[Show abstract][Hide abstract] ABSTRACT: Comparative in silico analyses of bacterial RNase P enzymes clustered their RNA subunits in type A RNA, found in E. coli, and in type B, found in B. subtilis. Zymomonas mobilis RNase P consists of one protein (Zmo-RnpA) and one type A RNA (RPR) subunit containing the P19 element, present in many RNase P RNAs of any structure class, but is lacking in the Escherichia coli RNase P RNA. To investigate the putative role of P19 stem, we constructed a P19 deletion RNA mutant (ΔP19RPR) and we performed detailed kinetic analysis of reconstituted enzymes in the presence of the homologous Zmo-RnpA protein or Eco-RnpA protein from E. coli. The deletion of P19 perturbs the monovalent ions requirements. The Mg2+ requirement for the ΔP19RPR holoenzyme was almost identical to that for the wtRPR holoenzyme at Mg2+ concentrations ≤ 25 mM. Interestingly, enzymes reconstituted with Eco-RnpA protein, relative to those assembled with Zmo-RnpA exhibited enhanced activity in the presence of ΔP19RPR, suggesting that Eco-RnpA protein can effectively replace its Z. mobilis counterpart. Homologous and heterologous reconstituted enzymes in the presence of ΔP19RPR exhibited differences in their Km values and catalytic efficacy. Overall, the presence of P19 stem points towards an adaption during the co-evolution of Zmo-RnpA and RPR which is essential for stabilizing the overall structure of the Z. mobilis RNase P. Finally, our results are in line with existing structural data on RNase P enzymes and provide biochemical support on the possible role of appended domains in RNase P RNA subunits.
[Show abstract][Hide abstract] ABSTRACT: MbeA and MbeC are two key proteins in plasmid ColE1 conjugal mobilization. Isothermal titration calorimetry was used to detect and quantify an interaction between MbeA and MbeC. As a result of this interaction, the affinity of MbeA for single stranded DNA increased. The interaction was confirmed in vivo using a bacterial two-hybrid system, which revealed that MbeA-MbeC complexes are formed through the amino-terminal region of MbeA and the carboxy-terminal region of MbeC. To the best of our knowledge, this is the first report of direct interactions between conjugative proteins encoded by a mobilizable plasmid.
[Show abstract][Hide abstract] ABSTRACT: A protein fraction exhibiting 1-hydroxy-2-naphthoic acid (1-H2NA) dioxygenase activity was purified via ion exchange, hydrophobic interactions, and gel filtration chromatography from Arthrobacter phenanthrenivorans sp. nov. strain Sphe3 isolated from a Greek creosote-oil-polluted site. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS-MS) analysis revealed that the amino acid sequences of oligopeptides of the major 45-kDa protein species, as analyzed by SDS-PAGE and silver staining, comprising 29% of the whole sequence, exhibited strong homology with 1-H2NA dioxygenase of Nocardioides sp. strain KP7. A BLAST search of the recently sequenced Sphe3 genome revealed two putative open reading frames, named diox1 and diox2, showing 90% nucleotide identity to each other and 85% identity at the amino acid level with the Nocardia sp. homologue. diox1 was found on an indigenous Sphe3 plasmid, whereas diox2 was located on the chromosome. Both genes were induced by the presence of phenanthrene used as a sole carbon and energy source, and as expected, both were subject to carbon catabolite repression. The relative RNA transcription level of the chromosomal (diox2) gene was significantly higher than that of its plasmid (diox1) homologue. Both diox1 and diox2 putative genes were PCR amplified, cloned, and overexpressed in Escherichia coli. Recombinant E. coli cells expressed 1-H2NA dioxygenase activity. Recombinant enzymes exhibited Michaelis-Menten kinetics with an apparent K(m) of 35 μM for Diox1 and 29 μM for Diox2, whereas they showed similar kinetic turnover characteristics with K(cat)/K(m) values of 11 × 10(6) M(-1) s(-1) and 12 × 10(6) M(-1) s(-1), respectively. Occurrence of two diox1 and diox2 homologues in the Sphe3 genome implies that a replicative transposition event has contributed to the evolution of 1-H2NA dioxygenase in A. phenanthrenivorans.
Applied and Environmental Microbiology 11/2011; 78(3):621-7. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 10/2011; 5(1):144-53. · 3.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 04/2011; 4(2):123-30. · 3.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 k(cat)/Km and/or k(cat) 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 pK(a)s, rate constants, activation energies and other important features were estimated from the profiles of parameters k(cat)/Km, k(cat) 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate the biochemical pathways induced during must fermentation employing the Saccharomyces cerevisiae, strain Z622, isolated from the traditional wine area of Zitsa (Epirus, Greece). Proteomic analysis (two-dimensional gel electrophoresis followed by nano-LC ESI-MS/MS) was used to assess the protein profile response of strain Z622 cultured in Debina grape must, yeast extract–malt extract (YM) or minimal medium (MM) under fermentation conditions, at the beginning and at the end of fermentation. The proteomic results and their analysis with Gene Ontology (GO) terms revealed that the intracellular proteins induced in yeast cells grown exclusively in Debina grape must during the early stage of fermentation were mainly involved in amino acid metabolism, glycolysis and sterol formation. These three pathways reflect the adaptation of the cell to the fermentative metabolism. During the late stage, several enzymes were induced due to starvation conditions or to participation in defence mechanisms against oxidative stress and protein degradation. Several of the identified enzymes have not previously been reported to be induced during wine fermentation and thus the results presented here can contribute to a better understanding of how S. cerevisiae cells adapt to wine fermentation.
