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Some microbial species are chemically homogenous, and the same secondary metabolites are found in all strains. In contrast, we previously found that five strains of P. luteoviolacea were closely related by 16S rRNA gene sequence but produced two different antibiotic profiles. The purpose of the present study was to determine whether such bioactivity differences could be linked to genotypes allowing methods from phylogenetic analysis to aid in selection of strains for biodiscovery. Thirteen P. luteoviolacea strains divided into three chemotypes based on production of known antibiotics and four antibacterial profiles based on inhibition assays against Vibrio anguillarum and Staphylococcus aureus. To determine whether chemotype and inhibition profile are reflected by phylogenetic clustering we sequenced 16S rRNA, gyrB and recA genes. Clustering based on 16S rRNA gene sequences alone showed little correlation to chemotypes and inhibition profiles, while clustering based on concatenated 16S rRNA, gyrB, and recA gene sequences resulted in three clusters, two of which uniformly consisted of strains of identical chemotype and inhibition profile. A major time sink in natural products discovery is the effort spent rediscovering known compounds, and this study indicates that phylogeny clustering of bioactive species has the potential to be a useful dereplication tool in biodiscovery efforts.

The H-NOX (Heme-Nitric oxide/OXygen binding) family of diatomic gas sensing hemoproteins has attracted great interest. Soluble guanylate cyclase (sGC), the well-characterized eukaryotic nitric oxide (NO) sensor is an H-NOX family member. When NO binds sGC at the ferrous histidine-ligated protoporphyrin-IX, the proximal histidine ligand dissociates, resulting in a 5-coordinate (5c) complex; formation of this 5c complex is viewed as necessary for activation of sGC. Characterization of other H-NOX family members has revealed that while most also bind NO in a 5c complex, some bind NO in a 6-coordinate (6c) complex or as a 5c/6c mixture. To gain insight into the heme pocket structural differences between 5c and 6c Fe(ii)-NO H-NOX complexes, we investigated the extended X-ray absorption fine structure (EXAFS) of the Fe(II)-unligated and Fe(II)-NO complexes of H-NOX domains from three species, Thermoanaerobacter tengcongensis, Shewanella woodyi, and Pseudoalteromonas atlantica. Although the Fe(II)-NO complex of TtH-NOX is formally 6c, we found the Fe-N(His) bond is substantially lengthened. Furthermore, although NO binds to SwH-NOX and PaH-NOX as a 5c complex, consistent with histidine dissociation, the EXAFS data do not exclude a very weakly associated histidine. Regardless of coordination number, upon NO-binding, the Fe-N(porphyrin) bond lengths in all three H-NOXs contract by ~0.07 Å. This study reveals that the overall heme structure of 5c and 6c Fe(II)-NO H-NOX complexes are substantially similar, suggesting that formal histidine dissociation may not be required to trigger NO/H-NOX signal transduction. The study has refined our understanding of the molecular mechanisms underlying NO/H-NOX signaling.

The growth behavior and the proteomic response after a cold shock were investigated in the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis. Remarkably, no cold-induced proteins were observed in the proteome, whereas some key proteins were repressed. This suggests noticeable differences in the cold shock response between a true psychrophile and mesophiles.

The marine bacterium Pseudoalteromonas flavipulchra JG1 can synthesize various antibacterial metabolites, including protein and small molecules. The draft genome of JG1 is about 5.36 Mb and harbors approximate 4,913 genes, which will provide further insight into the synthesis of antimicrobial agents and antagonistic mechanisms of P. flavipulchra against pathogens.

Bacteria employ two-component signaling to detect and respond to environmental stimuli. In essence, two-component signaling relies on a protein called a response regulator that can elicit a change in gene expression or protein function in response to phosphoryl transfer from a histidine kinase. Phosphorylation of the associated histidine kinase is regulated by detection of an environmental signal, thus linking sensing to cellular response. Recently, it has been suggested that H-NOX (Heme-nitric oxide/oxygen binding) proteins may act as nitric oxide (NO) sensors in two-component signaling systems. NO/H-NOX regulated histidine kinases have been reported, but their cognate response regulators have yet to be identified. In this work we provide biochemical characterization of a complete NO/H-NOX-regulated two-component signaling pathway in the biofilm-dwelling marine bacterium, Pseudoalteromonas atlantica. In P. atlantica, as is typical for bacteria that code for H-NOX, an hnoX gene is found in the same operon as a gene coding for a two-component signaling histidine kinase (H-NOX-associated histidine kinase; HahK). We find that HahK is capable of autophosphorylation in vitro and that NO-bound H-NOX inhibits HahK activity, implicating H-NOX as a selective NO sensor. The cognate response regulator, a protein annotated as a cyclic-di-GMP processing enzyme that we have named HarR (H-NOX-associated response regulator), was identified using bioinformatics tools. Phosphoryl transfer from HahK to HarR has been established. This report reveals the first biochemical characterization of an H-NOX-associated response regulator and contributes to a deeper understanding of NO/H-NOX signaling in bacteria., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The endogenous components of the thioredoxin system in the Antarctic eubacterium Pseudoalteromonas haloplanktis have been purified and characterised. The temperature dependence of the activities sustained by thioredoxin (PhTrx) and thioredoxin reductase (PhTrxR) pointed to their adaptation in the cold growth environment. PhTrxR was purified as a flavoenzyme and its activity was significantly enhanced in the presence of molar concentration of monovalent cations. The energetics of the partial reactions leading to the whole electron transfer from NADPH to the target protein substrate in the reconstituted thioredoxin system was also investigated. While the initial electron transfer from NADPH to PhTrxR was energetically favoured, the final passage to the heterologous protein substrate enhanced the energetic barrier of the whole process. The energy of activation of the heat inactivation process essentially reflected the psychrophilic origin of PhTrxR. Vice versa, PhTrx possessed an exceptional heat resistance (half-life, 4.4 h at 95 °C), ranking this protein among the most thermostable enzymes reported so far in psychrophiles. PhTrxR was covalently modified by glutathione, mainly by its oxidised or nitrosylated forms. A mutagenic analysis realised on three non catalytic cysteines of the flavoenzyme allowed the identification of C(303) as the target for the S-glutathionylation reaction.

Nowadays, emergence and prevalence of MRSA (Methicillin Resistant Staphylococcus aureus) strain have become a great global concern in 21st century, so, it is necessary to discover new antibiotics against this pathogen. The aim of this study was isolation and evaluation marine bacteria from the Persian Gulf in order to finding antibiotic compounds against some pathogenic bacteria. For this purpose, water and sediment samples were collected from the Persian Gulf during March to October 2009. The antibacterial activity of the isolated bacteria was assessed using disc diffusion method. The Growth Curve Interference (GCI) parameter against MRSA was determined for the high potential antibiotic producing strain. The most important factors affecting fermentation conditions in antibiotic production were also optimized. Definite identification of intended isolate was confirmed by 16S rRNA sequencing. Altogether, 51 bacterial colony was isolated and among them only 3 bacterium showed antibacterial activity. Pseudoalteromonas piscicida PG-01 isolated from a sediment sample was chosen as the best antibiotic producing strain. This strain was effective against all tested Gram-positive bacteria, had good anti-MRSA activity and also GCI value against MRSA was two times lower than MIC value. Among the optimized fermentation parameters, carbon and nitrogen sources play major role in efficacy of optimized antibiotic production. Ultrastructural study on the effect of intended antibiotic compounds on MRSA using TEM revealed that the target site for this compound is cell wall. Considering the antibacterial effect of PG-01 strain especially against MRSA, intended antibiotic compounds can gives hope for treatment of diseases caused by multi-drug resistant bacteria.

