Enhanced affinochromism of polydiacetylene monolayer in response to bacteria by incorporating US nano-crystallites

By incorporating bio-specific receptors, such as p-10,12-pentacosadiyne-1-N-(3,6,9-trioxaundecylamide)-alpha-D-mannopyranoside (MPDA), into 10,12-pentacosadiyonic acid (PDA) monolayer, the MPDA/PDA monolayer underwent affinochromatic transition in response to the bacteria binding to the receptor. Here, we described a new method to study the membrane/macromolececular interaction between Escherichia coli (E coli) and mannose and its relative affinochromism by modifying MPDA/PDA with CdS nano-crystallites (MPDA/PDA-CdS). CdS not only triggered the strong tropism of the bacteria but also reduced the rigidity of the MPDA/PDA backbone, resulting in the enhanced affinochromism. This discovery might be of significance in basic biophysical studies of membrane/macromolececular and designing novel biosensor.

Electrospray ionization multiple-stage tandem mass spectrometric analysis of diglycosyldiacylglycerol glycolipids from the bacteria Bacillus pumilus

Electrospray ionization (ESI) combined with multiple-stage tandem mass spectrometry (MSn) was used to directly analyze the glycolipid mixture from bacteria Bacillus pumilus without preliminary separation. Full scan ESI-MS revealed the composition of picomole quantities of glycerolglycolipid species containing C-14-C-19 fatty acids, some of which were monounsaturated, Two main components were identified from their molecular masses and fragmentation pathways. The fragmentation pathway of the known compound compared with the investigated compound verified the proposed structure as 1(3)-acyl-2-pentadecanoyl-3(1)-O-[beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-sn-glycerols. A comparison of the multiple tandem mass spectra of the different alkali-metal cation adducts indicates that the intensity of fragments and the dissociation pathways are dependent on the alkali-metal type, The basic structures of glycerolglycolipids were reflected clearly from the fragmentation patterns of the sodium cations, The intense fragments of the sugar residue from the precursor ions were obtained from the lithiated adduct ions. ESI-MSn spectra of [M + K](+) ions did not provide as much fragmentation as [M + Na](+) and [M + Li](+) adducts, but their spectra allow the position of glycerol acylation to be determined. On the basis of MS2 spectra of[M + K](+) ions, it was established that all components have a C-15:0 fatty acid at the sn-2 position of the glycerol backbone and C-14-C-19 acids at the sn-1 position of the glycerol backbone. Copyright (C) 1999 John Wiley & Sons, Ltd.

Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

Aims: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. Methods nand Results: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. Conclusions: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. Significance and Impact of the Study: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.

A serpin from Chinese shrimp Fenneropenaeus chinensis is responsive to bacteria and WSSV challenge

Arthropod defence responses (e.g. prophenoloxidase (proPO) activation and Toll pathway initiation) are mediated by serine proteinase cascades and regulated by serpins in haemolymph. A serpin (Fc-serpin) cDNA was cloned from the haemocytes of Fenneropenaeus chinensis by rapid amplification of cDNA ends (RACE) PCR and haemocyte cDNA library screening. The full-length cDNA consists of 1734 bp, encoding 411 amino acids with a calculated molecular mass of 46.55 kDa and a theoretical isoelectric point of 7.70. Fc-serpin contains a typical serpin-like homologue (serine proteinase inhibitors domain). The deduced protein contains a putative signal peptide of 19 amino acids and the serpin's signature sequence ((FHCNRPFLFLI389)-F-379). Fc-serpin showed some identity with Pacifastacus leniusculus serpin (42%) and Manduca sexta serpin-6 (34%). The reactive centre loop (RCL) sequences of Fc-serpin, P leniusculus serpin, M. sexta serpin-6 and Bombyx mori serpin-2 are highly similar. An Arg at the PI position of the reactive site indicates that Fc-serpin may have inhibitory activity against prophenoloxidase activating proteinase (PAP) and clotting enzyme. Transcripts of Fc-serpin mRNA were mainly detected in haemocytes and the lymphoid organ by RT-PCR. The variation of the mRNA transcription level in haemocytes followed by artificial infection with bacteria OF white spot syndrome virus (WSSV) was quantified by SYBR Green real-time PCR analysis. Expression profiles of Fc-serpin greatly fluctuated after challenge. This work represents the first report Of a serpin in penaeid shrimp. The data provide clues that Fc-serpin might play potential roles in the innate immunity of shrimp. (C) 2008 Elsevier Ltd. All rights reserved.

