Molecular characterisation of a 5.75-kb cryptic plasmid from Bifidobacterium breve NCFB 2258 and determination of mode of replication

A small cryptic plasmid originating from Bifidobacterium breve NCFB 2258 was cloned and its complete nucleotide sequence determined. pCIBb1 is a circular DNA molecule, 5750 bp in size with a GC composition of 57%. Computer-assisted analysis identified 10 possible open reading frames (ORFs), seven of which could be assigned no function from homology searches. One ORF, rep (380 amino acids), was postulated to encode a replication protein similar to known replication proteins of rolling circle replicons, particularly those of the pC194 family. Demonstration of single-stranded forms of the plasmid in cell lysates that could be specifically degraded by S1 nuclease provided experimental evidence to substantiate a replication mechanism via single-stranded intermediates. Two other ORFs, par (199 amino acids) and an ftsK-like gene (286 amino acids), were assigned putative functions based on the presence of conserved motifs in their deduced proteins.     

Characterization, sequence and mode of replication of plasmid pNB2 from the thermophilic bacterium Clostridium thermosaccharolyticum

 The 1882-bp nucleotide sequence of the cryptic plasmid pNB2 isolated from the thermophilic bacterium Clostridium thermosaccharolyticum was determined. pNB2 DNA has very low GC content (27%) and may serve as a model for studying the modes of maintenance and replication of AT-rich DNA under conditions of thermophilic growth. The plasmid sequence revealed three open reading frames (ORFs) which would encode polypeptides of 289, 68 and 59 amino acids, respectively, and these proteins were synthesized in E. coli extracts primed with the plasmid. We found that the product of ORF289 may be initiated at the non-ATG start codon, TTG, and has similarities with the conserved motifs of Rep proteins encoded by rolling circle (RC) plasmids of the pC194/pUB110 family. Southern hybridization analysis of lysates of C. thermosaccharolyticum cells harboring pNB2 revealed single-stranded intermediates, suggesting that this plasmid is able to replicate in clostridial cells via the RC mechanism. The most significant similarities are found between pNB2 Rep protein and the Rep proteins of three RC plasmids of the pC194 family (pTB913, pBC1 and pST1) isolated from thermophilic bacteria. Comparative analysis of these Rep proteins showed that despite the significant level of divergence, these Rep proteins share a high degree of similarity in the regions of five well-known conserved domains of RC Rep proteins and fall into two groups in accordance with the similarities found in their active sites. Received: 25 April 1996 / Accepted: 20 June 1996     

Native expression and purification of hormone-sensitive lipase from Psychrobacter sp. TA144 enhances protein stability and activity

Psychrobacter, a micro-organism originally isolated from Antarctic sea water, expresses an extremely active hormone-sensitive lipase (HSL) which catalyzes the hydrolysis of fatty acid esters at very low temperature and is therefore of great potential industrial and pharmaceutical interest. An insoluble form of the entire enzyme has previously been cloned and expressed in Escherichia coli, subsequently refolded and shown to be active, whilst a shorter but completely inactive version, lacking the N-terminal 98 amino acids has been expressed in soluble form. In this study the entire enzyme has been expressed as a fully soluble protein in E. coli in the presence of either the osmolyte trehalose, plus high salt concentration, or the membrane fluidizer benzyl alcohol. Trehalose promotes protein mono-dispersion by increasing the viscosity of the growth medium for bacterial cells, thereby helping circumvent protein aggregation, whilst the heat-shock inducer benzyl alcohol stimulates the production of a network of endogenous chaperones which actively prevent protein misfolding, whilst also converting recombinant aggregates to native, correctly folded proteins. The resultant recombinant protein proved to be more stable than its previously expressed counterpart, as shown by CD and enzymatic activity data which proved the enzyme to be more active at a higher temperature than its refolded counterpart. By light scattering analysis it was shown that the newly expressed protein was monomeric. The stability of the full length native protein will help in understanding the structure of PsyHSL and the role of its regulatory N-terminal for eventual application in a myriad of biotechnological processes.     