Objective: Fungal keratitis (FK) is a vision-threatening infection, whose treatment requires more effective and safer anti-fungal agent exploitation urgently. With this aim, we focused on the effect of an extracellular polysaccharide on fungal adhesion to human corneal epithelial cells., Methods: We performed the cytotoxicity assays of the extracellular polysaccharide EPS-II from an antarctic bacterium Pseudoaltermonas and evaluated its inhibitory effect on Candida albicans cells' adherence to human corneal epithelial cells (HCECs)., Results: EPS-II, which displayed minor cytotoxicity but also promoted proliferation of HCECs, could inhibit the adherence of yeast cells to HCECs in a dose-dependent manner. EPS-II could also suppress the subsequent PI3K/AKT signaling pathway, and thereby decrease the expression of early inflammatory cytokines., Conclusions: Extracellular polysaccharide EPS-II was suggested as a new natural agent for attenuating FK.

The cycloprodigiosin biosynthetic gene cluster has not been reported. We sequenced the genome of a cycloprodigiosin-producing bacterial strain, Pseudoalteromonas rubra ATCC 29570(T). Analysis revealed a probable cycloprodigiosin biosynthetic cluster, providing a good model for the study of cycloprodigiosin synthesis and regulation.

This review reports results from our laboratory on the development of an effective inducible expression system for the homologous/heterologous protein production in cold-adapted bacteria. Recently, we isolated and characterized a regulative genomic region from Pseudoalteromonas haloplanktis TAC125; in particular, a two-component regulatory system was identified. It is involved in the transcriptional regulation of the gene coding for an outer membrane porin (PSHAb0363) that is strongly induced by the presence of L: -malate in the growth medium.We used the regulative region comprising the two-component system located upstream the PSHAb0363 gene to construct an inducible expression vector - named pUCRP - under the control of L: -malate. Performances of the inducible system were tested using the psychrophilic β-galactosidase from P. haloplanktis TAE79 as model enzyme to be produced. Our results demonstrate that the recombinant cold-adapted enzyme is produced in P. haloplanktis TAC125 in good yields and in a completely soluble and catalytically competent form. Moreover, an evaluation of optimal induction conditions for protein production was also carried out in two consecutive steps: (1) definition of the optimal cellular growth phase in which the gene expression has to be induced; (2) definition of the optimal inducer concentration that has to be added in the growth medium.

Bacterial biofilms occur on all submerged structures in marine environments. The authors previously reported that the marine bacterium Pseudoalteromonas sp. 3J6 secretes antibiofilm activity. Here, it was discovered that another Pseudoalteromonas sp. strain, D41, inhibited the development of strain 3J6 in mixed biofilms. Confocal laser scanning microscope observations revealed that the culture supernatant of strain D41 impaired biofilm formation of strain 3J6 and another marine bacterium. A microtiter plate assay of the antibiofilm activity was set up and validated with culture supernatants of Pseudoalteromonas sp. 3J6. This assay was used to determine the spectra of action of strains D41 and 3J6. Each culture supernatant impaired the biofilm development of 13 marine bacteria out of 18. However, differences in the spectra of action and the physical behaviours of the antibiofilm molecules suggest that the latter are not identical. They nevertheless share the originality of being devoid of antibacterial activity against planktonic bacteria.

Compared to other analytical methods, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) presents several unique advantages for the structural characterization of degradation products of carbohydrates. Our final goal is to implement this technique as a high-throughput platform, with the aim of exploring natural bio-diversity to discover new carbohydrate depolymerizing enzymes. In this approach, a variety of carbohydrates will be used as enzymes substrates and MALDI-MS will be employed to monitor the oligosaccharides produced. One drawback of MALDI, however, is that the choice of the matrix is largely dependent on the chemical properties of the analyte. In this context, our objective in the present work was to find the smallest set of MALDI matrices able to detect chemically heterogeneous oligosaccharides. This was done through the performance evaluation of more than 40 MALDI matrices preparations. Homogeneity of analyte-matrix deposits was considered as a critical feature, especially since the final objective is to fully automate the analyses. Evaluation of the matrices was done by means of a rigorous statistical approach. Amongst all tested compounds, our work proposes the use of the DHB/DMA ionic matrix as the most generic matrix, for rapid detection of a variety of polysaccharides including neutral, anionic, methylated, sulfated, and acetylated compounds. The selected matrices were then used to screen crude bacterial incubation media for the detection of enzymatic degradation products., (Copyright © 2011 John Wiley & Sons, Ltd.)

The spectroscopic and ligand-binding properties of a 2/2 globin from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 have been studied in the ferrous state. It displays two major conformations characterized by CO-association rates that differ by a factor of 20, with relative fractions that depend on pH. A dynamic equilibrium is found between the two conformations, as indicated by an enhanced slower phase when lower CO levels were used to allow a longer time to facilitate the transition. The deoxy form, in the absence of external ligands, is a mixture of a predominant six-coordinate low spin form and a five-coordinate high-spin state; the proportion of low spin increasing at alkaline pH. In addition, at temperatures above the physiological temperature of 1 °C, an enhanced tendency of the protein to oxidize is observed., (Copyright © 2011 Wiley Periodicals, Inc.)

The proteomes expressed at 4°C and 18°C by the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis were compared using two-dimensional differential in-gel electrophoresis with special reference to proteins repressed by low temperatures. Remarkably, the major cold-repressed proteins, almost undetectable at 4°C, were heat shock proteins involved in folding assistance.

Violacein, a purple pigment produced by some Gram-negative bacteria, has various physiological properties, such as antitrypanosomal and antitumoral activities. A gene cluster that encodes five enzymes, VioA-VioE, is responsible for synthesizing violacein. The expression of these enzymes is known to be regulated by a quorum sensing mechanism in Chromobacterium violaceum and Pseudoalteromonas sp. 520P1. To clarify the molecular mechanism of regulation of violacein synthesis, we cloned and characterized the gene cluster from Pseudoalteromonas sp. 520P1. A fosmid library of strain 520P1 was constructed and clones containing the gene cluster were isolated. The gene cluster was 7383 bp in length and encoded five enzyme genes, vioA-vioE. A putative promoter sequence was predicted in the upstream region of the cluster. In the promoter region, two contiguous palindromic sequences, a possible quorum sensing regulatory site, were found. However, the isolated Escherichia coli clones harboring the gene cluster and its upstream region were unable to produce violacein probably due to the lack of quorum sensing machinery for expression. To further examine the ability of vioA-vioE genes to synthesize violacein in vivo, the upstream promoter region was removed from the cluster and heterologous expression of the treated cluster was performed in E. coli using a recombinant pET vector with T7 promoter. Purple pigment was expressed, and the pigment was identified to be violacein using ultraviolet and visible light and HPLC analysis. These results will contribute to further studies regarding violacein biosynthesis and its mass production.

