Cytochrome c' Isolated from Achromobacter xylosoxidans GIFU 1055

Cytochrome c' has been isolated from Achromobacter xylosoxidansGIFU 1055. The absorption spectra of the oxidized and the reducedforms are almost the same as those of cytochromes c' found sofar. The reduced form combines with CO and NO. The hemoproteinis a basic protein and consists of two identical subunits witha molecular weight of 14,000. It is readily autooxidizable withmidpoint redox potential of + 111 mV at pH 7.2. (Received December 2, 1985; Accepted February 20, 1986)     

Purification and some properties of cytochrome c' from a strain of Achromobacter xylosoxidans

Cytochrome c' was crystallized from Achromobacter xylosoxidans GIFU 543. The cytochrome was a basic protein and its molecular weight was 28,000. The pyridine ferrohemochrome showed absorption peaks at 415, 521, and 551 nm. The absorption spectra of the oxidized and reduced forms at neutral pH were almost the same as those of other cytochromes c' reported already. The reduced cytochrome c' reacted with CO and NO, and the NO complex showed a characteristic absorption spectrum. The midpoint redox potential of the hemoprotein was measured to be + 110 mV at pH 7.2.     

Degradation of p-nitrophenol by Achromobacter xylosoxidans Ns isolated from wetland sediment

Achromobacter xylosoxidans Ns strain, capable of utilizing p-nitrophenol (PNP) as the sole source of carbon, energy, and nitrogen, was isolated from wetland sediment and confirmed based on 16S rRNA gene sequence. The strain Ns could tolerate concentrations of PNP up to 1.8 mM, and degradation of PNP was achieved in 7 d at 30 °C in the dark under aerobic conditions. Biodegradation of PNP occurred quickly at an optimal pH of 7.0 and higher, and at ⩽0.5% salt (NaCl) contents. During bacterial growth on PNP, 4-nitrocatechol was observed as a key degradation intermediate using a combination of techniques, including HPLC, UV–visible spectra, and comparison with the authentic standard. In a similar way, a second degradation intermediate was identified to be 1,2,4-benzenetriol. Moreover, A. xylosoxidans Ns could also degrade 3-nitrophenol as the sole source of carbon, nitrogen, and energy, but 2-nitrophenol could not. The experimental results showed that bacteria indigenous to the wetland sediment are capable of degradading PNP and chemicals with similar structures.     

Combined nickel-cobalt-cadmium resistance encoded by the ncc locus of Alcaligenes xylosoxidans 31A.

The nickel-cobalt-cadmium resistance genes carried by plasmid pTOM9 of Alcaligenes xylosoxidans 31A are located on a 14.5-kb BamHI fragment. By random Tn5 insertion mutagenesis, the fragment was shown to contain two distinct nickel resistance loci, ncc and nre. The ncc locus causes a high-level combined nickel, cobalt, and cadmium resistance in strain AE104, which is a cured derivative of the metal-resistant bacterium Alcaligenes eutrophus CH34. ncc is not expressed in Escherichia coli. The nre locus causes low-level nickel resistance in both Alcaligenes and E. coli strains. The nucleotide sequence of the ncc locus revealed seven open reading frames designated nccYXHCBAN. The corresponding predicted proteins share strong similarities with proteins encoded by the metal resistance loci cnr (cnrYXHCBA) and czc (czcRCBAD) of A. eutrophus CH34. When different DNA fragments carrying ncc genes were heterologously expressed under the control of the bacteriophage T7 promoter, five protein bands representing NccA (116 kDa), NccB (40 kDa), NccC (46 kDa), NccN (23.5 kDa), and NccX (16.5 kDa) were detected.     

Complete genome sequence of the haloaromatic acid-degrading bacterium Achromobacter xylosoxidans A8

Achromobacter xylosoxidans strain A8 was isolated from soil contaminated with polychlorinated biphenyls. It can use 2-chlorobenzoate and 2,5-dichlorobenzoate as sole sources of carbon and energy. This property makes it a good starting microorganism for further development toward a bioremediation tool. The genome of A. xylosoxidans consists of a 7-Mb chromosome and two large plasmids (98 kb and 248 kb). Besides genes for the utilization of xenobiotic organic substrates, it contains genes associated with pathogenesis, toxin production, and resistance. Here, we report the complete genome sequence.     

Cloning,expression and characterization of d-aminoacylase from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173