In vivo definition of the functional origin of leading strand replication on the lactococcal plasmid pFX2

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism. Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S. pneumoniae. Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1. Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place. Received: 25 May 1998 / Accepted: 8 July 1998     

Thermal stabilization of psychrophilic enzymes: A case study of the cold-active hormone-sensitive lipase from Psychrobacter sp. TA144

Cold‐adapted enzymes possess high specific activity at low and moderate temperatures with respect to their mesophilic and thermophilic homologs; it is accepted that they have a less rigid and more flexible structure in the region surrounding the active site. However, the low stability of such molecules could represent the main barrier for their application in some industrial bioprocesses. The aim of this article was to investigate the ability of the naturally occurring osmolytes to increase the thermal stability and the specific activity of the cold‐active lipase from Psychrobacter sp. TA144 (PsyHSL), which belongs to the hormone‐sensitive lipase group. The effect of trimethylamine N‐oxide (TMAO), betaine, and L ‐proline addition on the activity and thermal stability of PsyHSL was investigated by means of biochemical and biophysical techniques. It turned out that in the presence of 3 M TMAO, the enzyme specific activity enhanced up to 250% at 50°C, while the addition of 1 M TMAO increased the thermostability fivefold at 45°C. Our experiments demonstrated that, even in the case of a psychrophilic enzyme, osmoprotectants, particularly TMAO, addition may be considered an efficient strategy to improve the protein thermal stability and specific activity at higher temperatures. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 946–952, 2012     

Characterization and expression analysis of three cold shock protein (CSP) genes under different stress conditions in the Antarctic bacterium Psychrobacter sp. G

Low temperature is one of the major environmental challenges that Antarctic bacteria must face. Detailed studies of cold shock responses of cold-adapted microorganisms are still insufficient. Here, we cloned three cold shock protein (CSP) genes (Csp1137, Csp2039, and Csp2531) in the Antarctic bacterium Psychrobacter sp. G and their regulatory sequences were identified. The three CSPs were highly conserved with other known CspAs. qRT-PCR was performed to evaluate their expression characteristics under stress conditions, and the potential influence of regulatory sequences also was analyzed. The expression of Csp1137 was enhanced both by low (0, 10?°C) and high temperature (30?°C). The expression of Csp2039 was enhanced by low temperature (0?°C), but was lower than that of Csp1137. This can be explained by the absence in Csp2039 of the AT-rich UP element. Different from Csp1137, the expression of Csp2531 was inhibited by low temperature (0?°C), even with the presence of AT-rich UP element, and it was not sensitive to high temperature (30?°C). The expression of Csp1137 was enhanced by high salinity (90, 120), whereas that of Csp2531was enhanced by low salinity (0, 15). At 0?°C and a salinity of 15, the expression of Csp1137 was repressed initially, but then it increased greatly during the next 10?h. The expressions of Csp2039 and Csp2531 were repressed significantly under four different combinations of stress conditions. Our results showed that the role of the upstream regulation sequences were much more complex than previously thought. Also, gene expressions were also affected by the environmental salinity. These are helpful in further clarification of the adaptation mechanism of Psychrobacter sp. G.     

Gene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipX_His with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipX_His is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipX_His lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.     

The Rep protein binding elements of the plasmid ColE2-P9 replication origin

The plasmid ColE2-P9 (ColE2) origin (32bp) is specifically recognized by the plasmid-specified Rep protein that initiates DNA replication. The ColE2 origin is divided into at least three functional subregions (I, II, and III), and three sites (a, b, and c) found in subregions I and II play important roles in Rep protein binding. We performed SELEX experiments of plasmid ColE2 to determine the optimal sequences for specific binding of the Rep protein. From these experiments, we obtained a common 16-bp sequence (5'-TGAGACCANATAAGCC-3'), which corresponds to about one half of the minimal ColE2 origin and contains sites a and b. Gel mobility shift assays using single-point mutant origins and the Rep protein further indicated that high affinity sequence-specific recognition by the Rep protein requires sites a, b, and c, but that mutations in site c were less disruptive to this recognition than those in sites a and b.     

Psychrobacter nivimaris sp. nov., a heterotrophic bacterium attached to organic particles isolated from the South Atlantic (Antarctica)

An aggregate-attached bacterium, strain 88/2-7, was isolated from samples of the Southern Ocean and investigated in a polyphasic approach. The novel marine isolate is an aerobic, Gram-negative, oxidase- and catalase-positive, non-motile short rod and grows in form of cream-colored colonies. Growth was observed at 5-35 degrees C. The bacterium tolerated concentrations of 0-13% (w/v) NaCl and utilized a relatively restricted spectrum of carbon sources. The analysis of the fatty acids revealed 18:1 cis 9 (18:1omega9c) as main fatty acid. The G+C content of the DNA was approximately 42 mol%. The sequence of the 16S rDNA assigned strain 88/2-7 to the gamma-subclass of Proteobacteria with a similarity of 99.65% to Psychrobacter proteolyticus (DSM 13887T). A DNA-DNA-hybridization study showed only 26.8% renaturation to the respective strain. Based on the morphological, physiological and molecular properties of the new isolate, the name Psychrobacter nivimaris sp. nov. (type strain 88/2-7T) is proposed.     

