysiology and microbiology of pulmonary disease in cystic fibrosis (CF) . The molecular basis of CF lung disease including the impact of defective cystic fibrosis transmembrane regulator (CFTR) protein function on airway physiology, mucociliary clearance, and establishment of Pseudomonas aeruginosa infection is described . An extensive review of the microbiology of CF lung disease with particular reference to infection with P . aeruginosa is provided . Other pathogens commonly associated with CF lung disease including Staphylococcal aureus, Burkholderia cepacia, Stenotrophomonas maltophilia, Achromobacter xylosoxidans and atypical mycobacteria are also described . Clinical presentation and assessment of CF lung disease including diagnostic microbiology and other measures of pulmonary health are reviewed . Current recommendations for management of CF lung disease are provided . An extensive review of antipseudomonal therapies in the settings of treatment for early P . aeruginosa infection, maintenance for patients with chronic P . aeruginosa infection, and treatment of exacerbation in pulmonary symptoms, as well as antibiotic therapies for other CF respiratory pathogens, are included . In addition, the article discusses infection control policies, therapies to optimize airway clearance and reduce inflammation, and potential future therapies.

Syst Appl Microbiol, 2003 Sep, 26(3), 445 - 52
Isolation and molecular characterization of thiosulfate-oxidizing bacteria from an Italian rice field soil; Graff A et al.; In rice paddy soils an active cycling of sulfur compounds takes place . To elucidate the diversity of thiosulfate-oxidizing bacteria these organisms were enriched from bulk soil and rice roots by the most probable number method in liquid medium . From the MPN enrichment cultures 21 bacterial strains were isolated on solid mineral medium, and could be further shown to produce sulfate from thiosulfate . These strains were characterized by 16S rDNA analyses . The isolates were affiliated to seven different phylogenetic groups within the alpha- and beta-subclass of Proteobacteria . Two of these phylotypes were already described as S-oxidizers in this environment (Xanthobacter sp . and Bosea sp . related strains), but five groups represented new S-oxidizers in rice field soil . These isolates were closely related to Mesorhizobium loti, to Hydrogenophaga sp., to Delftia sp., to Pandoraea sp . or showed sequence similarity to a strain of Achromobacter sp.

Infect Immun, 2003 Oct, 71(10), 5461 - 71
Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients; Kim YR et al.; Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo . We used the recently developed in vivo-induced antigen technology (IVIAT) to identify Vibrio vulnificus genes induced in vivo . An expression library of V . vulnificus was screened by colony blot analysis by using pooled convalescent-phase serum that had been thoroughly adsorbed with in vitro-expressed V . vulnificus whole cells and lysates . Twelve clones were selected, and the sequences of the insert DNAs were analyzed . The DNA sequences showed homologies with genes encoding proteins of diverse functions: these functions included chemotaxis (a methyl-accepting chemotaxis protein), signaling (a GGDEF-containing protein and a putative serine/threonine kinase), biosynthesis and metabolism (PyrH, PurH, and IlvC), secretion (TatB and plasmid Achromobacter secretion {PAS} factor), transcriptional activation (IlvY and HlyU), and the activity of a putative lipoprotein (YaeC) . In addition, one identified open reading frame encoded a hypothetical protein . Isogenic mutants of the 12 in vivo-expressed (ive) genes were constructed and tested for cytotoxicity . Cytotoxic activity of the mutant strains, as measured by lactate dehydrogenase release from HeLa cells, was nearly abolished in pyrH, purH, and hlyU mutants . The intraperitoneal 50% lethal dose in mice increased by ca . 10- to 50-fold in these three mutants . PyrH and PurH seem to be essential for in vivo growth . HlyU appears to be one of the master regulators of in vivo virulence expression . The successful identification of ive genes responsible for the in vivo bacterial virulence, as done in the present study, demonstrates the usefulness of IVIAT for the detection of new virulence genes.

Pathol Biol (Paris), 2003 Sep, 51(7), 405 - 11
{Phenotypic and genotypic characteristics of non fermenting atypical strains recovered from cystic fibrosis patients}; Ferroni A et al.; We used partial 16S rRNA gene (16S DNA) sequencing for the prospective identification of nonfermenting Gram-negative bacilli recovered from patients attending our cystic fibrosis center (hopital Necker-Enfants malades), which gave problematic results with conventional phenotypic tests . During 1999, we recovered 1093 isolates of nonfermenting Gram-negative bacilli from 702 sputum sampled from 148 patients . Forty-six of these isolates (27 patients) were not identified satisfactorily in routine laboratory tests . These isolates were identified by 16S DNA sequencing as Pseudomonas aeruginosa (19 isolates, 12 patients), Achromobacter xylosoxidans (10 isolates, 8 patients), Stenotrophomonas maltophilia (9 isolates, 9 patients), Burkholderia cepacia genomovar I/III (3 isolates, 3 patients), Burkholderia vietnamiensis (1 isolate), Burkholderia gladioli (1 isolate) and Ralstonia mannitolilytica (3 isolates, 2 patients) . Fifteen isolates (33%) were resistant to all antibiotics in routine testing . Sixteen isolates (39%) resistant to colistin were recovered on B . cepacia-selective medium: 2 P . aeruginosa, 3 A . xylosoxidans, 3 S . maltophilia and the 8 Burkholderia--Ralstonia isolates . The API 20NE system gave no identification for 35 isolates and misidentified 11 isolates (2 P . aeruginosa, 2 A . xylosoxidans and 1 S . maltophilia classified as B . cepacia ) . Control measures and/or treatment were clearly improved as a result of 16S DNA sequencing in three of these cases . This study confirms the weakness of phenotypic methods for identification of atypical nonfermenting Gram-negative bacilli recovered from cystic fibrosis patients . The genotypic methods, such as 16S DNA sequencing which allows identification of strains in routine practice, appears to have a small, but significant impact on the clinical management of CF patients.

Appl Environ Microbiol, 2003 Aug, 69(8), 4837 - 45
The biphenyl- and 4-chlorobiphenyl-catabolic transposon Tn4371, a member of a new family of genomic islands related to IncP and Ti plasmids; Toussaint A et al.; The nucleotide sequence of the biphenyl catabolic transposon Tn4371 has been completed and analyzed . It confirmed that the element has a mosaic structure made of several building blocks . In addition to previously identified genes coding for a tyrosine recombinase related to phage integrases and for biphenyl degradation enzymes very similar to those of Achromobacter georgiopolitanum KKS102, Tn4371 carries many plasmid-related genes involved in replication, partition, and other, as-yet-unknown, plasmid functions . One gene cluster contains most of the genes required to express a type IV secretion-mating pair formation apparatus coupled with a TraG ATPase, all of which are related to those found on IncP and Ti plasmids . Orthologues of all Tn4371 plasmid-related genes and of the tyrosine recombinase gene were found, with a very similar organization, in the chromosome of Ralstonia solanacearum and on the yet-to-be-determined genomic sequences of Erwinia chrysanthemi and Azotobacter vinelandii . In each of these chromosomal segments, conserved segments were separated by different groups of genes, which also differed from the Tn4371 bph genes . The conserved blocks of genes were also identified, in at least two copies, in the chromosome of Ralstonia metallidurans CH34 . Tn4371 thus appears to represent a new family of potentially mobile genomic islands with a broad host range since they reside in a wide range of soil proteobacteria, including plant pathogens.

Appl Microbiol Biotechnol, 2003 Oct, 62(5-6), 507 - 16 Epub 2003 Jun 24.
Isolation and characterization of a new strain of Achromobacter sp . with beta-lactam antibiotic acylase activity; Plhackova K et al.; A bacterial strain producing a beta-lactam antibiotic acylase, able to hydrolyze ampicillin to 6-aminopenicillanic acid more efficiently than penicillin G, was isolated from soil and characterized . The isolate was identified as Achromobacter sp . using the phenotypic characteristics, composition of cellular fatty acids and 16S rRNA gene sequence . The enzyme synthesis was fully induced by phenylacetic acid (PAA) at a concentration of 2 g l(-1) . PAA at concentrations up to 12 g l(-1) had no negative effect on the specific activity of acylase and biomass production, but slowed down the specific growth rate . Benzoic or 4-hydroxyphenylacetic acids can also induce synthesis of the enzyme . The inducers were metabolized in all cases . Acylase activity in cell-free extracts was determined with various substrates; ampicillin, cephalexin and amoxicillin were hydrolyzed 1.5- and 2-times faster than penicillin G . A high stability of acylase activity was observed over a wide range of pH (5.0-8.5) and at temperatures above 55 degrees C.

Ultramicroscopy, 2003 Oct-Nov, 97(1-4), 65 - 72
Ambient STM and in situ AFM study of nitrite reductase proteins adsorbed on gold and graphite: influence of the substrate on protein interactions; Contera SA et al.; Trimeric Achromobacter cycloclastes Cu-containing nitrite reductase (CuNIR) proteins adsorbed on gold and graphite have been studied by ambient STM and in situ AFM . STM resolves them individually and in layers, distinguishing the sub-molecular individual units of the trimer . The Cu atoms are not visible to STM . STM shows that individual CuNIR denatures as it adsorbs on Au, although a deformed trimeric shape can be identified in some cases . CuNIR forms disordered layers on gold . On graphite, ordered self-assembled layers of CuNIR have been resolved by in situ AFM and ambient STM forming parallel rows whose separation distance corresponds to the size of one of the units of the trimer, 5nm . Ambient STM can achieve better resolution than in situ AFM in the images of the layers . We observe differences between domains showing the parallel row structure and unstructured parts of the CuNIR layer by in situ phase imaging AFM.

Eur J Clin Microbiol Infect Dis, 2003 Jun, 22(6), 360 - 3 Epub 2003 May 16.
Achromobacter xylosoxidans bacteremia: a 10-year analysis of 54 cases; Gomez-Cerezo J et al.; Fifty-four cases of Achromobacter xylosoxidans bacteremia diagnosed over a 10-year period in patients from 2 months to 87 years of age were reviewed . Fifty-two episodes were nosocomial . The most frequent underlying condition was neoplasm (solid or hematological) . The source of infection was a contaminated intravenous catheter in 35 patients (60%) and pneumonia in 6 patients . Eight (15%) patients died . The only risk factors significantly associated with mortality were age over 65 years and neutropenia . The results of in vitro susceptibility studies of the isolates showed that antibiotic therapy with antipseudomonal penicillins or carbapenems would be a reasonable choice . An epidemiological study conducted in the hemodialysis unit showed Achromobacter xylosoxidans in tap water and on the hands of two healthcare workers but not in the hemodialysis systems . Patients were probably contaminated when healthcare workers manipulated the intravenous catheters without wearing gloves.

Biochem Biophys Res Commun, 2003 Apr 4, 303(2), 519 - 24
Characterization and function of Met150Gln mutant of copper-containing nitrite reductase from Achromobacter cycloclastes IAM1013; Kataoka K et al.; The mutant (M150Q-NIR) replacing the Met150 ligand of the type 1 Cu center in Achromobacter cycloclastes nitrite reductase (AcNIR) with Gln has been physicochemically and functionally characterized . The electronic absorption and CD spectra of M150Q-NIR are similar to those of mavicyanin and stellacyanin having the 2His, Cys, and Gln ligands, but the EPR signal has an axial character, although their blue copper proteins show rhombic EPR signals . The mutant has about 80% catalytic activity of AcNIR . Moreover, the midpoint potential (E(1/2)) of M150Q-NIR is +113 mV vs . NHE at pH 7.0, being negatively shifted compared to that of AcNIR (+240 mV) . Although the intermolecular electron-transfer process from Achromobacter cycloclastes pseudoazurin (pAz) to M150Q-NIR was not detected, the pAz mutant (M86Q-pAz) replacing the Met86 ligand with Gln transfers one electron to the NIR mutant with an intermolecular electron-transfer rate constant (k(ET)) of 2.3 x 10(5)M(-1)s(-1).

Biochem Biophys Res Commun, 2003 Mar 14, 302(3), 568 - 74
Crystal structure of a NO-forming nitrite reductase mutant: an analog of a transition state in enzymatic reaction; Liu SQ et al.; I257E was obtained by site directed mutagenesis of nitrite reductase from Achromobacter cycloclastes . The mutant has no enzyme activity . Its crystal structure determined at 1.65A resolution shows that the side-chain carboxyl group of the mutated residue, Glu257, coordinates with the type 2 copper in the mutant and blocks the contact between the type 2 copper and its solvent channel, indicating that the accessibility of the type 2 copper is essential for maintaining the activity of nitrite reductase . The carboxylate is an analog of the substrate, nitrite, but the distances between the type 2 copper and the two oxygen atoms of the side-chain carboxyl group are reversed in comparison to the binding of nitrite to the native enzyme . In the mutant, both the type 2 copper and the N epsilon atom on the imidazole ring of its coordinated residue His135 move in the substrate binding direction relative to the native enzyme . In addition, an EPR study showed that the type 2 copper in the mutant is in a reduced state . We propose that mutant I257E is in a state corresponding to a transition state in the enzymatic reaction.

Biochem Biophys Res Commun, 2002 Nov 29, 299(2), 173 - 6
Preliminary crystallographic studies of two C-terminally truncated copper-containing nitrite reductases from Achromobacter cycloclastes: changed crystallizing behaviors caused by residue deletion; Li HT et al.; The C-terminal segment of copper-containing nitrite reductase from Achromobacter cycloclastes (AcNiR) has been found essential for maintaining both the quaternary structure and the enzyme activity of AcNiR . C-terminal despentapeptide AcNiR (NiRc-5) and desundecapeptide AcNiR (NiRc-11) are two important truncated mutants whose activities and stability have been affected by residue deletion . In this study, the two mutants were crystallized using the hanging drop vapor diffusion method . Crystals of NiRc-5 obtained at pH 5.0 and 6.2 both belonged to the P2(1)2(1)2(1) space group with unit cell parameters a=99.0 A, b=117.4 A, c=122.8 A (pH 5.0) and a=98.9A, b=117.7A, c=123.0A (pH 6.2) . NiRc-11 was crystallized in two crystal forms: the tetragonal form belonged to the space group P4(1) with a=b=96.0A and c=146.6A; the monoclinic form belonged to the space group P2(1) with a=86.0A, b=110.1A, c=122.7A, and beta=101.9 degrees . The crystallizing behaviors of the two mutants differed from that of the native enzyme . Such change in combination with residue deletion is also discussed here.

J Am Chem Soc, 2002 Nov 20, 124(46), 13698 - 708
Metal-ligand interplay in blue copper proteins studied by 1H NMR spectroscopy: Cu(II)-pseudoazurin and Cu(II)-rusticyanin; Donaire A et al.; The blue copper proteins (BCPs), pseudoazurin from Achromobacter cycloclastes and rusticyanin from Thiobacillus ferrooxidans, have been investigated by (1)H NMR at a magnetic field of 18.8 T . Hyperfine shifts of the protons belonging to the coordinated ligands have been identified by exchange spectroscopy, including the indirect detection for those resonances that cannot be directly observed (the beta-CH(2) of the Cys ligand, and the NH amide hydrogen bonded to the S(gamma)(Cys) atom) . These data reveal that the Cu(II)-Cys interaction in pseudoazurin and rusticyanin is weakened compared to that in classic blue sites (plastocyanin and azurin) . This weakening is not induced by a stronger interaction with the axial ligand, as found in stellacyanin, but might be determined by the protein folding around the metal site . The average chemical shift of the beta-CH(2) Cys ligand in all BCPs can be correlated to geometric factors of the metal site (the Cu-S(gamma)(Cys) distance and the angle between the CuN(His)N(His) plane and the Cu-S(gamma)(Cys) vector) . It is concluded that the degree of tetragonal distortion is not necessarily related to the strength of the Cu(II)-S(gamma)(Cys) bond . The copper-His interaction is similar in all BCPs, even for the solvent-exposed His ligand . It is proposed that the copper xy magnetic axes in blue sites are determined by subtle geometrical differences, particularly the orientation of the His ligands . Finally, the observed chemical shifts for beta-CH(2) Cys and Ser NH protons in rusticyanin suggest that a less negative charge at the sulfur atom could contribute to the high redox potential (680 mV) of this protein.

J Biol Chem, 2003 Jan 24, 278(4), 2549 - 53 Epub 2002 Nov 04.
Identification of the active site residues of Pseudomonas aeruginosa protease IV . Importance of enzyme activity in autoprocessing and activation; Traidej M et al.; Protease IV is a lysine-specific endoprotease produced by Pseudomonas aeruginosa whose activity has been correlated with corneal virulence . Comparison of the protease IV amino acid sequence to other bacterial proteases suggested that amino acids His-72, Asp-122, and Ser-198 could form a catalytic triad that is critical for protease IV activity . To test this possibility, site-directed mutations by alanine substitution were introduced into six selected residues including the predicted triad and identical residues located close to the triad . Mutations at any of the amino acids of the predicted catalytic triad or Ser-197 caused a loss of enzymatic activity and absence of the mature form of protease IV . In contrast, mutations at His-116 or Ser-200 resulted in normal processing into the enzymatically active mature form . A purified proenzyme that accumulated in the His-72 mutant was shown in vitro to be susceptible to cleavage by protease IV purified from P . aeruginosa . Furthermore, similarities of protease IV to the lysine-specific endoprotease of Achromobacter lyticus suggested three possible disulfide bonds in protease IV . These results identify the catalytic triad of protease IV, demonstrate that autodigestion is essential for the processing of protease IV into a mature protease, and predict sites essential to enzyme conformation.

J Clin Microbiol, 2002 Nov, 40(11), 4051 - 5
Nosocomial infections caused by multidrug-resistant isolates of pseudomonas putida producing VIM-1 metallo-beta-lactamase; Lombardi G et al.; Successful carbapenem-based chemotherapy for the treatment of Pseudomonas infections has been seriously hindered by the recent appearance of IMP- and VIM-type metallo-beta-lactamases, which confer high-level resistance to carbapenems and most other beta-lactams . Recently, multidrug-resistant Pseudomonas putida isolates for which carbapenem MICs were >/=32 micro g/ml were recovered from cultures of urine from three inpatients in the general intensive care unit of the Ospedale di Circolo, Varese, Italy . Enzyme assays revealed production of a metallo-beta-lactamase activity, while molecular analysis detected in each isolate a bla(VIM-1) determinant carried by an apparently identical medium-sized plasmid . Conjugation experiments were unsuccessful in transferring the beta-lactamase determinant to Escherichia coli or Pseudomonas aeruginosa . Macrorestriction analysis by pulsed-field gel electrophoresis demonstrated that the isolates were of clonal origin . PCR mapping and sequencing of the variable region of the plasmid-borne class 1 integron carrying the bla(VIM-1) determinant (named In110) showed that the bla(VIM-1)-containing cassette was identical to that previously found in strains of different species from other Italian hospitals and that the cassette array of In110 was not identical but clearly related to that of In70 (a bla(VIM-1)-containing plasmid-borne integron from an Achromobacter xylosoxidans isolate), pointing to a common origin of this cassette and to a related evolutionary history of their cognate integrons.

Arch Biochem Biophys, 2002 Nov 1, 407(1), 72 - 82
Phosphorylation of calmodulin by Ca2+/calmodulin-dependent protein kinase IV; Ishida A et al.; Calmodulin-dependent protein kinase IV (CaM-kinase IV) phosphorylated calmodulin (CaM), which is its own activator, in a poly-L-Lys {poly(Lys)}-dependent manner . Although CaM-kinase II weakly phosphorylated CaM under the same conditions, CaM-kinase I, CaM-kinase kinase alpha, and cAMP-dependent protein kinase did not phosphorylate CaM . Polycations such as poly(Lys) were required for the phosphorylation . The optimum concentration of poly(Lys) for the phosphorylation of 1 microM CaM was about 10 microg/ml, but poly(Lys) strongly inhibited CaM-kinase IV activity toward syntide-2 at this concentration, suggesting that the phosphorylation of CaM is not due to simple activation of the catalytic activity . Poly-L-Arg could partially substitute for poly(Lys), but protamine, spermine, and poly-L-Glu/Lys/Tyr (6/3/1) could not . When phosphorylation was carried out in the presence of poly(Lys) having various molecular weights, poly(Lys) with a higher molecular weight resulted in a higher degree of phosphorylation . Binding experiments using fluorescence polarization suggested that poly(Lys) mediates interaction between the CaM-kinase IV/CaM complex and another CaM . The 32P-labeled CaM was digested with BrCN and Achromobacter protease I, and the resulting peptides were purified by reversed-phase HPLC . Automated Edman sequence analysis of the peptides, together with phosphoamino acid analysis, indicated that the major phosphorylation site was Thr44 . Activation of CaM-kinase II by the phosphorylated CaM was significantly lower than that by the nonphosphorylated CaM . Thus, CaM-kinase IV activated by binding Ca2+/CaM can bind and phosphorylate another CaM with the aid of poly(Lys), leading to a decrease in the activity of CaM.

Biochem J, 2003 Jan 15, 369(Pt 2), 275 - 85
Sulphoacetaldehyde acetyltransferase yields acetyl phosphate: purification from Alcaligenes defragrans and gene clusters in taurine degradation; Ruff J et al.; The facultatively anaerobic bacterium Alcaligenes defragrans NKNTAU was found to oxidize taurine (2-aminoethanesulphonate) with nitrate as the terminal electron acceptor . Taurine was transaminated to 2-sulphoacetaldehyde . This was not converted into sulphite and acetate by a "sulphoacetaldehyde sulpho-lyase" (EC 4.4.1.12), but into sulphite and acetyl phosphate, which was identified by three methods . The enzyme, which required the addition of phosphate, thiamin diphosphate and Mg(2+) ions for activity, was renamed sulphoacetaldehyde acetyltransferase (Xsc; EC 2.3.1.-) . Inducible Xsc was expressed at high levels, and a three-step 11-fold purification yielded an essentially homogeneous soluble protein, which was a homotetramer in its native form; the molecular mass of the subunit was found to be between about 63 kDa (SDS/PAGE) and 65.3 kDa (matrix-assisted laser-desorption ionization-time-of-flight MS) . The N-terminal and two internal amino acid sequences were determined, and PCR primers were generated . The xsc gene was amplified and sequenced; the derived molecular mass of the processed protein was 65.0 kDa . The downstream gene presumably encoded the inducible phosphate acetyltransferase (Pta) found in crude extracts . The desulphonative enzymes ("EC 4.4.1.12") from Achromobacter xylosoxidans NCIMB 10751 and Desulfonispora thiosulfatigenes GKNTAU were shown to be Xscs . We detected at least three subclasses of xsc in Proteobacteria and in Gram-positive bacteria, and they comprised a distinct group within the acetohydroxyacid synthase supergene family . Genome sequencing data revealed xsc genes in Burkholderia fungorum (80% sequence identity) and Sinorhizobium meliloti (61%) with closely linked pta genes . Different patterns of regulation for the transport and dissimilation of taurine were hypothesized for S . meliloti and B . fungorum.

J Clin Microbiol, 2002 Oct, 40(10), 3793 - 7
Use of 16S rRNA gene sequencing for identification of nonfermenting gram-negative bacilli recovered from patients attending a single cystic fibrosis center; Ferroni A et al.; During 1999, we used partial 16S rRNA gene sequencing for the prospective identification of atypical nonfermenting gram-negative bacilli isolated from patients attending our cystic fibrosis center . Of 1,093 isolates of nonfermenting gram-negative bacilli recovered from 148 patients, 46 (4.2%) gave problematic results with conventional phenotypic tests . These 46 isolates were genotypically identified as Pseudomonas aeruginosa (19 isolates, 12 patients), Achromobacter xylosoxidans (10 isolates, 8 patients), Stenotrophomonas maltophilia (9 isolates, 9 patients), Burkholderia cepacia genomovar I/III (3 isolates, 3 patients), Burkholderia vietnamiensis (1 isolate), Burkholderia gladioli (1 isolate), and Ralstonia mannitolilytica (3 isolates, 2 patients), a recently recognized species.

Eur J Biochem, 2002 Aug, 269(16), 4152 - 8
Electrostatic role of aromatic ring stacking in the pH-sensitive modulation of a chymotrypsin-type serine protease, Achromobacter protease I; Shiraki K et al.; Achromobacter protease I (API) has a unique region of aromatic ring stacking with Trp169-His210 in close proximity to the catalytic triad . This paper reveals the electrostatic role of aromatic stacking in the shift in optimum pH to the alkaline region, which is the highest pH range (8.5-10) among chymotrypsin-type serine proteases . The pH-activity profile of API showed a sigmoidal distribution that appears at pH 8-10, with a shoulder at pH 6-8 . Variants with smaller amino acid residues substituted for Trp169 had lower pH optima on the acidic side by 0-0.9 units . On the other hand, replacement of His210 by Ala or Ser lowered the acidic rim by 1.9 pH units, which is essentially identical to that of chymotrypsin and trypsin . Energy minimization for the mutant structures suggested that the side-chain of Trp169 stacked with His210 was responsible for isolation of the electrostatic interaction between His210 and the catalytic Asp113 from solvent . The aromatic stacking regulates the low activity at neutral pH and the high activity at alkaline pH due to the interference of the hydrogen bonded network in the catalytic triad residues.

FEMS Microbiol Lett, 2002 Jul 16, 213(1), 13 - 20
Lysobacter strain with high lysyl endopeptidase production; Chohnan S et al.; A new lysyl endopeptidase producing strain, Lysobacter sp . IB-9374, was isolated from soil . This strain secreted the endopeptidase to culture medium at 6-12-fold higher levels relative to Achromobacter lyticus and Lysobacter enzymogenes . The mature Lysobacter sp . enzyme was enzymatically identical to Achromobacter lysyl endopeptidase bearing lysyl bond specificity, a high peptidase activity, a wide pH optimum, and stability against denaturants . Nucleotide sequence analysis of the Lysobacter sp . lysyl endopeptidase gene revealed that the enzyme is synthesized as a precursor protein consisting of signal peptide (20 amino acids (aa)), pro-peptide (185 aa), mature enzyme (268 aa), and C-terminal extension peptide (198 aa) . The deduced amino acid sequence of the mature enzyme was totally identical to that of the Achromobacter enzyme . The Lysobacter sp . precursor protein has an 18-aa longer peptide chain following nine consecutive amino acid residues distinct from the Achromobacter counterpart at the C-terminus . Total precursor protein is 671 aa of which only 268 aa are in the finally processed exoenzyme.

Toxicon, 2002 Jul, 40(7), 959 - 70
Amino acid sequence of a thrombin like enzyme, elegaxobin, from the venom of Trimeresurus elegans (Sakishima-habu); Oyama E et al.; The amino acid sequence of a thrombin like enzyme, named elegaxobin, isolated from the venom of Trimeresurus elegans (Sakishima-habu) was determined by Edman sequencing of the peptides, derived from digests with cyanogen bromide, hydroxylamine, achromobacter protease I, trypsin, V8 protease, and chymotrypsin . Elegaxobin showed conservation of the catalytic amino acid residues (His, Asp, and Ser) of trypsin like serine protease in the amino acid sequence . The carboxy-terminal amino acid, Leu, was determined using carboxypeptidase Y . Elegaxobin consisted of 233 amino acids and had a calculated molecular weight of 25,439 . Elegaxobin was 53, 59, 26 and 40% homologous in sequence to ancrod, flavoxobin, bovine thrombin and trypsin, respectively.

Biosens Bioelectron, 2002 Aug, 17(8), 635 - 40
Bacteria-degraders as the base of an amperometric biosensor for detection of anionic surfactants; Taranova L et al.; Several strains belonging to genera Pseudomonas and Achromobacter and characterized by the ability to degrade anionic surfactants were tested as potential bases of microbial biosensors for surfactant detection . For each strain the substrate specificity and stability of sensor signals were studied . The total amount of the substrates tested (including carbohydrates, alcohols, aromatics, organic acids, etc.) was equal to 60; the maximal signals were observed towards the anionic surfactants . The lower limit of detection for sodium dodecyl sulfate used as a model surfactant was in the field of 1 microM for all the strains . The created microbial biosensor model can extend the practical possibilities for rapid evaluation of surfactants in water media.