d-Aminoacylase catalyzes the conversion of N-acyl-d-amino acids to d-amino acids and fatty acids. The aim of this study was to identify the d-aminoacylase gene from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173 and investigate the biochemical characterization of the enzyme. A previously uncharacterized d-aminoacylase gene (ADdan) from this organism was cloned and sequenced. The open reading frame (ORF) of ADdan was 1467 bp in size encoding a 488-amino acid polypeptide. ADdan, with a high amino acid similarity to N-acyl-d-aspartate amidohydrolase from Alcaligenes A6, showed relatively low sequence similarities to other characterized d-aminoacylases. The recombinant ADdan protein was expressed in Escherichia coli BL21 (DE3) using pET-28a with a T7 promoter. The enzyme was purified in a single chromatographic step using nickel affinity gel column. The molecular mass of the expressed protein, calculated by SDS–PAGE, was about 52 kDa. The purified ADdan showed optimal activity at pH 8.0 and 50 °C, and was stable at pH 6.0–8.0 and up to 45 °C. Its activity was inhibited by Cu²⁺, Fe²⁺, Ca²⁺, Mn²⁺, Ni²⁺, Zn²⁺ and Hg²⁺, whereas Mg²⁺ had no significant influence on this recombinant d-aminoacylase. This is the first report on the characterization of d-aminoacylase with activity towards both N-acyl derivatives of neutral d-amino acids and N-acyl-d-aspartate. The characteristics of ADdan could prove to be of interest in industrial production of d-amino acids.     

EPR spectra of ferric cytochromes c' from five strains of Achromobacter xylosoxidans at low temperature and their temperature dependence

The EPR spectra at low temperature (6 K) and their temperature dependence (10-93 K) for five ferric cytochromes c' isolated from chemoheterotrophic bacteria, Achromobacter xylosoxidans NCIB 11015 (formerly Alcaligenes sp. NCIB 11015), GIFU 543, GIFU 1048, GIFU 1051, and GIFU 1764 are reported. The EPR spectral results indicate that the ground state of the heme iron(III) of cytochromes c' from these chemoheterotrophic bacteria can appear to be in an admixed spin state which consists of predominant S = 5/2 with a slight S = 3/2 character. The EPR spectra were compared with those for ferric cytochromes c' from photosynthetic bacteria and the other ferric hemoproteins.     

Alkanesulfonate degradation by novel strains of Achromobacter xylosoxidans, Tsukamurella wratislaviensis and Rhodococcus sp., and evidence for an ethanesulfonate monooxygenase in A. xylosoxidans strain AE4

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.     

Swarming motility: a multicellular behaviour conferring antimicrobial resistance

Swarming is a type of social motility allowing the migration of highly differentiated bacterial cells. Swarming shares many similarities with biofilm communities, which are notable for their high resistance to antimicrobial agents. We investigate here if the swarming behaviour could also be associated with a widespread antimicrobial resistant phenotype. Challenged with 13 antibiotics from various classes, swarm cells of Pseudomonas aeruginosa , Escherichia coli , Serratia marcescens , Burkholderia thailandensis and Bacillus subtilis showed higher resistance than their planktonic counterparts to all the antibiotics tested, except for the antimicrobial peptides. Using P. aeruginosa as a model, this multiresistant phenotype was shown to be transient and intrinsically linked to the swarming state. Resistance of swarm cells towards other antimicrobial agents, such as triclosan and a heavy metal (arsenite), was also observed. Neither RND-type efflux pumps, including MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM, nor a biofilm-associated resistance mechanism involving periplasmic glucans, appear to account for the resistance of swarm cells. Together with the high resistance of biofilms, these results support the hypothesis that antimicrobial resistance is a general feature of bacterial multicellularity. Swarming motility might thus represent a form of social behaviour useful as a model to investigate biofilm antibiotic resistance.     

Cloning, overexpression, and characterization of a novel thermostable penicillin G acylase from Achromobacter xylosoxidans: probing the molecular basis for its high thermostability

The gene encoding a novel penicillin G acylase (PGA), designated pgaW, was cloned from Achromobacter xylosoxidans and overexpressed in Escherichia coli. The pgaW gene contains an open reading frame of 2586 nucleotides. The deduced protein sequence encoded by pgaW has about 50% amino acid identity to several well-characterized PGAs, including those of Providencia rettgeri, Kluyvera cryocrescens, and Escherichia coli. Biochemical studies showed that the optimal temperature for this novel PGA (PGA650) activity is greater than 60 degrees C and its half-life of inactivation at 55 degrees C is four times longer than that of another previously reported thermostable PGA from Alcaligenes faecalis (R. M. D. Verhaert, A. M. Riemens, J. V. R. Laan, J. V. Duin, and W. J. Quax, Appl. Environ. Microbiol. 63:3412-3418, 1997). To our knowledge, this is the most thermostable PGA ever characterized. To explore the molecular basis of the higher thermostability of PGA650, homology structural modeling and amino acid composition analyses were performed. The results suggested that the increased number of buried ion pair networks, lower N and Q contents, excessive arginine residues, and remarkably high content of proline residues in the structure of PGA650 could contribute to its high thermostability. The unique characteristic of higher thermostability of this novel PGA provides some advantages for its potential application in industry.     

