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, wer