J Clin Microbiol, 2002 Jun, 40(6), 2187 - 91
Prevalent bacterial species and novel phylotypes in advanced noma lesions; Paster BJ et al.; The purpose of this study was to determine the bacterial diversity in advanced noma lesions using culture-independent molecular methods . 16S ribosomal DNA bacterial genes from DNA isolated from advanced noma lesions of four Nigerian children were PCR amplified with universally conserved primers and spirochetal selective primers and cloned into Escherichia coli . Partial 16S rRNA sequences of approximately 500 bases from 212 cloned inserts were used initially to determine species identity or closest relatives by comparison with sequences of known species or phylotypes . Nearly complete sequences of approximately 1,500 bases were obtained for most of the potentially novel species . A total of 67 bacterial species or phylotypes were detected, 25 of which have not yet been grown in vitro . Nineteen of the species or phylotypes, including Propionibacterium acnes, Staphylococcus spp., and the opportunistic pathogens Stenotrophomonas maltophilia and Ochrobactrum anthropi were detected in more than one subject . Other known species that were detected included Achromobacter spp., Afipia spp., Brevundimonas diminuta, Capnocytophaga spp., Cardiobacterium sp., Eikenella corrodens, Fusobacterium spp., Gemella haemoylsans, and Neisseria spp . Phylotypes that were unique to noma infections included those in the genera Eubacterium, Flavobacterium, Kocuria, Microbacterium, and Porphyromonas and the related Streptococcus salivarius and genera Sphingomonas and TREPONEMA: Since advanced noma lesions are infections open to the environment, it was not surprising to detect species not commonly associated with the oral cavity, e.g., from soil . Several species previously implicated as putative pathogens of noma, such as spirochetes and Fusobacterium spp., were detected in at least one subject . However, due to the limited number of available noma subjects, it was not possible at this time to associate specific species with the disease.

J Clin Microbiol, 2002 Apr, 40(4), 1210 - 3
Ribosomal DNA-directed PCR for identification of Achromobacter (Alcaligenes) xylosoxidans recovered from sputum samples from cystic fibrosis patients; Liu L et al.; The opportunistic human pathogen Achromobacter (Alcaligenes) xylosoxidans has been recovered with increasing frequency from respiratory tract culture of persons with cystic fibrosis (CF) . However, confusion of this species with other closely related respiratory pathogens has limited studies to better elucidate its epidemiology, natural history, and pathogenic role in CF . Misidentification of A . xylosoxidans as Burkholderia cepacia complex is especially problematic and presents a challenge to effective infection control in CF . To address the problem of accurate identification of A . xylosoxidans, we developed a PCR assay based on a 16S ribosomal DNA sequence . In an analysis of 149 isolates that included 47 A . xylosoxidans and several related glucose-nonfermenting species recovered from CF sputum, the sensitivity and specificity of this PCR assay were determined to be 100 and 97%, respectively . The availability of this assay will enhance identification of A . xylosoxidans, thereby facilitating study of the pathogenic role of this species and improving infection control efforts in CF.

Antimicrob Agents Chemother, 2002 Apr, 46(4), 1105 - 7
Synergistic activities of macrolide antibiotics against Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans isolated from patients with cystic fibrosis; Saiman L et al.; Azithromycin and clarithromycin were paired with other antibiotics to test synergistic activity against 300 multidrug-resistant pathogens isolated from cystic fibrosis (CF) patients . Clarithromycin-tobramycin was most active against Pseudomonas aeruginosa and inhibited 58% of strains . Azithromycin-trimethoprim-sulfamethoxazole, azithromycin-ceftazidime, and azithromycin-doxycycline or azithromycin-trimethoprim-sulfamethoxazole inhibited 40, 20, and 22% of Stenotrophomonas maltophilia, Burkholderia cepacia complex, and Achromobacter (Alcaligenes) xylosoxidans strains, respectively.

Int J Syst Evol Microbiol, 2002 Jan, 52(Pt 1), 179 - 86
Brackiella oedipodis gen . nov., sp . nov., gram-negative, oxidase-positive rods that cause endocarditis of cotton-topped tamarin (Saguinus oedipus); Willems A et al.; A gram-negative, oxidase-positive, rod-shaped bacterium isolated from the heart of a cotton-topped tamarin was characterized by 16S rDNA sequence analysis, SDS-PAGE of whole-cell proteins, fatty acid analysis and biochemical tests . Outer-membrane proteins, iron-regulated outer-membrane proteins, lipopolysaccharides and siderophore production were studied . On the basis of the results, the organism belongs to the beta-Proteobacteria where it forms a separate line of descent, for which a novel genus and species are proposed, Brackiella oedipodis (LMG 19451T = DSM 13743T = NCIMB 13739T) . Nearest phylogenetic neighbours of the new genus are Taylorella, Pelistega, Bordetella, Alcaligenes and Achromobacter.

J Biochem (Tokyo), 2002 Feb, 131(2), 213 - 8
Contribution of an imidazole-indole stack to high catalytic potency of a lysine-specific serine protease, Achromobacter protease I; Shiraki K et al.; Achromobacter protease I (API), a lysine-specific serine-protease of the trypsin family, has an aromatic-ring stacking Trp 169-His 210 in close proximity to the reactive site . In order to investigate the role of this novel aromatic stacking, several mutants of the two residues were constructed and their kinetic parameters were determined . Three His 210 mutants showed lower activity by one order of magnitude than the wild-type with a peptide substrate of Ala-Ala-Lys-MCA (4-methylcoumaryl-7-amide), but 30-170% activity towards Val-Leu-Lys-MCA, suggesting that His 210 plays a role in keeping high activity toward various substrates by maintaining the active form of the substrate-binding subsite . Kinetic results of eight Trp 169 variants showed a roughly linear relation between k(cat) or K(m) values and the surface area at residue 169 . With increasing size of the side-chain, k(cat) values increased, while K(m) values decreased . A systematic kinetic analysis of the activities of Trp 169 mutants toward Lys-MCA, Ala-Lys-MCA, and Ala-Ala-Lys-MCA peptide substrates revealed that large side-chain, rather than aromaticity, plays an important role in retaining the high catalytic activity of API . Due to the presence of the aromatic stacking, API shows one order of magnitude higher activity than bovine trypsin.

J Clin Microbiol, 2002 Jan, 40(1), 26 - 30
Comparison of two culture methods for detection of tobramycin-resistant gram-negative organisms in the sputum of patients with cystic fibrosis; Van Dalfsen JM et al.; A culture method utilizing quantitative plating on antibiotic-containing media has been proposed as a technique for the detection of tobramycin-resistant organisms that is more sensitive than standard methods . Typical sputum culture methods quantitate the relative amounts of each distinct morphotype, followed by antibiotic susceptibility testing of a single colony of each morphotype . Sputum specimens from 240 cystic fibrosis patients were homogenized, serially diluted, and processed in parallel by the standard method (MacConkey agar and OF basal medium with agar, polymyxin, bacitracin, and lactose) and by plating on antibiotic-containing media (MacConkey agar with tobramycin added at 25 microg/ml {MAC-25} and 100 microg/ml {MAC-100}) . MICs of tobramycin were determined for all Pseudomonas aeruginosa isolates by broth microdilution . Growth of P . aeruginosa on MAC-25 was considered to be equivalent to a tobramycin MIC of > or = 16 microg/ml, and growth on MAC-100 was considered to be equivalent to a tobramycin MIC of > or = 128 microg/ml . Analysis of method-specific detection rates showed that tobramycin-containing medium was more sensitive than the standard method for the detection of tobramycin-resistant P . aeruginosa, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans but was less sensitive for the detection of Burkholderia cepacia than the standard method . When MICs for P . aeruginosa that grew on tobramycin-containing medium were tested by broth microdilution, the MICs for 28 of 121 strains (23%) growing on MAC-25 and 22 of 56 strains (39%) growing on MAC-100 were MICs < 16 and < 128 microg/ml, respectively . Addition of a tobramycin-containing MacConkey plate to the routine media for sputum culture may provide additional, clinically relevant microbiologic data.

Biochemistry, 2002 Jan 8, 41(1), 120 - 30
Effect of histidine 6 protonation on the active site structure and electron-transfer capabilities of pseudoazurin from Achromobacter cycloclastes; Sato K et al.; The paramagnetic (1)H NMR spectrum of Cu(II) pseudoazurin {PACu(II)} contains eight directly observed hyperfine-shifted resonances which we have assigned using saturation transfer experiments on a 1:1 mixture of PACu(I) and PACu(II) . The spectrum exhibits a number of similarities to those of other cupredoxins, but differences are found concerning the Cu-S(Met) interaction . The spectrum is dependent on pH* in the range 8.5-4.5 (pK(a)* 6.4), and a conformational change involving movement of the copper ion away from the Met toward the equatorial ligands, as a consequence of protonation of the surface His6 residue, is identified . Corresponding changes are also seen in the UV/vis spectrum . The protonation/deprotonation equilibrium of His6 influences the reduction potential of the protein in the same pH range . The self-exchange rate constant of PACu at pH* 6.0 (25 degrees C) is considerably smaller (1.1 x 10(3) M(-1) s(-1)) than the value obtained at pH* 7.6 (3.7 x 10(3) M(-1) s(-1)) . The effect on the self-exchange reactivity is mainly due to an alteration in the reorganization energy of the copper site brought about by the structural change resulting from His6 protonation.

Ying Yong Sheng Tai Xue Bao, 2000 Apr, 11(2), 249 - 52
{Isolation and selection of strains used to degrade organic chlorine pesticides and application effects}; Fang L; With the organic chlorine pesticides (666, DDT) as sole carbon resources, three strains, No . 153 (Bacillus), No . 411 (Achromobacter) and No . 512 (Pseudomonas), which can degrade 666 (BHC), were isolated and selected in Tonomura culture medium . The degradation rates of these strains were 59.6%, 56.9% and 56.0% for total amount of 666, and 55.9%, 57.6% and 56.9% for beta-666, respectively . The other strains, No . 288 (Alcaligenes), No . 410 and No . 411 (Achromobacter) to degrade DDT were also obtained . The degradation rates of the strains were 59.0%, 47.5% and 45.1% for total amount of DDT, and 59.9%, 57.6% and 49.6% for PP'-DDT, respectively . The degradation effects of the mixtures of the isolated strains in pot and field experiments were similar to those of the pure culture, indicating it is a feasible measure to apply the mixture of strains to degrade residual pesticides in fields.

Int J Cancer, 2001 Dec 1, 94(5), 662 - 8
Purification and identification of monoubiquitin-phosphoglycerate mutase B complex from human colorectal cancer tissues; Usuba T et al.; Ubiquitin-conjugated proteins in human colorectal cancer tissues were analyzed by the immunoprecipitation with the antibody FK2 against conjugated ubiquitin followed with SDS-PAGE . In these immunoprecipitable proteins, a 38-kDa protein was abundant in the tumor regions but almost absent in the adjacent normal regions in 17/26 patients, thus we attempted to purify it . Using immunoaffinity chromatography with the antibody FK2 followed by gel filtration and SDS-PAGE, approximately 10 pmol of this protein was separated from 34 g of the pooled cancerous tissue and transferred onto a PVDF membrane . The 38-kDa protein was further digested with Achromobacter protease I, resulting in several peptide fragments . Amino acid sequences of these peptides showed complete sequence identity to those derived from either ubiquitin or phosphoglycerate mutase-B, suggesting that the 38-kDa protein is monoubiquitinated phosphoglycerate mutase-B, whose calculated mass is 37,369 Da . Western blot using an antibody against phosphoglycerate mutase-B revealed the presence of the 38-kDa protein in the anti-ubiquitin immunoprecipitates derived from the tumor regions, but not from normal counterparts . In addition, part of non-ubiquitinated phosphoglycerate mutase-B (29 kDa) was also found in the anti-ubiquitin immunoprecipitates, whose levels were higher in the tumor regions than in the adjacent normal regions . These results suggest that monoubiquitination of phosphoglycerate mutase-B as well as formation of a noncovalent complex containing ubiquitin and phosphoglycerate mutase-B increases in colorectal cancer and novel modification of phosphoglycerate mutase-B might have a pathophysiological role .

Arch Microbiol, 2001 Dec, 176(6), 406 - 14 Epub 2001 Sep 14.
Alkanesulfonate degradation by novel strains of Achromobacter xylosoxidans, Tsukamurella wratislaviensis and Rhodococcus sp., and evidence for an ethanesulfonate monooxygenase in A . xylosoxidans strain AE4; Erdlenbruch BN et al.; Novel isolates of Achromobacter xylosoxidans, Tsukamurella wratislaviensis and a Rhodococcus sp . are described . These grew with short-chain alkanesulfonates as their sole source of carbon and energy . T . wratislaviensis strain SB2 grew well with C(3)-C(6) linear alkanesulfonates, isethionate and taurine, Rhodococcus sp . strain CB1 used C(3)-C(10) linear alkanesulfonates, taurine and cysteate, but neither strain grew with ethanesulfonate . In contrast, A . xylosoxidans strain AE4 grew well with ethanesulfonate, making it the first bacterium to be described which can grow with this compound . It also grew with unsubstituted C(3)-C(5) alkanesulfonates and isethionate . Hydrolysis was excluded as a mechanism for alkanesulfonate metabolism in these strains; and evidence is given for a diversity of uptake and desulfonatase systems . We provide evidence for an initial monooxygenase-dependent desulfonation in the metabolism of ethanesulfonate and propanesulfonate by A . xylosoxidans strain AE4.

J Drug Target, 2001, 9(4), 281 - 94
Preparation of Fab' from murine IgG2a for thiol reactive conjugation; Fowers KD et al.; Lysyl endopeptidase (LE) from Achromobacter lyticus M497-1 (EC 3.4.21.50) was utilized to prepare F(ab')2 fragments from mouse anti-P-glycoprotein IgG2a obtained from the UIC2 hybridoma . This report describes a novel single step purification procedure for F(ab')2 fragments that eliminates residual LE activity responsible for secondary cleavage of F(ab')2 to Fab fragments . The purification of F(ab')2 and Fc fragments was accomplished utilizing protein G affinity chromatography and either gradient or step changes in the pH/ionic strength for elution of the Fc and F(ab')2 fragments . Residual LE was eluted from the protein G column with buffer containing 200 mM L-lysine prior to elution of F(ab')2 and Fc fragments . The activity of LE was monitored using the fluorogenic substrate Boc-Val-Leu-Lys-7-amido 4-methyl coumarin . A similar purification procedure for F(ab')2 fragments produced following pepsin digestion of IgG2a is also outlined . The ability of Fab' fragments, from reduced F(ab')2 fragments following LE digestion of IgG2a, to conjugate to thiol reactive groups was demonstrated using N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-meso chlorin e6 mono (N-2-aminoethylamide) (Mce6) conjugates containing reactive maleimide groups . The biological activity of the Fab' targeted HPMA copolymer-Mce6 conjugates was tested against the P-glycoprotein expressing human ovarian carcinoma A2780/AD cell line utilizing a cell survival assay . Fab' targeted HPMA copolymer-Mce6 conjugate demonstrated significantly higher cytotoxicity than either a monoclonal antibody (mAb) targeted HPMA copolymer-Mce6 conjugate or a non-targeted HPMA copolymer-Mce6 conjugate, p < 0.05.

J Biol Chem, 2002 Jan 11, 277(2), 1310 - 5 Epub 2001 Oct 30.
Investigation of a peptide responsible for amyloid fibril formation of beta 2-microglobulin by achromobacter protease I; Kozhukh GV et al.; To obtain insight into the mechanism of amyloid fibril formation from beta(2)-microglobulin (beta2-m), we prepared a series of peptide fragments using a lysine-specific protease from Achromobacter lyticus and examined their ability to form amyloid fibrils at pH 2.5 . Among the nine peptides prepared by the digestion, the peptide Ser(20)-Lys(41) (K3) spontaneously formed amyloid fibrils, confirmed by thioflavin T binding and electron microscopy . The fibrils composed of K3 peptide induced fibril formation of intact beta2-m with a lag phase, distinct from the extension reaction without a lag phase observed for intact beta2-m seeds . Fibril formation of K3 peptide with intact beta2-m seeds also exhibited a lag phase . On the other hand, the extension reaction of K3 peptide with the K3 seeds occurred without a lag phase . At neutral pH, the fibrils composed of either intact beta2-m or K3 peptide spontaneously depolymerized . Intriguingly, the depolymerization of K3 fibrils was faster than that of intact beta2-m fibrils . These results indicated that, although K3 peptide can form fibrils by itself more readily than intact beta2-m, the K3 fibrils are less stable than the intact beta2-m fibrils, suggesting a close relation between the free energy barrier of amyloid fibril formation and its stability.

Ukr Biokhim Zh, 2001 Jan-Feb, 73(1), 148 - 52
{Use of Pseudomonas and Achromobacter species bacteria--degraders of surface-active agents--for detection and destruction of polycyclic aromatic hydrocarbons}; Ivashchenko GV et al.; The developed biosensor models were based on the use of immobilized Pseudomonas and Achromobacter cells for polycyclic aromatic hydrocarbons and surfactants detection . The responses of biosensors based on bacteria-degraders of anionic surfactants for organic substrates, which related to different classes of surfactants, aromatic and policyclic aromatic hydrocarbons (PAH) were investigated . The sensor showed the highest sensitivity to anionic surfactants and PAH . The lower limit of sodium dodecyl sulfate detection is within a range of 0.25-0.5 mg/l (0.86-1.73 microM) . The sensors showed the highest sensitivity to naphthalene (1-6 mM) and anthracene, fluorene, phenanthrene . All strains that have been investigated may be used as a receptor element of biosensors for detection of PAH and surfactants.

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1867 - 71
Pigmentiphaga kullae gen . nov., sp . nov., a novel member of the family Alcaligenaceae with the ability to decolorize azo dyes aerobically; Blumel S et al.; The taxonomic position of Pseudomonas strain K24, which was isolated previously after an aerobic enrichment with the azo compound 1-(4'-carboxyphenylazo)-4-naphthol as the sole source of carbon and energy, was investigated . The detection of a quinone system with ubiquinone Q-8 as the predominant compound and a polyamine pattern with putrescine and 2-hydroxyputrescine as the major polyamines present suggested that strain K24T belongs to the beta-subclass of the Proteobacteria . This was supported by sequencing the 16S rRNA gene, which demonstrated about 95-96% sequence similarity to different species of the genera Achromobacter, Alcaligenes and Bordetella . This suggested that strain K24T is a member of the family Alcaligenaceae . The G+C content of the DNA was 68.5 mol % . Different methods for the construction of phylogenetic dendrograms placed strain K24T separate from the genera Alcaligenes, Achromobacter and Bordetella . Analysis of the fatty acids demonstrated the presence of 10:0 3-OH and high concentrations of summed feature 7 (18:1omega7c, 18:1omega9t and/or 18:1omega12t) and 19:0 cycloomega8c, which is unique among previously described species of the genera Alcaligenes, Achromobacter and Bordetella . On the basis of the low 16S rRNA sequence similarities, the composition of the fatty acid profile and unique phenotypic properties, a new genus and species is proposed for strain K24T with the name Pigmentiphaga kullae gen . nov., sp . nov.

J Clin Microbiol, 2001 Oct, 39(10), 3597 - 602
Use of random amplified polymorphic DNA PCR to examine epidemiology of Stenotrophomonas maltophilia and Achromobacter (Alcaligenes) xylosoxidans from patients with cystic fibrosis; Krzewinski JW et al.; Stenotrophomonas maltophilia and Achromobacter (Alcaligenes) xylosoxidans have been increasingly recognized as a cause of respiratory tract colonization in cystic fibrosis (CF) . Although both organisms have been associated with progressive deterioration of pulmonary function, demonstration of causality is lacking . To examine the molecular epidemiology of S . maltophilia and A . xylosoxidans in CF, isolates from patients monitored for up to 2 years were fingerprinted using a PCR-based randomly amplified polymorphic DNA (RAPD-PCR) method . Sixty-one of 69 CF centers screened had 183 S . maltophilia culture-positive patients, and 46 centers had 92 A . xylosoxidans-positive patients . At least one isolate from each patient was genotyped, and patients with > or =10 positive cultures (12 S . maltophilia cultures, 15 A . xylosoxidans cultures) had serial isolates genotyped . In addition, centers with multiple culture-positive patients were examined for evidence of shared clones . There were no instances of shared genotypes among different CF centers . Some patients demonstrated isolates with a single genotype throughout the observation period, and others had intervening or sequential genotypes . At the six centers with multiple S . maltophilia culture-positive patients and the seven centers with multiple A . xylosoxidans-positive patients, there were three and five instances of shared genotypes, respectively . The majority of shared isolates were from pairs who were siblings or otherwise epidemiologically linked . These findings suggest RAPD-PCR typing can distinguish unique CF isolates of S . maltophilia and A . xylosoxidans, person-to-person transmission may occur, there are not a small number of clones infecting CF airways, and patients with long-term colonization may either have a persistent organism or may acquire additional organisms over time.

Antimicrob Agents Chemother, 2001 Oct, 45(10), 2838 - 44
Cathelicidin peptides inhibit multiply antibiotic-resistant pathogens from patients with cystic fibrosis; Saiman L et al.; Endogenous peptide antibiotics are under investigation as inhaled therapeutic agents for cystic fibrosis (CF) lung disease . The bactericidal activities of five cathelicidin peptides (LL37 {human}, CAP18 {rabbit}, mCRAMP {mouse}, rCRAMP {rat}, and SMAP29 {sheep}), three novel alpha-helical peptides derived from SMAP29 and termed ovispirins (OV-1, OV-2, and OV-3), and two derivatives of CAP18 were tested by broth microdilution assays . Their MICs were determined for multiply antibiotic-resistant Pseudomonas aeruginosa (n = 24), Burkholderia cepacia (n = 5), Achromobacter xylosoxidans (n = 5), and Stenotrophomonas maltophilia (n = 5) strains isolated from CF patients . SMAP29 was most active and inhibited mucoid and nonmucoid P . aeruginosa strains (MIC, 0.06 to 8 microg/ml) . OV-1, OV-2, and OV-3 were nearly as active (MIC, 0.03 to 16 microg/ml), but CAP18 (MIC, 1.0 to 32 microg/ml), CAP18-18 (MIC, 1.0 to >32 microg/ml), and CAP18-22 (MIC, 0.5 to 32 microg/ml) had variable activities . LL37, mCRAMP, and rCRAMP were least active against the clinical isolates studied (MIC, 1.0 to >32 microg/ml) . Peptides had modest activities against S . maltophilia and A . xylosoxidans (MIC range, 1.0 to > 32 microg/ml), but none inhibited B . cepacia . However, CF sputum inhibited the activity of SMAP29 substantially . The effects of peptides on bacterial cell membranes and eukaryotic cells were examined by scanning electron microscopy and by measuring transepithelial cell resistance, respectively . SMAP29 caused the appearance of bacterial membrane blebs within 1 min, killed P . aeruginosa within 1 h, and caused a dose-dependent, reversible decrease in transepithelial resistance within 5 h . The tested cathelicidin-derived peptides represent a novel class of antimicrobial agents and warrant further development as prophylactic or therapeutic agents for CF lung disease.

J Clin Microbiol, 2001 Sep, 39(9), 3104 - 9
Identification of strains of Alcaligenes and Agrobacterium by a polyphasic approach; Clermont D et al.; The number of stable discriminant biochemical characters is limited in the genera Alcaligenes and Agrobacterium, whose species are consequently difficult to distinguish from one another by conventional tests . Moreover, genomic studies have recently drastically modified the nomenclature of these genera; for example, Alcaligenes xylosoxidans was transferred to the genus Achromobacter in 1998 . Twenty-five strains of Achromobacter xylosoxidans, three strains of an Agrobacterium sp., five strains of an Alcaligenes sp., and four unnamed strains belonging to the Centers for Disease Control and Prevention group IVc-2 were examined . These strains were characterized by conventional tests, including biochemical tests . The assimilation of 99 carbohydrates, organic acids, and amino acids was studied by using Biotype-100 strips, and rRNA gene restriction patterns were obtained with the automated Riboprinter microbial characterization system after cleavage of total DNA with EcoRI or PstI restriction endonuclease . This polyphasic approach allowed the two subspecies of A . xylosoxidans to be clearly separated . Relationships between five strains and the Ralstonia paucula type strain were demonstrated . Likewise, three strains were found to be related to the Ochrobactrum anthropi type strain . We showed that substrate assimilation tests and automated ribotyping provide a simple, rapid, and reliable means of identifying A . xylosoxidans subspecies and that these two methods can be used as alternative methods to characterize unidentified strains rapidly when discriminant biochemical characters are missing.

J Basic Microbiol, 2001, 41(3-4), 159 - 70
Isolation and characterization of Achromobacter xylosoxidans T7 capable of degrading toluidine isomers; Hinteregger C et al.; A bacterial strain capable of utilizing toluidine isomers as its sole source of carbon and energy for growth was isolated from contaminated soil . The isolate was identified as Achromobacter xylosoxidans and was designated strain T7 . Strain T7 differs from other toluidine-degrading strains with respect to the use of all three toluidine isomers even as an equimolar mixture . Additionally, strain T7 harbours the ability to use aniline, phenol, and cresols as growth substrates . Utilization of the toluidine isomers was demonstrated by an increase in the bacterial biomass concomitant with a decrease of the respective toluidine concentration in liquid medium with this compound as sole source of carbon and energy . No accumulation of any intermediate was detectable by HPLC-analyses . Results of oxygen uptake experiments with resting cells of strain T7 pre-grown on the respective toluidine and enzymatic investigations in cell-free extracts indicate the metabolization of the toluidines via the respective methylcatechols as intermediates . These compounds are substrates for the meta-cleavage pathway initiated by inducible catechol 2,3-dioxygenase found in toluidine-grown cells of strain T7.

J Ind Microbiol Biotechnol, 2001 May, 26(5), 309 - 15
Conversion of gamma-butyrobetaine to L-carnitine by Achromobacter cycloclast; Naidu GS et al.; L-Carnitine is an ubiquitous substance that plays a major role in the transportation of long-chain fatty acids . We investigated crucial factors that influence microbial conversion of gamma-butyrobetaine to L-carnitine using an Achromobacter cycloclast strain . Two-stage culture results showed that gamma-butyrobetaine induced enzymes essential for the conversion, which suggests that the precursor should be present in the initial cell growth stage . The addition of yeast extract enhanced L-carnitine production whereas inorganic nitrogen sources inhibited it . Under nitrogen-limiting conditions, the cells accumulated poly-beta-hydroxybutyrate instead of L-carnitine . Among the trace elements tested, nickel addition enhanced L-carnitine production by almost twice that of the control and copper strongly inhibited the conversion . L-Carnitine production was reduced when the medium contained inorganic salts of sodium, potassium, and calcium at a concentration greater than 2 g l(-1) . A higher L-carnitine yield was achieved when cells were incubated in a lower culture volume . The optimal pH for L-carnitine production was 5 to 5.5, whereas that of growth was 7.0, indicating that a pH shift was required . Under optimal conditions, L-carnitine concentrations as high as 15 g l(-1) were obtained in 62 h with a 45% molar conversion yield.

J Biol Chem, 2001 Sep 28, 276(39), 36067 - 70 Epub 2001 Jul 31.
Oxidative modification of tryptophan 43 in the heme vicinity of the F43W/H64L myoglobin mutant; Hara I et al.; The F43W/H64L myoglobin mutant was previously constructed to investigate the effects of electron-rich tryptophan residue in the heme vicinity on the catalysis, where we found that Trp-43 in the mutant was oxidatively modified in the reaction with m-chloroperbenzoic acid (mCPBA) . To identify the exact structure of the modified tryptophan in this study, the mCPBA-treated F43W/H64L mutant has been digested stepwise with Lys-C achromobacter and trypsin to isolate two oxidation products by preparative fast protein liquid chromatography . The close examinations of the (1)H NMR spectra of peptide fragments reveal that two forms of the modified tryptophan must have 2,6-disubstituted indole substructures . The (13)C NMR analysis suggests that one of the modified tryptophan bears a unique hydroxyl group in stead of the NH(2) group at the amino-terminal . The results together with mass spectrometry (MS)/MS analysis (30 Da increase in mass of Trp-43) indicate that oxidation products of Trp-43 are 2,6-dihydro-2,6-dioxoindole and 2,6-dihydro-2-imino-6-oxoindole derivatives . Our finding is the first example of the oxidation of aromatic carbons by the myoglobin mutant system.

