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Mycobacterium sp. Spyr1 is a newly isolated strain that occurs in a creosote contaminated site in Greece. It was isolated by an enrichment method using pyrene as sole carbon and energy source and is capable of degrading a wide range of PAH substrates including pyrene, fluoranthene, fluorene, anthracene and acenapthene. Here we describe the genomic features of this organism, together with the complete sequence and annotation. The genome consists of a 5,547,747 bp chromosome and two plasmids, a larger and a smaller one with sizes of 211,864 and 23,681 bp, respectively. In total, 5,588 genes were predicted and annotated. Standards in Genomic Sciences

Arthrobacter phenanthrenivorans is the type species of the genus, and is able to metabolize phenanthrene as a sole source of carbon and energy. A. phenanthrenivorans is an aerobic, non-motile, and Gram-positive bacterium, exhibiting a rod-coccus growth cycle which was originally isolated from a creosote polluted site in Epirus, Greece. Here we describe the features of this organism, together with the complete genome sequence, and annotation. Standards in Genomic Sciences

Arthrobacter phenanthrenivorans is the type species of the genus, and is able to metabolize phenanthrene as a sole source of carbon and energy. A. phenanthrenivorans is an aerobic, non-motile, and Gram-positive bacterium, exhibiting a rod-coccus growth cycle which was originally isolated from a creosote polluted site in Epirus, Greece. Here we describe the fea-tures of this organism, together with the complete genome sequence, and annotation.

Mycobacterium sp.Spyr1 is a newly isolated strain that occurs in a creosote contaminated site in Greece. It was isolated by an enrichment method using pyrene as sole carbon and energy source and is capable of degrading a wide range of PAH substrates including pyrene, fluoran-thene, fluorene, anthracene and acenapthene. Here we describe the genomic features of this organism, together with the complete sequence and annotation. The genome consists of a 5,547,747 bp chromosome and two plasmids, a larger and a smaller one with sizes of 211,864 and 23,681 bp, respectively. In total, 5,588 genes were predicted and annotated.

A novel enzyme of molecular mass about 29 kDa was purified from the strain halo-alkaliphilic Bacillus sp. 17N-1 and designated protease-B-17N-1. This enzyme is likely to be a cysteine protease; it was found active in media containing EDTAK2 and dithiothreitol, it maintained considerable activity at temperatures 14 degrees C to 33 degrees C and pH 6.50 to 8.50 with optimum kcat/Km and/or kcat values at pH 7.00 and 25 degrees C. The activity of protease-B-17N-1 was strongly affected by the specific irreversible inhibitor of cysteine proteases E-64, while it remained unaffected by the 3,4-dichloro-isocoumarine, an irreversible inhibitor specific for serine proteases. Protease-B-17N-1 retained full activity at 25 degrees C after 30 min incubation at 8 degrees C or at 33 degrees C; moreover, it was found to be stable and active in the polar organic solvents DMSO and acetonitrile. The enzyme hydrolyzed the substrate Cbz-FR-pNA via Michaelis-Menten kinetics, while it showed insignificant activity for the substrate Suc-AAA-pNA. Valuable pKas, rate constants, activation energies and other important features were estimated from the profiles of parameters kcat/Km, kcat and Km, versus pH, temperature, and [NaCl]. In addition, interesting results were obtained from the effect of different metallic ions and polar organic solvents on the Michaelis-Menten parameters of protease-B-17N-1, showing that it performs catalysis via a (Cys)-S-/(His)-Im+H ion-pair, as well as its industrial and biotechnological potential, respectively. Environmental Technology

The bioremediation of petroleum-contaminated soil was investigated at laboratory scale, using three different approaches. The first approach comprised biostimulation of indigenous microorganisms. The second approach involved combination of biostimulation of indigenous microorganisms and bioaugmentation by inoculation with free cells of petroleum degrading Pseudomonas aeruginosa strain Spet. The third was a variation of the second, in which inoculation with encapsulated cells in starch and sodium alginate of P. aeruginosa strain Spet was applied. The bioremediation of the original hydrocarbon-contaminated soil (3.5% dry weight) and that of diluted with clean natural soil at 1:1 w/w were investigated. By providing sufficient moisture, nutrients and aeration by stirring in the original contaminated soil, total concentration of n-alkanes was reduced by 94% after 191 days of treatment and total concentration of 16 polycyclic aromatic compounds by 79%, while for the 1:1 diluted soils biodegradation reached 89% and 79%, respectively. The results showed that bioaugmentation with free or encapsulated P. aeruginosa cells and/or soil dilution had no significant effect on biodegradation. (C) 2010 Elsevier Ltd. All rights reserved. Bioresour Technol

