Identification of Poly(cis-1,4-Isoprene) Degradation Intermediates during Growth of Moderately Thermophilic Actinomycetes on Rubber and Cloning of a Functional lcp Homologue from Nocardia farcinica Strain E1

The enrichment and isolation of thermophilic bacteria capable of rubber [poly(cis-1,4-isoprene)] degradation revealed eight different strains exhibiting both currently known strategies used by rubber-degrading mesophilic bacteria. Taxonomic characterization of these isolates by 16S rRNA gene sequence analysis demonstrated closest relationships to Actinomadura nitritigenes, Nocardia farcinica, and Thermomonospora curvata. While strains related to N. farcinica exhibited adhesive growth as described for mycolic acid-containing actinomycetes belonging to the genus Gordonia, strains related to A. nitritigenes and T. curvata formed translucent halos on natural rubber latex agar as described for several mycelium-forming actinomycetes. For all strains, optimum growth rates were observed at 50°C. The capability of rubber degradation was confirmed by mineralization experiments and by gel permeation chromatography (GPC). Intermediates resulting from early degradation steps were purified by preparative GPC, and their analysis by infrared spectroscopy revealed the occurrence of carbonyl carbon atoms. Staining with Schiff's reagent also revealed the presence of aldehyde groups in the intermediates. Bifunctional isoprenoid species terminated with a keto and aldehyde function were found by matrix-assisted laser desorption ionization-time-of-flight and electrospray ionization mass spectrometry analyses. Evidence was obtained that biodegradation of poly(cis-1,4-isoprene) is initiated by endocleavage, rather than by exocleavage. A gene (lcp) coding for a protein with high homology to Lcp (latex-clearing protein) from Streptomyces sp. strain K30 was identified in Nocardia farcinica E1. Streptomyces lividans TK23 expressing this Lcp homologue was able to cleave synthetic poly(cis-1,4-isoprene), confirming its involvement in initial polymer cleavage.     

Identification and characterization of genes from Streptomyces sp. strain K30 responsible for clear zone formation on natural rubber latex and poly(cis-1,4-isoprene) rubber degradation

Streptomyces sp. strain K30 was isolated from soil next to a city high way in Münster (Germany) according to its ability to degrade natural and synthetic poly(cis-1,4-isoprene) rubber and to form clear zones on natural rubber latex agar plates. The clear zone forming phenotype was used to clone the responsible gene by phenotypic complementation of a clear zone negative mutant. An open reading frame (lcp) of 1,191 bp was identified, which was preceded by a putative signal sequence and restored the capability to form clear zones on natural rubber latex in the mutant. The putative translation product exhibited strong homologies (50% aa identity) to a putative secreted protein from Streptomyces coelicolor strain A3(2), another clear zone forming strain. Heterologous expression of lcp of Streptomyces sp. strain K30 in Streptomyces lividans strain TK23 enabled the latter to form clear zones on latex-overlay agar plates and to accumulate a degradation product of about 12 kDa containing aldehyde groups. Two ORFs putatively encoding a heterodimeric molybdenum hydroxylase (oxiAB) were identified downstream of lcp in Streptomyces sp. strain K30 strain which exerted a positive effect on clear zone formation and enabled the strain to oxidize the resulting aldehydes. Heterologous expression of a fragment harboring lcp plus oxiAB in S. lividans TK23 resulted in accumulation of aldehydes only in the presence of 10 mM tungstate. Determination of protein content during cultivation on poly(cis-1,4-isoprene) revealed an increase of the cellular protein, and gel permeation chromatography analysis indicated a shift of the molecular weight distribution of the rubber to lower values in the transgenic S. lividans strains and in the wild type, thus confirming utilization and degradation of rubber. Therefore, for the first time, genes responsible for clear zone formation on natural rubber latex and synthetic cis-1,4-polyisoprene degradation in Gram-positive bacteria were identified and characterized.     