J Biomed Sci, 2001 Jul-Aug, 8(4), 342 - 8
Isolation and partial characterization of a 46-kd allergen of Bermuda grass pollen; Wu WC et al.; 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 .

J Bacteriol, 2001 May, 183(9), 2803 - 7
NreB from Achromobacter xylosoxidans 31A Is a nickel-induced transporter conferring nickel resistance; Grass G et al.; There are two distinct nickel resistance loci on plasmid pTOM9 from Achromobacter xylosoxidans 31A, ncc and nre . Expression of the nreB gene was specifically induced by nickel and conferred nickel resistance on both A . xylosoxidans 31A and Escherichia coli . E . coli cells expressing nreB showed reduced accumulation of Ni(2+), suggesting that NreB mediated nickel efflux . The histidine-rich C-terminal region of NreB was not essential but contributed to maximal Ni(2+) resistance.

Medicina (B Aires), 2001, 61(1), 79 - 80
{Achromobacter xylosoxidans bacteremia in a patient with community-acquired pneumonia}; de Fernandez MI et al.; Achromobacter xylosoxidans is a rare cause of bacteremia, and little information on treatment is available . The majority of patients who have developed Achromobacter bacteremia have presented predisposing causes to the infection . A case of community-acquired pneumonia and bacteremia due to A . xylosoxidans in a previously healthy patient is reported . Achromobacter is usually resistant to ampicillin, cephalosporins (1st, 2nd, and 3rd generation), aminoglycosides, and fluoroquinolones . Piperacillin, piperacillin-tazobactam, and trimethoprim-sulfamethoxazole inhibit most isolates.

Antimicrob Agents Chemother, 2001 Apr, 45(4), 1249 - 53
In70 of plasmid pAX22, a bla(VIM-1)-containing integron carrying a new aminoglycoside phosphotransferase gene cassette; Riccio ML et al.; An Achromobacter xylosoxydans strain showing broad-spectrum resistance to beta-lactams (including carbapenems) and aminoglycosides was isolated at the University Hospital of Verona (Verona, Italy) . This strain was found to produce metallo-beta-lactamase activity and to harbor a 30-kb nonconjugative plasmid, named pAX22, carrying a bla(VIM-1) determinant inserted into a class 1 integron . Characterization of this integron, named In70, revealed an original array of four gene cassettes containing, respectively, the bla(VIM-1) gene and three different aminoglycoside resistance determinants, including an aacA4 allele, a new aph-like gene named aphA15, and an aadA1 allele . The aphA15 gene is the first example of an aph-like gene carried on a mobile gene cassette, and its product exhibits close similarity to the APH(3')-IIa aminoglycoside phosphotransferase encoded by Tn5 (36% amino acid identity) and to an APH(3')-IIb enzyme from Pseudomonas aeruginosa (38% amino acid identity) . Expression of the cloned aphA15 gene in Escherichia coli reduced the susceptibility to kanamycin and neomycin as well as (slightly) to amikacin, netilmicin, and streptomycin . Characterization of the 5' and 3' conserved segments of In70 and of their flanking regions showed that In70 belongs to the group of class 1 integrons associated with defective transposon derivatives originating from Tn402-like elements . The structure of the 3' conserved segment indicates the closest ancestry with members of the In0-In2 lineage . In70, with its array of cassette-borne resistance genes, can mediate broad-spectrum resistance to most beta-lactams and aminoglycosides.

Plant J, 2001 Feb, 25(3), 261 - 70
Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco; Ye GN et al.; Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants . However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type . We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs . We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) . Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes . Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves) . Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues . Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene . Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.

Biochemistry, 2001 Jan 30, 40(4), 1044 - 52
Molecular engineering of myoglobin: the improvement of oxidation activity by replacing Phe-43 with tryptophan; Ozaki S et al.; The F43W and F43W/H64L myoglobin (Mb) mutants have been constructed to investigate effects of an electron rich oxidizable amino acid residue in the heme vicinity on oxidation activities of Mb . The Phe-43 --> Trp mutation increases the rate of one-electron oxidation of guaiacol by 3-4-fold; however, the peroxidase activity for F43W/H64L Mb is less than that of the F43W single mutant because the absence of histidine, a general acid-base catalyst, in the distal heme pocket suppresses compound I formation . More than 15-fold improvement versus wild-type Mb in the two-electron oxidation of thioanisole and styrene is observed with the Phe-43 --> Trp mutation . Our results indicate that Trp-43 in the mutants enhances both one- and two-electron oxidation activities (i.e., F43W Mb > wild-type Mb and F43W/H64L Mb > H64L Mb) . The level of (18)O incorporation from H2(18)O2 into the epoxide product for the wild type is 31%; however, the values for F43W and F43W/H64L Mb are 75 and 73%, respectively . Thus, Trp-43 in the mutants does not appear to be utilized as a major protein radical site to form a peroxy protein radical in the oxygenation . The enhanced peroxygenase activity might be explained by the increase in the reactivity of compound I . However, the oxidative modification of F43W/H64L Mb in compound I formation with mCPBA prevents us from determining the actual reactivity of the catalytic species for the intact protein . The Lys-C achromobacter digestion of the modified F43W/H64L mutant followed by FPLC and mass analysis shows that the Trp-43-Lys-47 fragment gains a mass by 30 Da, which could correspond two oxygen atoms and loss of two protons.

J Clin Microbiol, 2001 Feb, 39(2), 808 - 10
Recurrent Achromobacter piechaudii bacteremia in a patient with hematological malignancy; Kay SE et al.; We describe a recurrent bacteremia caused by Achromobacter (formerly Alcaligenes) piechaudii in association with an intravenous catheter in an immunocompromised 73-year-old man . This is the first reported case of bacteremia due to A . piechaudii.

J Inorg Biochem, 2000 Nov, 82(1-4), 79 - 84
Spectroscopic and electrochemical properties of the Met86Gln mutant of Achromobacter cycloclastes pseudoazurin; Kataoka K et al.; The mutant replacing the Met86 ligand of Achromobacter cycloclastes pseudoazurin (Ac-pAz) with Gln has been prepared and spectroscopically and electrochemically characterized . Ac-pAz has four ligands (2His, Cys, and Met) and donates one electron to its cognate Cu-containing nitrite reductase (Ac-NIR) . The mutant ({Met86Gln}pAz) shows the electronic absorption and CD spectra considerably similar to those of zucchini mavicyanin (Mv) and lacquer and cucumber stellacyanins (St) having 2His, Cys, and Gln . The EPR signal of the mutant has an axial character, although those of Mv and St show rhombic signals . The findings indicate that the Cu site having Gln might be a distorted trigonal geometry . The half-wave potentials (E(1/2)) of {Met86Gln}pAz and the intermolecular electron-transfer rate constant (kET) from the mutant to Ac-NIR were determined by cyclic voltammetry at pH 7.0 and 25 degrees C . The E(1/2) is +134 mV (versus NHE) and the coordination of Gln instead of Met negatively shifts the E(1/2) of Ac-pAz (+260 mV (versus NHE)) . The kET of {Met86Gln}pAz (1.2x10(6) M(-1) s(-1)) is larger than that of the recombinant Ac-pAz (7.5x10(5) M(-1) s(-1)).

Clin Infect Dis, 2000 Nov, 31(5), 1183 - 7 Epub 2000 Nov 06.
Recurrent Achromobacter xylosoxidans bacteremia associated with persistent lymph node infection in a patient with hyper-immunoglobulin M syndrome; Weitkamp JH et al.; Achromobacter xylosoxidans (formerly Alcaligenes xylosoxidans) is a rare but important cause of bacteremia in immunocompromised patients, and strains are usually multiply resistant to antimicrobial therapy . We report an immunocompromised patient with hyper-immunoglobulin M syndrome who suffered from 14 documented episodes of A . xylosoxidans bacteremia . Each episode was treated and resulted in rapid clinical improvement, with blood cultures testing negative for bacteria . Between episodes, A . xylosoxidans was isolated from an excised right axillary lymph node, whereas the culture of the central venous catheter, removed at the same time, was negative . Multiple cultures from sputum, stool, and urine samples, as well as from gastrointestinal biopsies or environmental sources, were negative . Results from antibiotic sensitivity testing and pulsed-field gel electrophoresis suggested that a single strain of A . xylosoxidans caused the recurrent bacteremias in this patient; this strain originated from persistently infected lymph nodes . Lymphoid hyperplasia is a prominent characteristic of hyper-IgM syndrome and may serve as a source of bacteremia with low-pathogenicity organisms.

Biochim Biophys Acta, 2000 Oct 18, 1523(2-3), 254 - 60
Amino acid sequence and some properties of phytolacain G, a cysteine protease from growing fruit of pokeweed, Phytolacca americana; Uchikoba T et al.; A protease, phytolacain G, has been found to appear on CM-Sepharose ion-exchange chromatography of greenish small-size fruits of pokeweed, Phytolacca americana L, from ca . 2 weeks after flowering, and increases during fruit enlargement . Reddish ripe fruit of the pokeweed contained both phytolacain G and R . The molecular mass of phytolacain G was estimated to be 25.5 kDa by SDS-PAGE . Its amino acid sequence was reconstructed by automated sequence analysis of the peptides obtained after cleavage with Achromobacter protease I, chymotrypsin, and cyanogen bromide . The enzyme is composed of 216 amino acid residues, of which it shares 152 identical amino acid residues (70%) with phytolacain R, 126 (58%) with melain G, 108 (50%) with papain, 106 (49%) with actinidain, and 96 (44%) with stem bromelain . The amino acid residues forming the substrate binding S(2) pocket of papain, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Trp, Met, His, Ala, and Ser in phytolacain G, respectively . As a consequence of these substitutions, the S(2) pocket is expected to be less hydrophobic in phytolacain G than in papain.

Biochem Biophys Res Commun, 2000 Sep 16, 276(1), 23 - 8
Complete amino acid sequence of Japanese chestnut agglutinin; Nomura K et al.; 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 .

FEBS Lett, 2000 Aug 4, 478(3), 295 - 8
Zeta-crystallin catalyzes the reductive activation of 2,4,6-trinitrotoluene to generate reactive oxygen species: a proposed mechanism for the induction of cataracts; Kumagai Y et al.; Exposure to 2,4,6-trinitrotoluene (TNT) has been shown to cause induction of cataract in which oxidative stress plays a critical role . From bovine lens we purified to homogeneity and identified an enzyme that catalyzes the reduction of TNT, resulting in the production of reactive oxygen species . The final preparation of TNT reductase showed a single band with a subunit molecular weight of 38 kDa on SDS-PAGE . Sequence data from peptides obtained by digestion with lysylendopeptidase Achromobacter protease I (API) revealed that TNT reductase is identical to zeta-crystallin . Superoxide anions were formed during reduction of TNT by zeta-crystallin, though negligible enzyme activity or protein content for superoxide dismutase, a superoxide scavenging enzyme, was found in the lens . Thus, the present results suggest that the induction of cataracts by TNT may be associated with increased oxidative stress, as a result of reductive activation of TNT generating superoxide anions, there being minimal antioxidant enzyme activity for defense against reactive oxygen species exogenously produced in the lens.

Syst Appl Microbiol, 2000 Jun, 23(2), 292 - 9
Isolation and identification of cellulolytic bacteria involved in the degradation of natural cellulosic fibres; Lednicka D et al.; In search for bacterial cultures that are able to rapidly degrade cellulosic plant fibres in vitro, 77 cellulolytic strains were isolated from Belgian and Czech soils after enrichment on flax or sisal fibres as sole sources of carbon . The strains were characterized using fatty acid analysis, and 74 strains were grouped into three major clusters by numerical analysis . The first major cluster contained Cellulomonas strains . Within this cluster three subclusters could be delineated by principal component analysis, that were recognized by their fatty acid compositions as Cellulomonas gelida, Cellulomonas biazotea and Cellulomonas cellulans, containing 9, 8 and 13 strains respectively . The second major cluster, with 9 strains, was assigned to Flavobacterium johnsoniae . The 34 strains of the third cluster could not be identified by commercial identification systems on the basis of their fatty acid profiles and API 20NE profiles . On the basis of their phenotypic characteristics they met the description of the genus Cellvibrio, their fatty acid profiles were similar to those of four authentic Cellvibrio mixtus strains, and the 16S rRNA genes from four representatives showed up to 97.8% sequence similarity to 16S rDNA from Cellvibrio mixtus ACM 2603 . Three non-clustered strains were assigned to Curtobacterium flaccumfaciens, Achromobacter piechaudii and Pseudomonas mendocina . Two strains assigned to Cellvibrio were able to degrade several flax, broom and cotton fibres very rapidly in a standardized in vitro test, causing mass losses of 40 to 86% within 13 days of incubation, but not jute.

Electrophoresis, 2000 May, 21(9), 1755 - 65
Profiling of Caenorhabditis elegans proteins using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry; Kaji H et al.; The nematode Caenorhabditis elegans (C . elegans) is the first animal whose whole 97 Mb genome sequence, encoding ca . 19000 open reading frames (ORF's), has been essentially determined . We tried to establish a 2-DE map of the nematode proteome by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) . A soluble protein fraction of mixed stages of the worm, wild-type strain N2, was applied to 2-D PAGE . After Coomassie Brilliant Blue (CBB) staining, 1200 spots were detected and 140 major spots were excised from the gel and subjected to in-gel digestion with Achromobacter protease I (lysyl endopeptidase) . Resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting for protein identification . With this approach we have obtained a two-dimensional electrophoresis (2-DE) protein map in which 69 spots were localized as landmarks for comparison of expression profiles to elucidate the basis of various biological events.

FEMS Microbiol Lett, 2000 Jul 1, 188(1), 41 - 6
Isolation and characterization of an Achromobacter xylosoxidans strain B3 and other bacteria capable to degrade the synthetic chelating agent iminodisuccinate; Reinecke F et al.; Three bacterial strains were isolated, which used the synthetic chelating agent iminodisuccinate (IDS) as sole carbon source for growth in mineral salts media (MSM) . Taxonomic analysis and 16S rDNA sequence analysis identified one of these isolates (B3), which was isolated from sewage sludge, as a strain of Achromobacter xylosoxidans subsp . xylosoxidans . It exhibited a doubling time of approximately 3 h in liquid MSM supplemented with IDS and grew even in the presence of 1.0% (w/v) IDS . Since photometric and high performance liquid chromatography analysis showed that IDS, which came onto the market only recently as an alternative for ethylenediaminetetraacetate, was completely degraded by axenic cultures of bacteria; it will probably be readily degraded in the environment.

Can J Microbiol, 2000 Mar, 46(3), 229 - 36
Stimulation of the ionic transport system in Brassica napus by a plant growth-promoting rhizobacterium (Achromobacter sp.); Bertrand H et al.; A plant growth-promoting rhizobacterium belonging to the genus Achromobacter was isolated from the oil-seed-rape (Brassica napus) root . Growth promotion bioassays were performed with oilseed rape seedlings in a growth chamber in test tubes containing attapulgite and mineral nutrient solution, containing NO3- as N source . The presence of this Achromobacter strain increased shoot and root dry weight by 22-33% and 6-21%, respectively . Inoculation of young seedlings with the Achromobacter bacteria induced a 100% improvement in NO3- uptake by the whole root system . Observations on the seminal root of seedlings 20 h after inoculation showed that there was an enhancement of both the number and the length of root hairs, compared to non-inoculated seedlings . Electrophysiological measurements of NO3- net flux with ion-selective microelectrodes showed that inoculation resulted in a specific increase of net nitrate flux in a root zone morphologically similar in inoculated and non-inoculated plants . The root area increased due to root hair stimulation by the Achromobacter bacteria, which might have contributed to the improvement of NO3- uptake by the whole root system, together with the enhancement of specific NO3- uptake rate . Moreover, inoculated plants showed increased potassium net influx and proton net efflux . Overall, the data presented suggest that the inoculation of oilseed-rape with the bacteria Achromobacter affects the mineral uptake.

Biochim Biophys Acta, 1999 Sep 14, 1434(1), 18 - 30
Helix 6 of subdomain III A of human serum albumin is the region primarily photolabeled by ketoprofen, an arylpropionic acid NSAID containing a benzophenone moiety; Chuang VT et al.; It is well known that the subdomain III A (site II) of human serum albumin (HSA) binds a variety of endogenous and exogenous substances . However, the nature of the microenvironment of the binding site remains unclear . Ketoprofen (KP), an arylpropionic acid NSAID which contains a benzophenone moiety, was used as a photoaffinity labeling agent to label the binding region . Subsequent CNBr cleavage of the photolabeled HSA revealed that the 11.6 kDa and 9.4 kDa fragments contained most of the incorporated radioactivity . Competition experiments showed that the 11.6 kDa fragment contains the common binding region for site II ligands . This fragment was redigested with Achromobacter lyticus protease I (AP-I) and the amino acid sequence of the photolabeled peptide was determined to be XCTESLVNRR, which corresponds to the sequence 476C-485K of HSA . The complete amino acid sequence of the corresponding AP-I digested HSA peptide encompasses residues 476 to 499, which form helices 5 and 6 of subdomain III A . The HSA-Myr X-ray crystallography data showed that helix 5 is involved to the least extent in ligand binding . A docking model provided further support that helix 6 represents the photolabeled region of KP.

J Pept Sci, 1999 Aug, 5(8), 352 - 9
Reactivity of Lys(NH2)-containing peptides toward endopeptidases; Samson F et al.; Lys(NH2)-containing peptides were subjected to various proteolytic enzymes which were selected for their well-documented specificity for arginyl and/or lysyl peptide bonds . Lys(NH2)-containing peptides were cleaved more rapidly by clostripain than the corresponding lysyl peptides . On the other hand, they proved to be resistant to Achromobacter protease I hydrolysis . The modified peptides synthesized in this study were more stable than the arginyl and lysyl analogues when incubated with trypsin or thrombin . The same tendency was observed when Lys(NH2)-containing peptides were incubated in diluted human serum, suggesting that the replacement of Arg or Lys by Lys(NH2) could be used to increase the stability of peptides in vivo.

Biodegradation, 1999 Apr, 10(2), 105 - 12
Batch culture biodegradation of methylhydrazine contaminated NASA wastewater; Nwankwoala AU et al.; The batch culture degradation of NASA wastewater containing mixtures of citric acid, methylhydrazine, and their reaction product was studied . The organic contaminants present in the NASA wastewater were degraded by Achromobacter sp., Rhodococcus B30 and Rhodococcus J10 . While the Achromobacter sp . showed a preference for the degradation of the citric acid, the Rhodococcus species were most effective in reducing the methylhydrazine and the reaction product . Removals of more than 50% were observed for citric acid, methylhydrazine and the reaction product when the NASA wastewater was inoculated with the microbes in batch cultures . Simulation and chemical characterization of citric acid and hydrazine mixtures show that the interaction is partly of a chemical nature and leads to the formation of a conjugated UV/Visible absorbing compound . An 'azo' carbonyl derivative of the citric acid, consistent with the spectral data obtained from the investigation, has been proposed as the possible product.

J Biochem (Tokyo), 1999 Aug, 126(2), 395 - 401
Production of homo- and hetero-dimeric isozymes from two aldehyde oxidase genes of Arabidopsis thaliana; Akaba S et al.; Polyclonal antibodies were raised against synthetic peptides or recombinant polypeptides encoded by Arabidopsis atAO-1 and atAO-2 cDNAs, which have sequences similar to maize and animal aldehyde oxidase (AO) cDNAs . Anti-atAO-1 antibodies recognized AOalpha and AObeta among the three isoforms, AOalpha, AObeta, and AOgamma, detected in Arabidopsis seedlings after native PAGE, while anti-atAO-2 antibodies reacted with AObeta and AOgamma . The polypeptide specifically recognized by each antibody was collected as the Protein-A/IgG/antigen complex . The 150- and 145-kDa polypeptides were purified by SDS-PAGE and digested with Achromobacter Protease I . From the amino acid sequences and molecular masses of the derivative peptides, it was revealed that the 150- and 145-kDa polypeptides were the products of atAO-1 and atAO-2, respectively . Molecular masses of the native forms of AOalpha, AObeta, and AOgamma were estimated as approximately 290-300 kDa . These results suggest that AOalpha and AOgamma are homodimers consisting of atAO-1 and atAO-2 products, respectively, and that AObeta is a heterodimer of the atAO-1 and atAO-2 products.

J Biochem (Tokyo), 1999 Jul, 126(1), 26 - 33
Amino acid sequence and some properties of phytolacain R, a cysteine protease from full-growth fruits of pokeweed, Phytolacca americana; Yonezawa H et al.; A cysteine protease, phytolacain R from full-growth greenish fruits of pokeweed, Phytolacca americana L, was purified to electrophoretic homogeneity by a simple purification procedure employing CM-Sepharose ion-exchange chromatography . The enzyme was present in low content in the young fruits about 50 d after flowering but gradually accumulated in growing fruits . Its molecular mass was estimated to be ca . 23 kDa by SDS-PAGE, and its sugar content was zero . Its amino acid sequence was established by automated sequence analysis of the peptides obtained by cleavage with Achromobacter protease I, chymotrypsin, trypsin, and cyanogen bromide . The enzyme is composed of 218 amino acid residues, of which it shares 110 residues (50%) with papain, 104 (47%) with actinidain, and 87 (40%) with stem bromelain . The amino acid residues forming the substrate-binding the S2 pocket of papain, Tyr61, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Gly, Trp, Met, His, Ala, and Met in phytolacain R, respectively . As a consequence of these substitutions, the S2 pocket is expected to be less hydrophobic in phytolacain R than in papain.

Eur J Cardiothorac Surg, 1999 Apr, 15(4), 490 - 4; discussion 495
Lung infections in pediatric lung transplantation: experience in 49 cases; Metras D et al.; OBJECTIVES: Pulmonary infections, and particularly cytomegalovirus (CMV) infections, are a major cause of morbidity after lung transplantation . We report here our results in 49 pediatric lung transplantations . METHODS: Between may 1988 and 1997, we have done 49 lung transplantations in 42 children (en bloc double lung transplantation (DLT):10, HLTx:7, sequential bilateral sequential-lung transplantation (BSLT):31, single-lung transplantation (SLT): 1) . In seven, it was a retransplantation . Among these, 34 were cystic fibrosis (CF) patients, all with multiresistant organisms (Pseudomonas aeruginosa, Burkholderia cepacia, Achromobacter xylososydans, Staphylococcus aureus) . All patients were treated with multiantibiotic prophylaxy adapted to the preoperative cultures . Donor-recipient CMV matching was possible in only 31 cases . CMV prophylaxy and immunosuppression protocols have evolved with time, with a current protocol of IV Gancyclovir prophylaxy for 3 months and triple drug immunosuppression without post-operative rabbit anti-thymocyte globulin (RATG) induction . There was no perioperative mortality in the primary transplantations and three early deaths in the whole group (6.1%) . RESULTS: Only five patients had no pulmonary infection . The patients presented 3.2 infection episodes per year, 75% localized on the lungs, 41% during the first 3 months . Among the 13 deaths in the 1st year, 10 were directly related to infection, 60% due to CMV . After the 1st year, in all patients dying of pulmonary dysfunction or obliterative bronchiolitis (OB), bacterial infections were associated . There was no serious fungal infection . Actuarial survival at 3 months, 1, 3, 5 years were 85, 65.7, 47.5 and 28.5%, respectively . There was a significant difference in 3 year survival between patients receiving CMV negative organs (40%) and CMV positive organs (17%) . CONCLUSION: In our experience, as in other's, pulmonary infection risk is important in lung transplantation . Bacterial infections were mainly an aggravating factor of secondary pulmonary dysfunction or OB, and were not the primary cause of death . CMV infections have been very severe and lead us, despite the scarcity of donors, to avoid positive donors in negative recipients, this leads to disastrous mid-term results in our experience, despite prophylaxis.

Biochim Biophys Acta, 1999 Apr 19, 1427(2), 133 - 44
Structure of the glucan-binding sugar chain of Tip1p, a cell wall protein of Saccharomyces cerevisiae; Fujii T et al.; Tip1p is one of the major cell wall mannoproteins of Saccharomyces cerevisiae and is presumed to be synthesized as a glycosylphosphatidylinositol (GPI)-anchored form . We purified Tip1p from a glucanase extract of yeast cell walls and analyzed the sugar chain involved in the cell wall linkage . One mol of glucanase-extracted Tip1p contained 7.5 mol of glucose derived from glucan and 1 mol of ethanolamine, a component of the GPI anchor . One mol of the C-terminal peptide of Tip1p digested with Achromobacter protease I also contained 7.9 mol of glucose and 1 mol of ethanolamine . On the other hand, Tip1p contained no glucosamine, which is a component of the GPI anchor . The glucan-binding sugar chain of Tip1p was released by hydrazinolysis and isolated . This sugar chain contained ethanolamine with a free amino group and a glucose reducing end, but no mannose reducing end . Phosphodiesterase treatment eliminated the free amino group from this sugar chain, suggesting that a phosphodiester bond exists between the ethanolamine and the glucan remnant . These results indicate (1) the glucan-binding sugar chain of Tip1p is a GPI derivative, and (2) the GPI anchor is cleaved at the glycosyl moiety, and the resultant mannose reducing end is probably used to link Tip1p to cell wall glucan.

Heart Lung, 1999 Mar-Apr, 28(2), 145 - 6
Ochrobactrum anthropi bacteremia; Jelveh N et al.; Ochrobactrum Anthropi (O . anthropi ), formerly known as Achromobacter CDC group Vd, is a gram-negative bacillus that is aerobic, oxidase producing, and nonlactose fermenting . This organism has been found in environmental and hospital water sources and has pathogenic potential in humans . Most reports in the literature of O . anthropi bacteremia are associated with intravenous line infections . We describe a case of bacteremia with O . anthropi in a 33-month-old boy with acute osteomyelitis . O . anthropi bacteremia also has been reported in immunocompromised hosts . Rarely, O . anthropi has been a cause of soft tissue or bone infection.

J Biochem (Tokyo), 1999 Mar, 125(3), 443 - 8
A monoclonal antibody, 3A10, recognizes a specific amino acid sequence present on a series of developmentally expressed brain proteins; Harada A et al.; Immunoblotting showed that a monoclonal antibody, 3A10, binds to a series of rat brain-specific antigens with molecular masses of 150-, 120-, 118-, 106-, 104-, 79-, and 77-kDa . The expression of 3A10 antigens is dependent on the developmental stage of the brain; only the 106-kDa antigen is detected during embryonic stages of rat brain development, while the expression of the remaining 6 antigens starts after birth and reaches a maximum during postnatal days 15-21 . Detection of the 3A10 antigens in cultured neuronal and glial cells derived from cerebral cortices of rat brain at embryonic day 18 showed that the 77-, 79-, 106-, and 150-kDa antigens are specifically expressed in neuronal cells . The 77-kDa antigen was purified and identified as synapsin I by amino acid sequence analyses of the peptide fragments isolated after Achromobacter protease I treatment . During the isolation of 3A10-reactive proteins by immunological screening of cDNA libraries constructed from adult rat brain, we found that all of the 3A10-reactive clones contain nucleotide sequences encoding the unique amino acid sequence TRSP(S, R,G)P . Analyses of 3A10-binding to various synthetic peptides showed that the monoclonal antibody recognizes a specific conformational structure formed by either the TRSPXP sequence or similar amino acid sequences that are expressed on a series of developmentally expressed brain proteins.

Biochem Biophys Res Commun, 1999 Feb 16, 255(2), 427 - 31
Cloning, sequencing, and transcriptional studies of the gene encoding copper-containing nitrite reductase from Alcaligenes xylosoxidans NCIMB 11015; Suzuki E et al.; Gene encoding of the blue copper-containing nitrite reductase (nir) from Alcaligenes xylosoxidans NCIMB 11015 has been cloned and characterized . The nir is translated into a polypeptide of 360 amino acid residues as a precursor, and the N-terminal 24 residues are subsequently removed upon transport into the periplasm as a mature protein . A specific transcription product of nir was detected only in the presence of nitrate . The aeration level of the culture medium did not show a significant effect on the transcriptional level . A varsigma54 binding sequence is identified upstream of the transcriptional initiation at 53 to 26 nucleotides . A putative fnr box has also been identified in the sequence of the upstream region . The mature polypeptide showed 70% sequence identity with those of the Achromobacter cycloclastes enzyme . The transcriptional start point has been determined at 92 nucleotides upstream of the initiation codon and is preceded by the binding sites for varsigma54 and the fnr box . These results suggest that gene expression depends on the presence of nitrate and is stimulated under an anaerobic environment .