[Show abstract][Hide abstract] ABSTRACT: (2-methyl-4,5-ditridecyl-1,4-cyclohexadienyl)methanol has been isolated from the lipidic fraction of the ethanologenic bacterium Zymomonas mobilis. The amphiphilic feature of this novel lipid is compatible with interactions within a phospholipid bilayers and suggests a function in membrane metabolism of the bacterium.
[Show abstract][Hide abstract] ABSTRACT: The spontaneous alcoholic fermentation of grape must is a complex microbiological process involving a large number of various yeast species, to which the flavour of every traditional wine is largely attributed. Whilst Saccharomyces cerevisiae is primarily responsible for the conversion of sugar to alcohol, the activities of various non-Saccharomyces species enhance wine flavour. In this study, indigenous yeast strains belonging to Metschnikowia pulcherrima var. zitsae as well as Saccharomyces cerevisiae were isolated and characterized from Debina must (Zitsa, Epirus, Greece). In addition, these strains were examined for their effect on the outcome of the wine fermentation process when used sequentially as starter cultures. The resulting wine, as analyzed over three consecutive years, was observed to possess a richer, more aromatic bouquet than wine from a commercial starter culture. These results emphasize the potential of employing indigenous yeast strains for the production of traditional wines with improved flavour.
Journal of Industrial Microbiology 10/2009; 37(1):85-93. · 1.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel halophilic bacterium, designated strain MSS4(T), was isolated from the solar salterns of Mesolongi, Greece. The micro-organism, a motile, Gram-stain-positive, aerobic rod, proliferated at salinities of 1.0-4.0 M NaCl, with optimal growth at 2.5 M NaCl. Endospores were not observed. Strain MSS4(T) showed optimal growth at 37 degrees C and pH 8.0. The G+C content of its DNA was 47.2 mol%. The polar lipid pattern of strain MSS4(T) consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidic acid and phosphatidylethanolamine. It possessed anteiso-C(15 : 0), C(18 : 0), C(16 : 0) and anteiso-C(17 : 0) as the major fatty acids (altogether representing 84.7 % of the total). The predominant isoprenoid quinone was MK-7. The cell-wall peptidoglycan contained meso-diaminopimelic acid. 16S rRNA gene sequence analysis showed that the new isolate has 96.1 % similarity to Bacillus qingdaonensis CM1(T) and Bacillus aidingensis 17-5(T), 95.5 % to Bacillus salarius BH169(T) and lower similarity to other Bacillus species. These results justify the assignment of strain MSS4(T) to a novel species within the genus Bacillus, for which the name Bacillus halochares sp. nov. is proposed. The type strain is MSS4(T) (=LMG 24571(T) =DSM 21373(T)).
International journal of systematic and evolutionary microbiology 09/2009; 60(Pt 6):1432-6. · 2.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel phenanthrene-degrading bacterium, designated strain Sphe3(T), was isolated from a creosote-contaminated soil in Greece. Cells were non-motile, Gram-positive, aerobic, and rod- to coccus-shaped. The strain was isolated on the basis of formation of a clear zone on agar plates sprayed with phenanthrene. Optimal growth occurred at 30 degrees C. The G+C content of the DNA was 65.7 mol%. The polar lipid pattern of strain Sphe3(T) consisted of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The dominant fatty acids were iso-C(15 : 0), anteiso-C(15 : 0), iso-C(16 : 0), C(16 : 0) and anteiso-C(17 : 0), representing >86 % of the total fatty acids. The predominant isoprenoid quinone of strain Sphe3(T) was menaquinone-8 (MK-8). Based on 16S rRNA gene sequence analysis, strain Sphe3(T) showed 99 and 98.9 % similarity to the type strains of Arthrobacter oxydans and Arthrobacter polychromogenes, respectively. Strain Sphe3(T) showed 91 % similarity to homologues of A. oxydans and A. polychromogenes based on recA gene sequence analysis. Based on 16S rRNA and recA gene sequence analysis and DNA-DNA hybridization analysis, as well as physiological and chemotaxonomic characteristics, it is concluded that strain Sphe3(T) represents a novel species of the genus Arthrobacter, for which the name Arthrobacter phenanthrenivorans sp. nov. is proposed. The type strain is Sphe3(T) (=DSM 18606(T) =LMG 23796(T)).