Background: Pseudoalteromonas is an important genus widespread in marine environment, and a lot of psychrophilic Pseudoalteromonas strains thrive in deep sea and polar sea. By now, there are only a few genetic systems for Pseudoalteromonas reported and no commercial Pseudoalteromonas genetic system is available, which impedes the study of Pseudoalteromonas, especially for psychrophilic strains. The aim of this study is to develop a heterologous expression system for psychrophilic Pseudoalteromonas., Results: A cryptic plasmid pSM429 isolated from psychrophilic Pseudoalteromonas sp. BSi20429 from the Arctic sea ice, was sequenced and characterized. The plasmid pSM429 is 3874 bp in length, with a G+C content of 28%. Four putative open reading frames (ORFs) were identified on pSM429. Based on homology, the ORF4 was predicted to encode a replication initiation (Rep) protein. A shuttle vector (Escherichia coli, Pseudoalteromonas), pWD, was constructed by ligating pSM429 and pUC19 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. To determine the minimal replicon of pSM429 and to check the functionality of identified ORFs, various pWD derivatives were constructed. All derivatives except the two smallest ones were shown to allow replication in Pseudoalteromonas sp. SM20429, a plasmid-cured strain of Pseudoalteromonas sp. BSi20429, suggesting that the orf4 and its flanking intergenic regions are essential for plasmid replication. Although not essential, the sequence including some repeats between orf1 and orf2 plays important roles in segregational stability of the plasmid. With the aid of pWD-derived plasmid pWD2, the erythromycin resistance gene and the cd gene encoding the catalytic domain of a cold-adapted cellulase were successfully expressed in Pseudoalteromonas sp. SM20429., Conclusions: Plasmid pSM429 was isolated and characterized, and the regions essential for plasmid replication and stability were determined, helping the development of pSM429-based shuttle vectors. The shuttle vectors pWD and its derivatives could be used as cloning vectors for Pseudoalteromonas, offering new perspectives in the genetic manipulation of Pseudoalteromonas strains. With the aid of pWD-derived vector and its host, the erythromycin resistance gene and the cd gene of a cold-adapted protein were successfully expressed, indicating that the potential use of this system for recombinant protein production, especially for cold-adapted proteins.

The induction of larval attachment and metamorphosis of benthic marine invertebrates is widely considered to rely on habitat specific cues. While microbial biofilms on marine hard substrates have received considerable attention as specific signals for a wide and phylogenetically diverse array of marine invertebrates, the presumed chemical settlement signals produced by the bacteria have to date not been characterized. Here we isolated and fully characterized the first chemical signal from bacteria that induced larval metamorphosis of acroporid coral larvae (Acropora millepora). The metamorphic cue was identified as tetrabromopyrrole (TBP) in four bacterial Pseudoalteromonas strains among a culture library of 225 isolates obtained from the crustose coralline algae Neogoniolithon fosliei and Hydrolithon onkodes. Coral planulae transformed into fully developed polyps within 6 h, but only a small proportion of these polyps attached to the substratum. The biofilm cell density of the four bacterial strains had no influence on the ratio of attached vs. non-attached polyps. Larval bioassays with ethanolic extracts of the bacterial isolates, as well as synthetic TBP resulted in consistent responses of coral planulae to various doses of TBP. The lowest bacterial density of one of the Pseudoalteromonas strains which induced metamorphosis was 7,000 cells mm(-2) in laboratory assays, which is on the order of 0.1-1% of the total numbers of bacteria typically found on such surfaces. These results, in which an actual cue from bacteria has been characterized for the first time, contribute significantly towards understanding the complex process of acroporid coral larval settlement mediated through epibiotic microbial biofilms on crustose coralline algae.

A polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) with an inner arrangement consisting of eight equidistantly spaced rectangular strips mounted radially on a circular disk to provide additional surface area for microbial attachment and ports to allow air supply was employed for melanin production by Shewanella colwelliana and antibiotic production by Pseudoalteromonas rubra. The design allowed comparison of production between (1) CCF with hydrophobic surface (PMMA-CCF), (2) CCF with hydrophilic glass surface (GS-CCF), and (3) standard unbaffled Erlenmeyer flask (EF). Melanin production in the PMMA-CCF was higher by at most 33.5% and growth of S. colwelliana by at most 309.2% compared to the other vessels. Melanin synthesis was positively correlated with reactor surface area and hydrophobicity, suspended cell growth, and biofilm formation. Antibiotic production in the EF was higher by at most 83.3%, but growth of P. rubra was higher in the PMMA-CCF by at most 54.5% compared to the other vessels. A hydrophilic vessel surface, abundant air supply, but low shear stress enhanced antibiotic production. The CCF together with the EF allowed identification of the crucial parameters (vessel surface characteristics, growth, biofilm formation, and aeration) influencing productivity, knowledge of which in the initial stages of process development will facilitate informed decisions at the later phases.

Background: Understanding how complex antibiotics are synthesised by their producer bacteria is essential for creation of new families of bioactive compounds. Thiomarinols, produced by marine bacteria belonging to the genus Pseudoalteromonas, are hybrids of two independently active species: the pseudomonic acid mixture, mupirocin, which is used clinically against MRSA, and the pyrrothine core of holomycin., Methodology/principal Findings: High throughput DNA sequencing of the complete genome of the producer bacterium revealed a novel 97 kb plasmid, pTML1, consisting almost entirely of two distinct gene clusters. Targeted gene knockouts confirmed the role of these clusters in biosynthesis of the two separate components, pseudomonic acid and the pyrrothine, and identified a putative amide synthetase that joins them together. Feeding mupirocin to a mutant unable to make the endogenous pseudomonic acid created a novel hybrid with the pyrrothine via "mutasynthesis" that allows inhibition of mupirocin-resistant isoleucyl-tRNA synthetase, the mupirocin target. A mutant defective in pyrrothine biosynthesis was also able to incorporate alternative amine substrates., Conclusions/significance: Plasmid pTML1 provides a paradigm for combining independent antibiotic biosynthetic pathways or using mutasynthesis to develop a new family of hybrid derivatives that may extend the effective use of mupirocin against MRSA.

The red-pigmented marine bacterium Pseudoalteromonas sp. sf57 forms a biofilm that deters larval settlement of the tube-building polychaete Hydroides elegans. To investigate the correlation between pigmentation and larval settlement deterrence, mutants of sf57 with deficient or altered pigmentation were generated by transposon mutagenesis. Five groups of pigmented mutants were obtained, viz. white, yellow, pink, dark red, and white-to-red. The white mutant WM1, which exhibited a substantial increase in bacterial density in the biofilm, became inductive to larval settlement. The other mutants that showed a lesser increase in bacterial density in their biofilms either retained their deterrence or induced higher larval settlement rates, but did not become inductive strains. Analysis of the disrupted genes in these mutants suggests that the type II secretion pathway, the LysR transcriptional regulator, NAD(P)-binding proteins, exonuclease, pyruvate metabolism, flagella assembly, and cell membrane processes may play a role in the regulation of pigmentation in sf57.