Heme-dependent rubber oxygenase RoxA of Xanthomonas sp. cleaves the carbon backbone of poly(cis-1,4-Isoprene) by a dioxygenase mechanism

Oxidative cleavage of poly(cis-1,4-isoprene) by rubber oxygenase RoxA purified from Xanthomonas sp. was investigated in the presence of different combinations of (16)O(2), (18)O(2), H(2)(16)O, and H(2)(18)O. 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD; m/z 236) was the main cleavage product in the absence of (18)O-compounds. Incorporation of one (18)O atom in ODTD was found if the cleavage reaction was performed in the presence of (18)O(2) and H(2)(16)O. Incubation of poly(cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with H(2)(18)O in the presence of (16)O(2) indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyl-trideca-4,8-diene-1-ol (HDTD; m/z 240) during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene). In the presence of (18)O(2), H(2)(16)O, and alcohol dehydrogenase/NADH, incorporation of two atoms of (18)O into the reduced metabolite HDTD was found (m/z 244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly(cis-1,4-isoprene) is proposed.     

Isolation and characterization of Streptomyces, Actinoplanes, and Methylibium strains that are involved in degradation of natural rubber and synthetic poly(cis-1,4-isoprene)

Rubber-degrading bacteria were screened for the production of clearing zones around their colonies on latex overlay agar plates. Novel three bacteria, Streptomyces sp. strain LCIC4, Actinoplanes sp. strain OR16, and Methylibium sp. strain NS21, were isolated. To the best of our knowledge, this is the first report on the isolation of a Gram-negative rubber-degrading bacterium other than γ-proteobacteria. Gel permeation chromatography analysis revealed that these strains degraded poly(cis-1,4-isoprene) to low-molecular-weight products. The occurrence of aldehyde groups in the degradation products by NS21 was suggested by staining with Schiff's reagent and 1H-nuclear magnetic resonance spectroscopy. The lcp gene of LCIC4, which showed 99% amino acid sequence identity with that of Streptomyces sp. strain K30, was cloned, and contained a putative twin-arginine motif at its N terminus. It is located next to oxiB, which is estimated to be responsible for oxidation of degradation intermediate of rubber in K30. Southern hybridization analysis using LCIC4 lcp probe revealed the presence of a lcp-homolog in OR16. These results suggest that the lcp-homologs are involved in rubber degradation in LCIC4 and OR16.     

Isolation and characterization of Streptomyces,Actinoplanes, and Methylibium strains that are involved in degradation of natural rubber and synthetic poly(cis-1,4-isoprene)

Rubber-degrading bacteria were screened for the production of clearing zones around their colonies on latex overlay agar plates. Novel three bacteria, Streptomyces sp. strain LCIC4, Actinoplanes sp. strain OR16, and Methylibium sp. strain NS21, were isolated. To the best of our knowledge, this is the first report on the isolation of a Gram-negative rubber-degrading bacterium other than γ-proteobacteria. Gel permeation chromatography analysis revealed that these strains degraded poly(cis-1,4-isoprene) to low-molecular-weight products. The occurrence of aldehyde groups in the degradation products by NS21 was suggested by staining with Schiff's reagent and ¹H-nuclear magnetic resonance spectroscopy. The lcp gene of LCIC4, which showed 99% amino acid sequence identity with that of Streptomyces sp. strain K30, was cloned, and contained a putative twin-arginine motif at its N terminus. It is located next to oxiB, which is estimated to be responsible for oxidation of degradation intermediate of rubber in K30. Southern hybridization analysis using LCIC4 lcp probe revealed the presence of a lcp-homolog in OR16. These results suggest that the lcp-homologs are involved in rubber degradation in LCIC4 and OR16.     