Protein Expr Purif, 1998 Dec, 14(3), 309 - 16
Prepro-leaders lacking N-linked glycosylation for secretory expression in the yeast Saccharomyces cerevisiae; Kjeldsen T et al.; 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 .

Biochim Biophys Acta, 1998 Dec 22, 1443(3), 369 - 74
Cloning of a Lysobacter enzymogenes gene that encodes an arginyl endopeptidase (endoproteinase Arg-C); Wright DS et al.; Screening an expression library of Lysobacter enzymogenes DNA allowed us to clone a gene encoding a serine protease that cleaves synthetic substrates C-terminal to Arg and, to a lesser extent, Lys residues . The gene product, which shares sequence homology with the lysyl endopeptidases from L . enzymogenes and Achromobacter lyticus, consists of a signal sequence (24 residues), pro-region ( approximately 195 residues), and catalytic domain ( approximately 244 residues) . Downstream of this gene is an open reading frame that lacks a promoter and appears to encode an inactive type I subtilase.

Kansenshogaku Zasshi, 1998 Oct, 72(10), 1070 - 5
{Two cases of Achromobacter xylosoxidans sepsis}; Kobayashi A et al.; Achromobacter xylosoxidans is a gram-negative bacterium whose natural habitat has not been clearly defined . It has been isolated from ear discharge and the large intestine of humans and from various hospital or environmental water sources . Infection with A . xylosoxidans in humans has been documented, and resulting illnesses include meningitis, pneumonia, cholecystitis, peritonitis and urinary tract infection . Bacteremia due to A . xylosoxidans is rare, and little information on treatment is available . Two cases of bacteremia due to A . xylosoxidans in patients with hemapoietic malignancies are reported herein . Case 1 involved a 70-yr . male whose clinical diagnosis was IgA lambda-type plasmacytoma . Case 2 involved 72-yr . male whose clinical diagnosis was acute lymphatic leukemia (L2) . Both patients had been catheterized . Neutropenia was noted and the white blood cell counts were 20/microliter in case 1 and 35/microliter in case 2 when A . xylosoxidans was isolated from the blood culture . We suggest that bacteremia due to A . xylosoxidans may have been related to the presence of the catheter and neutropenia.

Biochem Biophys Res Commun, 1998 Sep 29, 250(3), 782 - 5
The C-terminal segment is essential for maintaining the quaternary structure and enzyme activity of the nitric oxide forming nitrite reductase from Achromobacter cycloclastes; Chang WC et al.; We have constructed and expressed a series of mutated nitrite reductase (NIR) mutants based on the sequence of NIR from Achromobacter cycloclastes . Deleting a pentapeptide, an undecapeptide, or a heptadecapeptide from the C-terminus of NIR resulted in a series of C-terminal deletion mutated proteins designated as NIR-5, NIR-11, and NIR-17, respectively . A C-terminally extended mutated protein, NIR+8, was also produced, which contains an extra octapeptide attached to the C-terminus of the wild-type NIR . An SDS-PAGE system using tris-tricine buffer could retain the native NIR in its trimeric form, thus offering a convenient method to check the quaternary structure of NIR analogs . By using this system it was found that NIR-5 was maintained as trimer and retained 72% of wild-type enzyme activity . However, both NIR-11 and NIR-17 behaved as monomers in the SDS-PAGE and lost all their enzyme activity . Although NIR+8 maintained its trimeric structure it was enzymatically inactive . These results clearly indicate that the C-terminal undecapeptide is essential for maintaining the quaternary structure as well as the full enzymatic activity, as expected from the X-ray crystallography studies .

J Inorg Biochem, 1998 Jul, 70(3-4), 155 - 69
The nos (nitrous oxide reductase) gene cluster from the soil bacterium Achromobacter cycloclastes: cloning, sequence analysis, and expression; McGuirl MA et al.; The nitrous oxide (N2O) reductase (nos) gene cluster from Achromobacter cycloclastes has been cloned and sequenced . Seven protein coding regions corresponding to nosR, nosZ (structural N2O reductase gene), nosD, nosF, nosY, nosL, and nosX are detected, indicating a genetic organization similar to that of Rhizobium meliloti . To aid homology studies, nosR from R . meliloti has also been sequenced . Comparison of the deduced amino acid sequences with corresponding sequences from other organisms has also allowed structural and functional inferences to be made . The heterologous expression of NosD, NosZ (N2O reductase), and NosL is also reported . A model of the CuA site in N2O reductase, based on the crystal structure of this site in bovine heart cytochrome c oxidase, is presented . The model suggests that a His residue of the CuA domain may be a ligand to the catalytic CuZ site . In addition, the origin of the spectroscopically-observed Cys coordination to CuZ is discussed in terms of the sequence alignment of seven N2O reductases.

Appl Environ Microbiol, 1998 Aug, 64(8), 2822 - 30
Isolation of broad-host-range replicons from marine sediment bacteria; Sobecky PA et al.; Naturally occurring plasmids isolated from heterotrophic bacterial isolates originating from coastal California marine sediments were characterized by analyzing their incompatibility and replication properties . Previously, we reported on the lack of DNA homology between plasmids from the culturable bacterial population of marine sediments and the replicon probes specific for a number of well-characterized incompatibility and replication groups (P . A . Sobecky, T . J . Mincer, M . C . Chang, and D . R . Helinski, Appl . Environ . Microbiol . 63:888-895, 1997) . In the present study we isolated 1.8- to 2.3-kb fragments that contain functional replication origins from one relatively large (30-kb) and three small (<10-kb) naturally occurring plasmids present in different marine isolates . 16S rRNA sequence analyses indicated that the four plasmid-bearing marine isolates belonged to the alpha and gamma subclasses of the class Proteobacteria . Three of the marine sediment isolates are related to the gamma-3 subclass organisms Vibrio splendidus and Vibrio fischeri, while the fourth isolate may be related to Roseobacter litoralis . Sequence analysis of the plasmid replication regions revealed the presence of features common to replication origins of well-characterized plasmids from clinical bacterial isolates, suggesting that there may be similar mechanisms for plasmid replication initiation in the indigenous plasmids of gram-negative marine sediment bacteria . In addition to replication in Escherichia coli DH5alpha and C2110, the host ranges of the plasmid replicons, designated repSD41, repSD121, repSD164, and repSD172, extended to marine species belonging to the genera Achromobacter, Pseudomonas, Serratia, and Vibrio . While sequence analysis of repSD41 and repSD121 revealed considerable stretches of homology between the two fragments, these regions do not display incompatibility properties against each other . The replication origin repSD41 was detected in 5% of the culturable plasmid-bearing marine sediment bacterial isolates, whereas the replication origins repSD164 and repSD172 were not detected in any plasmid-bearing bacteria other than the parental isolates . Microbial community DNA extracted from samples collected in November 1995 and June 1997 and amplified by PCR yielded positive signals when they were hybridized with probes specific for repSD41 and repSD172 replication sequences . In contrast, replication sequences specific for repSD164 were not detected in the DNA extracted from marine sediment microbial communities.

J Biochem (Tokyo), 1998 Aug, 124(2), 332 - 9
Bacteriolytic activity and specificity of Achromobacter beta-lytic protease; Li S et al.; Achromobacter beta-lytic protease (blp), one of the bacteriolytic proteases secreted by Achromobacter lyticus, exhibited both peptidase and bacteriolytic activities at alkaline pH . The protease was strongly inhibited by 1,10-phenanthroline, and one zinc atom was detected in the molecule by ion-spray mass spectrometry . The zinc-protease specifically cleaved Gly-X bonds in peptides and possibly possessed subsites S2, S1, S1', and S2' for binding substrate {Schecter, I . and Berger, A . (1967) Biochem . Biophys . Res . Commun . 27, 157-162} . Blp lysed Staphylococcus aureus and Micrococcus luteus cells more efficiently than Achromobacter alpha-lytic protease (alp) and lysozyme, thus being responsible for the high bacteriolytic activity of A . lyticus . In the lysis of bacterial cell walls, blp hydrolyzed both the D-Ala-Gly/Ala bond at the linkage between the peptide subunit and the interpeptide and the Gly-Gly bond in the interpeptide bridge . These results indicate that blp is a highly active bacteriolytic enzyme with a broad bacteriolytic spectrum, which acts primarily by splitting the linkage between the peptide subunit and the interpeptide in the peptidoglycan.

Biochem J, 1998 Aug 1, 333 ( Pt 3), 735 - 9
Amino acid sequence of glutathione S-transferase rGSTM5* from rat testis; Tam MF et al.; Glutathione S-transferase rGSTM5* was isolated from rat testis with a combination of glutathione affinity column and reverse-phase column chromatography . The protein was digested with Achromobacter protease I or endoproteinase Arg-C . The peptide fragments were isolated for electrospray MS and N-terminal peptide sequencing analyses . The primary amino acid sequence of rGSTM5* comprises 217 residues and has a calculated average molecular mass of 25495.3 Da . The result is identical to that obtained for rGSTM5* with liquid chromatography-MS from a mixture of rat testicular GSTs . Therefore, rGSTM5* has not been post-translationally modified.

J Biol Chem, 1998 Jul 24, 273(30), 19190 - 7
Isolation and characterization of rhamnose-binding lectins from eggs of steelhead trout (Oncorhynchus mykiss) homologous to low density lipoprotein receptor superfamily; Tateno H et al.; Two L-rhamnose-binding lectins named STL1 and STL2 were isolated from eggs of steelhead trout (Oncorhynchus mykiss) by affinity chromatography and ion exchange chromatography . The apparent molecular masses of purified STL1 and STL2 were estimated to be 84 and 68 kDa, respectively, by gel filtration chromatography . Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry of these lectins revealed that STL1 was composed of noncovalently linked trimer of 31.4-kDa subunits, and STL2 was noncovalently linked trimer of 21.5-kDa subunits . The minimum concentrations of STL1, a major component, and STL2, a minor component, needed to agglutinate rabbit erythrocytes were 9 and 0.2 microg/ml, respectively . The most effective saccharide in the hemagglutination inhibition assay for both STL1 and STL2 was L-rhamnose . Saccharides possessing the same configuration of hydroxyl groups at C2 and C4 as that in L-rhamnose, such as L-arabinose and D-galactose, also inhibited . The amino acid sequence of STL2 was determined by analysis of peptides generated by digestion of the S-carboxamidomethylated protein with Achromobacter protease I or Staphylococcus aureus V8 protease . The STL2 subunit of 195 amino acid residues proved to have a unique polypeptide architecture; that is, it was composed of two tandemly repeated homologous domains (STL2-N and STL2-C) with 52% internal homology . These two domains showed a sequence homology to the subunit (105 amino acid residues) of D-galactoside-specific sea urchin (Anthocidaris crassispina) egg lectin (37% for STL2-N and 46% for STL2-C, respectively) . The N terminus of the STL1 subunit was blocked with an acetyl group . However, a partial amino acid sequence of the subunit showed a sequence similarity to STL2 . Moreover, STL2 also showed a sequence homology to the ligand binding domain of the vitellogenin receptor . We have also employed surface plasmon resonance biosensor methodology to investigate the interactions between STL2 and major egg yolk proteins from steelhead trout, lipovitellin, and beta'-component, which are known as vitellogenin digests . Interestingly, STL2 showed distinct interactions with both egg yolk proteins . The estimated values for the affinity constant (Ka) of STL2 to lipovitellin and beta' component were 3.44 x 10(6) and 4.99 x 10(6), respectively . These results suggest that the fish egg lectins belong to a new family of animal lectin structurally related to the low density lipoprotein receptor super- family.

Biochem Biophys Res Commun, 1998 Jun 29, 247(3), 734 - 40
The covalent structure of the blue copper-containing nitrite reductase from Achromobacter xylosoxidans; Vandenberghe IH et al.; The complete amino acid sequence of the blue copper-containing nitrite reductase enzyme (NiR) from Achromobacter xylosoxidans has been determined by chemical analysis, supported by high precision mass analysis . The polypeptide chain contains 336 residues with an overall charge of 0, including the +2 state of each of the copper ions . The two NiR enzymes for which the three-dimensional structures have been solved are green in color and have different absorption spectra than those of the blue-colored protein from A . xylosoxidans . The ligands to the two copper atoms are conserved . Therefore, the difference between the blue and the green NiR must depend on subtle changes in the geometry of the type I copper-sulfur bond . Both overall protein charge and active site charge are different in A . xylosoxidans NiR which may reflect the use of azurin as electron donor as opposed to the other enzymes that use pseudoazurin.

Biosci Biotechnol Biochem, 1998 Apr, 62(4), 778 - 81
In search of circular permuted variants of Escherichia coli dihydrofolate reductase; Iwakura M; A circularized form of a Cys-free mutant of Escherichia coli dihydrofolate reductase (DHFR) was used to search for a proteolytic site that gave new N- and C-termini on circularized DHFR with enzyme activity . Of the six site-specific proteolytic enzymes tested, three proteases, Achromobacter protease I (lysine-specific endopeptidase), asparaginylendopeptidase, and Staphylococcus aureus V8 protease, cleaved a single site of the circularized DHFR to form circular permuted variants . Twenty-four possible sites for cleavage were found formation of eight circular permuted variants was suggested by results of N-terminal sequence analysis of the linearized proteins isolated by gel filtration in the presence of 5 M guanidine hydrochloride . Mapping of the predicted cleavage sites on the DHFR molecule suggested that they were not all at a specific loop and, therefore, there are many possible circular permuted variants.

Biochem Biophys Res Commun, 1998 May 8, 246(1), 20 - 5
Genetic structure of the bphG gene encoding 2-hydroxymuconic semialdehyde dehydrogenase of Achromobacter xylosoxidans KF701; Kang E et al.; 2-Hydroxymuconic semialdehyde dehydrogenase catalyzes the conversion of 2-hydroxymuconic semialdehyde (HMS) to an enol form of 4-oxalocrotonate which is a step in the catechol meta-cleavage pathway . A bphG gene encoding HMS dehydrogenase of A . xylosoxidans KF701, a soil bacterium degrading biphenyl, was identified at between catechol 2,3-dioxygenase gene and HMS hydrolase gene, and its sequence was analyzed . An open reading frame (ORF) corresponding to bphG gene was consisted of 1461 nucleotides with ATG initiation codon and TGA termination codon . The ORF exhibited 66% of G + C content, and a putative ribosome-binding sequence, AGAGA, was identified at about 10 nucleotides upstream initiation codon of the bphG gene . The bphG gene can encode a polypeptide of molecular weight 52 kDa containing 486 amino acid residues . A deduced amino acid sequence of HMS dehydrogenase encoded in bphG gene from A . xylosoxidans KF701 exhibited the highest 94% homology with that of corresponding enzyme encoded in xylG from P . putida mt-2, 63% to 90% homology with those of other reported HMS dehydrogenases, and 29% to 42% homology with those of betaine aldehyde dehydrogenase, 5-carboxy-HMS dehydrogenase, aldehyde dehydrogenase, indole-3-acetaldehyde dehydrogenase, succinic semialdehyde dehydrogenase, methylmalonate semialdehyde dehydrogenase, and succinylglutamate 5-semialdehyde dehydrogenase . From an alignment of amino acid sequence of HMS dehydrogenase from A xylosoxidans KF701 with other reported dehydrogenases, putative cofactor NAD(+)-binding regions and catalytic residues were identified.

Haematologica, 1998 Mar, 83(3), 284 - 5
Achromobacter xylosoxidans bacteremia in patients with hematologic malignancies; Hernandez JA et al.; We report nine cases of Achromobacter xylosoxidans bacteremia diagnosed in patients with hematologic malignancies . There was not an obvious epidemiologic link between cases and the organism was not isolated from any source . Outcome was cure in all nine cases . In our experience, catheter removal is generally required for eradication of A . xylosoxidans.

J Virol, 1998 Apr, 72(4), 3107 - 16
Bombyx mori nucleopolyhedrovirus encodes a DNA-binding protein capable of destabilizing duplex DNA; Mikhailov VS et al.; A DNA-binding protein (designated DBP) with an apparent molecular mass of 38 kDa was purified to homogeneity from BmN cells (derived from Bombyx mori) infected with the B . mori nucleopolyhedrovirus (BmNPV) . Six peptides obtained after digestion of the isolated protein with Achromobacter protease I were partially or completely sequenced . The determined amino acid sequences indicated that DBP was encoded by an open reading frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the BmNPV genome (GenBank accession no . L33180) . This ORF (designated dbp) is a homolog of Autographa californica multicapsid NPV ORF25, whose product has not been identified . BmNPV DBP is predicted to contain 317 amino acids (calculated molecular mass of 36.7 kDa) and to have an isoelectric point of 7.8 . DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA . When bound to oligonucleotides, DBP protected them from hydrolysis by phage T4 DNA polymerase-associated 3'-->5' exonuclease . The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer . DBP, but not BmNPV LEF-3, was capable of unwinding partial DNA duplexes in an in vitro system . This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication.

J Biochem (Tokyo), 1997 Oct, 122(4), 772 - 8
Purification, staphylolytic activity, and cleavage sites of alpha-lytic protease from Achromobacter lyticus; Li S et al.; Alpha-lytic protease (alp) was purified from a bacteriolytic agent, Achromopeptidase from Achromobacter lyticus M497-1, and has been shown to possess staphylolytic activity . Cleavage sites of this enzyme on the peptidoglycan of Staphylococcus aureus were determined by N-terminal amino acid sequence and amino acid composition analyses . Alp cleaved the N-acetylmuramoyl-L-alanine amide bond, the junction between the polysaccharide and peptide moieties, in addition to the D-Ala-Gly and Gly-Gly peptide bonds, implying that this enzyme recognizes the amino acid of D-configuration at the P1 site and possesses N-acetylmuramoyl-L-alanine amidase activity . However, alp could not cleave the D-Ala-Gly peptide bond in a synthetic peptide, suggesting that this hydrolytic activity of alp is peptidoglycan-specific . The results obtained from different consecutive actions of alp and glycosidase on S . aureus peptidoglycan indicate that the presence of polysaccharide in the peptidoglycan is necessary for the bacteriolytic activity of alp.

Int Arch Allergy Immunol, 1997 Nov, 114(3), 258 - 64
Using monoclonal antibodies to characterize a sequential epitope on the group I allergen of Bermuda grass pollen; Chang ZN et al.; BACKGROUND: Cyn d 1, the group I allergen of Bermuda grass pollen, had been purified and characterized . METHODS: A sequential B cell epitope on Cyn d 1 was studied with monoclonal antibodies (MoAbs) . Cyn d 1 was cleaved by Achromobacter protease I into fragments, and the resulting peptides were fractionated on reversed-phase columns before being reacted with anti-Cyn d 1 MoAbs in a radioimmunoassay . A Cyn d 1 fragment recognized by its MoAb was selected for Edman degradation . A synthetic peptide was constructed according to the determined sequence . RESULTS: The epitope on Cyn d 1 recognized by MoAb 18-53 was found to be conformation independent, since its activity was not changed after sodium periodate, guanidine or urea treatment . The enzyme-cleaved fragment containing this epitope was determined to be DVDKPPFDGMTACGNEPIF which corresponds to the N-terminal 46-64 residues of Cyn d 1 . The presence of this sequence in the epitope recognized by MoAb 18-53 was demonstrated by enzyme immunoassay and further confirmed by inhibition of binding enzyme immunoassay with synthetic peptides . Some cross-reactivity with the N-terminal 45-63 residues of Lol p 1 was also found . CONCLUSIONS: The primary structure of a sequential epitope on Cyn d 1 was determined, and its activity was confirmed with peptides synthesized according to the determined sequence.

Arch Biochem Biophys, 1997 Oct 1, 346(1), 15 - 20
Purification, characterization, and amino acid sequencing of DNase gamma from rat spleen; Shiokawa D et al.; An endonuclease named DNase gamma was purified to apparent homogeneity from rat splenocyte nuclei and its properties were characterized . We also determined the NH2-terminal and partial amino acid sequences of the proteolytic internal peptides . The molecular mass of gamma DNase was 33,000 daltons as determined by SDS-polyacrylamide gel electrophoresis . A native molecular mass of 30,000 was estimated by gel filtration . Purified DNase gamma is active in the presence of both Ca2+ and Mg2+ or Mn2+ alone and inhibited by Co2+, Ni2+, Cu2+, and especially Zn2+ . Maximal activity was achieved at pH 7.2 in Mops-NaOH buffer . The sequence data on the NH2-terminal and seven internal peptides obtained by sequential digestions with Achromobacter protease I and endoproteinase Asp-N revealed that DNase gamma is a novel endonuclease that shows sequence homology with DNase I.

Biochemistry, 1997 Oct 7, 36(40), 12071 - 9
Structure of a covalently cross-linked form of core histones present in the starfish sperm; Shimizu T et al.; The post-translational modification of core histones plays an essential role in chromatin remodeling processes . We recently reported the occurrence of a novel histone modification, involving a epsilon-(gamma-glutamyl)lysine cross-link between a glutamine residue of histone H2B and a lysine residue of histone H4 in the testis of the starfish, Asterina pectinifera{Shimizu, T., Hozumi, K., Horiike, S., Nunomura, K., Ikegami, S., Takao, T., and Shimonishi, Y . (1996) Nature 380, 32} . In order to determine the complete structure of the modified histone heterodimer, p28 from both testis and sperm was purified . p28 was digested with Achromobacter lyticus protease I or Staphylococcus aureus V8 protease to give proteolytic fragments that were separated by HPLC . Amino acid analysis, sequencing, and mass spectrometric analysis of the fragments showed that the amino acid sequences of these fragments are identical to those of both histones H2B and H4, except for two NH2-terminal peptides obtained by digestion with A . lyticus protease I . One of the peptides, K8, was identical to that reported previously, and the other was a here-to-fore unidentified peptide, which was designated K10 . Amino acid and positive-ion FAB-MS/MS analyses of K10 showed that it to be a fragment, derived from Gly8-Lys10 of histone H2B and Gly9-Lys16 of histone H4 . The yields of K8 and K10 were calculated to be 47 and 42%, respectively, expressed as the percent of the total amount of p28 used in the experiment . Based on these data, the structure of p28 was determined to be a heterodimer, composed of histones H2B and H4, formed through a transglutaminase-catalyzed acyl transfer reaction between Gln9 of histone H2B and Lys5 or Lys12 of histone H4.

Biochem Biophys Res Commun, 1997 Sep 18, 238(2), 430 - 5
Characterization of the gene encoding catechol 2,3-dioxygenase from Achromobacter xylosoxidans KF701; Moon J et al.; Catechol 2,3-dioxygenase (C23O) catalyzes a meta cleavage of the aromatic ring in catechol to form 2-hydroxymuconic semialdehyde . A C23O gene was cloned from chromosomal DNA of A . xylosoxidans KF701, a soil bacterium degrading biphenyl, and expressed in E . coli HB101 . In substrate specificity to catechol and its analogs, the C23O exhibited the highest aromatic ring-fission activity to catechol, and its relative activity to other dihydroxylated aromatics was 4-chlorocatechol > 4-methylcatechol > 3-methylcatechol >> 2, 3-dihydroxybiphenyl . Aromatic ring-fission activity of the C23O to catechol was about 40-fold higher than that to 2,3-dihydroxybiphenyl . Nucleotide sequence analysis of the C23O gene from A . xylosoxidans KF701 revealed an open reading frame consisting of 924 base pairs, and identified a putative ribosome-binding sequence (AGGTGA) at about 10 nucleotides upstream from the initiation codon . The open reading frame can encode a polypeptide chain with molecular weight of 34 kDa containing 307 amino acid residues . The deduced amino acid sequence of the C23O exhibited the highest homology with that of C23O from Pseudomonas sp . IC with 96% identity, and the least homology with that of C23O from P . putida F1 with 22% identity among reported C23O sequences . Furthermore, comparison of the C23O sequence with other extradiol dioxygenases has led to identification of evolutionally conserved amino acid residues whose possible catalytic and structural roles are proposed .

Biochem Biophys Res Commun, 1997 Aug 18, 237(2), 262 - 5
Sulfated lipids as inhibitors of pancreatic trypsin and chymotrypsin in epithelium of the mammalian digestive tract; Iwamori M et al.; Cholesterol sulfate and I3SO3-GalCer, which were commonly contained in the epithelia of mammalian digestive tracts, were found to inhibit the activities of typical digestive enzymes, pancreatic trypsin and chymotrypsin, but steroid sulfates, gangliosides and the other membrane constituents did not show any inhibitory activity . The preincubation of trypsin with I3SO3-GalCer at 37 degrees C for 10min was necessary to inhibit the activity of trypsin, but cholesterol sulfate showed its inhibitory activity without preincubation . Sulfated lipid-treated enzyme gave the same Km as and lower Vmax than those of the original enzymes . Also, both sulfated lipids inhibited pronase from Streptomyces griseus, but not lysyl endopeptidase from Achromobacter lyticus . These findings indicate that cholesterol sulfate and I3SO3-GalCer in the digestive tract function as epithelial inhibitors to prevent tissue injury by endogenous and exogenous proteases.

J Vet Med Sci, 1997 Jul, 59(7), 575 - 9
Domon L, an immunopotentiating agent, acts as an NF-kappaB activator; Tanaka S et al.; Domon L, a heat-treated component of gram-negative bacterium Achromobacter stenohalis, is used for the treatment of infectious diseases of animals . Here, we investigated the immunopotentiating potential of Domon L . In vitro studies showed that Domon L enhanced nitric oxide (NO) formation from murine macrophage RAW264.7 cells in concert with interferon (IFN)-gamma . The effect of Domon L on NF-kappaB activation was investigated, in order to understand the molecular mechanisms of enhanced NO formation by Domon L . Domon L induced translocation of NF-kappaB to the nucleus in RAW264.7 cells . Induction of NF-kappaB dependent gene expression by Domon L was further confirmed using a transfectant containing an NF-kappaB-luciferase reporter gene . In vivo injection of Domon L elevated both serum IL-6 and mucoprotein, whose gene expression is partly under the control of NF-kappaB . The spleen cells of rats treated with Domon L produced much more NO when stimulated with LPS + IFN gamma than spleen cells of untreated rats . These results suggest that Domon L acts as an anti-infectious agent via NF-kappaB activation.

Heart Lung, 1997 Jul-Aug, 26(4), 335 - 6
Intravenous line infection due to Ochrobactrum anthropi (CDC Group Vd) in a normal host; Gill MV et al.; Ochrobactrum anthropi, formerly known as Achromobacter species (CDC group Vd), is an aerobic, gram-negative bacillus widely distributed in aquatic environments . Most important, it has been implicated as a cause of intravenous line infection in immunocompromised hosts with solid tumors or hematologic malignancies . Trimethoprim-sulfamethoxazole and aminoglycosides are usually active against O . anthropi, but this organism is usually resistant to beta-lactam antibiotics . Because O . anthropi is a low-virulence organism, patients with intravenous-line infections have been cured without removal of the intravenous catheter . We describe a case of intravenous-line infection in a normal host that was successfully resolved alter catheter removal.

Biochim Biophys Acta, 1997 May 24, 1360(3), 196 - 202
Evidence for increased hydroxylation of pyrrolidone amino acid residues in the cuticle of mature Onchocerca volvulus; Sakwe AM et al.; To determine whether morphologic changes are accompanied by variations in the biochemical and antigenic properties of the cuticle of Onchocerca volvulus during development, we isolated and compared the 2-mercaptoethanol soluble cuticular proteins and the insoluble cuticlin from the predominant life-cycle stages occurring in man . SDS-PAGE analysis, before and after digestion with collagenase from Achromobacter iophagus, revealed that the polypeptide composition of the 2-mercaptoethanol-solubilised extracts from adult males and nodular microfilariae are quite distinct and that these extracts contained predominantly collagen-like proteins . Demonstrated by immunoblotting with a hyper immune patient serum pool (n = 107), five strongly reactive antigens with apparent molecular weights of 126, 68, 43, 37 and 33 kDa were detected in the extracts from adult males, while at least eight prominent and several weakly reactive components were detected in the extracts from nodular microfilariae . The overall amino acid composition of the cuticular extracts from the various stages demonstrates that: (a) the cuticle of the adult male stage is rich in glycine, pyrrolidone amino acids, and acidic amino acids or their amides, (b) eggshells are particularly poor in proline but rich in serine residues (14.5%), (c) nodular microfilariae cuticular extracts are poor in proline but rich in valine (9.0%) and lysine (7.3%) and (d) hydroxyproline and hydroxylysine are present in the cuticle of adults but absent in the juvenile life-cycle stages (nodular microfilariae and eggs) . This study firstly, indicates that the composition of the cuticle of O . volvulus may thus, be quite distinct from one parasite stage to another and secondly, that the maturation of the parasite in the human host may be accompanied by the extensive hydroxylation of prolyl residues and to a lesser extent of lysyl residues in the predominantly collagen-like cuticular proteins.