The psychrophilic model bacterium Pseudoalteromonas haloplanktis is characterized by remarkably fast growth rates under low-temperature conditions in a range from 5°C to 20°C. In this study the proteome of cellular compartments, the cytoplasm and periplasm, of P. haloplanktis strain TAC125 was analyzed under exponential growth conditions at a permissive temperature of 16°C. By means of two-dimensional protein gel electrophoresis and mass spectrometry, a first inventory of the most abundant cytoplasmic and periplasmic proteins expressed in a peptone-supplemented minimal medium was established. By this approach major enzymes of the amino acid catabolism of this marine bacterium could be functionally deduced. The cytoplasmic proteome showed a predominance of amino acid degradation pathways and tricarboxylic acid (TCA) cycle enzymes but also the protein synthesis machinery. Furthermore, high levels of cold acclimation and oxidative stress proteins could be detected at this moderate growth temperature. The periplasmic proteome was characterized by a significant abundance of transporters, especially of highly expressed putative TonB-dependent receptors. This high capacity for protein synthesis, efficient amino acid utilization, and substrate transport may contribute to the fast growth rates of the copiotrophic bacterium P. haloplanktis in its natural environments.

The genome of the cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct monomeric hemoglobins exhibiting a 2/2 α-helical fold. In the present work, one of these hemoglobins is studied by resonance Raman, electronic absorption and electronic paramagnetic resonance spectroscopies, kinetic measurements, and different bioinformatic approaches. It is the first cold-adapted bacterial hemoglobin to be characterized. The results indicate that this protein belongs to the 2/2 hemoglobin family, Group II, characterized by the presence of a tryptophanyl residue on the bottom of the heme distal pocket in position G8 and two tyrosyl residues (TyrCD1 and TyrB10). However, unlike other bacterial hemoglobins, the ferric state, in addition to the aquo hexacoordinated high-spin form, shows multiple hexacoordinated low-spin forms, where either TyrCD1 or TyrB10 can likely coordinate the iron. This is the first example in which both TyrCD1 and TyrB10 are proposed to be the residues that are alternatively involved in heme hexacoordination by endogenous ligands.

Marine bacteria are a rich, yet underexplored, resource of compounds with inhibitory bioactivity against a range of eukaryotic target organisms. Identification of those inhibitors, however, requires a culturable or genetically tractable producer strain, a prerequisite that is not often fulfilled. This study describes a novel functional genomic screen that is based on expression of inhibitors in a heterogeneous recombinant host (i.e., Escherichia coli). Functional libraries were screened by selective grazing by the nematode Caenorhabditis elegans, in a simple, rapid, high-throughput manner. We applied our approach to discover inhibitors of C. elegans produced by the marine bacterium Pseudoalteromonas tunicata D2, a model organism for exploring a range of antagonistic activities between bacteria and eukaryotes and a known producer of several toxic compounds. Expression of P. tunicata DNA in E. coli and grazing selection by the nematode Caenorhabditis elegans identified two clones, with slow- and fast-killing modes of action. Genomic analysis of the slow-killing clone revealed that the activity was due to a small molecule, tambjamine, while the fast-killing activity involved a gene encoding for a novel protein. Microscopic analysis showed substantial colonization of the intestinal lumen, or rapid death of the nematode without colonization, for the two activities, respectively. The novel functional genomic screen presented here therefore detects new eukaryotic inhibitors with different chemical structures, kinetics, and predicted modes of actions.

A strictly aerobic, Gram-negative, non-pigmented bacterial strain, designated LMEB 39(T), was isolated from a seawater sample collected from the Yangtze River estuary near the East China Sea and was examined physiologically, chemotaxonomically and phylogenetically. The novel isolate was motile by a single polar flagellum and positive for nitrate reduction and decomposition of casein, gelatin, Tween 20 and Tween 80, but negative for indole production. Chemotaxonomic analysis revealed ubiquinone-8 as the predominant respiratory quinone and phosphatidylethanolamine and phosphatidylglycerol as major polar lipids. The major fatty acids were C(16 : 1) ω 7c/iso-C(15 : 0) 2-OH, C(16 : 0), C(18 : 1) ω 7c, C(12 : 0) 3-OH, C(17 : 1)ω 8c and C(17 : 0). The genomic DNA G+C content was 42.3 mol%. Comparative 16S rRNA gene sequence analysis revealed that the isolate belongs to the genus Pseudoalteromonas. Strain LMEB 39(T) exhibited the closest phylogenetic affinity to Pseudoalteromonas byunsanensis JCM 12483(T) (97.4 % sequence similarity). The DNA-DNA reassociation values between strain LMEB 39(T) and P. byunsanensis JCM 12483(T) and Pseudoalteromonas undina DSM 6065(T) (97.2 % sequence similarity) were 31.7 and 30.3 %, respectively. On the basis of phenotypic and genotypic data, strain LMEB 39(T) represents a novel species of the genus Pseudoalteromonas, for which the name Pseudoalteromonas lipolytica sp. nov. is proposed; the type strain is LMEB 39(T) (=CGMCC 1.8499(T)=JCM 15903(T)).

The 2-on-2 haemoglobins, previously named truncated, are monomeric, low-molecular weight oxygen-binding proteins that share the overall topology with vertebrate haemoglobins. Although several studies on 2-on-2 haemoglobins have been reported, their physiological and biochemical functions are not yet well defined, and various roles have been suggested. The genome of the psychrophilic Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is endowed with three genes encoding 2-on-2 haemoglobins. To investigate the function played by one of the three trHbs, PhHbO, a PhTAC125 genomic mutant strain was constructed, in which the encoding gene was knocked-out. The mutant strain was grown under controlled conditions and several aspects of bacterium physiology were compared with those of wild-type cells when dissolved oxygen pressure in solution and growth temperature were changed. Interestingly, inactivation of the PhHbO encoding gene makes the mutant bacterial strain sensitive to high solution oxygen pressure, to H(2)O(2), and to a nitrosating agent, suggesting the involvement of PhHbO in oxidative and nitrosative stress resistance., (Copyright 2010 Elsevier Masson SAS. All rights reserved.)