Draft genome sequence of Halomonas sp. strain KM-1, a moderately halophilic bacterium that produces the bioplastic poly(3-hydroxybutyrate)

We report the draft genome sequence of Halomonas sp. strain KM-1, which was isolated in Ikeda City, Osaka, Japan, and which produces the bioplastic poly(3-hydroxybutyrate). The total length of the assembled genome is 4,992,811 bp, and 4,220 coding sequences were predicted within the genome. Genes encoding proteins that are involved in the production and depolymerization of poly(3-hydroxybutyrate) were identified. The identification of these genes might be of use in the production of the bioplastic poly(3-hydroxybutyrate) and its monomer 3-hydroxybutyrate.     

Identification of the functional periplasmic nitrate reductase (nap) gene cluster from the deep-sea denitrifier Pseudomonas sp. strain MT-1

The nap gene cluster encoding periplasmic nitrate reductase was identified from Pseudomonas sp. strain MT-1, a deep-sea denitrifier isolated from the Mariana Trench. The ORFs identified were highly homologous with those of Pseudomonas stutzeri, but the cluster included only four ORFs (napDABC), less than those in other organisms. For other bacteria, some additional small ORFs (such as napE, napF, napG, napH, and napK) are found in the nap gene cluster, although their physiological function is still unclear. The soluble fraction of MT-1 grown under denitrifying condition showed significant nitrate reductase activity. This observation suggests that the periplasmic nitrate reductase encoded by the gene cluster identified in this study is functional. The activity was highest when the organism was grown under denitrifying conditions, suggesting that the enzyme participates in dissimilatory nitrite reduction.     

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Gene cloning and molecular characterization of an extracellular poly(L-lactic acid) depolymerase from Amycolatopsis sp. strain K104-1

We have isolated a polylactide or poly(L-lactic acid) (PLA)-degrading bacterium, Amycolatopsis sp. strain K104-1, and purified PLA depolymerase (PLD) from the culture fluid of the bacterium. Here, we cloned and expressed the pld gene encoding PLD in Streptomyces lividans 1326 and characterized a recombinant PLD (rPLD) preparation. We also describe the processing mechanism from nascent PLD to mature PLD. The pld gene encodes PLD as a 24,225-Da polypeptide consisting of 238 amino acids. Biochemical and Western immunoblot analyses of PLD and its precursors revealed that PLD is synthesized as a precursor (prepro-type), requiring proteolytic cleavage of the N-terminal 35-amino-acid extension including the 26-amino-acid signal sequence and 9-residue prosequence to generate the mature enzyme of 20,904 Da. The cleavage of the prosequence was found to be autocatalytic. PLD showed about 45% similarity to many eukaryotic serine proteases. In addition, three amino acid residues, H57, D102, and S195 (chymotrypsin numbering), which are implicated in forming the catalytic triad necessary for cleavage of amide bond of substrates in eukaryotic serine proteases, were conserved in PLD as residues H74, D111, and S197. The G193 residue (chymotrypsin numbering), which is implicated in forming an oxyanion hole with residue S195 and forms an important hydrogen bond for interaction with the carbonyl group of the scissile peptide bond, was also conserved in PLD. The functional analysis of the PLD mutants H74A, D111A, and S197A revealed that residues H74, D111, and S197 are important for the depolymerase and caseinolytic activities of PLD and for cleavage of the prosequence from pro-type PLD to form the mature one. The PLD preparation had elastase activity which was not inhibited by 1 mM elastatinal, which is 10 times higher than needed for complete inhibition of porcine pancreatic elastase. The rPLD preparation degraded PLA with an average molecular mass of 220 kDa into lactic acid dimers through lactic acid oligomers and finally into lactic acid. The PLD preparation bound to high polymers of 3-hydoxybutyrate, epsilon-caprolacton, and butylene succinate as well as PLA, but it degraded only PLA.     