Alteration of metallothionein mRNA in bay scallop Argopecten irradians under cadmium exposure and bacteria challenge

Metallothionein (MT) is a superfamily of cysteine-rich proteins contributing to metal metabolism, detoxification of heavy metals, and immune response such as protecting against ionizing radiation and antioxidant defense. A metallothionein (designated AiMT2) gene was identified and cloned from bay scallop, Argopecten irradians. The full length cDNA of AiMT2 consisted of an open reading frame (ORF) of 333 bp encoding a protein of 110 amino acids. with nine characteristic Cys-X-Cys, five Cys-X-X-Cys, five Cys-X-X-X-Cys and two Cys-Cys motif arrangements and a conserved structural pattern Cys-x-Cys-x(3)-Cys-Tyr-x(3)Cys-x-Cys-x(3)-Cys-x-Cys-Arg at the C-terminus. The cloned ANT showed about 50% identity in the deduced amino acid sequence with previously published MT sequences of mussels and oysters. The conserved structural pattern and the close phylogenetic relationship of AiMT2 shared with MTs from other mollusc especially bivalves indicated that AiMT2 was a new member of molluscan MT family. The mRNA transcripts in hemolymph of AiMT2 under cadmium (Cd) exposure and bacteria challenge were examined by real-time RT-PCR. The mRNA expression of AiMT2 was up-regulated to 3.99-fold at 2 h after Listonella anguillarum challenge, and increased drastically to 66.12-fold and 126.96-fold at 16 and 32 h post-challenge respectively. Cadmium ion exposure could induce the expression of AiMT2, and the expression level increased 2.56-fold and 6.91-fold in hemolymph respectively after a 10-day exposure of 100 mu g L-1 and 200 mu g L-1 CdCl2. The sensitivity of AiMT2 to bacteria challenge and cadmium stress indicated it was a new Cd-dependent MT in bay scallop and also regulated by an immune challenge. The changes in the expression of AiMT2 could be used as an indicator of exposure to metals in pollution monitoring programs and oxidative stress, and bay scallop as a potential sentinel organism for the cadmium contamination in aquatic environment. (C) 2008 Elsevier Inc. All rights reserved.

Diverse tetracycline resistant bacteria and resistance genes from coastal waters of Jiaozhou Bay

Environmental microbiology investigation was carried out in Jiaozhou Bay to determine the source and distribution of tetracycline-resistant bacteria and their resistance mechanisms. At least 25 species or the equivalent molecular phylogenetic taxa in 16 genera of resistant bacteria could be identified based on 16S ribosomal deoxyribonucleic acid sequence analysis. Enterobacteriaceae, Pseudomonadaceae, and Vibrionaceae constituted the majority of the typical resistant isolates. Indigenous estuarine and marine Halomonadaceae, Pseudoalteromonadaceae, Rhodobacteraceae, and Shewanellaceae bacteria also harbored tetracycline resistance. All the six resistance determinants screened, tet(A)-(E) and tet(G), could be detected, and the predominant genes were tet(A), tet(B), and tet(G). Both anthropogenic activity-related and indigenous estuarine or coastal bacteria might contribute to the tet gene reservoir, and resistant bacteria and their molecular determinants may serve as bioindicators of coastal environmental quality. Our work probably is the first identification of tet(E) in Proteus, tet(G) in Acinetobacter, tet(C) and tet(D) in Halomonas, tet(D) and tet(G) in Shewanella, and tet(B), tet(C), tet(E), and tet(G) in Roseobacter.

Phylogenetic analysis of epiphytic marine bacteria on Hole-Rotten diseased sporophytes of Laminaria japonica

During an occurrence of Hole-Rotten Disease of Laminaria japonica in a cultivating farm in Ma Shan Shandong province, China, 42 Gram-negative epiphytic marine bacteria were isolated and purified on Zobell 2216E marine agar medium. Morphological and biochemical characteristics of each isolated bacterium were studied, and molecular identification of bacterial strains was conducted with polymerase chain reaction amplification to 16S rRNA gene sequence analysis. Based on nearly full length of 16S rRNA gene sequence analysis, the isolated strains were bacteria that belong to genus Pseudoalteromonas, Vibrio, Halomonas and Bacillus. The percentage of each group was 61.9%, 28.6%, 7.1% and 2.4% respectively. The results of pathogenicity assay showed that 12 strains could cause the disease symptoms in sporophytes of L. japonica. They belonged to the genera Pseudoalteromonas, Vibrio and Halomonas with 58.3%, 33.3%, 8.3% respectively. The results suggest that these bacteria are the dominant marine bacteria on diseased sporophytes of L. japonica and may be the potential pathogenic bacteria associated with Hole-Rotten Disease of L. japonica.