Isolation and characterization of a conditional replication mutant of the antibiotic resistance factor R1 affected in the gene of the replication protein RepA

Summary In vitro mutagenesis with hydroxylamine of a ParD^- miniderivative of R1, pAB174, yielded mutants that were less stable in the cell than pAB174. Some of these mutants had a thermosensitive phenotype. The replication of pAB2623, one of the thermosensitive mutants, was inhibited in the cell at the restrictive temperature of 42° C. The efficiency of the RepA protein of pAB2623 to promote replication of R1 in an in vitro assay was greatly reduced. Sequence analysis indicated that the repA gene of pAB2623 contains, close to its 3 end, two GC-AT transitions, separated by a single base, that change two consecutive codons of the gene. These results indicate that the phenotype of the mutant is the consequence of a mutated RepA protein and is consistent with the requirement of RepA for the in vivo replication of this plasmid.     

Molecular characterization of a cryptic plasmid from the psychrotrophic antarctic bacterium Pseudoalteromonas sp. 643A

We report the identification and nucleotide sequence analysis of pKW1, a plasmid of the psychrotrophic bacterium Pseudoalteromonas sp. 643A isolated from the stomach of Antarctic krill Euphasia superba. pKW1 consists of 4583 bp, has a G+C content of 43% and seven putative open reading frames (ORFs). The deduced amino acid sequence from ORF-1 shared significant similarity with the plasmid replicase protein of Psychrobacter cryohalolentis, strain K5. The DNA region immediately downstream of the ORF-1 showed some homology with the Rep-binding sequence of the theta-replicating ColE2-type plasmids. The ORF-3 amino acid sequence revealed amino acid sequence homology with the mobilization protein of Psychrobacter sp. PRwf-1 and Moraxella catarrhalis, with identities of 28% and 25%, respectively. The ORF-4 showed 46% amino acid sequence homology with the putative relaxase/mobilization nuclease MobA of Hafnia alvei and 44% homology with the putative mobilization protein A of Pasterulla multocida. The copy number of pKW1 in Pseudoalteromonas sp. 643A was estimated of 15 copies per chromosome.     

Clostridium vincentii sp. nov., a new obligately anaerobic, saccharolytic, psychrophilic bacterium isolated from low-salinity pond sediment of the McMurdo Ice Shelf, Antarctica

A gram-positive, motile, rod-shaped, strictly anaerobic bacterium was isolated from an enrichment initiated with sediment taken from below the cyanobacterial mat of a low-salinity pond on the McMurdo Ice Shelf, Antarctica. The organism grew optimally at 12° C, at pH 6.5, and at an NaCl concentration of < 0.5% (w/v). It survived freeze-thawing at low salt concentrations, but not exposure to temperatures over 25° C for more than 20 h or short-term exposure to temperatures > 50° C. Out of a variety of polysaccharides tested as growth substrates, only xylan supported growth. The organism also grew on a variety of mono- and disaccharides including the cyanobacterial cell wall constituent, N-acetyl glucosamine. Fermentation products on a mol product per 100 mol of hexose monomer fermented basis were: acetate, 72; formate, 72; butyrate, 55; hydrogen, 114; and CO₂, 100. Not detectable in the culture medium (< 2 mol per 100 mol of monomer) were lactate, propionate, ethanol, n-propanol, n-butanol, and succinate. The G+C content of the DNA from the bacterium was 33 mol%, and a phylogenetic analysis indicated that it grouped closely with members of the RNA-DNA homology group 1 of the genus Clostridium. It differed from other species of this genus with regard to growth temperature optimum, substrate range, and fermentation pattern, and is therefore designated as a new species of Clostridium for which the name Clostridium vincentii is proposed. The type strain is lac-1 (DSM 10228). Received: 6 August 1996 / Accepted: 30 October 1996     

The hormone-sensitive lipase from Psychrobacter sp. TA144: New insight in the structural/functional characterization

Cold-adapted esterases and lipases have been found to be dominant activities throughout the cold marine environment, indicating their importance in bacterial degradation of the organic matter. lip2 Gene from Psychrobacter sp. TA144, a micro-organism isolated from the Antarctic sea water, was cloned and over-expressed in Escherichia coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from which it was recovered in active form, purified to homogeneity and deeply characterised. Temperature dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 °C at pH 8.0. The enzyme resulted to be active on pNP-esters of fatty acids with acyl chain length from C₂ to C₁₂ and the preferred substrate was pNP-pentanoate showing a k_cat = 26.2 ± 0.1 s⁻¹, K_M = 0.122 ± 0.006 mM and a k_cat/K_M = 215 ± 11 mM⁻¹ s⁻¹. The enzyme was strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Fe³⁺, Mn²⁺ ions and it resulted to be activated in presence of methanol and acetonitrile, with calculated C₅₀ values of 1.98 M and 0.92 M, respectively.     