International journal of systematic and evolutionary microbiology 02/2009; 59(Pt 2):275-9. · 2.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MbeC is a 13-kDa ColE1-encoded protein required for efficient mobilization of ColE1, a plasmid widely used in cloning vector technology. MbeC protein was purified and used for in vitro DNA binding, which showed that it binds specifically double-stranded DNA (dsDNA) containing the ColE1 oriT. Amino acid sequence comparison and secondary structure prediction imply that MbeC is related to the ribbon-helix-helix (RHH) protein family. Alignment with RHH members pointed to a conserved arginine (R13 in MbeC) that was mutated to alanine. The mutant MbeC(R13A) was unable to bind either single-stranded DNA or dsDNA. Limited proteolysis fragmented MbeC in two stable folding domains: the N-terminal domain, which contains the RHH motif, and the C-terminal domain, which comprises a signature shared by nicking accessory proteins. The results indicate that MbeC plays a similar role in conjugation as TraY and TrwA of plasmids F and R388, respectively. Thus, it appears that an extended, possibly universal mechanism of DNA conjugative processing exists, in which oriT-processing is carried out by relaxases assisted by homologous nicking accessory proteins. This mechanism seems to be shared by all major conjugative systems analyzed thus far.
Journal of bacteriology 01/2009; 191(5):1446-55. · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A polycyclic aromatic hydrocarbon (PAH)-degrading bacterial strain Spyr1 was isolated from Greek creosote polluted soil by an enrichment method using pyrene as sole carbon and energy source. Spyr1 was identified as Mycobacterium sp. based on 16S rDNA analysis and it was capable of degrading pyrene, fluoranthene, fluorene, anthracene, and acenaphthene. The effect of entrapment in glass beads and alginate/starch mixtures on the survival and pyrene degradation ability of Spyr1 cells in liquid suspensions and soil microcosms was tested and compared with that of freely suspended cells. In general, free cells showed higher degradation of pyrene and other PAH than immobilized cells. However, immobilized cells could better tolerate PAH and they maintained their viability and PAH degradation capability for at least 1 year after storage at 4 degrees C. Entrapped cells in glass beads exhibited better pyrene biodegradation performance than alginate/starch entrapped cells in liquid suspensions and could be used effectively for at least ten repeated cycles. Alginate/starch entrapped cells exhibited better yields than glass beads entrapped cells for removing pyrene as well as mixtures of PAH in soil microcosms.
Applied biochemistry and biotechnology 01/2009; 159(1):155-67. · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A hyperthermophilic alpha-amylase encoding gene from Pyrococcus woesei was transferred and expressed in Xanthomonas campestris ATCC 13951. The heterologous alpha-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 alpha-amylase was found to be stable at 90 degrees C for 4 h and within the same period it retained more than 50% of its initial activity at 110 degrees C. Furthermore, X. campestris transformants produced similar levels of recombinant alpha-amylase activity regardless of the carbon source present in the growth medium, whereas the native X. campestris alpha-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 alpha-amylase and xanthan gum.
Applied Biochemistry and Biotechnology 06/2008; 149(2):99-108. · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This report describes phenanthrene uptake as well as the effect of phenanthrene on the membrane phospholipid and fatty acid composition in a newly isolated bacterial strain, Sphe3, that we taxonomically identified as Arthrobacter sp. Strain Sphe3 is able to utilize phenanthrene as a carbon source at high rates and appears to internalize phenanthrene with two mechanisms: a passive diffusion when cells are grown on glucose, and an inducible active transport system when cells are grown on phenanthrene as a sole carbon source. Active transport followed Michaelis-Menten kinetics, and it was amenable to inhibition by 2,4-dinitrophenol and sodium azide. Evidence provided here indicates that apart from inducing an active PAH uptake, the presence of phenanthrene elicits significant changes in membrane fluidity.
Applied Microbiology and Biotechnology 10/2007; 76(3):709-17. · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel protease designated protease-A-17N-1, was purified from the halo-alkalophilic Bacillus sp. 17N-1, and found active in media containing dithiothreitol and EDTAK(2). This enzyme maintained significant activity from pH 6.00 to 9.00, showed optimum k(cat)/K(m) value at pH 7.50 and 33 degrees C. It was observed that only specific inhibitors of cysteine proteinases inhibited its activity. The pH-(k(cat)/K(m)) profile of protease-A-17N-1 was described by three pK(a)s in the acid limb, and one in the alkaline limb. Both are more likely due t3o the protonic dissociation of an acidic residue, and the development and subsequent deprotonation of an ion-pair, respectively, in its catalytic site, characteristic for cysteine proteinases. Moreover, both the obtained estimates of rate constant k(1) and the ratio k(2)/k(-1) at 25 degrees C, from the temperature-(k(cat)/K(m)) profile of protease-A-17N-1, were found similar to those estimated from the proton inventories of the same parameter, verifying the reliability of the latter methodology. Besides, the bowed-downward proton inventories of k(cat)/K(m), as well as the large inverse SIE observed for this parameter, in combination with its dependence versus temperature, were showed unambiguously that k(cat)/K(m) = k(1). Such results suggest that the novel enzyme is more likely to be a cysteine proteinase functioning via a general acid-base mechanism.
Journal of Biochemistry 09/2007; 142(2):293-300. · 3.07 Impact Factor