Febs Journal

Febs Journal

Journal URL: http://www.springerlink.com/content/120548/ A hyperthermophilic α-amylase encoding gene from Pyrococcus woesei was transferred and expressed in Xanthomonas campestris ATCC 13951. The heterologous α-amylase activity was detected in the intracellular fraction of X. campestris and presented similar thermostability and catalytic properties with the native P. woesei enzyme. The recombinant α-amylase was found to be stable at 90 °C for 4 h and within the same period it retained more than 50% of its initial activity at 110 °C. Furthermore, X. campestris transformants produced similar levels of recombinant α-amylase activity regardless of the carbon source present in the growth medium, whereas the native X. campestris α-amylase production was highly dependent on starch availability and it was suppressed in the presence of glucose or other reducing sugars. On the other hand, xanthan gum yield, which appeared to be similar for both wild type and recombinant X. campestris strains, was enhanced at higher starch or glucose concentrations. Evidence presented in this study supports that X. campestris is a promising cell factory for the co-production of recombinant hyperthermophilic α-amylase and xanthan gum. © 2007 Humana Press Inc.

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Febs Journal

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Iubmb Life

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Febs Journal

Febs Journal

Three bacterial strains, designated as Wphe1, Sphe1, and Ophe1, were isolated from Greek soils contaminated with polycyclic aromatic hydrocarbon (PAH)-containing waste from the wood processing, steel, and oil refinery industries. Wphe1, Sphe1, and Ophe1 were characterized and identified as species of Pseudomonas, Microbacterium, and Paracoccus, respectively, based on Gram staining, biochemical tests, phospholipid analysis, FAME analysis, G+C content and 16S rRNA gene sequence analysis. The results of gas chromatography showed that strain Wphe1 degraded naphthalene, phenanthrene, and m-cresol over a wide temperature range; strain Sphe1 was a degrader of phenanthrene and n-alkanes; most interestingly, strain Ophe1 degraded anthracene, phenanthrene, fluorene, fluoranthene, chrysene, and pyrene, as well as cresol compounds and n-alkanes as sole carbon source. This is the first report of a representative of the genus Paracoccus capable of degrading PAHs with such versatility. These three strains may be useful for bioremediation applications. Appl Microbiol Biotechnol

Chemistry and Physics of Lipids

Chemistry and Physics of Lipids

MbeA is a 60 kDa protein encoded by plasmid ColE1. It plays a key role in conjugative mobilization. MbeA*, a slightly truncated version of MbeA, was purified for in vitro analysis. MbeA* catalysed DNA cleavage and strand-transfer reactions using oligonucleotides embracing the ColE1 nic site, which was mapped to 5'-(1469)CTGG/CTTA(1462)-3'. Thus MbeA is the relaxase for ColE1 conjugal mobilization, in spite of the fact that it lacks a three histidine motif considered the invariant signature of conjugative relaxases. Amino acid sequence comparisons suggest MbeA is nevertheless related to the common relaxase protein family. For instance, MbeA residue Y19 could correspond to the invariant tyrosine in Motif I, whereas H97, E104 and N106 may constitute the equivalent residues to the histidine triad in Motif III. This hypothesis was tested by site-directed mutagenesis. MbeA amino acid residues Y19, H97, E104 and N106 were changed to alanine. MbeA mutant N106A showed reduced oligonucleotide cleavage and strand-transfer activities, whereas mutation in the other three residues resulted in proteins without detectable activity, suggesting they are directly implicated in catalysis of DNA-cleavage and strand-transfer reactions. A double substitution of E104 and N106 by histidines, therefore reconstituting the canonical histidine triad, restored relaxase activities to 1% of wild type. Thus, MbeA is a variant of the common relaxase theme with a HEN signature motif, which has to be added to the canonical three histidine motif of previously reported relaxases. Mol Microbiol

Indian Journal of Biotechnology

Two recombinant plasmids, expressing ice nucleation activity, were constructed and named pCPP30inaZ and pCPP38inaZ. They were transferred to the ice-negative, xanthan-producing Xanthomonas campestris pv. campestris by electroporation. The transformants were used for co-production of xanthan gum and ice nuclei from sugar beet molasses. The highest values obtained were 20 g l(-1) and 10(18) ice nuclei ml(-1), respectively. The above values fulfil the criteria for industrial manipulation. This is the first report on co-formation of two products by a transformed X. campestris strain. Biotechnology Letters