Appl Microbiol Biotechnol, 1997 Apr, 47(4), 347 - 51
D-lysine production from L-lysine by successive chemical racemization and microbial asymmetric degradation; Takahashi E et al.; 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.

Exp Cell Res, 1997 Feb 1, 230(2), 393 - 8
Characterization and distribution of O-glycosylated carbohydrates in the cell adhesion molecule, contact site A, from Dictyostelium discoideum; Yoshida M et al.; 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.

Eur J Biochem, 1996 Dec 15, 242(3), 627 - 35
Location of cysteine and cystine residues in S-ribonucleases associated with gametophytic self-incompatibility; Ishimizu T et al.; S-Ribonucleases (S-RNases) that cosegregate with S-alleles in the styles of solanaceous and rosaceous plants are associated with gametophytic self-incompatibility (GSI) . The amino acid sequences of many S-RNases have been derived from cDNA sequences, but the state of half-cystines has not been clarified . We report the locations of the two free cysteine residues and four disulfide bridges of tobacco S6-RNase and of the four disulfide bridge of Japanese pear S4-RNase . The protein was first S-pyridylethylated at a low pH to selectively modify the free cysteines without thiol-disulfide exchange . The S-pyridylethylated protein (PE-protein) was digested with Achromobacter protease I (API) at pH 6.5 then analyzed by liquid chromatography/electrospray-ionization mass spectrometry (LC/ESI-MS) . This analysis showed that tobacco S6-RNase has two free cysteine residues, Cys77 and Cys95, and four disulfide bonds at Cys16-Cys21, Cys45-Cys94, Cys153-Cys182 and Cys165-Cys176 . Similarly, four disulfide bonds were identified for pear S4-RNase, which bears no free cysteine, at Cys15-Cys22, Cys48-Cys92, Cys156-Cys195 and Cys172-Cys183 . The eight cysteines forming these four disulfide bonds are conserved in all the known S-RNases, indicative that these cross-links are important in stabilizing the tertiary structures of the self-incompatibility-associated glycoproteins in the solanaceous and rosaceous families . The LC/ ESI-MS analysis also provided some structural informations regarding sugar chains attached to the S-RNases.

J Bacteriol, 1996 Nov, 178(22), 6608 - 17
A substitution at His-120 in the LasA protease of Pseudomonas aeruginosa blocks enzymatic activity without affecting propeptide processing or extracellular secretion; Gustin JK et al.; The LasA protease of Pseudomonas aeruginosa can degrade elastin and is an important contributor to the pathogenesis of this organism . LasA (20 kDa) is a member of the beta-lytic endopeptidase family of extracellular bacterial proteases, and it shows high-level staphylolytic activity . We sequenced the lasA gene from strain FRD1 and overexpressed it in Escherichia coli . The lasA gene encodes a precursor, known as pre-proLasA, of 45,582 Da . Amino-terminal sequence analysis allowed the identification of the signal peptidase cleavage site and revealed that the 31-amino-acid signal peptide was removed in E . coli . The remaining proLasA (42 kDa) did not undergo autoproteolytic processing and showed little staphylolytic activity . However, it was readily processed to a 20-kDa active staphylolytic protease by incubation with trypsin or with the culture filtrate of a P . aeruginosa lasAdelta mutant . Thus, removal of the propeptide (22 kDa) was required to convert proLasA into an active protease . Although LasA protease was critical for staphylolytic activity, other proteases like elastase were found to enhance staphylolysis . Under the control of an inducible trc promoter, lasA was overexpressed in P . aeruginosa and the processing intermediates were examined . Compared with wild-type cells, the overproducing cells accumulated more 42-kDa proLasA species, and the culture supernatants of the overproducing cells showed increased levels of active 20-kDa LasA protease . Small amounts of a 25-kDa extracellular LasA-related protein, which could represent a potential processing intermediate, were also observed . To better understand the structure-function relationships in LasA protease, we tested whether His-120-X-His-122 in the mature portion of LasA plays a role in activity . This motif and surrounding sequences are conserved in the related beta-lytic protease of Achromobacter lyticus . Oligonucleotide-directed mutagenesis was used to change His-120 to Ala-120, thus forming the lasA5 allele . The product of lasA5 expressed from the chromosome of P . aeruginosa was processed to a stable, secreted 20-kDa protein (designated LasA-H120A) which was devoid of staphylolytic activity . This suggests that His-120 is essential for LasA activity and favors the possibility that proLasA processing and secretion in P . aeruginosa can proceed via mechanisms which do not involve autoproteolysis.

Clin Infect Dis, 1996 Sep, 23(3), 569 - 76
Achromobacter xylosoxidans bacteremia: report of four cases and review of the literature; Duggan JM et al.; Seventy-seven cases of bacteremia due to Achromobacter xylosoxidans were reviewed, and susceptibility studies were performed on 11 clinical isolates of A . xylosoxidans . Nosocomial bacteremia was noted in 54 of 77 patients (70%), and 28 (36%) had infection associated with an outbreak or acquired from a discrete point source . The most common underlying illnesses were malignancies (30%) and cardiac disease (21%); immunosuppression affected 27% . The most common clinical syndromes were primary and catheter-associated bacteremia (19% each) and pneumonia (16%) . The case-fatality rate was 30%; only 3% of patients with primary or catheter-associated bacteremia died, but 65% of patients with meningitis, endocarditis, and pneumonia died . The case-fatality rate in neonates was 80% . Susceptibility studies showed that all strains were resistant to aminoglycosides, most were resistant to quinolones, and all were susceptible to broad-spectrum penicillins, imipenem, ceftazidime, and trimethoprim-sulfamethoxazole . Two-disk approximation and time-kill studies showed synergy or additive effects for the combination of gentamicin and piperacillin against most strains.

Enferm Infecc Microbiol Clin, 1996 Aug-Sep, 14(7), 436 - 40
{Bacteremia caused by Alcaligenes (Achromobacter) xylosoxidans . Description of 3 cases and review of the literature}; Ramos JM et al.; BACKGROUND: Alcaligenes (Achromobacter) xylosoxidans occasionally cause infections, mainly in immunocompromised hosts . METHODS: Three cases of bacteremia due to A . xylosoxidans observed at the Fundacion Jimenez Diaz between 1985-1994 were described . Moreover, 38 single cases of bacteremia due to A . xylosoxidans and 21 episodes associated with outbreak were reviewed by using computerized bibliography data base MEDLINE (1970-december 1994) . RESULTS: From 41 patients with bacteremia (including our 3 cases) reviewed, 27 were immunosuppressed hosts (twenty had neoplasia disease) . The most common clinical presentation was primary bacteremia (11 cases, 27%) and pneumonia (10, 24%), followed by catheter-associated bacteremia (8, 20%), meningitis (4), bacteremia from abdominal cavity (4), endocarditis (3) and pyelonephritis (1) . The mortality rate was higher (39%), specially in patients whom were a intensive care unit acquisition (87%), and illness with endocarditis (100%) . No patient with catheter-related bacteremia died . All of 21 outbreak episodes of bacteremia had a autolimited form and low mortality (4.8%) . CONCLUSION: A . xylosoxidans is a microorganism with demonstrated capacity of cause bacteremia, mainly in immunocompromised hosts, with high mortality rates . Sometimes, it causes outbreaks of bacteremia with low mortality.

J Biochem (Tokyo), 1996 Aug, 120(2), 326 - 34
Identification and partial amino acid sequences of seven S-RNases associated with self-incompatibility of Japanese pear, Pyrus pyrifolia Nakai; Ishimizu T et al.; S-allele-specific proteins (S-proteins) were separated and identified by two-dimensional (2D) gel electrophoresis from the style extract of 14 cultivars of Japanese pear, Pyrus pyrifolia Nakai, which exhibits gametophytic self-incompatibility . These S-proteins were 30-32 kDa basic proteins with putative pIs of 9.6-10.1 and were distinct from the other proteins, which were common for all cultivars examined . Each S-protein was assigned to a given S-genotype based on electrophoretic mobility and the partial amino acid sequence . For S1- to S7-proteins, five different N-terminal amino acid sequences sharing the YFQFTQQY sequence were determined . Since the same N-terminal amino acid sequences were found for both S1- and S7-proteins, and for S3- and S5-proteins, the two S-proteins of each pair were distinguished based on their electrophoretic behavior . The internal amino acid sequences of S2- and S4-proteins, determined for Achromobacter protease I (API) digests, revealed that these proteins are S2- and S4-RNases, respectively . In the cultivar Nijisseiki, these two RNases were expressed from the white bud to mature flower stages when the cultivar acquires and enforces self-incompatibility . Osa-nijisseiki, a self-compatible mutant of Nijisseiki, produced S2-RNase, but did not produce S4-RNase . The absence of S4-RNase was also observed in self-compatible offsprings derived from Osa-Nijisseiki . These results suggest that Japanese pear in the family Rosaceae possesses a gametophytic self-incompatibility system involving an S-RNase, and that a reduction or lack of expression of S4-RNase in the style is responsible for the self-compatibility of Osa-Nijisseiki.

J Biochem (Tokyo), 1996 Jul, 120(1), 29 - 34
Systematic peptide fragmentation of polyvinylidene difluoride(PVDF)-immobilized proteins prior to microsequencing; Iwamatsu A et al.; The sequential in situ digestion of proteins immobilized on polyvinylidene difluoride (PVDF) has been systematically designed and optimized . The method consists of immobilization of the proteins on PVDF, S-carboxymethylation, and then successive in situ digestions using specific proteases . In order to obtain high yields of the peptide fragments, from which specific amino acid residues connected to the N- or C-terminal of the resulting digestion fragments can be deduced, the cleavages are performed in the following order: (1) Achromobacter protease I (API), (2) endoproteinase Asp-N, and (3) trypsin . Procedures for recovering the numerous fragments remaining on the PVDF membrane after the third digestion with trypsin are also discussed . Application of sequential in situ digestion for the acquisition of fragments suitable for sequencing from digests of large-molecular-weight proteins is also presented.

Cancer Res, 1996 Jun 15, 56(12), 2752 - 7
Human colorectal carcinomas specifically accumulate Mr 42,000 ubiquitin-conjugated cytokeratin 8 fragments; Nishibori H et al.; Recent studies have shown that various tumor cells accumulate ubiquitin (Ub)-conjugated proteins, the profiles of which differ from those of normal cells . To identify the Ub-conjugated proteins accumulated specifically by human carcinoma cells, a two-dimensional immunoblot analysis of 31 surgically resected human primary colorectal carcinoma tissues was performed using an anti-Ub monoclonal antibody, KM691 . Two distinct Mr 42,000 and 45,000 proteins in the Triton X-insoluble fractions of carcinoma tissues reacted with this antibody, whereas only one Mr 45,000 protein reacted in normal tissues . The Mr 42,000 Ub-conjugated proteins were specific to carcinoma tissues from 25 patients (80.6%) . One of the purified Mr 42,000 proteins was digested with Achromobacter protease I . This protein was identified as a cytokeratin 8 (CK 8) fragment based on both molecular mass determination and molecular mass searching of Achromobacter protease I-digested fragments of proteins registered in a protein sequence data base . Two-dimensional immunoblot analysis with an anti-CK 8 antibody confirmed that all of the Mr 42,000 proteins were CK 8 degradation products . These results demonstrate that human colorectal carcinomas specifically accumulate Mr 42,000 Ub-conjugated CK 8 fragments . This accumulation was observed frequently not only in advanced (18/22, 81.8%), but also in early stage cases (7/9, 77.8%), suggesting that it occurs even in the early stages of colorectal carcinoma progression.

J Biol Chem, 1996 May 3, 271(18), 10635 - 9
Complete amino acid sequence and identification of the platelet glycoprotein Ib-binding site of jararaca GPIb-BP, a snake venom protein isolated from Bothrops jararaca; Kawasaki T et al.; Jararaca GPIb-BP, a snake venom protein composed of alpha and beta subunits purified from Bothrops jararaca, binds to platelet glycoprotein (GP)Ib and functions as a receptor blocker for von Willebrand factor binding to GPIb (Fujimura, Y., Ikeda, Y., Miura, S., Yoshida, E., Shima, H., Nishida, S., Suzuki, M., Titani, K., Taniuchi, Y., and Kawasaki, T . (1995) Thromb . Haemostasis 74, 743-750) . We present here the entire 142- and 123-residue amino acid sequence of the respective alpha and beta subunits and also demonstrate that the platelet GPIb-binding site resides on the beta and not on the alpha subunit based on an enzyme-linked immunosorbent assay using biotin-labeled jararaca GPIb-BP and competing ligands . Sequences of the alpha and beta subunits were determined by analysis of the intact S-pyridylethylated proteins and their peptides generated by digestion with Achromobacter protease I, Staphyloccocus aureus V8 protease, pepsin, endoproteinase Asp-N, or L-1-tosylamino-2-phenylethyl chloromethyl ketone-trypsin . A 38-39% identity of amino acid sequence between the alpha and beta subunits of jararaca GPIb-BP was observed, as well as a high degree of sequence identities (38-64%) with the respective subunits of botrocetin (Usami, Y., Fujimura, Y., Suzuki, M., Ozeki, Y., Nishio, K., Fukui, H., and Titani, K (1993) Proc . Natl . Acad . Sci . U . S . A . 90, 928-932) and the beta-chain of echicetin (Peng, M., Holt, J . C., and Niewiarowski, S . (1994) Biochem . Biophys . Res . Commun . 205, 68-72).

Mikrobiologiia, 1996 May-Jun, 65(3), 293 - 304
{Collagenolytic enzymes synthesized by microorganisms}; Demina NS et al.; The review uses data obtained by the authors and available in the literature to discuss microbial collagenolytic enzymes, widely employed in scientific research, biotechnology, and medicine . Collagenases differing in their structure and the specificity of their action on collagen fibrils were isolated from bacteria (including marine isolates), actinomycetes, and fungi . Collagenases produced by Clostridium histolyticum and Achromobacter iophagus are the best studied enzymes; both are metalloenzymes that contain Zn2+ in their active site and retain collagenolytic activity in the presence of Ca2+ . Serine-type collagenolytic proteases were also found in microorganisms . These enzymes differ from "true" collagenases by the structure of their active site . Both serine proteases and metalloproteases with high collagenolytic activities were isolated from the culture liquids of Streptomyces and Actinomyces strains . The biosynthesis of collagenases is induced by the addition of various collagen-containing substrates to the cultivation medium.

Electrophoresis, 1996 May, 17(5), 907 - 17
S-pyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically separated proteins: a rapid mass spectrometric method for identifying cysteine-containing peptides; Moritz RL et al.; In-gel proteolytic digestion of acrylamide-gel separated proteins is a method widely used for generating peptide fragments for the purpose of identifying proteins by Edman degratation, tandem mass spectrometry, and peptide-mass fingerprinting . However, it is well recognised for disulfide-bonded proteins electrophoresed under reducing conditions that if no precautions are taken to minimise disulfide bond formation during protein digestion or peptide isolation, complex peptide maps can result . Here, we describe an improved method for in-gel protein digestion . It consists of first reducing and S-pyridylethylating Coomassie Brilliant Blue R-250-stained proteins immobilised in the whole gel slab with dithiothreitol and 4-vinylpyridine, excising the individual stained and alkylated proteins, and then digesting them in situ in the gel matrix with trypsin or Achromobacter lyticus protease I . Peptide fragments generated in this manner are extracted from the gel piece and purified to homogeneity by a rapid (< or = 12 min) reversed-phase high performance liquid chromatography (HPLC) procedure, based upon conventional silica supports . Recoveries of peptides are increased by S-pyridylethylation of acrylamide-immobilised proteins prior to in-gel digestion . Further, the levels of gel-related contaminants, which otherwise result in suppression of sample signals during electrosprayionisation mass spectrometry, are greatly reduced by the reduction/alkylation step . Additionally, we demonstrate that S-beta-(4-pyridylethyl)-cysteine containing peptides can be readily identified during reversed-phase HPLC by absorbance at 254 nm, and during electrospray ionisation tandem mass spectrometry by the appearance of a characteristic-pyridylethyl fragment ion of 106 Da . The position of cysteine residues in a sequence can be determined as phenylthiohydantoin S-beta-(4-pyridylethyl)-cysteine during Edman degradation, and by tandem mass spectrometry.

Gene, 1996 Apr 17, 170(1), 107 - 12
A removable spacer peptide in an alpha-factor-leader/insulin precursor fusion protein improves processing and concomitant yield of the insulin precursor in Saccharomyces cerevisiae; Kjeldsen T et al.; An alpha-factor leader/insulin precursor fusion protein was produced in Saccharomyces cerevisiae and metabolically labeled in order to analyse the efficiency of maturation and secretion . A substantial fraction of the secreted material was found in a hyperglycosylated unprocessed form, indicating incomplete Kex2p endopeptidase maturation . Introduction of a spacer peptide (EAEAEAK) after the dibasic Kex2p site, creating a N-terminal extension of the insulin precursor, greatly increased the Kex2p catalytic efficiency and the fermentation yield of insulin precursor . The N-terminal extension features a Lys to allow subsequent proteolytic removal by trypsin or the Achromobacter lyticus Lys-specific protease . Dipeptidyl aminopeptidase A (DPAPA) activity removing Glu-Ala dipeptides from the extension was inhibited by adding a Glu N-terminally to the extension . Unexpectedly, this modified N-terminal extension (EEAEAEAK) was partially cleaved after the Lys during fermentation . This monobasic proteolytic activity was demonstrated to be associated with Yap3p . Yap3p cleavage could be prevented by insertion of a Pro before the Lys (EEAEAEAPK).

Biochim Biophys Acta, 1996 Feb 8, 1292(2), 289 - 92
The 30 kDa protein co-purified with chick liver glutathione S-transferases is a carbonyl reductase; Tsai SP et al.; An unidentified 30 kDa protein was co-purified with chick liver glutathione S-transferases from S-hexylglutathione affinity column . The protein was isolated to apparent homogeneity with chromatofocusing . The molecular mass of the protein was determined to be 30 277 +/- 3 dalton by mass spectrometry . The protein was digested with Achromobacter proteinase I . Amino-acid sequence analyses of the resulting peptides show a high degree of identity with those of human carbonyl reductase . The protein is active with menadione as substrate . Thus, it is identified as chick liver carbonyl reductase.

J Biochem (Tokyo), 1995 Dec, 118(6), 1224 - 31
Dephosphorylation of fetal-tau and paired helical filaments-tau by protein phosphatases 1 and 2A and calcineurin; Yamamoto H et al.; We have reported that many sites of tau in fetal brain (fetal-tau) as well as in paired helical filaments (PHF-tau) are phosphorylated . In the present study, we used site-specific antibodies and peptide mapping to examine protein phosphatases involved in dephosphorylation of fetal-tau and PHF-tau . Immunoblot analysis and electrophoretic mobility showed that protein phosphatases 1 and 2A and calcineurin could dephosphorylate fetal-tau and PHF-tau . Phosphoserines 199, 202, 396, and 413 and phosphothreonine 231, numbered according to the longest human tau isoform, were dephosphorylated, as shown by the immunoblot analysis . Phosphoserine 422 was dephosphorylated by protein phosphatase 2A and calcineurin, but not by protein phosphatase 1 . Peptide mapping with Achromobacter lyticus protease 1 showed that phosphoserines 199, 202, 235, and 396 and phosphothreonine 231 were dephosphorylated by protein phosphatases . Fetal-tau was more rapidly dephosphorylated by protein phosphatase 2A and calcineurin than PHF-tau . Interestingly, PHF-tau which had not been solubilized with guanidine HCl was little dephosphorylated by protein phosphatases . Thus, PHF-tau in neurofibrillary tangles of Alzheimer's disease brain is likely to be resistant to dephosphorylation by protein phosphatases.

J Biol Chem, 1995 Nov 17, 270(46), 27458 - 74
The structure of copper-nitrite reductase from Achromobacter cycloclastes at five pH values, with NO2- bound and with type II copper depleted; Adman ET et al.; High resolution x-ray crystallographic structures of nitrite reductase from Achromobacter cycloclastes, undertaken in order to understand the pH optimum of the reaction with nitrite, show that at pH 5.0, 5.4, 6.0, 6.2, and 6.8, no significant changes occur, other than in the occupancy of the type II copper at the active site . An extensive network of hydrogen bonds, both within and between subunits of the trimer, maintains the rigidity of the protein structure . A water occupies a site approximately 1.5 A from the site of the type II copper in the structure of the type II copper-depleted structure (at pH 5.4), again with no other significant changes in structure . In nitrite-soaked crystals, nitrite binds via its oxygens to the type II copper and replaces the water normally bound to the type II copper . The active-site cavity of the protein is distinctly hydrophobic on one side and hydrophilic on the other, providing a possible path for diffusion of the product NO . Asp-98 exhibits thermal parameter values higher than its surroundings, suggesting a role in shuttling the two protons necessary for the overall reaction . The strong structural homology with cupredoxins is described.

Biochem J, 1995 Nov 15, 312 ( Pt 1), 91 - 8
Amino acid sequencing, molecular cloning and modelling of the chick liver class-theta glutathione S-transferase CL1; Hsiao CD et al.; Glutathione S-transferase CL1-2 heterodimers purified from 1-day-old chick livers were digested with Achromobacter proteinase I . The resulting fragments were separated for amino acid sequence analysis . Oligonucleotide probes were constructed based on sequence similarity to class-Theta glutathione S-transferases for PCR using a chicken liver cDNA library as template . A full-length clone (1725 bp) encoding a polypeptide comprising 261 amino acids was isolated . Including conservative substitutions, this protein has 70-73% sequence similarity with other mammalian class-Theta glutathione S-transferases . Based on known X-ray crystal structures of class-Alpha, -Mu and -Pi glutathione S-transferases, a model is constructed for the N-terminal 232 residues of CL1.

Toxicon, 1995 Nov, 33(11), 1469 - 78
Purification and primary structure of a myotoxic lysine-49 phospholipase A2 with low lipolytic activity from Trimeresurus gramineus venom; Nakai M et al.; Four acidic phospholipase A2 (PLA2) isozymes named PLA2-I, II, III and IV have previously been isolated from Trimeresurus gramineus (green habu snake) venom and sequenced {Oda et al . (1991) Toxicon 29, 157; Fukagawa et al . (1992) Toxicon 30, 1131; Fukagawa et al . (1993) Toxicon 31, 957} . They contain aspartate-49 which is known to bind Ca2+, essential for catalysis . In the present study, a basic PLA2 named PLA2-V containing lysine-49 was newly isolated from the same snake venom . Its isoelectric point was 9.4 and considerably higher than those (c . 4.5) of PLA2-I-IV . PLA2-V was 1.1% as active as PLA2-I toward egg-yolk emulsion but exhibited strong myotoxicity . The amino acid sequence of PLA2-V was determined by sequencing the S-carboxamidomethylated derivative and its peptide fragments produced by enzymatic (clostripain, chymotrypsin, Achromobacter protease I and Staphylococcus aureus V8 protease) cleavages . PLA2-V consists of 122 amino acid residues and is highly homologous (72-78%) to Lys-49 PLA2s so far isolated from Viperidae snake venoms but less homologous (52%) to PLA2-I . The presence of Asn-28, which is characteristic of Lys-49 PLA2s, was confirmed.

J Biol Chem, 1995 Oct 27, 270(43), 25733 - 8
Spectroscopic and electrochemical studies on active-site transitions of the type 1 copper protein pseudoazurin from Achromobacter cycloclastes; Kohzuma T et al.; The single type 1 copper protein pseudoazurin from Achromobacter cycloclastes gives reversible electrochemical behavior at a (4-pyridyl)disulfide-modified gold electrode . Measurements carried out at 25.0 degrees C indicate a midpoint reduction potential of E 1/2 = 260 mV versus normal hydrogen electrode at pH 7.0 and a peak-to-peak separation of delta Ep = 59 mV . The diffusion coefficient and heterogeneous electron transfer rate constant are estimated to be 2.23 x 10(-6) cm2 s-1 and 3.7 x 10(-2) cm s-1, respectively . Also, controlled potential electrolysis indicates a 1-electron transfer process and a formal reduction potential of 259 mV versus normal hydrogen electrode for the Cu(II)/Cu(I) couple . The heterogeneous electron transfer rate constant determined at the (4-pyridyl)disulfide-modified gold electrode at pH 4.6 is 6.7 x 10(-3) cm s-1, consistent with a slower process at the positively charged electrode surface . At pH 11.3, UV-visible, EPR, and resonance Raman spectra indicate a conversion of the distorted tetrahedral copper geometry to a trigonal structure . The trigonal form has elongated axial bonding and an axial EPR spectrum . At pH 11.3, the reduction potential is further decreased, and Cu-S bands in resonance Raman spectra at 330-460 cm-1 are shifted to higher energy (approximately 10 cm-1), consistent with a stronger Cu-S bond.

Biochem Biophys Res Commun, 1995 Sep 5, 214(1), 163 - 70
The complete amino acid sequence of a mannose-binding lectin from "Kidachi Aloe" (Aloe arborescens Miller var . natalensis Berger); Koike T et al.; The complete amino acid sequence of a mannose-binding lectin purified from the leaf skin of "Kidachi Aloe" (Aloe arborescens Miller var . natalensis Berger) is presented . The 109-residue sequence of the subunit was determined by analysis of peptides of the intact or S-pyridylethylated protein generated by digestion with cyanogen bromide, BNPS-skatole, Achromobacter protease I, or trypsin . The subunit contains an intrachain disulfide bridge . The sequence is highly homologous to that of a mannose-binding lectin from snowdrop bulb.

Eur J Biochem, 1995 Aug 15, 232(1), 106 - 10
Identification of the electrophilic substrate-binding site of glutathione S-transferase P by photoaffinity labeling; Nishihira J et al.; We determined the electrophilic substrate-binding site of rat glutathione S-transferase P (GST-P) by photoaffinity labeling using the photosensitive compound S-{2-(2-fluoro-4-nitrophenoxy)ethyl}glutathione . This photosensitive glutathione analogue inhibited the catalytic activity in a competitive manner against both glutathione and 1-chloro-2,4-dinitrobenzene, a putative electrophilic substrate . The enzyme kinetics indicated that the photoactivatable glutathione analogue was specifically bound at the active site, which consisted of glutathione-binding (G-site) and the electrophilic substrate-binding (H-site) regions . The procedure involved the following steps: S-{2-(2-fluoro-4-nitrophenoxy)ethyl}glutathione was photochemically reacted with a purified recombinant GST-P expressed in Escherichia coli using ultraviolet irradiation for 30 min on ice . After the reaction, only the GST-P complexed with the glutathione analogue was prepared with glutathione-immobilized agarose . The GST-P covalently bound with the analogue was digested with lysyl endopeptidase (Achromobacter protease I), and the peptides were separated by high-performance liquid chromatography . Only a single major peak with appreciable absorbance at 340 nm was observed by peptide mapping . The peptide was collected and analyzed using an automated peptide sequencer (ABI 477A) . Amino acid sequence analysis showed that this peptide consisted of seven amino acid residues corresponding to the sequence at positions 122-128 of GST-P (Ala-Leu-Pro-Gly-Xaa-Leu-Lys) . No appreciable phenylthiohydantoin-amino acid was detected at the fifth cycle, which indicated that His126 was chemically labeled with the photosensitive glutathione analogue . It was concluded that His126 was one of the amino acid residues forming the electrophilic substrate-binding site of GST-P.

Appl Microbiol Biotechnol, 1995 Jul, 43(3), 508 - 13
Screening and characterization of microorganisms with glutaryl-7ADCA acylase activity; Franzosi G et al.; 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.