The purpose of the present study was to isolate marine culturable bacteria with antibacterial activity and hence a potential biotechnological use. Seawater samples (244) and 309 swab samples from biotic or abiotic surfaces were collected on a global Danish marine research expedition (Galathea 3). Total cell counts at the seawater surface were 5 x 10(5) to 10(6) cells/ml, of which 0.1-0.2% were culturable on dilute marine agar (20 degrees C). Three percent of the colonies cultured from seawater inhibited Vibrio anguillarum, whereas a significantly higher proportion (13%) of colonies from inert or biotic surfaces was inhibitory. It was not possible to relate a specific kind of eukaryotic surface or a specific geographic location to a general high occurrence of antagonistic bacteria. Five hundred and nineteen strains representing all samples and geographic locations were identified on the basis of partial 16S rRNA gene sequence homology and belonged to three major groups: Vibrionaceae (309 strains), Pseudoalteromonas spp. (128 strains), and the Roseobacter clade (29 strains). Of the latter, 25 strains were identified as Ruegeria mobilis or pelagia. When re-testing against V. anguillarum, only 409 (79%) retained some level of inhibitory activity. Many strains, especially Pseudoalteromonas spp. and Ruegeria spp., also inhibited Staphylococcus aureus. The most pronounced antibacterial strains were pigmented Pseudoalteromonas strains and Ruegeria spp. The inhibitory, pigmented Pseudoalteromonas were predominantly isolated in warmer waters from swabs of live or inert surfaces. Ruegeria strains were isolated from all ocean areas except for Arctic and Antarctic waters and inhibitory activity caused by production of tropodithietic acid.

The peptide transporter from a cold-adapted bacterium has never been reported. In the present study, the dpp operon from the psychrophilic bacterium Pseudoalteromonas sp. strain SM9913 was cloned and analyzed. The dipeptide binding protein DppA of SM9913 was overexpressed in Escherichia coli, and its cold adaptation characteristics were studied. The recombinant DppA of SM9913 (PsDppA) displayed the highest ligand-binding affinity at 15 degrees C, whereas the recombinant DppA of E. coli (EcDppA) displayed the highest ligand-binding affinity at 35 degrees C. Thermal and guanidium hydrochloride unfolding analyses indicated that PsDppA has more structural instability than EcDppA. Six domain-exchanged mutants of PsDppA were expressed and purified. Analyses of these mutants indicated that domains III, I-2, and I-3 of PsDppA were less stable than those from EcDppA and that domains III and I-2 made a significant contribution to the high binding affinity of PsDppA at low temperatures. Structural and sequence analyses suggested that the state transition-involved regions in domain III and the alpha part of domain I-2 are the hot spots of optimization during cold adaptation and that decreasing the side-chain size in these regions is an important strategy for the cold adaptation of PsDppA.

Microbial degradation of aromatic hydrocarbons has been studied with the aim of developing applications for the removal of toxic compounds. Efforts have been directed toward the genetic manipulation of mesophilic bacteria to improve their ability to degrade pollutants, even though many pollution problems occur in sea waters and in effluents of industrial processes which are characterized by low temperatures. From these considerations the idea of engineering a psychrophilic microorganism for the oxidation of aromatic compounds was developed.In a previous paper it was demonstrated that the recombinant Antarctic Pseudoalteromonas haloplanktis TAC125 (PhTAC/tou) expressing a toluene-o-xylene monooxygenase (ToMO) is able to convert several aromatic compounds into corresponding catechols. In our work we improved the metabolic capability of PhTAC/tou cells by combining action of recombinant ToMO enzyme with that of the endogenous P. haloplanktis TAC125 laccase-like protein. This strategy allowed conferring new and specific degradative capabilities to a bacterium isolated from an unpolluted environment; indeed engineered PhTAC/tou cells are able to grow on aromatic compounds as sole carbon and energy sources. Our approach demonstrates the possibility to use the engineered psychrophilic bacterium for the bioremediation of chemically contaminated marine environments and/or cold effluents., (© 2010 Landes Bioscience)

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter, which has potential biotechnology applications. We examined the ultrastructure of extracellular matter from five Antarctic bacteria (Shewanella livingstonensis NF22(T), Shewanella vesiculosa M7(T), Pseudoalteromonas sp. M4.2, Psychrobacter fozii NF23(T), and Marinobacter guineae M3B(T)) by transmission electron microscopy after high-pressure freezing and freeze substitution. All analyzed extracellular matter appeared as a netlike mesh composed of a capsular polymer around cells and large numbers of membrane vesicles (MVs), which have not yet been described for members of the genera Psychrobacter and Marinobacter. MVs showed the typical characteristics described for these structures, and seemed to be surrounded by the same capsular polymer as that found around the cells. The analysis of MV proteins from Antarctic strains by SDS-PAGE showed different banding profiles in MVs compared to the outer membrane, suggesting some kind of protein sorting during membrane vesicle formation. For the psychrotolerant bacterium, S. livingstonensis NF22(T), the growth temperature seemed to influence the amount and morphology of MVs. In an initial attempt to elucidate the functions of MVs for this psychrotolerant bacterium, we conducted a proteomic analysis on membrane vesicles from S. livingstonensis NF22(T) obtained at 4 and 18 degrees C. At both temperatures, MVs were highly enriched in outer membrane proteins and periplasmic proteins related to nutrient processing and transport in Gram-negative bacteria suggesting that MVs could be related with nutrient sensing and bacterial survival. Differences were observed in the expression of some proteins depending on incubation temperature but further studies will be necessary to define their roles and implications in the survival of bacteria in the extreme Antarctic environment.

Genome mining and identification of natural product gene clusters typically relies on the presence of canonical nonribosomal polypeptide synthetase (NRPS) or polyketide synthase (PKS) domains. Recently, other condensation enzymes, such as the ATP-grasp ligases, have been recognized as important players in natural product biosynthesis. In this study, sequence based searching for homologues of DdaF, the ATP-grasp amide ligase from dapdiamide biosynthesis, led to the identification of a previously unannotated biosynthetic gene cluster in Pseudoalteromonas tunicata. Heterologous expression of the cluster in Escherichia coli allowed for the production and structure determination of two new 3-formyl tyrosine metabolites.

Marine bacterial isolates cultured from the digestive tracts of blue mussels (Mytilus edulis) contaminated with paralytic shellfish toxins (PSTs) were screened for the ability to reduce the toxicity of a PST mixture. Seven isolates reduced the overall toxicity of the algal extract by > or = 90% within 3 days. These isolates shared at least 99% 16S rRNA gene sequence similarity with five Pseudoalteromonas spp. Phenotypic tests suggested that all are novel strains of Pseudoalteromonas haloplanktis.

A marine bacterium was isolated from seawater near the Korean south coast for efficient saccharification from alginate. Based on 16S rDNA sequence, the isolated strain was identified as Pseudoalteromonas agarovorans. Various environmental factors affecting saccharification of alginate using P. agarovorans CHO-12 have been investigated in flask cultures. The optimum concentration of sugar was obtained at 30 rpm and 29 degrees C. Among various NaCl concentrations, when NaCl concentration was increased from 10 to 30 g/l, the cell concentration sharply increased, while there is no increase at above 40 g/l. The maximum sugar concentration was obtained at 13.8 when 30 g/l of NaCl was used. Yeast extract and corn steep liquor were the best nitrogen source for efficient saccharification. Especially, the sugar concentration of 14.9 g/l was obtained after 3 days of culture using a mixture of 1.0 g/l of yeast extract and 1.5 g/l of corn steep liquor. Scale up was carried out at 50 l of reactor for 3 days using P. agarovorans CHO-12 and Stenotrophomonas maltophilia sp. When S. maltophilia was used, cell concentration was about twofold higher than that of P. agarovorans CHO-12. On the other hand, when P. agarovorans CHO-12 was used, the maximum saccharification rate was obtained, 7.5 g/l/day after 2 days of culture, which was about tenfold higher than that of S. maltophilia.