Replication of the promiscuous plasmid pLSI: a region encompassing the minus origin of replication is associated with stable plasmid inheritance

Deletion of a region of the promiscuous plasmid pLS1 encompassing the initiation signals for the synthesis of the plasmid lagging strand led to plasmid instability in Streptococcus pneumoniae and Bacillus subtilis. This defect could not be alleviated by increasing the number of copies (measured as double-stranded plasmid DNA) to levels similar to those of the wild-type plasmid pLS1. Our results indicate that in the vicinity of, or associated with the single-stranded origin region of pLS1 there is a plasmid component involved in its stable inheritance. Homology was found between the DNA gyrase binding site within the par region of plasmid pSC101 and the pLS1 specific recombination site RS_R.     

Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp. strain Fi:7

A marine psychrotolerant bacterium from the Antarctic Ocean showing high chitinolytic activity on chitin agar at 5 degrees C was isolated. The sequencing of the 16S rRNA indicates taxonomic affiliation of the isolate Fi:7 to the genus Vibrio. By chitinase activity screening of a genomic DNA library of Vibrio sp. strain Fi:7 in Escherichia coli, three chitinolytic clones could be isolated. Sequencing revealed, for two of these clones, the same open reading frame of 2,189 nt corresponding to a protein of 79.4 kDa. The deduced amino acid sequence of the open reading frame showed homology of 82% to the chitinase ChiA from Vibrio harveyi. The chitinase of isolate Fi:7 contains a signal peptide of 26 amino acids. Sequence alignment with known chitinases showed that the enzyme has a chitin-binding domain and a catalytic domain typical of other bacterial chitinases. The chitinase ChiA of isolate Fi:7 was overexpressed in E. coli BL21(DE3) and purified by anion-exchange and hydrophobic interaction chromatography. Maximal enzymatic activity was observed at a temperature of 35 degrees C and pH 8. Activity of the chitinase at 5 degrees C was 40% of that observed at 35 degrees C. Among the main cations contained in seawater, i.e., Na+, K+, Ca2+, and Mg2+, the enzymatic activity of ChiA could be enhanced twofold by the addition of Ca2+.     

Phenotypic and genomic characterization of the Antarctic bacterium Gillisia sp. CAL575, a producer of antimicrobial compounds

Microorganisms from Antarctica have evolved particular strategies to cope with cold. Moreover, they have been recently reported as producers of antimicrobial compounds, which inhibit the growth of other bacteria. In this work we characterized from different viewpoints the Gillisia sp. CAL575 strain, a psychrotrophic bacterium that produces microbial volatile organic compounds involved in the growth inhibition of Burkholderia cepacia complex members. Sequencing and analysis of the whole genome of Gillisia sp. CAL575 revealed that it includes genes that are involved in secondary metabolite production, adaptation to cold conditions, and different metabolic pathways for the production of energy. All these features make Gillisia sp. CAL575 a possible tool for biotechnology.     

Isolation and characterization of chitinase from a marine bacterium Vibrio sp.

A chitinase was separated from the culture broth of Vibrio sp. 11211 isolated from sediment from the South China Sea. The chitinase was purified 18.3-fold with 33% recovery by ammonium sulphate precipitation and chromatography. The subunit molecular weight of the enzyme was estimated by SDS-PAGE to be about 30kDa. The enzyme showed optimum pH at 6.5 and optimum temperature at 50^°C, and was stable in the pH range of 4 to 9 and at the temperature below 40^°C.     

Response of Heat-Shock Protein (HSP) Genes to Temperature and Salinity Stress in the Antarctic Psychrotrophic Bacterium Psychrobacter sp. G

Temperature and salinity fluctuations are two of the most important factors affecting the growth of polar bacteria. In an attempt to better understand the function of heat-shock proteins (HSPs) in the adaptive mechanisms of the Antarctic psychrotrophic bacterium Psychrobacter sp. G to such conditions, genes Hsp845, Hsp2538, Hsp2666, and Hsp2667 were cloned on the basis of the draft genome. The expression characteristics of these HSP genes under different stress conditions were analyzed by the qRT-PCR method. Expression of Hsp845 and Hsp2667 was inhibited significantly by low temperature (0 and 10 °C, respectively). There was no difference of expression when Hsp2538 and Hsp2666 were exposed to 0 °C but the expression of Hsp2666 was inhibited when exposed to 10 °C. Expression of Hsp2538 and Hsp2667 was not sensitive but expression of Hsp845 and Hsp2666 was increased at low salinity (0 and 15, respectively). Expression of the four HSP genes was enhanced at high salinity (90 and 120) and at high temperature independent of salinity. By contrast, low temperature had no significant effect independent of salinity.