Identification and functional characterization of the 2-hydroxy fatty N-acyl-Delta3(E)-desaturase from Fusarium graminearum

Delta3(E)-unsaturated fatty acids are characteristic components of glycosylceramides from some fungi, including also human- and plant-pathogenic species. The function and genetic basis for this unsaturation is unknown. For Fusarium graminearum, which is pathogenic to grasses and cereals, we could show that the level of Delta3-unsaturation of glucosylceramide (GlcCer) was highest at low temperatures and decreased when the fungus was grown above 28 degrees C. With a bioinformatics approach, we identified a new family of polypeptides carrying the histidine box motifs characteristic for membrane-bound desaturases. One of the corresponding genes was functionally characterized as a sphingolipid-Delta3(E)-desaturase. Deletion of the candidate gene in F. graminearum resulted in loss of the Delta3(E)-double bond in the fatty acyl moiety of GlcCer. Heterologous expression of the corresponding cDNA from F. graminearum in the yeast Pichia pastoris led to the formation of Delta3(E)-unsaturated GlcCer.     

Gene cloning and functional analysis of yellow green leaf3 (ygl3) gene during the whole-plant growth stage in rice

Chlorophyll is an important photosynthetic pigment in the process of photosynthesis in plants and photosynthetic bacteria. Genes involved in chlorophyll biosynthesis in Arabidopsis and photosynthetic bacteria have been well documented. In rice, however, these genes have not been fully annotated. In this paper, a yellow-green leaf gene, yellow green leaf3 (ygl3) was cloned and analyzed. ygl3 encodes magnesium chelation ChlD (D) subunit, a key enzyme for chlorophyll synthesis, resulting in a yellow-green leaf phenotype in all growth stages in rice. Expression content of ygl3 is highest in the leaf blades, followed by the leaf sheaths, while there is virtually no expression of the gene in the stems and seeds. The sub-cellular structure and protein content of the photosynthetic system of the ygl3 mutant were revealed by transmission electron microscopy, BN-PAGE, and western blotting. The results show that the mutation of the ygl3 gene indirectly leads to a decrease in the protein content of the photosynthetic system and severely obstructs the formation of granum thylakoids.     

Isolation and characterisation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degrading actinomycetes and purification of PHBV depolymerase from newly isolatedStreptoverticillium kashmirense AF1

Streptoverticillium kashmirense AF1 with the ability to degrade a natural polymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was isolated from municipal sewage sludge by soil burial technique. The PHBV film was degraded by the action of extracellular enzymes secreted by the microorganisms. Degradation of PHBV was evident by the formation of clear zones of hydrolysis on the polymer containing mineral salt agar plates. The extent of PHBV degradation increased up to 30 days of incubation. Maximum production of PHBV depolymerase was observed both at pH 8 and pH 7, 45 °C, 1% substrate concentration and in the presence of lactose as an additional carbon source. Two types of extracellular PHBV depolymerases were purified fromS. kashmirense AF1 by gel permeation chromatography using Sephadex G-75. The molecular weights of the two proteins were found to be 35 and 45 kDa approximately, as determined by SDS-PAGE. The results of the Sturm test also showed more CO₂ production as a result of PHBV degradation, in the test as compared to control. The present findings indicated the degradation capabilities ofS. kashmirense AF1.     

Identification, characterisation and cDNA cloning of two caseins from the common brushtail possum (Trichosurus vulpecula)1

Two major caseins have been isolated from the milk of the common brushtailed possum (Trichosurus vulpecula). These have been identified as alpha- and beta-casein on the basis of the similarity of their N-terminal sequences to those of the caseins of another marsupial (Macropus eugenii). Both proteins appear to exist in multiple forms. Possum alpha-casein is glycosylated mainly in the form of sialic acid residues and was shown by electrospray mass spectrometry to have multiply phosphorylated forms of three families with molecular masses 22700 and 23200 Da that may represent genetic variants. Two-dimensional electrophoresis showed that beta-casein exists as a complex of five or six proteins of identical N-terminal sequence but differing pI. Electrospray mass spectrometry indicated that the beta-caseins also are multiply phosphorylated with masses between 32300 and 32600 Da. A subfamily with mass values 1530 greater was also detected. The patterns were not affected by stage of lactation and quantitative analysis of two-dimensional gels of whole milk shows that alpha- and beta-caseins are present at a constant ratio throughout lactation. cDNA clones for the possum alpha- and beta-caseins have been isolated from an early lactation mammary cDNA library and sequenced.     