Cloning, Overexpression, and Characterization of a Novel Thermostable Penicillin G Acylase from Achromobacter xylosoxidans: Probing the Molecular Basis for Its High Thermostability

The gene encoding a novel penicillin G acylase (PGA), designated pgaW, was cloned from Achromobacter xylosoxidans and overexpressed in Escherichia coli. The pgaW gene contains an open reading frame of 2,586 nucleotides. The deduced protein sequence encoded by pgaW has about 50% amino acid identity to several well-characterized PGAs, including those of Providencia rettgeri, Kluyvera cryocrescens, and Escherichia coli. Biochemical studies showed that the optimal temperature for this novel PGA (PGA650) activity is greater than 60°C and its half-life of inactivation at 55°C is four times longer than that of another previously reported thermostable PGA from Alcaligenes faecalis (R. M. D. Verhaert, A. M. Riemens, J. V. R. Laan, J. V. Duin, and W. J. Quax, Appl. Environ. Microbiol. 63:3412-3418, 1997). To our knowledge, this is the most thermostable PGA ever characterized. To explore the molecular basis of the higher thermostability of PGA650, homology structural modeling and amino acid composition analyses were performed. The results suggested that the increased number of buried ion pair networks, lower N and Q contents, excessive arginine residues, and remarkably high content of proline residues in the structure of PGA650 could contribute to its high thermostability. The unique characteristic of higher thermostability of this novel PGA provides some advantages for its potential application in industry.     

A novel locus conferring fluoroquinolone resistance in Staphylococcus aureus.

Fluoroquinolones such as ciprofloxacin and ofloxacin are potent antimicrobial agents that antagonize the A subunit of DNA gyrase. We selected and mapped a novel fluoroquinolone resistance gene on the Staphylococcus aureus chromosome. Resistant mutants were selected with ciprofloxacin or ofloxacin and were uniformly localized to the A fragment of chromosomal DNA digested with SmaI and arrayed by pulsed-field gel electrophoresis. Several mutants (cfxB, ofxC) were genetically mapped between the thr and trp loci in the A fragment. A majority of A fragment fluoroquinolone resistance mutations were associated with reduced susceptibility to novobiocin, an antagonist of the B subunit of DNA gyrase. Two genes previously associated with fluoroquinolone resistance, the gyrA gene of DNA gyrase and the norA gene (associated with decreased drug accumulation), were localized to the G and D fragments, respectively. Thus, the fluoroquinolone resistance mutations in the A fragment are distinct from previously identified fluoroquinolone resistance mutations in gyrA and norA. Whether mutations in the A fragment after a second topoisomerase or another gene controlling supercoiling or affect drug permeation is unknown.     

TetX is a flavin-dependent monooxygenase conferring resistance to tetracycline antibiotics

The tetracycline antibiotics block microbial translation and constitute an important group of antimicrobial agents that find broad clinical utility. Resistance to this class of antibiotics is primarily the result of active efflux or ribosomal protection; however, a novel mechanism of resistance has been reported to be oxygen-dependent destruction of the drugs catalyzed by the enzyme TetX. Paradoxically, the tetX genes have been identified on transposable elements found in anaerobic bacteria of the genus Bacteroides. Overexpression of recombinant TetX in Escherichia coli followed by protein purification revealed a stoichiometric complex with flavin adenine dinucleotide. Reconstitution of in vitro enzyme activity demonstrated a broad tetracycline antibiotic spectrum and a requirement for molecular oxygen and NADPH in antibiotic degradation. The tetracycline products of TetX activity were unstable at neutral pH, but mass spectral and NMR characterization under acidic conditions supported initial monohydroxylation at position 11a followed by intramolecular cyclization and non-enzymatic breakdown to other undefined products. TetX is therefore a FAD-dependent monooxygenase. The enzyme not only catalyzed efficient degradation of a broad range of tetracycline analogues but also conferred resistance to these antibiotics in vivo. This is the first molecular characterization of an antibiotic-inactivating monooxygenase, the origins of which may lie in environmental bacteria.     

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.     

A novel enzyme conferring streptothricin resistance alters the toxicity of streptothricin D from broad-spectrum to bacteria-specific

Streptothricins (STs) produced by Streptomyces strains are broad-spectrum antibiotics. All STs consist of a carbamoylated D-gulosamine to which the beta-lysine homopolymer (1 to 7 residues) and the amide form of the unusual amino acid streptolidine (streptolidine lactam) are attached. Although many ST-resistance genes have been identified in bacteria, including clinically isolated pathogens and ST-producing Streptomyces strains, only one resistance mechanism has been identified to date. This mechanism involves the modification of the ST molecule by monoacetylation of the moiety of the beta-lysine(s). In this study, we successfully isolated a novel ST-resistance gene (sttH) from Streptomyces albulus, which is a known ST nonproducer. The in vitro analysis of SttH demonstrated that this enzyme catalyzes the hydrolysis of the amide bond of streptolidine lactam, thereby conferring ST resistance. Interestingly, the selective toxicity of ST-D possessing 3x beta-lysine moiety was altered from broad-spectrum to bacteria-specific by the hydrolysis of streptolidine lactam, although ST-F (1 x beta-lysine) was detoxified by SttH in both prokaryotes and eukaryotes (yeasts). STs have not been clinically developed due to their toxicities; however, in this study, we showed that hydrolyzed ST-D (ST-D-acid) exhibits potent antibacterial activity even when its toxicity against eukaryotic cells is reduced by SttH. This suggests that ST-D-acid is a potential candidate for clinical development or for use as a new lead compound for drug discovery.