High-molecular-mass polysaccharides were released by mild acid degradation of the lipopolysaccharides of two wild-type Vibrio vulnificus strain, a flagellated motile strain CECT 5198 and a non-flagellated non-motile strain S3-I2-36. Studies by sugar analysis and partial acid hydrolysis along with (1)H and (13)C NMR spectroscopies showed that the polysaccharides from both strains have the same trisaccharide repeating unit of the following structure: -->4)-beta-d-GlcpNAc3NAcylAN-(1-->4)-alpha-l-GalpNAmA-(1-->3)-alpha-d-QuipNAc-(1--> where QuiNAc stands for 2-acetamido-2,6-dideoxyglucose, GalNAmA for 2-acetimidoylamino-2-deoxygalacturonic acid, GlcNAc3NAcylAN for 2-acetamido-3-acylamino-2,3-dideoxyglucuronamide and acyl for 4-d-malyl ( approximately 30%) or 2-O-acetyl-4-d-malyl ( approximately 70%). The structure of the polysaccharide studied resembles much that of a marine bacterium Pseudoalteromonas rubra ATCC 29570 reinvestigated in this work. The latter differs in (i) the absolute configuration of malic acid (l vs d), (ii) 3-O-acetylation of GalNAmA and (iii) replacement of QuiNAc with its 4-keto biosynthetic precursor.

Reversible insertion of IS492 at a site within epsG on the Pseudoalteromonas atlantica chromosome controls peripheral extracellular polysaccharide production and biofilm formation by P. atlantica. High-frequency precise excision of IS492 from epsG requires 5 and 7 bp of flanking DNA, suggesting that IS492 transposition involves a site-specific recombination mechanism. The site specificity of IS492 insertion was examined in P. atlantica and shown to be specific for a 7-bp target, 5'-CTTGTTA-3'. Characterization of numerous insertion events at the target site in epsG indicated that insertion is also orientation specific. The frequency of IS492 insertion at the epsG target site (2.7 x 10(-7)/cell/generation), determined by quantitative PCR, is 4 to 5 orders of magnitude lower than the frequency of IS492 precise excision from the same site. Comparison of insertion sites for IS492 and the highly related ISPtu2 from Pseudoalteromonas tunicata suggests DNA sequence and/or structural features that may contribute to site recognition and recombination by the transposase of IS492.

The alginate lyase encoding gene (alyPI) of marine bacterium Pseudoalteromonas sp. CY24 was cloned using a battery of PCR techniques. Gene alyPI was composed of a 1575 bp open reading frame encoding a protein of 57.4 kDa containing 524 amino acid residues with a signal peptide of 23 amino acids. The AlyPI protein was expressed in Escherichia coli with a His-tag sequence fused at the C-terminal end and purified to electrophoretic homogeneity using Ni-sepharose affinity chromatography. AlyPI was most active at 40 degrees C and pH 7.0 in the presencce of 0.1 mol/L NaCl and stable over a broad range of pH, 6.0-10.6. The presence of Na+, K+, Mn2+, Ca2+, and Fe3+ can enhance the enzyme activity. The alginate lyase consensus region YFKAGXYXQ, regarded as a striking feature at the C termini of several alginate lyase of ~30 kDa, was found in AlyPI, which belongs to the ~60 kDa group. Another nine amino acid consensus region, YXRSELREM, only found in G-specific alginate lyases previously existed in AlyPI, which could degrade sodium alginate, M blocks, and G blocks and appeared to be a broad substrate-specific alginate lyase.

A psychrotrophic petroleum-degrading bacterium Pseudoalteromonas sp. P29 was isolated from marine sediment, which was collected during 2nd Chinese Arctic Scientific Expedition. The phenotypic character and biodegradation efficiency on mixed oil or vacuum oil were tested at low temperature. The strain Pseudoalteromonas sp. P29 grew in a range of temperature from 5 to 35 degrees C and the optimum temperature was 25 degrees C. Gas chromatography analysis indicated that the strain might preferentially metabolize shorter-chain alkanes. The biodegradation efficiency were nearly 90 and 80%, respectively, after incubation at 5 degrees C for 28 days in the mineral medium supplement with mixed oil or vacuum oil as the sole carbon and energy source. The results showed a possible exploitation of the strain in future biotechnological processes especially in cold contaminated environments.

The chitinase gene cluster of the marine bacterium Pseudoalteromonas sp. S91, chiABC, which produces the major chitinases of this sp., was transcribed as an operon and from each individual gene. chiA, chiB and chiC were found to possess multiple transcriptional start points (TSPs), the use of which was determined by the nutrient regime used for S91 growth. In minimal medium containing glutamate, chiA, chiB and chiC each used 3, 1 and 1 TSP, respectively. Upon the addition of the chitin monomer N-acetylglucosamine, the number of chiA TSPs was unaffected. However, chiB used an additional 4 TSPs, and chiC used four new TSPs excluding the TSP used in glutamate only. In addition, the cluster was transcribed as an operon from TSP A1 of chiA. All TSPs were potentially associated with either a sigma(70)- or sigma(54)-dependent promoter. Under the growth conditions used, no TSPs were detected for chiB or chiC in S91CX, a chiA transposon mutant. The transcription of the S91 chiABC gene cluster produced at least four polycistronic mRNAs. In addition, the occurrence of operon transcription of chiABC, and identification of an additional 12 putative TSPs within the gene cluster, gave an indication that each gene appeared to be transcribed from more than one promoter region upstream of each in-frame translation start codon. Questions arose regarding the reason for this complexity of transcription within the gene cluster, leading to a re-evaluation of the Chi protein domains. By bioinformatic review, ChiA, ChiB and ChiC were found to potentially possess additional putative domains.

In this study, we examined the algal-lytic activities and biological control mechanisms of Pseudoalteromonas haloplanktis AFMB-08041, which was isolated from surface seawater obtained at Masan Bay in Korea. In addition, we assessed whether AFMB-08041 could be used as a biocontrol agent to regulate harmful dinoflagellate Prorocentrum minimum. From these experiments, we found that the inoculation of AFMB-08041 at a final density of 2.5 x 10(4) cfu ml(-1) caused P. minimum cells to degrade (>90%) within 5 days. The algal cells were lysed through an indirect attack by the AFMB-08041 bacterial strain. Our results also suggest that the algal-lytic compounds produced by AFMB-08041 may have beta-glucosidase activity. However, P. haloplanktis AFMB-08041 was not able to suppress the growth of other alga such as Alexandrium tamarense, Akashiwo sanguinea, Cochlodinium polykrikoides, Gymnodinium catenatum, and Heterosigma akashiwo. Moreover, we observed that the growth of Prorocentrum dentatum, which has a very similar morphological structure to P. minimum, was also effectively suppressed by P. haloplanktis AFMB-08041. Therefore, the effect of AFMB-08041 on P. minimum degradation appears to be species specific. When testing in an indoor mesocosms, P. haloplanktis AFMB-08041 reduced the amount of viable P. minimum cells by 94.5% within 5 days after inoculation. The combined results of this study clearly demonstrate that this bacterium is capable of regulating the harmful algal blooms of P. minimum. In addition, these results will enable us to develop a new strategy for the anthropogenic control of harmful algal bloom-forming species in nature.