Pol Arch Med Wewn, 1995 Apr, 93(4), 322 - 8
{Infection in the dialysis unit as a contributing factor to the problem of hospital acquired infections}; Sosnowska A et al.; Hospital acquired infections are especially dangerous for patients with chronic renal insufficiency . An epidemic of hospital acquired infection of dramatic course in described in 13 patients (8 being on haemodialysis (HD) and 5 treated conservatively), with 15 episodes of septic syndrome (12 cases) or septic shock (3 cases) . Initial cultures of blood, urine and pharyngeal swabs were positive only in 5 patients treated conservatively . Initial cultures of dialysing fluid from all 12 monitors of HD in our Dialysis Unit revealed the presence of Gram-negative bacteria (Achromobacter) in blood of 3 patients as well as in the dialysing fluid compartments from 2 monitors of Gambro AK 10/100 . The possible role of Gram-negative, apparently benign water rods, in the pathogenesis of hospital acquired opportunistic infections in Dialysis Unit were discussed.

J Biochem (Tokyo), 1995 Jan, 117(1), 147 - 57
Site-specific glycosylation of bovine butyrophilin; Sato T et al.; Our previous studies showed that the N-linked sugar chains of most bovine glycoproteins from milk fat globule membranes (MFGM) contain the GalNAc beta 1-->4GlcNAc group {Sato et al . (1993) J . Biochem . 114, 890-900} . Since expression of the disaccharide structure is influenced by peptide sequences near the glycosylation sites {Smith and Baenziger (1992) Proc . Natl . Acad . Sci . USA 89, 329-333}, the site-specificity of the N-acetylgalactosaminylated sugar chains was investigated using bovine butyrophilin, a major MFGM glycoprotein with known primary structure . Two glycopeptide fragments which contained the N-linked sugar chains linked to either Asn-55 or Asn-215 residue were obtained by digestion of the protein with Achromobacter protease I . The sugar chains released from each glycopeptide by hydrazinolysis were reduced with NaB3H4 . Structural analyses of the oligosaccharides by sequential exoglycosidase digestion and methylation analysis revealed that only complex-type sugar chains with the GalNAc beta 1-->4GlcNAc structure are included in Asn-55-linked oligosaccharides, while only novel hybrid-type sugar chains detected previously in bovine MFGM glycoproteins are included in Asn-215-linked oligosaccharides . The results show that the glycosylation of butyrophilin occurs in a site-specific manner.

Biochem J, 1994 Dec 15, 304 ( Pt 3), 849 - 52
Delta 3, delta 2-enoyl-CoA isomerase is the protein that copurifies with human glutathione S-transferases from S-hexylglutathione affinity matrices; Takahashi Y et al.; An unidentified 30 kDa protein frequently copurifies with human glutathione S-transferases from S-hexyl-glutathione affinity matrices . Application of two-step sequential affinity chromatographic methods yielded a homogeneous preparation of that protein from human liver specimens . The protein was digested with Achromobacter protease I, and sequences of peptides resolved by h.p.l.c . showed a high degree of identity with those of rat mitochondrial delta 3, delta 2-enoyl-CoA isomerase . The human protein also exhibited catalytic activity with delta 3-cis-octenyl CoA as a substrate . Thus it is identified as liver delta 3, delta 2-enoyl-CoA isomerase.

J Bacteriol, 1994 Nov, 176(21), 6489 - 96
Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase; Yoshihara K et al.; The colH gene encoding a collagenase was cloned from Clostridium histolyticum JCM 1403 . Nucleotide sequencing showed a major open reading frame encoding a 116-kDa protein of 1,021 amino acid residues . The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, HEXXH . A 116-kDa collagenase and a 98-kDa gelatinase were copurified from culture supernatants of C . histolyticum . While the former degraded both native and denatured collagen, the latter degraded only denatured collagen . Peptide mapping with V8 protease showed that all peptide fragments, except a few minor ones, liberated from the two enzymes coincided with each other . Analysis of the N-terminal amino acid sequence of the two enzymes revealed that their first 24 amino acid residues were identical and coincided with those deduced from the nucleotide sequence . These results indicate that the 98-kDa gelatinase is generated from the 116-kDa collagenase by cleaving off the C-terminal region, which could be responsible for binding or increasing the accessibility of the collagenase to native collagen fibers . The role of the C-terminal region in the functional and evolutional aspects of the collagenase was further studied by comparing the amino acid sequence of the C . histolyticum collagenase with those of three homologous enzymes: the collagenases from Clostridium perfringens and Vibrio alginolyticus and Achromobacter lyticus protease I.

J Biochem (Tokyo), 1994 Nov, 116(5), 1025 - 9
Evidence for conformational change of fatty acid-binding protein accompanying binding of hydrophobic ligands; Honma Y et al.; Conformational change of rat liver fatty acid-binding protein was investigated by making use of change of protease-susceptibility in the presence or absence of bound oleic acid and clofibrate . Delipidated fatty acid-binding protein was rapidly digested by Achromobacter lysyl endopeptidase, bovine alpha-chymotrypsin, and Staphylococcal V8 protease, while protein recombined with oleic acid was strongly refractory to proteolysis . This observation indicates that a striking change in the conformational state of the protein occurred upon lipid binding, and seems to support the recent hypothesis that the large segment homologous to fatty acid-binding proteins found in a fatty acid-regulated ion channel constitutes a regulatory domain of the channel {Petrou, S., Ordway, R.W., Singer, J.J., and Walsh, J.V., Jr . (1993) Trends Biochem . Sci . 18, 41-42} . Clofibrate, which is a potent peroxisome proliferator and structurally unrelated to oleic acid, also conferred similar protease resistance upon the protein . A possible physiological aspect of the present observation is that the cellular level of free fatty acids, which have metabolic importance and cytotoxicity as well, regulates the turnover rate of fatty acid-binding proteins by modulating the protease susceptibility of the protein.

PDA J Pharm Sci Technol, 1994 Nov-Dec, 48(6), 299 - 303
Metabolic and structural changes in Pseudomonas aeruginosa, Achromobacter CDC and Agrobacterium radiobacter cells injured in parenteral fluids; Papapetropoulou M et al.; The long term metabolic changes of three oxidase positive microorganisms (Pseudomonas aeruginosa, Agrobacterium radiobacter and Achromobacter CDC) all isolated from aquatic environment, were defined after they were inoculated in three parenteral fluids: Lactated Ringer's solution, Sodium Chloride 0.9% and Dextrose 5% . The number of microorganisms introduced into the parenteral products was adjusted to 10(5) bacteria/ml and left at room temperature (20-22 degrees C) for 30 days . Their enzymatic and protein profile as compared with their initial characteristics after they were grown in broth, were measured using API 20NE batteries of tests and gel electrophoresis . In L-R and NaCl 0.9% fluids, P . aeruginosa and Ag . radiobacter lost the ability to hydrolyse urea while Ac CDC retained this ability . In Dextrose 5% fluid the microorganisms lost most of their metabolic characters . The protein patterns in SDS-PAGE of samples prepared from cells of the tested microorganisms showed marked differences (in P . aeruginosa) to minor differences (in Ag . radiobacter and Ac CDC) while new proteins with M(r) > 66KDa revealed Ag . radiobacter cells . The gelatinolytic zymogram shows also differences between bacterial cells grown in nutrient broth and those that remained in parenteral fluids . These changes reflect the stress of the tested bacteria in an unfavorable condition . The alterations of injured bacteria could render them unable to grow on routine, for sterilization testing, culture media.

Biosci Biotechnol Biochem, 1994 Nov, 58(11), 2004 - 8
Property and amino acid sequence of a subtilisin inhibitor from seeds of beach canavalia (Canavalia lineata); Katayama H et al.; A subtilisin inhibitor was purified from the seeds of Canavalia lineata by ammonium sulfate precipitation, ultrafiltration on a YM-30 membrane, column chromatography on DEAE-Toyopearl and SP-Toyopearl, followed by reverse-phase HPLC . The inhibitor (CLSI-I) is a low molecular weight protein (M(r) about 6500) containing no half-cystine residue, and quite stable as to extreme heat and pH treatment . CLSI-I inhibited subtilisin-type serine proteases including S . griseus alkaline protease . The amino acids of CLSI-I were sequenced by manual Edman degradation after enzymatic digestion with Achromobacter lyticus lysyl endopeptidase and Staphylococcus aureus V8 protease . CLSI-I contains 65 amino acid residues and showed a high homology to potato inhibitor I family proteins.

J Biol Chem, 1994 Sep 23, 269(38), 23708 - 15
Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule; Ishiwata N et al.; To demonstrate the presence of a soluble isoform of P-selectin predicted from cDNA sequencing (Johnston, G.I., Bliss, G.A., Newman, P.J., and McEver, R.P . (1990) J . Biol . Chem . 265, 21381-21385), we immunoisolated and compared structurally P-selectin from fresh frozen human plasma with that from washed intact platelets . Plasma P-selectin was reactive with rabbit antiserum to a synthesized peptide (residues 762-774 of mature P-selectin) but was significantly less reactive with antibody to a peptide (residues 747-760) . In contrast, platelet P-selectin reacted with both antibodies . S-Pyridylethylated plasma P-selectin was fractionated by reversed phase-high performance liquid chromatography into two major species . From platelets, two virtually identical species were separated . Sequential digestion with Achromobacter protease I and then Staphylococcus V8 protease produced peptides assigned to the tail region of the protein including the putative spliced site . From the more hydrophilic species in both plasma and platelets, a peptide completely lacking the sequence of the putative spliced site was identified . In contrast, the more hydrophobic species yielded a peptide with an intact transmembrane sequence . Hence, these results provide direct evidence that the previously predicted soluble isoform of P-selectin is actually synthesized in vivo and is present as a circulating molecule.

Eur J Biochem, 1994 Aug 1, 223(3), 727 - 32
Cloning and expression of the gene for hydroxypyruvate reductase (D-glycerate dehydrogenase from an obligate methylotroph Hyphomicrobium methylovorum GM2; Yoshida T et al.; The gene encoding hydroxypyruvate reductase, catalyzing the asymmetric reduction of hydroxypyruvate to D-glycerate, and its flanking regions were isolated from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2 . Nucleotide sequencing of the recombinant plasmids revealed that the hydroxypyruvate-reductase gene codes for the 322-amino-acid protein with calculated molecular mass 35,726 Da . The sequence was confirmed by sequencing the intact enzyme and peptides obtained by digestion of the enzyme with Achromobacter proteinase I . The amino acid sequence of the enzyme showed similarity to members of the D-isomer-specific 2-hydroxyacid dehydrogenase family . The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223-3, was introduced into Escherichia coli HB101 . The recombinant enzyme purified from the transformed E . coli cells was indistinguishable from the enzyme isolated from H . methylovorum GM2 by immunological and enzymological analyses.

Am J Infect Control, 1994 Aug, 22(4), 228 - 30
Handwashing machines, handwashing compliance, and potential for cross-contamination; Wurtz R et al.; Although handwashing is considered an important factor in the prevention of nosocomial infections, the optimal technique has not been determined and compliance is often difficult to obtain . Handwashing compliance is particularly important in intensive care areas of the hospital . In an effort to improve HW compliance, the surgical intensive care unit in our hospital purchased three handwashing machines . Four months after installation of the handwashing machines, an outbreak of methicillin-resistant Staphylococcus aureus occurred in the intensive care unit . As part of evaluating the outbreak, we cultured the handwashing machines, including the portholes and the paper towel dispenser . Cultures were positive for methicillin-resistant Staphylococcus epidermidis, Achromobacter species, and Streptococcus viridans . The design of the handwashing machines made contamination of sleeves and already-washed hands possible . An observational study revealed that handwashing compliance was poor but improved from 22% to 38% when the handwashing machines were in use . Nurses preferred handwashing at the sink and physicians preferred the handwashing machine . Handwashing machines may increase handwashing compliance because of their novelty, but they may also result in novel problems.

J Biol Chem, 1994 Jun 24, 269(25), 17025 - 9
Identification of three catalytic triad constituents and Asp-225 essential for function of lysine-specific serine protease, Achromobacter protease I; Norioka S et al.; Achromobacter protease I is a lysine-specific serine protease that Achromobacter lyticus M497-1 extracellularly secretes . The structural aspects necessary for the protease to function were investigated by means of site-directed mutagenesis to identify the constituents of the catalytic triad and the amino acid residue responsible for lysine specificity . The precursor molecules, which were produced by substitution of His-57, Asp-113, or Ser-194 for alanine, could not be converted to the mature form . In contrast, a precursor of a mutant in which either His-56 or Ser-193 is converted to alanine was perfectly processed autocatalytically and attained full protease activity . Substitution of Glu-190, one of the two candidates for determining lysine specificity, to glutamine, aspartic acid, or leucine had no or little effect on both proteolytic activity and substrate specificity . However, the kinetic parameters were subtly different from one another, depending on the nature of substituents in these mutants . The substitution of the other candidate, Asp-225, for asparagine or leucine resulted in the failure of maturation to the active forms . However, the precursor of the D225E mutant slowly matured and was essentially inactive . The observed reduction of protease activity is largely due to a decrease in the affinity of lysine to the protease . These results suggest that His-57, Asp-113, and Ser-194 are the three constituents of the catalytic triad in Achromobacter protease I and that Asp-225 plays a critical role in restricted substrate specificity as a lysyl endopeptidase.

J Biol Chem, 1994 Mar 4, 269(9), 6671 - 6
Crucial role of intralobe peptide-peptide interactions in the uptake and release of iron by ovotransferrin; Kurokawa H et al.; The mechanism for reversible iron binding in the N-terminal lobe of ovotransferrin was investigated by a protein fragmentation approach . The iron-saturated N-terminal half-molecule of ovotransferrin was proteolyzed into large 30-kDa (1-279) and small 6-kDa (280-332) fragments by a single cleavage with Achromobacter protease I, producing a stable nicked form . For the separation of the two fragments, denaturing conditions were required . The isolated large fragment contained all four iron-coordinating ligands and the anion-binding ligand, and according to spectroscopic titration analysis, it showed iron binding capacity . The iron-bound large fragment, however, showed a blue-shifted visible absorption spectrum and a much decreased iron stability compared with those of the intact half-molecule . Analyses by ion-exchange chromatography revealed that the large fragment reassociates with the small fragment . In order for reassociation to occur, iron must be bound to the large fragment; upon reassociation, the large fragment regained its stable iron binding capacity as well as a visible absorption spectrum almost indistinguishable from those of the intact half-molecule . These data are consistent with a two-step sequential pathway for reversible iron binding in the N-terminal lobe of ovotransferrin that includes an initial iron binding intermediate having a perturbed metal environment and its transformation into the stable iron-bound holoform by peptide-peptide interactions between the large coordinating and small noncoordinating segments.

Biochemistry, 1994 Feb 22, 33(7), 1843 - 9
Purification and characterization of bothrombin, a fibrinogen-clotting serine protease from the venom of Bothrops jararaca; Nishida S et al.; A fibrinogen-clotting enzyme (bothrombin) was purified from the venom of Bothrops jararaca . Bothrombin showed M(r) values of 33,000 under nonreducing and 35,000 under reducing conditions on SDS polyacrylamide gel electrophoresis and specific fibrinogen-clotting activity equivalent to 814-904 NIH alpha-thrombin units/mg . Diisopropyl fluorophosphate totally abolished its activity, but hirudin, a specific alpha-thrombin inhibitor, had negligible effect on bothrombin activity . Unlike alpha-thrombin, bothrombin split off fibrinopeptide A without releasing fibrinopeptide B . Bothrombin activated blood coagulation factor VIII, but its activity was about 950 times less than that of alpha-thrombin . Bothrombin did not induce aggregation or serotonin release of washed normal platelets by itself, but did aggregate platelets in the presence of exogenous fibrinogen . This latter activity was completely inhibited by either anti-glycoprotein (GP) IIb/IIIa monoclonal antibody (which blocks fibrinogen binding to GP IIb/IIIa) or anti-GP Ib monoclonal antibody (which specifically inhibits alpha-thrombin binding to GP Ib) . Prostaglandin E1 (1 microM) and EDTA (10 mM) also abolished platelet aggregation without affecting clotting activity . Washed platelets from a patient with Bernard-Soulier syndrome did not respond to bothrombin even in the presence of exogenous fibrinogen, suggesting that the initial binding of bothrombin on platelets is GP Ib, but not a recently cloned thrombin receptor . The complete amino acid sequence of bothrombin was determined by analysis of (S)-pyridylethylated protein and peptides generated by digestion with cyanogen bromide and Achromobacter protease I, respectively . Bothrombin is composed of 232 amino acid residues and contains three Asn-linked oligosaccharide chains.(ABSTRACT TRUNCATED AT 250 WORDS)

J Chemother, 1994 Feb, 6(1), 20 - 4
Survey on antibiotic resistance in gram-negative non-fermentative bacteria other than pseudomonas in Italy; Nicoletti G et al.; A survey was conducted to investigate the incidence and antibiotic resistance of clinical isolates of Gram-negative non-fermentative bacteria, isolated during a one-year period in Italy . Overall, these microorganisms account respectively for 11.47% and 20.5% of all Gram-negative bacteria . The most representative species isolated during the study were: Acinetobacter, Flavobacterium, Alcaligenes, Achromobacter and Xanthomonas species . As regards their antimicrobial pattern of resistance, this survey demonstrated the wide differences existing within each group . The antimicrobial agents usually active against Pseudomonas aeruginosa do not seem to be useful against these microorganisms.

Biosci Biotechnol Biochem, 1994 Feb, 58(2), 376 - 9
Amino acid sequences of double-headed proteinase inhibitors from the seeds of Canavalia lineata; Terada S et al.; The amino acids of two Bowman-Birk type proteinase inhibitors (CLTI-I and -II) from the seeds of Canavalia lineata were sequenced by a manual Edman degradation using the DABITC/PITC double coupling method after enzymatic digestions with Achromobacter lyticus lysyl endopeptidase, Staphylococcus aureus V8 protease, and chymotrypsin . CLTI-I contains 75 amino acid residues . CLTI-II has an identical sequence to CLTI-I except an extra Asp residue attached at the C-terminus . The inhibitors showed a homology (40-70%) to other Bowman-Birk inhibitors . The reactive-site peptide bonds were estimated to be Lys21-Ser22 and Leu48-Ser49 against trypsin and chymotrypsin, respectively . An inhibitory active fragment containing only the chymotrypsin-reactive site was also described.

J Bacteriol, 1994 Jan, 176(1), 149 - 56
Purification and characterization of Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene; Matsushita O et al.; Clostridium perfringens type C NCIB 10662 produced various gelatinolytic enzymes with molecular masses ranging from approximately 120 to approximately 80 kDa . A 120-kDa gelatinolytic enzyme was present in the largest quantity in the culture supernatant, and this enzyme was purified to homogeneity on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The purified enzyme was identified as the major collagenase of the organism, and it cleaved typical collagenase substrates such as azocoll, a synthetic substrate (4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg {Pz peptide}), and a type I collagen fibril . In addition, a gene (colA) encoding a 120-kDa collagenase was cloned in Escherichia coli . Nested deletions were used to define the coding region of colA, and this region was sequenced; from the nucleotide sequence, this gene encodes a protein of 1,104 amino acids (M(r), 125,966) . Furthermore, from the N-terminal amino acid sequence of the purified enzyme which was found in this reading frame, the molecular mass of the mature enzyme was calculated to be 116,339 Da . Analysis of the primary structure of the gene product showed that the enzyme was produced with a stretch of 86 amino acids containing a putative signal sequence . Within this stretch was found PLGP, the amino acid sequence constituting the Pz peptide . This sequence may be implicated in self-processing of the collagenase . A consensus zinc-binding sequence (HEXXH) suggested for vertebrate Zn collagenases is present in this bacterial collagenase . Vibrio alginolyticus collagenase and Achromobacter lyticus protease I showed significant homology with the 120-kDa collagenase of C . perfringens, suggesting that these three enzymes are evolutionarily related.

Biosci Biotechnol Biochem, 1994 Jan, 58(1), 215 - 6
Hydrolysis of S-2-aminoethylcysteinyl peptide bond by Achromobacter protease I; Masaki T et al.; The substrate specificity of Achromobacter protease I (API) was examined for S-2-aminoethyl(AE)cysteinyl bonds in Bz-AEC-OMe/OEt, Bz-AEC-NH2, and AE-insulin B chain . The protease hydrolyzed all of the tested AE-cysteinyl bonds at the same rate as that of lysyl bonds . Kinetic parameters were estimated for this hydrolysis reaction.

Biochemistry, 1993 Nov 23, 32(46), 12455 - 64
Resonance Raman spectroscopy of the azurin His117Gly mutant . Interconversion of type 1 and type 2 copper sites through exogenous ligands; den Blaauwen T et al.; The copper center of the Pseudomonas aeruginosa His117Gly azurin mutant is accessible to exogenous ligands through an aperture in its surface created by the removal of the endogenous imidazole ligand . Depending on the exogenous ligand, a surprising variety of type 1 and type 2 copper sites can be obtained that are readily distinguished by electronic, EPR, and resonance Raman (RR) spectroscopy . The RR spectrum of type 1 H117G with exogenous imidazole is nearly identical to that of wild-type azurin, indicating that the trigonal geometry and short Cu-S(Cys) bond of approximately 2.15 A have been maintained . With anionic ligands (e.g., Cl-, Br-, N3-), the RR spectra show increased intensity at 370 and 400 cm-1 and a corresponding decrease in intensity at 410 cm-1, suggesting a lengthening of the Cu-S(Cys) bond as the site achieves a more tetrahedral character . An extreme example is the hydroxide adduct of H117G which is green in color and has optical and RR spectra reminiscent of the tetrahedral type 1 site in Achromobacter cycloclastes nitrite reductase . The fact that the basic RR pattern is little changed in most of the type 1 adducts indicates that the RR spectrum is due primarily to vibrations of the Cu-cysteinate moiety and that its coplanar conformation is conserved . Type 2 H117G proteins are formed by the addition of bidentate exogenous ligands such as histidine and histamine . They have their absorption maxima blue-shifted to 400 nm and their EPR A parallel values increased to approximately 160 x 10(-4) cm-1, both of which are characteristic of tetragonal Cu sites with Cu-S(thiolate) bonds of > 2.25 A . The RR spectra of the type 2 H117G proteins are still dominated by multiple cysteinate-related vibrational modes . However, the vibrational modes with the greatest intensity and Cu-S(Cys) stretching character have shifted approximately 100 cm-1 to lower energy compared to the type 1 sites, consistent with a longer (Cys)S-Cu bond . It is proposed that the tetragonal type 2 character of the bidentate ligand complexes is due to the addition of a fourth strong ligand in the equatorial ligand plane.

Appl Environ Microbiol, 1993 Oct, 59(10), 3339 - 49
Isolation and characterization of an N-methylcarbamate insecticide-degrading methylotrophic bacterium; Topp E et al.; A gram-negative bacterium which hydrolyzed aryl N-methylcarbamate insecticides was isolated from an agricultural soil which quickly degraded these pesticides . This organism, designated strain ER2, grew on carbofuran as a sole source of carbon and nitrogen with a doubling time of 3 h in a mineral salts medium . The aromatic nucleus of the molecule was not metabolized, and carbofuran 7-phenol accumulated as the end product of metabolism . The insecticides carbaryl, bendiocarb, and propoxur were similarly hydrolyzed, with each yielding the corresponding phenol . Strain ER2 contained two plasmids (120 and 130 kb) . A probe cloned from the pDL11 plasmid of Achromobacter sp . strain WM111, which encodes the carbofuran hydrolase (mcd) gene (P . H . Tomasek and J . S . Karns, J . Bacteriol . 171:4038-4044, 1989), hybridized to the 120-kb plasmid . Restriction fragment profiles of pDL11 and strain ER2 plasmid DNAs suggested that the 120-kb plasmid of strain ER2 is very similar to pDL11 . On the basis of the results of biochemical tests, 16S rRNA sequence analysis, and membrane lipid analyses, strain ER2 was found to be a phylogenetically unique type II methylotroph . The constitutive carbofuran hydrolase activity in glucose-grown cells increased sevenfold when strain ER2 was grown in the presence of 100 mg of carbofuran per liter as the sole source of carbon and nitrogen or as the sole nitrogen source in the presence of glucose . Growth on carbofuran resulted in the induction of enzymes required for methylamine-dependent respiration and the serine pathway of formaldehyde assimilation . These results indicate that the carbofuran hydrolase mcd gene is conserved on a plasmid found in organisms from different geographic areas and that the specific activity of carbofuran degradation may increase in response to carbofuran treatment.

Infection, 1993 Sep-Oct, 21(5), 306 - 10
Ochrobactrum anthropi bacteremia: report of four cases and short review; Kern WV et al.; 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.

Eur J Biochem, 1993 Aug 15, 216(1), 25 - 38
Sweet potato beta-amylase . Primary structure and identification of the active-site glutamyl residue; Toda H et al.; The complete amino acid sequence of a subunit of sweet potato beta-amylase, a homotetramer, was established by sequence analysis of peptides obtained by digestions with Achromobacter protease I and Staphylococcus aureus V8 protease and by cyanogen bromide cleavage of the S-carboxymethylated subunit . The subunit of the enzyme is a single polypeptide consisting of 498 amino acid residues . It showed 50-60% identity in the amino acid sequence with those of beta-amylases from soybean and barley, while it about 25% with those of three bacterial beta-amylases deduced from the cDNA sequences . Sweet potato beta-amylase was completely inactivated with 2,3-epoxypropyl alpha-D-{U-14C}glucopyranoside . Sequence analysis of the inactivated enzyme revealed that Glu187 was specifically esterified by the affinity labeling with the above reagent, proposing that Glu187 is a potent candidate involved directly in the catalysis with this plant beta-amylase.

Nippon Eiseigaku Zasshi, 1993 Aug, 48(3), 707 - 20
{Relationship between water pollution and bacterial flora in river water}; Wada M; To clarify the relationship between water pollution and bacterial flora in rivers, 132 samples of river water were collected at eleven stations of the Chikuma-Sai river system from April 1985 to March 1986, and 29 biological-physicochemical examinations for indices of water pollution were done using these samples . Species and genus of bacteria in 485 isolates from bacterial flora were identified . Although variations of bacterial flora were seen among sampling stations and sampling seasons, most isolated bacteria were Gram-negative (96.7-100%) with Pseudomonas (25.0-90.0%), Acinetobacter (0-43.8%) and Aeromonas (0-12.5%) occurring predominantly . The principal-component analysis of the data from water pollution indices indicated that the water pollution could be divided into two groups: One was categorized as organic pollution because of the strong relationship to the total plate count, the Bacillus number, coliforms, yeast and dissolved oxygen; the other as visible pollution because of strong relationship to transparency, chemical oxygen demand and suspended solids . Cluster analysis was applied to 19 indices of water pollution with strong relationships to the river pollution, indicating that five stages of river water pollution could be shown visually . The studies on the relationship between water pollution and bacterial flora in river showed that Pseudomonas, the coliform group, Pasteurella, Aeromonas, Acinetobacter, Achromobacter and Bacillus were strongly related to the organic pollution, and Flavobacterium, Moraxella and Pseudomonas (non-growth on MacConkey agar) to the visible pollution . It seemed that the more organic substances exceeded the capacity for self-purification of the river, the more the bacterial populations of Aeromonas, Pasteurella, Pseudomonas and the coliform group increased.

Toxicon, 1993 Aug, 31(8), 957 - 67
Purification, sequencing and characterization of single amino acid-substituted phospholipase A2 isozymes from Trimeresurus gramineus (green habu snake) venom; Fukagawa T et al.; Two phospholipases A2 named PLA2-III and IV were newly isolated from Trimeresurus gramineus (green habu snake) venom in addition to PLA2-I and II reported previously {ODA et al . (1991) Toxicon 29, 157; Fukagawa et al . (1992) Toxicon 30, 133} . Their isoelectric points were determined to be about 4.5 . PLA2-III and IV exhibited almost unchanged lipolytic activity toward egg-yolk when compared with PLA2-I . The amino acid sequences were determined by sequencing the native proteins and the peptides produced by enzymatic (Achromobacter protease I and clostripain) and chemical (hydroxylamine) cleavages of the S-carboxamidomethylated derivative of the proteins . Both proteins consisted of 122 amino acid residues . When compared with PLA2-I, PLA2-III showed only a single amino acid substitution at the N-terminal position; namely from His to Asn . PLA2-IV also showed a single substitution from Ala to Asp at position 72 . It was inferred that these amino acid substitutions between PLA2-I and PLA2-III or IV are due to the single base substitution at the corresponding codons of genes, which might be preserved independently . The unique presence of Phe at position 28, where Tyr is commonly located and assumed to be a part of the Ca(2+)-binding loop, was conserved in both PLA2-III and IV as in PLA2-I . There was no significant difference in the dissociation constants (4.3-5.2 x 10(-4) M) for Ca2+ between these PLA2S and Tyr-28-containing PLA2S . These results suggested that the p-hydroxy group of Try-28 does not play a crucial role in binding of PLA2S to Ca2+.