Identification of infectious salmon anaemia virus in Atlantic salmon from Nova Scotia (Canada): evidence for functional strain differences

Infectious salmon anaemia (ISA) is a serious disease responsible for high morbidity in farmed Atlantic salmon Salmo salar in Norway, Scotland and New Brunswick, Canada. Recent attempts to identify different strains of ISA virus (ISAV) based on nucleotide sequence variation have shown that the Norwegian and Scottish samples are similar to one another but markedly different from New Brunswick samples. These data may suggest the presence of different strains on each side of the Atlantic but no functional difference has been found with either strain. We describe the first identification and characterisation of ISAV in Atlantic salmon from Nova Scotia, Canada. Further, salmon infected with the Nova Scotia ISAV do not show typical ISAV pathology or mortality. Sequencing of this new strain showed it to possess greater similarity to ISAV from Norway and Scotland than to ISAV from New Brunswick. These findings are discussed in terms of a possible origin of the Nova Scotia ISAV strain and the existence of an avirulent ISAV strain. The impact of current strain variation studies on our knowledge of ISAV is also discussed.     

Identification and cloning of a key insecticide-metabolizing glutathione S-transferase (MdGST-6A) from a hyper insecticide-resistant strain of the housefly Musca domestica

Strains of the housefly, Musca domestica, highly resistant to organophosphate (OP) and other insecticides are known because they overproduce glutathione S-transferases (GSTs). Previous work has shown that overproduction in these strains involved numerous isozymes with glutathione conjugating activities (Pesticide Biochem. Physiol., 25 (1986) 169; Mol. General Genetics, 227 (1991) 355; J. Biol. Chem., 267 (1992) 1840; Mol. General Genetics, 245 (1994) 236; J. Mol. Evol., 43 (1996) 236). The current work describes the purification and identification of a M. domestica GST isozyme (pI 7.1) broadly specific for substrates from a housefly strain, Cornell-HR, that is highly resistant against OP-insecticides, and the isolation of two new MdGST genes using the antibody made against it. This isozyme, which was identified from amongst more than 20 isoelectric forms of GSTs of the same subunit size, was highly active for conjugating GSH to the model substrate 3,4-dichloronitrobenzne (DCNB). When expressed in Escherichia coli, one of the cloned GSTs, MdGST-6A, produces an enzyme that conjugates glutathione to the insecticides methyl parathion and lindane. On indication that it was the most active isozyme toward several xenobiotics among several MdGSTs tested, we advance the notion that MdGST-6A probably plays an important role in M. domestica Cornell-HR's resistance towards OP-insecticides. MdGST-6A and a second closely related one found in this work, MdGST-6B, are members of the traditional insect class I family (theta-class) and share the greatest homologies with a cluster of Drosophila GSTs on locus 55. In addition to having the unusually broad substrate specificity, the sequence of the new group of enzymes reveals that it has a highly diverged hydrophobic motif in its active site as compared to other class I GSTs from insects.     