Two-component systems are widespread in nature and constitute the most common mechanism of transmembrane signal transduction in bacteria. Recently, a functionally active two-component system consisting of malS and malR genes possibly involved in the expression of a C4-dicarboxylate transporter system (dctAB operon) was identified in the marine Antarctic bacterium Pseudoalteromonas haloplanktis TAC125. In this paper, we performed a functional analysis of the two-component system and demonstrated its involvement in the regulation of the expression of C4-dicarboxylate transporter genes. The expression of the C4-dicarboxylate transporter genes was induced by l-malate with the promoter element located upstream of the dctA gene being active only in the presence of the inducer. A sigma(54) promoter responsible for the l-malate dependent transcription regulation was identified and functionally characterized. The molecular mechanism involves an inverted repeat sequence located upstream the sigma(54) promoter that was shown to bind regulatory proteins only in the presence of l-malate. The protein factor responsible for the induction of the dctAB operon expression was eventually identified as the transcriptional regulatory protein MalR. MalR is the first transcriptional factor identified in P. haloplanktis TAC125 and one of the few transcriptional modulators reported so far in cold adapted bacteria.

Thioredoxin and thioredoxin reductase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis were obtained as recombinant His-tagged proteins (rPhTrx and rPhTrxR, respectively). rPhTrxR is organised as a homodimeric flavoenzyme, whereas rPhTrx is a small monomeric protein, both containing a functional disulfide bridge. However, three additional cysteines are present as free thiols in purified rPhTrxR. When individually tested in specific assays, rPhTrxR and rPhTrx display a full activity at low temperatures, an indispensable requirement for cold-adapted proteins. In particular, rPhTrxR catalyses the NADPH dependent reduction of DTNB and rPhTrx provokes the insulin precipitation in the presence of DTT. The analysis of the effect of temperature on these reactions indicates that rPhTrxR is more cold-adapted than rPhTrx, having a higher psychrophilicity. The combined activity of rPhTrxR and rPhTrx, tested in a reconstituted assay containing NADPH as electrons donor and human insulin as the thioredoxin substrate, demonstrates a direct functional interaction between the purified recombinant components of the thioredoxin system of P. haloplanktis. Furthermore, the NADPH-dependent reduction of rPhTrx catalysed by rPhTrxR is fully reversible and allows the determination of its redox potential, whose value is in the range of other bacterial and archaeal thioredoxins. The analysis of the thermostability of rPhTrxR points to its discrete heat resistance. However, rPhTrx is much more heat resistant, with a half inactivation time of about 4 h at 95 degrees C. This exceptional heat resistance for a psychrophilic protein is significantly decreased by the reduction of the disulfide bridge of rPhTrx. Functionality, thermodependence and thermostability of the P. haloplanktis thioredoxin system point to the relevance of this key mechanism for the preservation of the reduced state of cytoplasmic proteins even in a cold-adapted source.

In this paper production of a cold-active esterase EstA from the Antarctic bacterium Pseudoalteromonas sp. 643A in E. coli expression system was described. The purification and biochemical characteristic of EstA were performed in the presence of urea and then compared with results obtained for the esterase with no addition of urea and isolated from the native source. In both cases the cold-active enzyme displayed similar properties. However, the differences concerning thermal activity were observed. The optimal temperature for recombinant esterase in the presence of urea (1 M) was about 15 degrees C lower in comparison with enzyme isolated from the native source. Furthermore, the EstA was found to be more thermolabile in denaturant conditions. The differences were presumably caused by slightly changed protein structure in the presence of urea. The preservation of activity of EstA dissolved in buffer containing 8 M urea suggests that the protein structure is retained and it does not undergo dramatic changes due to high urea concentration. This thesis was confirmed with FT-IR data.

Biosorption of Pb2+ and Cu2+ by exopolysaccharide (EPS) from the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913 was investigated. The influence of EPS dosage, pH, equilibrium time and coexisted-ions on adsorption property and the adsorption isotherm of this EPS were studied. The results show that the equilibrium adsorption capacity (Qe) decreases with the increase of EPS dosage. The optimum pH for the adsorption of Pb2+ is 4.5-5.5, and for the adsorption of Cu2+ is 4.5-6.0. The adsorption equilibrium of Cu2+ onto the EPS is attained within 90 min, while the Pb2+ adsorption equilibrium time is 180 min. Qe of Pb2+ decreases with the addition of coexisted-ions such as Ca2+, Mg2+, Na+ and K+. Addition of Ca2+, Mg2+ also decreases the Qe of Cu2+, but low dosage of Na+ and tested dosage of K+ increase the Qe of Cu2+. Both Freundlich and Dubinin-Radushkevich isotherm equations could well describe the thermodynamics process of Pb2+ and Cu2+ adsorbed onto the EPS. The maximum adsorption capacity of the EPS determined by Dubinin-Radushkevich isotherm equation for Pb2+ and Cu2+ are 243.3 mg/g (10 degrees C) and 36.7 mg/g (40 degrees C), respectively. IR analysis demonstrates that the group of C--O--C, acetyl and hydroxyl of polysaccharide are the main functional groups for binding metal ions.

Collagens are the most abundant proteins in marine animals and their degradation is important for the recycling of marine nitrogen. However, it is rather unclear how marine collagens are degraded because few marine collagenolytic proteases are studied in detail. Deseasins are a new type of multidomain subtilases. Here, the collagenolytic activity of deseasin MCP-01, the type example of deseasins, was studied. MCP-01 had broad substrate specificity to various type collagens from terrestrial and marine animals. It completely decomposed insoluble collagen into soluble peptides and amino acids, and was more prone to degrade marine collagen than terrestrial collagen. Thirty-seven cleavage sites of MCP-01 on bovine collagen chains were elucidated, showing the cleavage is various but specific. As the main extracellular cold-adapted protease from deep-sea bacterium Pseudoalteromonas sp. SM9913, MCP-01 displayed high activity at low temperature and alkaline range. Our data also showed that the C-terminal polycystic kidney disease (PKD) domain of MCP-01 was able to bind insoluble collagen and facilitate the insoluble collagen digestion by MCP-01. Site-directed mutagenesis demonstrated that Trp-36 of the PKD domain played a key role in its binding to insoluble collagen. It is the first time that the structure and function of a marine collagenolytic protease, deseasin MCP-01, has been studied in detail. Moreover, the PKD domain was experimentally proven to bind to insoluble protein for the first time. These results imply that MCP-01 would play an important role in the degradation of deep-sea sedimentary particulate organic nitrogen.