Biochemistry, 1993 Jul 27, 32(29), 7360 - 6
X-ray scattering using synchrotron radiation shows nitrite reductase from Achromobacter xylosoxidans to be a trimer in solution; Grossmann JG et al.; We demonstrate here the applicability of X-ray scattering for studying molecular conformation of multimeric proteins in solution by using synchrotron radiation to extend the range of data collection to include medium angles (ca . 3-4 degrees) . We have been able to define the solution structure of the dissimilatory nitrite reductase of Achromobacter xylosoxidans (AxNiR), an enzyme for which there are conflicting reports as to the nature of its multimeric structure . Quantitative interpretation of the X-ray scattering profile, based on a modeling study using the high-resolution crystal structure data for the nitrite reductase from the related organism Achromobacter cycloclastes (AcNiR), provides a detailed model for the trimeric structure of AxNiR in solution . Sedimentation equilibrium centrifugation gave an M(r) of 103,000, consistent with such a trimeric structure.

Biochim Biophys Acta, 1993 Jun 4, 1163(3), 243 - 9
Pro-major basic protein has three types of sugar chains at the pro-portion; Shikata Y et al.; The amino-acid sequence of purified recombinant pro-major basic protein from Chinese hamster kidney cells was determined to verify the primary structure and glycosylation sites . Reduced and S-carboxamidemethylated protein was first digested with Achromobacter proteinase I . Each peptide was characterized by amino-acid analysis and amino-acid sequence analysis . We could identify all the peptides which were expected from the pro-major basic protein cDNA sequence . Sequence analysis and deglycosylation study revealed that Ser-8, Thr-9, Ser-46 and Asn-70 were glycosylated . The results indicated that proMBP has three types of sugar chains, O-glycoside, N-glycoside and glycosaminoglycan, in the pro-portion.

J Clin Microbiol, 1993 Apr, 31(4), 1007 - 8
Cellular fatty acid compositions of "Achromobacter groups B and E"; Holmes B et al.; Strains of "Achromobacter groups B and E" were examined for cellular fatty acid (CFA) composition to evaluate their chemical relatedness to known bacterial species and groups . The CFAs were liberated from whole cells by base hydrolysis, methylated, and analyzed by capillary gas-liquid chromatography . The CFA profiles of the two groups were identical and were distinct from CFA profiles of all other bacteria we have previously tested . These data provide support for results from whole-cell protein pattern analysis and DNA-DNA and rRNA-DNA hybridization studies, which show that "Achromobacter groups B and E" are biotypes of a single new genus and species.

Proc Natl Acad Sci U S A, 1993 Feb 1, 90(3), 928 - 32
Primary structure of two-chain botrocetin, a von Willebrand factor modulator purified from the venom of Bothrops jararaca; Usami Y et al.; The complete amino acid sequence and location of the disulfide bonds of two-chain botrocetin, which promotes platelet agglutination in the presence of von Willebrand factor, from venom of the snake Bothrops jararaca are presented . Sequences of the alpha and beta subunits were determined by analysis of peptides generated by digestion of the S-pyridylethylated protein with Achromobacter protease I or alpha-chymotrypsin and by chemical cleavage with cyanogen bromide or 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine . Two-chain botrocetin is a heterodimer composed of the alpha subunit (consisting of 133 amino acid residues) and the beta subunit (consisting of 125 amino acid residues) held together by a disulfide bond . Seven disulfide bonds link half-cystine residues 2 to 13, 30 to 128, and 103 to 120 of the alpha subunit; 2 to 13, 30 to 121, and 98 to 113 of the beta subunit; and 80 of the alpha subunit to 75 of the beta subunit . In terms of amino acid sequence and disulfide bond location, two-chain botrocetin is homologous to echinoidin (a sea urchin lectin) and other C-type (Ca(2+)-dependent) lectins.

Zoolog Sci, 1993 Feb, 10(1), 65 - 72
Mouse gamma-casein cDNA: PCR cloning and sequence analysis; Sasaki T et al.; A partial amino acid sequence of mouse gamma-casein was determined on a gas-phase amino acid sequencer . The N-terminal sequence (23 residues) was obtained using native gamma-casein, and the C-terminal sequence (13 residues) was determined with a corresponding peptide . The C-terminal peptide was isolated by affinity chromatography on an anhydrotrypsin agarose column after digestion of gamma-casein with Achromobacter lysyl-endopeptidase . A cDNA (507 base pairs) encoding the mature protein of gamma-casein was produced by polymerase chain reaction (PCR) amplification of lactating mammary gland first strand cDNA with oligonucleotide primers designed from the N-terminal (KHEIKDK-) and the C-terminal (-AHYTRFY) amino acid sequences . The full-length cDNA including 5'- and 3'-noncoding regions (920 base pairs) was cloned by the rapid amplification of cDNA ends (RACE) method of Frohman et al . Comparison of full-length cDNAs of mouse gamma-casein and rat gamma-casein showed 80% identity at the nucleotide level . The amino acids in the coding region of both gamma-caseins were 75% identical.

Acta Microbiol Bulg, 1993, 29, 3 - 8
Phosphatidylcholine-specific phospholipase C from Achromobacter xylosoxidans; Ivanov A et al.; A new phosphatidylcholine-specific phospholipase C (EC 3.1.4.3.) has been isolated from the culture broth of Achromobacter xylosoxidans . Growth conditions were optimized for maximum production of the enzyme . A temperature-sensitive synthesis was established . Chromatography on CM Sephadex and polyacrylamide gel electrophoresis of the native enzyme revealed a number of isozymes . The water soluble substrate for phospholipase p-nitrophenylphosphorylcholine was not hydrolysed by the enzyme.

Refract Corneal Surg, 1993 Jan-Feb, 9(1), 71 - 3
Achromobacter xylosoxidans keratitis following penetrating keratoplasty; Siganos DS et al.; BACKGROUND: Achromobacter xylosoxidans is a bacterium not previously reported in a corneal infection . We present a case of infectious keratitis caused by this organism, occurring 1 month following penetrating keratoplasty . METHODS: Identification of the organism was accomplished by staining, culture, and sensitivity testing of the corneal scraping obtained from the involved area . The infection responded to subconjunctival ticarcillin, piperacillin eye drops, and I.V . azlocillin . RESULTS: Complete healing with a resultant corneal opacity involving mostly the anterior and middle stroma was achieved 1 month later . CONCLUSIONS: A . xylosoxidans should be considered as a potential pathogen in instances of early postkeratoplasty infectious keratitis.

Toxicon, 1992 Nov, 30(11), 1331 - 41
Sequence determination and characterization of a phospholipase A2 isozyme from Trimeresurus gramineus (green habu snake) venom; Fukagawa T et al.; In addition to phospholipase A2-I (PLA2-I) reported previously (ODA et al., 1991, Toxicon 29, 157), a new PLA2 named PLA2-II was isolated from Trimeresurus gramineus (green habu snake) venom, and its amino acid sequence was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I and clostripain) cleavages of the carboxamidomethylated derivative of the protein . The protein consisted of 122 amino acid residues and His-47, Asp-48, and Asp-98 which have been assumed to be essential for PLA2 activity were conserved . Its sequence similarity to PLA2-I was 79%, with 26 residual differences . In contrast to the unique presence of Phe-28 in PLA2-I, PLA2-II contains Tyr-28 as seen in most of other PLA2s . There was no significant difference between the dissociation constants of PLA2-I and PLA2-II for Ca2+ . Secondary structure compositions of PLA2-II were similar to those of PLA2-I and Crotalus atrox PLA2 . A striking difference was found between these isozymes in contractile activity of isolated smooth muscle preparation of guinea-pig ileum . PLA2-II was over ten times more potent than PLA2-I, although its lipolytic activity toward egg-yolk was even slightly weaker (73%) than that of PLA2-I . The difference in contractile activities of PLA2-I and PLA2-II could be assumed to be due to discriminative lipid recognition brought about by different amino acid residues at the 58th position (Asp for PLA2-I and Asn for PLA2-II).

Glycobiology, 1992 Oct, 2(5), 419 - 27
Characterization of recombinant murine interleukin 5 expressed in Chinese hamster ovary cells; Kodama S et al.; We have purified recombinant murine interleukin 5 (rmIL-5) from the supernatant of Chinese hamster ovary cells . Each peptide fragment of the purified rmIL-5 generated by Achromobacter protease I digestion was characterized and glycosylation sites were determined . Although rmIL-5 contains three potential sites of N-linked glycosylation (Asn-26, Asn-55 and Asn-69), Asn-69 is not glycosylated . The oligosaccharides released from the protein by hydrazinolysis were fractionated by paper electrophoresis, lectin column chromatography and gel permeation chromatography, and their structures were analysed by sequential exoglycosidase digestion in combination with methylation analysis . The results indicated that they are a mixture of bi-, tri- and tetraantennary complex-type sugar chains with and without a fucose at the C-6 position of the proximal N-acetylglucosamine residue and high-mannose-type sugar chains . Although > 80% of the sugar chains are neutral oligosaccharides similar to recombinant human IL-5 (rhIL-5; Kodama, S., Endo, T., Tsuroka, N., Tsujimoto, M . and Kobata, A . (1991) J . Biochem., 110, 693-701), rmIL-5 has more tetraantennary oligosaccharides than rhIL-5 . A site differential study revealed that Asn-55 has more tetraantennary oligosaccharides than Asn-26.

Biosci Biotechnol Biochem, 1992 Oct, 56(10), 1604 - 7
Inhibition of Achromobacter protease I by lysinal derivatives; Masaki T et al.; Z-Val-, Z-Pro-, Z-Leu-Leu-, and Z-Leu-Pro-lysinals and BZ-DL-lysinal were chemically synthesized and tested as novel inhibitors for Achromobacter protease I (API), a lysine-specific serine protease . Among the lysinal derivatives tested, Z-Val-lysinal was the most potent competitive inhibitor, its Ki being estimated as 6.5 nM in an esterolytic assay with Tos-Lys-OMe . In an amidolytic assay, Z-Leu-Leu-lysinal was the most potent inhibitor and the apparent mode of inhibition was non-competitive . The Kis of the other lysinal derivatives in both esterolytic and amidolytic assays were more than 10(3) times lower than that of leupeptin . Z-Val-lysinol, lacking the aldehyde group, was a poor competitive inhibitor . These results suggest that acyl-, acylaminoacyl-, and acylpeptidyllysinals function as a transition-state inhibitor for Achromobacter protease I.

Biochem Biophys Res Commun, 1992 Sep 30, 187(3), 1529 - 35
Evidence that the type 2 copper centers are the site of nitrite reduction by Achromobacter cycloclastes nitrite reductase; Libby E et al.; Methods have been developed for selective depletion and reconstitution of the Type 2 Cu (non-blue) sites in the nitrite reductase from A . cycloclastes, resulting in preparations ranging from 0.5 to 2.6 Type Cu per trimer; the Type 1 Cu content is invariant at 3.0 per trimer . The activity of the enzyme is directly proportional to the Type 2 content as measured by direct metal determination or by analysis of the EPR spectra . These results indicate that an earlier report that the A . cycloclastes enzyme contains only Type 1 Cu sites is incorrect, and that the Type 2 Cu centers constitute the site at which NO2- is reduced . Furthermore, they suggest that other Cu nitrite reductases that are reported to contain only Type 1 Cu sites and exhibit relatively low activity may actually be largely Type 2 Cu-depleted forms of the enzymes.

Biochem Biophys Res Commun, 1992 Sep 16, 187(2), 609 - 14
Molecular cloning and characterization of catechol 2,3-dioxygenases from biphenyl/polychlorinated biphenyls-degrading bacteria; Chang H et al.; Catechol 2,3-dioxygenases were cloned from Alcaligenes sp . KF711, Pseudomonas putida KF715, and Achromobacter xylosoxidans KF701 which are biphenyl/polychlorinated biphenyls-degrading bacteria . All of the cloned enzymes were purified by preparative polyacrylamide gel electrophoresis (PAGE) . The purified catechol 2,3-dioxygenases were significantly different from one another in ring-fission activities to catechol and its derivatives . The catechol 2,3-dioxygenase from Alcaligenes sp . KF711 exhibited higher ring-fission activity to 4-chlorocatechol than those from P . putida KF715 and A . xylosoxidans KF701 . In electrophoretic mobilities, the three enzymes were different from one another on nondenaturing PAGE but the same on SDS-PAGE.

Curr Opin Rheumatol, 1992 Aug, 4(4), 509 - 15
Bacterial arthritis; Ho G Jr; The 1991 literature on septic arthritis included a concise review of adult septic arthritis, examples of pseudoseptic arthritis, and two interesting animal studies . One animal study examined the induction of acute synovitis by the intra-articular injection of bacterial endotoxin and the cytokines tumor necrosis factor-alpha, and interleukin-1 beta; and the other studied the effects of early and delayed synovectomy in the management of septic arthritis . The predispositions to septic arthritis can be divided into local joint abnormalities, systemic factors, or both . Examples of the local joint abnormalities include osteoarthritis of the hip and apatite-associated arthropathy . Septic arthritis in a patient with rheumatoid arthritis, in a patient with diabetes mellitus and hip arthropathy associated with hemochromatosis, or in a patient with acquired immunodeficiency syndrome and hemophilic arthropathy are examples of how systemic predisposition is coupled with local joint pathology to increase the vulnerability of the host to joint infection . Other examples of systemic disease that predispose to septic arthritis are systemic lupus erythematosus, hypogammaglobulinemia, and human immunodeficiency virus infection, as well as intravenous drug abuse . Unusual microorganisms causing septic arthritis in the adult include Achromobacter xylosoxidans, Moraxella catarrhalis, meningococci, and diphtheroids . Uncommon pathogenesis is represented by a case of intra-articular inoculation of Mycobacterium gastri into the small joint of the hand and a case of mixed bacterial infection of the hip resulting from an extension of a contiguous pelvic infection associated with trauma . Two cases of immune complex glomerulonephritis illustrate the extra-articular complications of septic arthritis: one due to group G streptococcus and the other due to pneumococcus . Finally, septic bursitis is reviewed from the community practice perspective.

Biochim Biophys Acta, 1992 Jul 13, 1122(1), 93 - 8
Proteolytic processing of human lysosomal arylsulfatase A; Fujii T et al.; Arylsulfatase A purified from human placenta contained an unreported component with an apparent molecular mass of 7 kDa in addition to the two known components with apparent molecular masses of 58 and 50 kDa . The detailed relationship between the 58 kDa component and the 50 kDa component is as yet unknown . The present study was undertaken to define the structure of the subunits of the sulfatase . The N-terminal sequence of the 50 kDa component was identical to that of the 58 kDa component . Furthermore, the peptide maps of the 50 kDa component, which was separately digested with trypsin and Achromobacter proteinase I, were quite similar to those of the 58 kDa one . Through sequence analysis of the incompatible peaks in the peptide maps, the 50 kDa component was found to lack a sequence from Val-445 to the C-terminus . On the other hand, the N-terminal sequence of the 7 kDa component began with Ala-448, though there was a minor sequence commencing with Thr-449 . These observations suggest that the 50 and 7 kDa components were produced by limited proteolysis near the C-terminus of the 58 kDa component . Through analysis using unreducing SDS-PAGE, the 58 and the 7 kDa components were found to be linked by disulphide bonds . Arylsulfatase A purified from human liver was also composed of the same subunits as the placental one . This finding suggests that human arylsulfatase A undergoes similar proteolytic processing regardless of the tissue involved.

J Rheumatol, 1992 Jun, 19(6), 992 - 3
Prosthetic knee infection due to Achromobacter xylosoxidans; Taylor P et al.; Achromobacter xylosoxidans is an aerobic gram negative organism that has been infrequently implicated in clinical infections in a variety of anatomical sites . We describe a case of a prosthetic knee infection due to Achromobacter xylosoxidans in a patient with rheumatoid arthritis receiving high dose prednisone.

Bioconjug Chem, 1992 May-Jun, 3(3), 262 - 8
Construction of protein analogues by site-specific condensation of unprotected fragments; Gaertner HF et al.; The extreme sensitivity to periodate of 1-amino, 2-hydroxy compounds permits the selective conversion of N-terminal serine and threonine to an aldehydic group . We have used this reaction to construct analogues of human granulocyte colony stimulating factor (G-CSF) by allowing such oxidized peptides to react with others that have had a hydrazide derivative attached to the C-terminus by reversed proteolysis . Two recombinant analogues of G-CSF were used as starting materials . Both had only a single lysine residue (at position 62 and 75, respectively) followed immediately by a serine . Digestion of each analogue by the lysine-specific protease from Achromobacter lyticus gave two fragments, one of which could be N-terminally oxidized and the other converted to the C-terminal hydrazide derivative by reversed proteolysis using the same enzyme . After preliminary studies with model peptides, we first reacted the corresponding peptide pairs together and then, in order to eliminate the 64-74 disulfide loop, fragment 1-62 from the first analogue with fragment 76-174 from the second . Reactions are efficient (up to 80% product based on the oxidized fragment) and take place under very mild conditions . The hydrazone bond can easily be stabilized by reduction with NaBH3CN . This method represents a new, reasonably general route for the construction of large protein chimeras of precisely controlled structure.

Clin Infect Dis, 1992 Apr, 14(4), 902 - 7
Catheter-associated sepsis caused by Ochrobactrum anthropi: report of a case and review of related nonfermentative bacteria; Cieslak TJ et al.; Ochrobactrum anthropi, formerly known as CDC group Vd, is an oxidase-producing, gram-negative, non-lactose-fermenting bacillus that oxidizes glucose and grows readily on MacConkey agar . Only occasionally isolated from human clinical specimens, this organism has rarely been found to be pathogenic . We describe the first reported case of infection due to O . anthropi in a child, that of bacteremia in a 3-year-old girl undergoing chemotherapy for retinoblastoma . In addition, we review the literature concerning cases of infection due to this and closely related bacterial species, namely Alcaligenes xylosoxidans subspecies xylosoxidans, Agrobacterium radiobacter, and "Achromobacter" group B . Finally, we attempt to clarify the confusing history and taxonomy of these organisms as well as make recommendations regarding antimicrobial therapy for infections caused by them.

Biochem Biophys Res Commun, 1992 Feb 28, 183(1), 77 - 82
Characterization of catechol 2,3-dioxygenases; Kim Y et al.; Three catechol 2,3-dioxygenases for biphenyl, naphthalene/salicylate, and toluene/xylene oxidation were cloned from Achromobacter xylosoxidans KF701, Pseudomonas putida (NAH7), and Pseudomonas sp . (pWWO) . The cloned catechol 2,3-dioxygenases were identified by enzymatic activity assay in addition to yellow bands on polyacrylamide gel after electrophoresis and activity staining . All of the cloned catechol 2,3-dioxygenases exhibited their highest activities on catechol as a substrate compared with catechol derivatives including 4-chlorocatechol, 3-methylcatechol, and 4-methylcatechol . The cloned catechol 2,3-dioxygenases are not fused proteins but were significantly different from one another in their electrophoretic mobilities on nondenaturing 7.5%-polyacrylamide gel.

Clin Infect Dis, 1992 Feb, 14(2), 479 - 84
Bacteremia due to Achromobacter xylosoxidans in patients with cancer; Legrand C et al.; Bacteremia due to Achromobacter xylosoxidans is rare, and little information on treatment is available . Between 1983 and 1988, A . xylosoxidans was recovered from 26 cultures of blood from 10 patients with cancer and clinical signs of infection, including one patient with septic shock and two with pneumonia . Neutropenia did not seem to be a predisposing factor . The infection may have been catheter related in four patients and associated with gastrointestinal pathology in four others . Probable cause was not determined in the remaining two . In vitro studies of susceptibility showed that the isolates were susceptible to trimethoprim-sulfamethoxazole (TMP-SMZ), the antipseudomonal penicillins, ceftazidime, cefoperazone, and imipenem; moderately susceptible to ciprofloxacin; and resistant to ceftriaxone, cefotaxime, cefoxitin, ceftizoxime, aztreonam, and amikacin . All patients receiving therapy recovered, including those six who received TMP-SMZ or a beta-lactam antibiotic as a single agent . A . xylosoxidans bacteremia is a significant infection and may be catheter related or associated with gastrointestinal pathology . The infection usually responds to therapy with TMP-SMZ or an appropriate beta-lactam antibiotic.

Biosci Biotechnol Biochem, 1992 Feb, 56(2), 180 - 5
Evidence for the existence of isozymes of glucose isomerase from Streptomyces phaeochromogenes; Basuki W et al.; Glucose isomerase from Streptomyces phaeochromogenes was purified from a commercial preparation, Swetase, by DEAE-cellulose, Bio-Gel A-0.5 m, and hydroxyapatite column chromatographies . It was found to be 2 fractions; F-A, not adsorbed on hydroxyapatite and F-B, adsorbed on hydroxyapatite . They were homogeneous in ordinary and SDS-PAGE and had similarities in some enzymatic and physico-chemical properties . The differences, however, were found in the N-terminal amino acid, which was only serine for F-A while it was serine and alanine for F-B, and also in their peptide mapping patterns of digests with trypsin, Achromobacter protease I, and cyanogen bromide . The results suggest that glucose isomerase from S . phaeochromogenes was composed of the two kinds of isozymes and that each of isozymes was a tetramer constituted of non-identical subunits.

Biochem J, 1992 Jan 15, 281 ( Pt 2), 545 - 51
Cloning and expression of a chick liver glutathione S-transferase CL 3 subunit with the use of a baculovirus expression system; Chang LH et al.; Glutathione S-transferase CL 3 subunits purified from 1-day-old-chick livers were digested with Achromobacter proteinase I and the resulting fragments were isolated for amino acid sequence analysis . An oligonucleotide probe was constructed accordingly for cDNA library screening . A cDNA clone of 1342 bases, pGCL301, encoding a protein of 26209 Da was isolated and sequenced . Including conservative substitutions, this protein has 75-79% sequence similarity to other Alpha family glutathione S-transferases . The coding sequence of pGCL301 was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells . The expressed protein has a high relative activity with ethacrynic acid (47% of the specific activity with 1-chloro-2,4-dinitrobenzene) . The enzyme has a subunit molecular mass of 25.2 +/- 1.2 kDa (by SDS/PAGE), a pI of 9.45 and an absorption coefficient A1%1cm of 13.0 +/- 0.5 at 280 nm.

Matrix Suppl, 1992, 1, 127 - 33
Vibrio alginolyticus ("Achromobacter") collagenase: biosynthesis, function and application; Keil B; Bacterial collagenase from aerobic non-pathogenic Vibrio alginolyticus chemovar iophagus ("Achromobacter" collagenase, EC 3.4.24.08) is an inducible extracellular metallo-proteinase . Production of Vibrio collagenase is induced specifically by collagen or by its macromolecular fragments . On the cell surface is expressed a specific receptor recognizing collagen structure . The study of natural inducers led to synthetic peptides with inducing properties . Vibrio collagenase cleaves collagen helical chains preferentially at 3/4 from the N-terminal . Its specific activity on synthetic substrate, 180,000 ukat/mg, represents the highest value for known collagenases . Its specificity differs from that of Clostridium: The enzyme cleaves preferentially sequences with Gly or Ala in position P'1 and Pro in position P2 or P'2 . Highly specific cleavages were obtained in beta-casein, prolactin, myosin, adenylate kinase and fibronectin . Autolysis yields partially degraded forms still active on native collagen and peptide substrate . The determination of the sequence of Vibrio collagenase is nearly achieved; the enzyme was not yet obtained in crystalline form . On basis of the already known sequence and structure of Hypoderma collagenase (EC 3.4.21.49), a hypothesis is advanced on the character of collagen binding site loops . Vibrio collagenase can be produced in kilogram quantities at low cost . It was found highly efficient in debridement of necrotic burns, ulcers and decubitus.

J Biochem (Tokyo), 1991 Dec, 110(6), 971 - 5
Identification of lysyl residues at the AMP-binding site of biodegradative threonine deaminase from Escherichia coli; Hirose K et al.; The biodegradative threonine deaminase from Escherichia coli is activated allosterically by AMP . To identify the residues interacting with the phosphate group of AMP at the binding site, we used the affinity labeling reagent, adenosine diphosphopyridoxal (AP2-PL) . In the absence of AMP, the enzyme formed the Schiff base with AP2-PL and Scatchard plot analysis showed a biphasic pattern, the respective Kd values for the high- and low-affinity binding phases being 20 and 110 microM . The former value is comparable to the Kd value of the enzyme for AMP . In the presence of AMP, the Schiff base formation was greatly reduced . Although the maximal activating effect of adenosine diphosphopyridoxine, a non-reactive derivative of AP2-PL, was about 13% of that of AMP, the half-saturation concentration was almost the same . These findings suggest that AP2-PL specifically labeled the lysyl residue(s) at the AMP-binding site of the enzyme . To identify the labeled residue(s), we reduced the modified enzyme with sodium borohydride, then cleaved it with cyanogen bromide and Achromobacter lyticus protease I . Reverse-phase HPLC was used to isolate two labeled peptides from the digest . Their amino acid compositions and sequences showed that Lys-111 and Lys-113 were labeled . We conclude that these two lysyl residues are located around the phosphate group of AMP at the allosteric regulation site of the enzyme.

Biochemistry, 1991 Oct 29, 30(43), 10451 - 7
Complete amino acid sequence of human liver cytosolic alanine aminotransferase (GPT) determined by a combination of conventional and mass spectral methods; Ishiguro M et al.; The complete amino acid sequence of human liver cytosolic alanine aminotransferase (GPT) (EC 2.6.1.2) is presented . Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively . Isolated peptides were analyzed with a protein sequencer or with a plasma desorption time of flight mass spectrometer and placed in the sequence on the basis of their molecular mass and homology to the sequence of rat GPT . The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues . The Mr of the subunit was calculated to be 54,479, which was in good agreement with a Mr of 55,000 estimated by SDS-PAGE, and also indicated that the active enzyme with a Mr of 114,000 was a homodimer composed of two identical subunits . The amino acid sequence is highly homologous to that of rat GPT (87.9% identity) recently determined {Ishiguro, M., Suzuki, M., Takio, K., Matsuzawa, T., & Titani, K . (1991) Biochemistry 30, 6048-6053} . All of the crucial amino acid residues are conserved in human GPT, which seem to be hydrogen bonding to pyridoxal 5'-phosphate in rat GPT by the sequence homology to other alpha-aminotransferases with known tertiary structures.

FEBS Lett, 1991 Oct 7, 291(1), 41 - 4
Evidence for a NO-rebound mechanism for production of N2O from nitrite by the copper-containing nitrite reductase from Achromobacter cycloclastes; Jackson MA et al.; Reduction of NO2- by the Cu-containing nitrite reductase from Achromobacter cycloclastes produces NO as the primary product initially, but as NO accumulates, NO production levels-off and N2O production becomes significant . Reaction of the enzyme with NO2- in the presence of NO increases the amount of N2O product significantly, while trapping the NO product as nitrosylhemoglobin or rapid removal of NO by sparging results in no detectable N2O production . Reaction of the enzyme with 15NO2- in the presence of 14NO results in rapid formation of the mixed isotope product (14N, 15N)O in ca . 45% yield . In contrast, the presence or absence of NO has no effect on N2O production by a prototypical heme cd1-containing nitrite reductase . These results are consistent with formation of a labile Cu(+)-NO+ species in the copper enzyme, which normally decomposes to NO . Production of N2O requires that the released NO must rebind to the enzyme to combine with a second NO2- or a species derived therefrom.