Sphingobacterium sp. strain PM2-P1-29 harbours a functional tet(X) gene encoding for the degradation of tetracycline

Aims:  The tet (X) gene has previously been found in obligate anaerobic Bacteroides spp., which is curious because tet (X) encodes for a NADP-dependent monooxygenase that requires oxygen to degrade tetracycline. In this study, we characterized a tetracycline resistant, aerobic, Gram-negative Sphingobacterium sp. strain PM2-P1-29 that harbours a tet (X) gene.
Methods and Results:  Sphingobacterium sp. PM2-P1-29 demonstrated the ability to transform tetracycline compared with killed controls. The presence of the tet (X) gene was verified by PCR and nucleotide sequence analysis. Additional nucleotide sequence analysis of regions flanking the tet (X) gene revealed a mobilizable transposon-like element (Tn 6031 ) that shared organizational features and genes with the previously described Bacteroides conjugative transposon CTnDOT. A circular transposition intermediate of the tet (X) region, characteristic of mobilizable transposons, was detected. However, we could not demonstrate the conjugal transfer of the tet (X) gene using three different recipient strains and numerous experimental conditions.
Conclusions:  This study suggests that Sphingobacterium sp. PM2-P1-29 or a related bacterium may be an ancestral source of the tet (X) gene.
Significance and Impact of the Study:  This study demonstrates the importance of environmental bacteria and lateral gene transfer in the dissemination and proliferation of antibiotic resistance among bacteria.     

Molecular cloning of cDNA encoding a ubiquitin-activating enzyme (E1) from goldfish (Carassius auratus) and expression analysis of the cloned gene

Destruction of cyclin B is required to the mitotic and meiotic cycles. A cyclin-specific ubiquitinating system, including ubiquitin-activating enzyme (E1), is thought to be responsible for cyclin B destruction. Here we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, E1 from goldfish ovary. The cloned cDNA is 4069 bp long and encodes 1059 amino acids. The deduced amino acid sequence is highly homologous to E1 from other species. Recombinant goldfish E1 could transfer ubiquitin to cyclin-selective ubiquitin-conjugating enzyme. Tissue distribution revealed a single 4.0-kb message ubiquitous among tissues.     

一株有色金属矿山1-亚硝基-2-萘酚降解菌的筛选、鉴定和降解特性

1-亚硝基-2-萘酚是一类有色选矿药剂代表性的新型浮选药剂,在采选冶行业为了提高低品位矿物资源的利用率,被大量投入使用,该药剂的高稳定性进一步增大了矿山环境中重金属与有机选冶药剂复合污染的治理难度。微生物修复作为稠环芳烃(polycyclic aromatic hydrocarbons,PAHs)类污染物重要技术手段之一,具有安全、经济、高效和无二次污染等特点。【目的】本研究从我国广西河池市周边的典型有色金属尾矿环境中分离出1株高效降解1-亚硝基-2-萘酚的菌株,并分析其降解特性及其潜在的代谢途径,从而探究矿山复合污染生态系统中稠环芳烃类污染物的微生物修复技术的应用前景。【方法】从有色金属尾矿样本中筛选到以1-亚硝基-2-萘酚为唯一碳源的菌株,经16S rRNA基因序列鉴定,结合气相色谱-质谱联合(gas chromatography-mass spectrometer,GC-MS)检测分析菌株对1-亚硝基-2-萘酚的降解特性及中间代谢产物,并推测可能的代谢途径。【结果】筛选获得1株高效降解1-亚硝基-2-萘酚的Pseudomonas putida CUGB-JL11菌株,经鉴定为革兰氏阴性杆的恶臭假单胞菌。该菌株最适温度为30 ℃左右,最适pH值范围为6-8条件下,菌株5 d内对40 mg/L 1-亚硝基-2-萘酚的降解率高达81%。降解中间代谢产物主要为苯环类物质：苯甲羟肟酸甲酯和苯丙胺,但其母体及大部分中间产物都降解为小分子物质或者被完全降解。【结论】恶臭假单胞菌P. putida CUGB-JL11具有良好的1-亚硝基-2-萘酚降解能力和较强的环境适应性,有进一步被开发为微生物菌剂以用于稠环芳烃类污染修复的巨大潜力,为有色金属矿山生态系统中重金属和有机选冶药剂复合污染的微生物修复研究提供了理论依据和可利用的微生物资源。