Metal complexes of thiamine pyrophosphate (IPP) of the general formula [M(2)(TPPH)(2)Cl(2)]. 4H(2)O (M = Zn(2+), Cd(2+)) were isolated from methanolic solutions and characterized by elemental analysis, FT-IR, and multinuclear NMR spectroscopies. The data provide evidence for the bonding of the metals to the N(1') atom of the pyrimidine ring and to the pyrophosphate group. The stability constant measurements of TPP and 2-(alpha-hydroxyethyl)thiamine pyrophosphate (HETPP) metal complexes in aqueous solution imply the formation of dimeric complex species similar to the isolated solid products. They indicate also that HETPP forms more stable metal complexes than does TPP. To evaluate the coenzyme action of TPP and HETPP metal complexes, enzymic studies have been done using pyruvate decarboxylase apoenzyme. TPP metal complexes do not bind to the apoenzyme, unlike the Zn(II)-HETPP complex which can act as coenzyme. Considering these results, possible functional implications for thiamine involvement in catalysis are discussed. Journal of Biological Inorganic Chemistry

An extracellular alpha-amylase gene from the hyperthermophilic archaeon Pyrococcus woesei has been cloned and sequenced. The 1.4-kb protein-coding sequence is identical to that of the corresponding alpha-amylase gene of the closely related species P. furiosus. By using a shuttle cloning vector for halophilic bacteria, the P. woesei alpha-amylase was expressed in the moderate halophile Halomonas elongata, under the control of a native H. elongata promoter. The hyperthermophilic amylase activity expressed in the halophilic host was recovered completely in the crude membrane fraction of cell homogenates, suggesting the formation of inclusion bodies or that the secretion machinery of H. elongata may fail to recognize and release the pyrococcal alpha-amylase to the extracellular medium. However, thermal stability, metal ion interactions, optimal temperature and pH values for the crude and purified recombinant alpha-amylase were comparable with those of the native pyrococcal enzyme. The P. woesei amylase activity expressed in H. elongata was consistently detected in the cells upon growth on a wide range of NaCl concentrations (0.7-2.5 mol l-1). To our knowledge, this is the first report on the expression of an archaeal gene (P. woesei alpha-amylase) in a moderate halophilic host which serves as a cell factory able to grow under extreme salt conditions and with very simple nutritional requirements. J Appl Microbiol

The complete nucleotide sequences of two small cryptic Zymomonas mobilis ATCC 10988 plasmids (pZMO1 and pZMO2) were determined. The plasmids showed 67% homology to each other at their nucleotide level. Plasmid pZMO1 was 1651 bp long with 38% G + C content and contained an open reading frame (ORFZMO1) of 1044 nucleotides. ORFZMO1 is predicted to encode a polypeptide of 348 amino acids and shows a high degree of homology with gram-negative replication proteins of rolling circle replicating plasmids, which belong to the pC194/pUB110 family. Plasmid pZMO2 was found to be 1669 bp long, with a 38.5% G + C content, and it contained an ORF of 552 nucleotides (ORFZMO2) encoding a putative polypeptide of 184 amino acids. This polypeptide also shows a high degree of homology with the replication proteins of RCR plasmids of gram-negative bacteria, but only at their N-termini. The region necessary for replication of both plasmids was determined by stability tests under nonselective conditions, following cloning in pBR325 and introduction in Z. mobilis ATCC 10988 by pRK2013 assisted conjugation. Double- and single-strand origin regions were predicted by sequence analysis. Detection of single-stranded DNA in the extract of exponentially growing cells confirmed experimentally the rolling circle replication mode of at least pZMO2. (C) 2000 Academic Press. Plasmid

The 2.7-kb Zymomonas mobilis ATCC10988 plasmid pZMO3 contains a coding region (ORF1) indispensable for mobilization. A cis-acting 409-bp sequence between ORF2 (C-terminal) and ORF1 (N-terminal) conferred mobilization activity to pUC19, when the product of ORF1 was provided iii trans. In this area, two segments showed homology with previously characterized oriT regions. (C) 1999 Academic Press. Plasmid

A new recombinant plasmid, pBZIP1, was constructed for heterologous expression of high levels of ice nucleation activity in ethanol-producing Zymomonas mobilis strains CP4 and NCIB 11163. The plasmid construct contained the mobilization region and tetracycline resistance gene of pBR325, the replication region of the Z. mobilis native plasmid pZMO3 and the Pseudomonas syringae ice nucleation gene under the control of the Z. mobiiis CP4 pyruvate decarboxylase promoter (P-pdc). Z. mobilis transconjugants retained the plasmid stably, expressed ice nucleation activity up to 0.73 log [ice nuclei/cell] and can be used as improved sources of ice nuclei for industrial applications. Biotechnology Letters

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