J Parasitol, 1991 Oct, 77(5), 784 - 6
Bacteria associated with tegument of Clinostomum marginatum (Digenea); Aho JM et al.; Adults of Clinostomum marginatum freshly collected from a heron, Ardea herodias, were examined using transmission electron microscopy . Specimens from the mouth of the bird were encrusted with bacteria that were not removed by washing unless the saline contained antibiotics . There was no evidence that the attached bacteria were damaging to the trematode tegument . Three species of Gram-negative bacteria were isolated from the worm surfaces and identified; Achromobacter sp . was present in pure culture on 4 of 6 original cultures and in mixed culture with Edwardsiella tarda and Enterobacter agglomerans in 2 cultures . These species and 3 unidentified species of bacteria were isolated from the oral epithelium of the heron . Microorganisms were not seen attached to the surfaces of worms recovered from the esophagus . Because E . tarda and E . agglomerans were the only species isolated from the heron esophagus, the intimate bacterial-worm association in the heron mouth may be due specifically to Achromobacter sp.

Eur J Biochem, 1991 Sep 15, 200(3), 651 - 61
Primary structure of nuclease P1 from Penicillium citrinum; Maekawa K et al.; The primary structure of nuclease P1, which cleaves both RNA and single-stranded DNA, from Penicillium citrinum was elucidated . The complete amino acid sequence consisting of 270 residues was determined by analysis of peptides obtained by digestion with Achromobacter protease I of the reduced and S-aminoethylated protein and by digestion with Staphylococcus aureus V8 protease of the reduced and S-carboxymethylated protein . Four half-cystine residues were assigned to Cys72-Cys217 and Cys80-Cys85 . N-Glycosylated asparagine residues were identified at positions 92, 138, 184 and 197 . Fast-atom-bombardment and laser-ionization MS were successfully used to confirm the determined amino acid sequences of peptides and to estimate the molecular mass of this glycoprotein having heterogenous sugar moieties, respectively . Comparison of the amino acid sequence of nuclease P1 with other nucleases revealed that the protein has a high degree of sequence identity (50%) with nuclease S1 from Aspergillus oryzae . The His-Phe-Xaa-Asp-Ala sequence (positions 60-64) is similar to the sequence (His-Phe-Asp-Ala) involving the active-site His119 of bovine pancreatic RNase A, and the Pro-Leu-His sequence (positions 124-126) is identical with the sequence involving the active-site His134 of porcine pancreatic DNase I.

J Chromatogr, 1991 Aug 23, 568(2), 315 - 24
Purification and some properties of phospholipase C from Achromobacter xylosoxidans; Kostadinova S et al.; A non-haemolytic phospholipase C (EC 3.1.4.3) was purified from the culture medium of Achromobacter xylosoxidans with a 5% yield and a purification factor of 330 . A combination of ultrafiltration, acetone precipitation and two subsequent affinity chromatographic steps was used . The affinity chromatography is a new application of 2-(4-aminophenylsulphonyl)ethyl-cellulose, a sorbent that has previously been used for the purification of phospholipase C from Bacillus cereus . The purified enzyme gave four distinct bands on polyacrylamide gel electrophoresis, and each band was catalytically active . Under our experimental conditions, the phospholipids examined were hydrolysed in the following order: phosphatidylcholine, phosphatidylethanolamine, sphingomyelin . Neither the synthetic substrate p-nitrophenylphosphorylcholine nor phosphatidylinositol was hydrolysed under different experimental conditions . For maximal hydrolytic activity toward phosphatidylcholine, the enzyme required Triton X-100 and Ca2+ ions . EDTA was inhibitory, but the enzyme activity was almost completely restored by Zn2+ . The molecular mass of the phospholipase C, estimated by gel permeation, was 34,000 daltons.

Biochem J, 1991 Aug 15, 278 ( Pt 1), 293 - 7
Cysteine-86 is not needed for the enzymic activity of glutathione S-transferase 3-3; Hsieh JC et al.; Recombinant glutathione S-transferase 3-3 expressed in Spodoptera frugiperda (SF9) cells with the use of a baculovirus expression system was modified with 1 mM-iodoacetamide . Amino acid analysis indicated that 0.79 +/- 0.15 cysteine residue was modified per enzyme subunit . The S-carbaminomethylated protein retains the GSH-conjugating activity . Glutathione S-transferase 3-3 modified with iodo{14C}acetamide was digested with Achromobacter proteinase I and the resulting peptides were separated by h.p.l.c . The modified peptides were pooled and further digested with Staphylococcus aureus V8 proteinase . Isotope-labelled peptides were isolated and collected for N-terminal sequence analysis . By this procedure, cysteine-86 was identified as the major S-carbaminomethylated residue . Verification of this findings came from the use of site-directed mutagenesis in which this cysteine was replaced by serine (C86S mutant) . The C86S mutant is enzymically active . Therefore cysteine-86 is not needed for the conjugation of GSH with electrophilic compounds on glutathione S-transferase 3-3.

Am J Clin Pathol, 1991 Aug, 96(2), 211 - 4
Achromobacter xylosoxidans . An unusual neonatal pathogen; Hearn YR et al.; Perinatal acquisition of a rare pediatric pathogen, Achromobacter xylosoxidans, with evidence for in utero transmission, is described . Cultures from the mother and neonate demonstrated A . xylosoxidans . An ascending bacterial infection in the mother with clinical chorioamnionitis is presented as the probable mode of transmission . Postmortem examination of the infant confirmed Achromobacter meningitis . In contrast to the current case with transmission from mother to neonate, previously published neonatal cases of Achromobacter infections indicate that nosocomial transmission of the organism is most common (79%) . In addition, the literature review revealed a high mortality associated with meningitis (77%), frequent hydrocephalus, and subsequent neurologic sequelae (36%) . To the authors' knowledge, this is the first documented case of maternal-fetal transfer of A . xylosoxidans.

Appl Environ Microbiol, 1991 Aug, 57(8), 2251 - 4
Detection and identification of groundwater bacteria capable of escaping entrapment on 0.45-micron-pore-size membrane filters; Shirey JJ et al.; Rural drinking water systems supplied by untreated groundwater were examined to determine whether coliform or heterotrophic plate count bacteria are capable of escaping entrapment on standard porosity (0.45-micron-pore-size) membrane filters . Filterable bacteria were present in 42% of the 24 groundwater sources examined by using nonselective media (R2A, full strength m-HPC, and 0.1x m-HPC agars) . Pseudomonads were the most frequently identified group of filterable bacteria detected . Flavobacterium, Alcaligenes, Acinetobacter, and Achromobacter isolates were also identified . Total coliforms were not recovered from any of the 24 groundwater samples following filtration through 0.45-micron-pore-size membrane filters by using selective M-Endo LES agar or mT7 agar . In addition, none of the isolates identified from nonselective media were coliforms . Similarly, neither total coliforms nor specifically Escherichia coli were detected in these filtrates when Colilert P/A medium was used.

Science, 1991 Jul 26, 253(5018), 438 - 42
The 2.3 angstrom X-ray structure of nitrite reductase from Achromobacter cycloclastes; Godden JW et al.; The three-dimensional crystal structure of the copper-containing nitrite reductase (NIR) from Achromobacter cycloclastes has been determined to 2.3 angstrom (A) resolution by isomorphous replacement . The monomer has two Greek key beta-barrel domains similar to that of plastocyanin and contains two copper sites . The enzyme is a trimer both in the crystal and in solution . The two copper atoms in the monomer comprise one type I copper site (Cu-I; two His, one Cys, and one Met ligands) and one putative type II copper site (Cu-II; three His and one solvent ligands) . Although ligated by adjacent amino acids Cu-I and Cu-II are approximately 12.5 A apart . Cu-II is bound with nearly perfect tetrahedral geometry by residues not within a single monomer, but from each of two monomers of the trimer . The Cu-II site is at the bottom of a 12 A deep solvent channel and is the site to which the substrate (NO2-) binds, as evidenced by difference density maps of substrate-soaked and native crystals.

Biochemistry, 1991 Jul 23, 30(29), 7180 - 5
Amino acid sequence of nitrite reductase: a copper protein from Achromobacter cycloclastes; Fenderson FF et al.; The amino acid sequence of the copper-containing nitrite reductase (EC 1.7.99.3) from Achromobacter cycloclastes strain IAM 1013 has been determined by using peptides derived from digestion with Achromobacter protease I (Lys), Staphylococcus aureus V8 protease (Glu), cyanogen bromide, and BNPS-skatole in acetic acid . The subunit contains 340 amino acids . The identity of the first seven amino acids is tentative . The sequence has been instrumental in the X-ray structure determination of this molecule; in conjunction with the X-ray structure, ligands to a type I copper atom and a type II copper atom (one of each per subunit) have been identified . Comparison of the sequence to those of multi-copper oxidases such as ascorbate oxidase, laccase, and ceruloplasmin {Messerschmidt, A., & Huber, R . (1990) Eur . J . Biochem . 187, 341-352} reveals that each of two domains seen in the X-ray structure is similar to the oxidases and also to the small blue copper-containing proteins such as plastocyanin . The combination of sequence and structural similarity to ascorbate oxidase and sequence similarity to ceruloplasmin leads to a plausible model for the domain structure of ceruloplasmin.

J Biol Chem, 1991 Jul 5, 266(19), 12639 - 45
The posttranslationally modified C-terminal structure of bovine aortic smooth muscle rhoA p21; Katayama M et al.; rhoA p21, a ras p21-like small GTP-binding protein, has the same C-terminal consensus motif of Cys-A-A-X (A is an aliphatic amino acid and X is any amino acid) as ras p21s, which is posttranslationally processed . We here determine the posttranslationally processed C-terminal structure of the rhoA p21 purified from bovine aortic smooth muscle . Incubation of rhoA p21-expressing insect cells with exogenous {3H}mevalonolactone caused the labeling of rhoA p21, suggesting that rhoA p21 is prenylated . Consistently, Raney nickel treatment of rhoA p21 released a geranylgeranyl moiety as estimated by gas chromatography/mass spectrometry . No lipid moiety was released by KOH or NH2OH treatment . Extensive digestion of rhoA p21 with Achromobacter protease I yielded a C-terminal peptide, Ser-Gly-Cys190, that lacked the three C-terminal amino acids predicted from the cDNA but was geranylgeranylated and carboxyl methylated at the cysteine residue . Bovine brain cytosol geranylgeranylated the bacterial rhoA p21 having the three C-terminal amino acids predicted from the cDNA but not the protein lacking the three C-terminal amino acids . Bovine brain membranes methylated the synthetic C-terminal peptide with 10 amino acids of rhoA p21 which was geranylgeranylated at its C-terminal cysteine residue but not the peptide which was not geranylgeranylated . These results suggest that rhoA p21 is first geranylgeranylated followed by removal of the three C-terminal amino acids and the subsequent carboxyl methylation of the exposed cysteine residue.

J Biochem (Tokyo), 1991 Jul, 110(1), 151 - 8
Amino acid sequence of nuclease S1 from Aspergillus oryzae; Iwamatsu A et al.; The amino acid sequence of nuclease S1, a nuclease which cleaves both single-stranded DNA and RNA, from Aspergillus oryzae was determined . Reduced and S-carboxymethylated or S-aminoethylated nuclease S1 was digested with Achromobacter protease I, Staphylococcus aureus V8 protease, or endoproteinase Asp-N . Peptides thus obtained were purified by reverse-phase high-performance liquid chromatography and sequenced, and the complete primary structure was established . Nuclease S1 consists of a single peptide chain of 267 amino acid residues bearing N-glycosylated Asns 92 and 228 . Five half-cystine residues are present at positions 25, 72, 80, 85, and 216, and the latter four residues are implicated in the formation of disulfide bonds by analogy with those in nuclease P1 . Two short stretches of sequences involving His 60 and His 125 are shown to be identical with those involving active site His 119 in bovine ribonuclease A and active-site His 134 in porcine deoxyribonuclease I, respectively.

Biochemistry, 1991 Jun 18, 30(24), 6048 - 53
Complete amino acid sequence of rat liver cytosolic alanine aminotransferase; Ishiguro M et al.; The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented . Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively . The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues . The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits . No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme {Tanase, S., Kojima, H., & Morino, Y . (1979) Biochemistry 18, 3002-3007}, which was almost identical with that of residues 303-322 of the rat liver enzyme . In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%) . Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase . This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.

Biochim Biophys Acta, 1991 May 23, 1058(1), 79 - 82
Spectral properties of Achromobacter xylosoxidans cytochromes c' and their NO complexes; Iwasaki H et al.; Cytochromes c' have been isolated from six strains of Achromobacter xylosoxidans: NCIB 11015 (formerly Alcaligenes sp . NCIB 11015), GIFU 543, 1048, 1051, 1055 and 1764 . They are dimeric proteins with more positive redox potentials than those of cytochromes c' from phototrophic bacteria at neutral pH . The electronic absorption, EPR and MCD spectra on NO-ferrous cytochromes c' at physiological pH showed that the major part of the heme-iron of nitrosylheme was penta-coordinated . The EPR spectral results indicated that the ground state of the heme-iron of ferric cytochromes c' appears to be in an admixed spin states which consists of predominant high-spin with a slight intermediate-spin character at pH 7.2 . These spectra were compared with those for cytochromes c' from phototrophic bacteria and the other hemoproteins.

Biochemistry, 1991 Mar 5, 30(9), 2391 - 4
Amino acid sequence and molecular characterization of a D-galactoside-specific lectin purified from sea urchin (Anthocidaris crassispina) eggs; Ozeki Y et al.; The complete amino acid sequence of a 11.5-kDa subunit of D-galactoside binding lectin purified from sea urchin (Anthocidaris crassispina) eggs is presented . The 105-residue sequence of the subunit was determined by analysis of the intact S-carbamoylmethylated protein and peptides generated by digestion with Achromobacter protease I or Staphylococcus aureus V8 protease . The lectin exists as a disulfide-linked homodimer of two subunits; the dimeric form is essential for hemagglutination activity . However, the monomeric form obtained by partial reduction retains the carbohydrate binding capacity . Neither Ca2+ nor SH reagent is essential for hemagglutination or carbohydrate binding . The sequence has no similarity to that of any known protein and apparently represents a new type of galactoside binding lectin.

Mol Cell Biol, 1991 Mar, 11(3), 1438 - 47
Role of the C-terminal region of smg p25A in its interaction with membranes and the GDP/GTP exchange protein; Araki S et al.; smg p25A is a ras p21-like small GTP-binding protein which is implicated in the regulated secretory processes . We have recently found that bovine brain smg p25A is geranylgeranylated at its C-terminal region . In this study, we examined the function(s) of the C-terminal region of smg p25A . Limited proteolysis of bovine brain smg p25A with Achromobacter protease I produced an N-terminal fragment and a C-terminal tail . The Mrs of intact smg p25A, the N-terminal fragment, and the C-terminal tail were estimated to be about 24,000, 20,000, and less than 2,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The N-terminal fragment contained the consensus amino acid sequences for GDP/GTP-binding and GTPase activities and showed these activities with kinetic properties similar to those of the intact protein but did not bind to plasma membranes or phosphatidylserine-linked Affigel under conditions in which the intact protein bound to them . The C-terminal tail neither contained the consensus amino acid sequences for GDP/GTP-binding and GTPase activities nor bound to plasma membranes or phosphatidylserine-linked Affigel . The GDP/GTP exchange protein specific for smg p25A, named GDP dissociation inhibitor (GDI), made a complex with the GDP-bound form of the intact smg p25A at a molar ratio of 1:1 and thereby inhibited its GDP/GTP exchange reaction but neither made a complex with the N-terminal fragment or the C-terminal tail nor affected the GDP/GTP exchange reaction of the N-terminal fragment . We expressed smg p25A in Escherichia coli and purified it to near homogeneity . This bacterial protein was not geranylgeranylated . Bacterial smg p25A did not bind to plasma membranes or phosphatidylserine-linked Affigel . smg p25A GDI neither made a complex with bacterial smg p25A nor affected its GDP/GTP exchange reaction . These results suggest that the N-terminal region of smg p25A has GDP/GTP-binding and GTPase activities but lacks the ability to interact with membranes and smg p25A GDI, that the C-terminal region of smg p25A plays important roles in its interaction with membranes and smg p25A GDI, and that some modifications of the C-terminal region, such as geranylgeranylation, which are absent in bacterial smg p25A, are important for these interactions.

Arch Biochem Biophys, 1991 Mar, 285(2), 270 - 5
Rapid purification and characterization of homoserine dehydrogenase from Saccharomyces cerevisiae; Yumoto N et al.; Homoserine dehydrogenase of Saccharomyces cerevisiae has been rapidly purified to homogeneity by heat and acid treatments, ammonium sulfate fractionation, and chromatography on Matrex Gel Red A and Q-Sepharose columns . The final preparation migrated as a single entity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr of 40,000 . The Mr of the native enzyme was 81,000 as determined by gel filtration, suggesting that the enzyme is composed of two identical subunits . This feature was also confirmed by cross-linking analysis using the bifunctional reagent dimethyl suberimidate . Feedback inhibition by L-methionine and L-threonine was observed using the purified enzyme . The enzyme was markedly stabilized against heat treatment at high salt concentrations . Additions of feedback inhibitors or high concentrations of salts failed to cause any dissociation or aggregation of the enzyme subunits unlike enzymes from other sources such as Rhodospirillum rubrum . The enzyme denatured in 3 M guanidine-HCl was refolded by simple dilution with a concomitant restoration of the activity . Cross-linking analysis of the renaturation process suggested that the formation of the dimer is required for activity expression . Amino acid sequence analysis of peptides obtained by digestion of the enzyme protein with Achromobacter lyticus protease I revealed that several amino acid residues are strictly conserved among homoserine dehydrogenases from S . cerevisiae, Escherichia coli, and Bacillus subtilis.

Mikrobiol Zh, 1991 Mar-Apr, 53(2), 17 - 20
{The DNA nucleotide composition of bacteria oxidizing diethylene glycols}; Sedina SA et al.; Nucleotide composition of DNA has been analyzed in bacteria composing the diethylene glycol-oxidizing association . It is shown that the microorganisms under study are representatives of genera Alkaligenes and Achromobacter.

Biopolymers, 1991 Feb 15, 31(3), 305 - 17
Proton and tritium NMR relaxation studies of peptide inhibitor binding to bacterial collagenase: conformation and dynamics; Dive V et al.; The interaction of succinyl-Pro-Ala, a competitive inhibitor of Achromobacter iophagus collagenase, with the enzyme was studied by longitudinal proton and tritium relaxation . Specific deuterium and tritium labeling of the succinyl part at vicinal positions allowed the measurement of the cross-relaxation rates of individual proton or tritium spin pairs in the inhibitor-enzyme complex as well as in the free inhibitor . Overall correlation times, internuclear distances, and qualitative information on the internal mobility in Suc1 (as provided by the generalized order parameter S2) could be deduced by the comparison of proton and tritium cross-relaxation of spin pairs at complementary positions in the -CH2- CH2- moiety as analyzed in terms of the model-free approach by Lipari and Szabo . The conformational and motional parameters of the inhibitor in the free and enzyme-bound state were directly compared by this method . The measurement of proton cross-relaxation in the Ala residue provided additional information on the inhibitor binding . The determination of the order parameter in different parts of the inhibitor molecule in the bound state indicates that the succinyl and alanyl residues are primarily involved in the interaction with the enzyme activity site . The succinyl moiety, characterized in solution by the conformational equilibrium among the three staggered rotamers--i.e., trans: 50%; g+: 20%; g-: 30%--adopted in the bound state the unique trans conformation.

Int J Pept Protein Res, 1991 Feb, 37(2), 122 - 7
Human recombinant CuZn-superoxide dismutase . Amino acid sequence and location of the disulfide bond; Peretz M et al.; The complete amino acid sequence of recombinant human Cu-Zn superoxide dismutase (CuZnSOD) is presented . The S-carboxymethylated protein was cleaved at lysine residues (with Achromobacter protease I) to provide a set of nine non-overlapping fragments accounting for 90% of the sequence . These fragments were then overlapped and aligned, and the sequence was completed by using peptides generated by cleavage at glutamic acid residues (with S . aureus V8 protease) and at arginine (with clostripain) . The recombinant protein contains a single disulfide bond between cysteine residues 57 and 146 . The primary sequence of recombinant human CuZnSOD is identical to that predicted by its cDNA sequence.

Oral Microbiol Immunol, 1991 Feb, 6(1), 62 - 4
In vitro activity of chlorhexidine against enteric rods, pseudomonads and acinetobacter from human periodontitis; Slots J et al.; Fifty-eight periodontal isolates of the families Enterobacteriaceae and Pseudomonadaceae, and the genera Acinetobacter and Achromobacter were studied to determine their susceptibility to Peridex (0.12% chlorhexidine digluconate solution; Procter & Gamble) . In an agar dilution assay, about 50% of the study strains grew in the presence of 70 micrograms/ml of chlorhexidine . Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas cepacia, and Serratia marcescens comprised the most resistant species . Studies are needed to determine the microbiological and clinical effects of chlorhexidine usage in patients infected with enteric rods and pseudomonads.

Neurology, 1991 Feb, 41(2 ( Pt 1)), 306 - 10
A prion protein missense variant is integrated in kuru plaque cores in patients with Gerstmann-Sträussler syndrome; Kitamoto T et al.; Kuru plaques are the pathologic hallmark in Gerstmann-Straussler syndrome (GSS) . To demonstrate that prion protein (PrP) is a component of kuru plaque cores, we fractionated and sequenced kuru plaque core derived peptides, following digestion with Achromobacter lyticus protease I . We identified 3 PrP-derived peptides by reverse-phase high-performance liquid chromatography and found a fragment of digests derived from a missense variant of PrP . The variant PrP was also present in the prion rod fraction in patients with GSS . This substitution may play a major role in cerebral amyloidogenesis.

Toxicon, 1991, 29(2), 157 - 66
Amino acid sequence of a phospholipase A2 from the venom of Trimeresurus gramineus (green habu snake); Oda N et al.; Two phospholipases A2, named phospholipases A2-I and A2-II, were purified to homogeneity from the venom of Trimeresurus gramineus (green habu snake) . The complete amino acid sequence of phospholipase A2-I was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I, clostripain, and chymotrypsin) and chemical (hydroxylamine) cleavages of the S-pyridylethylated derivative of the protein . The protein consisted of 122 amino acid residues and was similar in sequence to phospholipases A2 from the venoms of crotalid snakes which belong to the category of Group II . A most striking feature of this protein is that tyrosine at the 28th position which is common in phospholipases A2 and is assumed to be a part of the Ca2(+)-binding loop is replaced by phenylalanine . Such replacement is the first finding in Group II phospholipases A2 . Secondary structure compositions of phospholipase A2-I are similar to those of Crotalus atrox phospholipase A2 . No appreciable Ca2(+)-induced difference spectrum was observed, due probably to the absence of the effective chromophoric groups in the neighborhood of the Ca2+ binding site although Ca2+ is bound with affinity similar to that for T . flavoviridis phospholipase A2.

Dev Comp Immunol, 1991 Winter, 15(1-2), 9 - 16
A novel lipopolysaccharide-binding hemagglutinin isolated from hemocytes of the solitary ascidian, Halocynthia roretzi: it can agglutinate bacteria; Azumi K et al.; A hemagglutinin was isolated from hemocytes of the ascidian, Halocynthia roretzi, by a procedure including extraction and ion-exchange chromatography on CM-cellulose . The molecular weight of the hemagglutinin was estimated to be 120,000 by gel filtration . It was resistant to acid treatment but sensitive to alkali or heat treatment . The hemagglutinating activity was inhibited by heparin, chondroitin sulfate, and lipopolysaccharide (LPS), but not by mono- and disaccharides such as N-acetyl-galactosamine, galactose, and melibiose . The hemagglutinin showed binding ability to heparin and LPS, as demonstrated by heparin-Sepharose chromatography and centrifugation experiments, respectively . It was also found that the hemagglutinin can bind to various bacteria such as Escherichia coli, Bacillus subtilis, Vibrio anguillarum, Pseudomonas perfectomarinus, Achromobacter aquamarinus, and Alteromonas putrefaciens, and can agglutinate all of them.

Acta Microbiol Hung, 1991, 38(3-4), 283 - 91
beta-D-glucuronidase (BDG) activity of gram-negative bacteria; Ralovich B et al.; BDG is an inducible enzyme that is encoded by the uidA gene in Escherichia coli . Genetic sequences of this gene are present in most if not all E . coli strains regardless of the BDG phenotype . Expression of BDG activity can be influenced by lactose-induced catabolite repression or genetic mutations . Salmonella, Shigella and Yersinia strains frequently exhibit positive BDG reaction . BDG activity of strains belonging to genus Edwardsiella, Serratia, Yersinia, Vibrio, Erwinia, Alcaligenes, Acinetobacter, Moraxella, Plesiomonas, Achromobacter, Flavobacterium, Chromobacterium and Pasteurella awaits examination.

Biochem Biophys Res Commun, 1990 Dec 14, 173(2), 627 - 31
The complete amino acid sequence of the mature form of rat sepiapterin reductase; Oyama R et al.; The partial amino acid sequence of rat sepiapterin reductase was determined using peptides generated by cleavage of the S-carboxyamidomethylated protein with Achromobacter protease I, cyanogen bromide, chymotrypsin or BNPS-skatole . The protein began with N-acetyl methionyl residue at the N-terminus and ended with isoleucyl residue at the C-terminus . The present results essentially coincided with the amino acid sequence predicted from the nucleotide sequence of the cDNA recently reported by Citron et al . (Proc . Natl . Acad . Sci . USA 87, 6436-6440 (1990)), clarified the processing event during the biosynthesis and provided the complete amino acid sequence of the mature form of the enzyme.

Epidemiol Infect, 1990 Dec, 105(3), 541 - 51
Differentiation of Achromobacter-like strains from human blood by DNA restriction endonuclease digest and ribosomal RNA gene probe patterns; Holmes B et al.; Variation amongst Achromobacter-like strains was examined by DNA restriction endonuclease digestion and rDNA gene patterns generated using a non-radioactive probe . Chromosomal DNA was extracted from 12 cultures representing Achromobacter groups B, E and F, all from human blood cultures . DNA fingerprinting using EcoRI, Hae III or HindIII sub-divided the strains in a similar manner to that obtained by their protein patterns . The HaeIII patterns, with their small number of bands, were the easiest to interpret . The EcoRI patterns included a species-species triplet of bands but minor band patterns allowed further differentiation . The Achromobacter group F strains comprised a separate taxon and were distinct from the group B and E strains by all techniques examined . The study demonstrates that, in addition to total DNA digest analysis, rDNA gene restriction patterns provide a simple but discriminatory electrophoretic method for distinguishing within Achromobacter groups B and E.

Pathol Biol (Paris), 1990 Dec, 38(10), 999 - 1004
{Role of a collagenase in the latent proteolytic activity of fibronectin}; Imhoff JM et al.; Fibronectin fragments generated by Achromobacter iophagus collagenase exhibit a gelatinolytic activity . This activity is inhibited by phenyl-methyl-sulfonyl fluoride and pepstatin A . After separation of this collagenase digest of fibronectin on heparin Ultrogel, a laminase activity was also evidenced using laminin and the synthetic peptide Gly-Pro-Ala-Gly-Pro-Arg as substrates . Different results were obtained with a cathepsin D digest of fibronectin that exhibited gelatinolytic and laminolytic activities only after incubation with Ca++ . This suggests that the proteinases produced by hydrolysis of fibronectin enhance the effect of collagenase on extracellular matrix proteins.

J Bacteriol, 1990 Nov, 172(11), 6506 - 11
Molecular cloning and nucleotide sequence of the beta-lytic protease gene from Achromobacter lyticus; Li SL et al.; Two bacteriolytic enzymes secreted by Achromobacter lyticus M497-1 were purified and identified as being very similar (considering their amino acid composition and N-terminal sequence) to alpha- and beta-lytic proteases from Lysobacter enzymogenes . A 1.8-kb EcoRI fragment containing the structural gene for beta-lytic protease was cloned from A . lyticus chromosomal DNA . The protein sequence deduced from the nucleotide sequence was identical to the known sequence of beta-lytic protease, except for six residues . The nucleotide sequence revealed that the mature enzyme is composed of 179 amino acid residues with an additional 195 amino acids at the amino-terminal end of the enzyme, which includes the signal peptide, thus indicating that the enzyme is synthesized as a precursor protein.

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