Intracellular alpha-L-rhamnosidase (EC 3.2.1.40) from the psychrotolerant Pseudoalteromonas sp. 005NJ showed a dose-dependent inhibition for L-rhamnose (IC(50) = 20 mM) and D-ribose (IC(50) = 95 mM), whereas D-glucose and L-fucose presented a lower inhibition, with IC(50) values as high as >0.5 and >0.2 M, respectively. On the other hand, D-fructose enhanced enzyme activity threefold, reaching a plateau of maximum specific activity between 0.2 and 0.4 M of this monosaccharide. Both effects, low inhibition and stimulation, caused by key fruit sugars (glucose and fructose), make this biocatalyst an interesting system in terms of its potential application for debittering fruit juices.

Aims: The nature of exopolymers involved in the adhesion of a marine biofilm-forming bacterium Pseudoalteromonas sp. D41 was investigated to evaluate and understand the antifouling potential of subtilisin., Methods and Results: The exopolymers of D41 produced by fermentation were analysed by FTIR and SDS-PAGE showing the presence of polysaccharides, glycoproteins and proteins. A high content of proteins was detected both in soluble and capsular fractions. The microscopic observations of fluorescamine and calcofluor stained adhered D41 indicated mainly the presence of proteins in exopolymers produced during adhesion. Subtilisin, the broad spectrum protease, tested in natural sea water and in polystyrene microplates showed that antifouling activity was higher in the prevention of bacterial adhesion than in the detachment of adhered D41 cells., Conclusions: Overall, these results demonstrate the involvement of proteins in Pseudoalteromonas sp. D41 adhesion and confirm the high antifouling potential of subtilisin., Significance and Impact of the Study: This study emphasizes the major role of proteins instead of polysaccharides, thus extending our knowledge regarding the nature of extracellular polymers involved in bacterial adhesion. Furthermore, the high antifouling potential of subtilisin evaluated in the very first stages of fouling, bacterial adhesion, could lead to less toxic compounds than organometallic compounds in antifouling paint.

In this study, we describe the isolation and characterization of a new exopolymer that exhibits high emulsifying activities against a range of oil substrates and demonstrates a differential capacity to desorb various mono-, di-, and trivalent metal species from marine sediment under nonionic and seawater ionic-strength conditions. This polymer, PE12, was produced by a new isolate, Pseudoalteromonas sp. strain TG12 (accession number EF685033), during growth in a modified Zobell's 2216 medium amended with 1% glucose. Chemical and chromatographic analysis showed it to be a high-molecular-mass (>2,000 kDa) glycoprotein composed of carbohydrate (32.3%) and protein (8.2%). PE12 was notable in that it contained xylose as the major sugar component at unusually high levels (27.7%) not previously reported for a Pseudoalteromonas exopolymer. The polymer was shown to desorb various metal species from marine sediment-a function putatively conferred by its high content of uronic acids (28.7%). Seawater ionic strength (simulated using 0.6 M NaCl), however, caused a significant reduction in PE12's ability to desorb the sediment-adsorbed metals. These results demonstrate the importance of electrolytes, a physical parameter intrinsic of seawater, in influencing the interaction of microbial exopolymers with metal ions. In summary, PE12 may represent a new class of Pseudoalteromonas exopolymer with a potential for use in biotechnological applications as an emulsifying or metal-chelating agent. In addition to the biotechnological potential of these findings, the ecological aspects of this and related bacterial exopolymers in marine environments are also discussed.

Aim: To evaluate the effect of temperature on growth parameters and on extracellular polymeric substance (EPS) production for Pseudoalteromonas antarctica NF(3)., Methods and Results: For this purpose, three growth parameters, lag time (lambda), maximum growth rate (mu) and maximum population density (A), were calculated with the predictive Gompertz model. To evaluate the variations in mu with respect to temperature, the secondary Arrhenius and the square root models were used. Below the optimal growth temperature (17.5 degrees C), the growth of P. antarctica was separated into two domains at the critical temperature of 12 degrees C. Within the suboptimal domain (12-17.5 degrees C), the temperature characteristic was the lowest (5.29 kcal mol(-1)). Growth population densities were maintained over the entire physiological portion assayed (5-17.5 degrees C). Higher crude EPS production was found at temperatures included in the cold domain (5-12 degrees C)., Conclusions: All calculated parameters revealed an optimal adaptation of this strain to cold temperatures., Significance and Impact of the Study: The knowledge of the influence of temperature on growth parameters of P. antarctica NF(3) and on EPS production could improve the production of this extracellular polymeric substance that is currently being used in the cosmetic and pharmaceutical industries.

Objectives: To optimize the culture conditions of Pseudoalteromonas sp. AJ5 for a higher production of extracellular kappa-carrageenase., Methods: A kappa-carrageenan-degrading bacterium AJ5, capable of utilizing kappa-carrageenan as sole source of carbon and energy, was isolated from the intestine of holothurian Apostichopus japonicus by enrichment culture technique. The strain was identified as the genus Pseudoalteromonas sp. according to its morphological and physiological characterization and 16S rRNA gene analysis. Culture conditions for the bacterium were standardized for the maximal productivity of the extracellular kappa-carrageenase by the single factor and orthogonal tests., Results: According to the single factor test, the optimal culture conditions were: 75 mL medium in 250 mL Erlenmeyer flask, shaking speed of 150 r/min, inoculum's volume 7%, and pH 8.0. Based on the single factor and orthogonal tests the optimal medium components were: kappa-carrageenan (1 g/L), beef extract (2 g/L ), NaCl (20 g/L), K2HPO4.3H2O (1 g/L), MgSO4.7H2O (0.5 g/L), MnCl2.4H2O (0.2 g/L), FePO4.4H2O (0.01 g/L), with the incubation temperature and time of 28 degrees C and 28 h., Conclusion: Pseudoalteromonas sp. AJ5 secreted an extracellular kappa-carrageenase. Under the optimal culture conditions, four-fold increase in kappa-carrageenase activity was achieved as compared to the control.

LipL32 is the major outer membrane protein in pathogenic Leptospira. It is highly conserved throughout pathogenic species and is expressed in vivo during human infection. While these data suggest a role in pathogenesis, a function for LipL32 has not been defined. Outer membrane proteins of gram-negative bacteria are the first line of molecular interaction with the host, and many have been shown to bind host extracellular matrix (ECM). A search for leptospiral ECM-interacting proteins identified the major outer membrane protein, LipL32. To verify this finding, recombinant LipL32 was expressed in Escherichia coli and was found to bind Matrigel ECM and individual components of ECM, including laminin, collagen I, and collagen V. Likewise, an orthologous protein found in the genome of Pseudoalteromonas tunicata strain D2 was expressed and found to be functionally similar and immunologically cross-reactive. Lastly, binding activity was mapped to the C-terminal 72 amino acids. These studies show that LipL32 and an orthologous protein in P. tunicata are immunologically cross-reactive and function as ECM-interacting proteins via a conserved C-terminal region.