Characterization of proteolytic bacteria from the Aleutian deep-sea and their proteases

Six deep-sea proteolytic bacteria taken from Aleutian margin sediments were screened; one of them produced a cold-adapted neutral halophilic protease. These bacteria belong to Pseudoalteromonas spp., which were identified by the 16S rDNA sequence. Of the six proteases produced, two were neutral cold-adapted proteases that showed their optimal activity at pH 7-8 and at temperature close to 35 degrees C, and the other four were alkaline proteases that showed their optimal activity at pH 9 and at temperature of 40-45 degrees C. The neutral cold-adapted protease E1 showed its optimal activity at a sodium chloride concentration of 2 M, whereas the activity of the other five proteases decreased at elevated sodium chloride concentrations. Protease E1 was purified to electrophoretic homogeneity and its molecular mass was 34 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of protease E1 was determined to be 32,411 Da by mass spectrometric analysis. Phenylmethyl sulfonylfluoride (PMSF) did not inhibit the activity of this protease, whereas it was partially inhibited by ethylenediaminetetra-acetic acid sodium salt (EDTA-Na). De novo amino acid sequencing proved protease E1 to be a novel protein.

CfLGBP, a pattern recognition receptor in Chlamys farreri involved in the immune response against various bacteria

Lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) is a kind of pattern recognition receptor, which can recognize and bind LPS and beta-1, 3-glucan, and plays curial roles in the innate immune defense against Gram-negative bacteria and fungi. In this study, the functions of LGBP from Zhikong scallop Chlamys farreri performed in innate immunity were analyzed. Firstly, the mRNA expression of CfLGBP in hemocytes toward three typical PAMPS stimulation was examined by realtime PCR. It was up-regulated extremely (P < 0.01) post stimulation of LPS and beta-glucan, and also exhibited a moderate up-regulation (P < 0.01) after PGN injection. Further PAMPs binding assay with the polyclonal antibody specific for CfLGBP proved that the recombinant CfLGBP (designated as rCfLGBP) could bind not only LPS and beta-glucan, but also PGN in vitro. More importantly, rCfLGBP exhibited obvious agglutination activity towards Gram-negative bacteria Escherichia coil, Gram-positive bacteria Bacillus subtilis and fungi Pichia pastoris. Taking the results of immunofluorescence assay into account, which displayed CfLGBP was expressed specifically in the immune cells (hemocytes) and vulnerable organ (gill and mantle), we believed that LGBP in C farreri, serving as a multi-functional PRR, not only involved in the immune response against Gram-negative and fungi as LGBP in other invertebrates, but also played significant role in the event of anti-Gram-positive bacteria infection. As the first functional research of LGBP in mollusks, our study provided new implication into the innate immune defense mechanisms of C. farreri and mollusks. (C) 2010 Elsevier Ltd. All rights reserved.

Molecular characterizations of oxytetracycline resistant bacteria and their resistance genes from mariculture waters of China

Oxytetracycline-resistant bacteria were isolated from a mariculture farm in China, and accounted for 32.23% and 5.63% of the total culturable microbes of the sea cucumber and the sea urchin rearing waters respectively. Marine vibrios, especially strains related to Vibrio splendidus or V. tasmaniensis, were the most abundant resistant isolates. For oxytetracycline resistance, tet(A), tet(B) and tet(D) genes were detected in both sea cucumber and sea urchin rearing ponds. The dominant resistance type for V. tasmaniensis-like strains was the combination of both tet(A) and tet(B) genes, while the major resistance type for V. splendidus-like strains was a single tet(D) gene. Most of the sea cucumber tet-positive isolates harbored a chloramphenicol-resistance gene, either cat IV or cat II, while only a few sea urchin tet-positive isolates harbored a cat gene, actually cat IV. The coexistence of tet and cat genes in the strains isolated from the mariculture farm studied was helpful in explaining some of the multi-resistance mechanisms. (c) 2006 Elsevier Ltd. All rights reserved.

Concurrence of cat and tet genes in multiple antibiotic-resistant bacteria isolated from a sea cucumber and sea urchin mariculture farm in China

A basic understanding of abundance and diversity of antibiotic-resistant microbes and their genetic determinants is necessary for finding a way to prevent and control the spread of antibiotic resistance. For this purpose, chloramphenicol and multiple antibiotic-resistant bacteria were screened from a mariculture farm in northern China. Both sea cucumber and sea urchin rearing ponds were populated with abundant antibiotic-resistant bacteria, especially marine vibrios. Sixty-five percent chloramphenicol-resistant isolates from sea cucumber harbored a cat gene, either cat IV or cat II, whereas 35% sea urchin isolates harbored a cat gene, actually cat II. The predominant resistance determinant cat IV gene mainly occurred in isolates related to Vibrio tasmaniensis or Pseudoalteromonas atlantica, and the cat II gene mainly occurred in Vibrio splendidus-like isolates. All the cat-positive isolates also harbored one or two of the tet genes, tet(D), tet(B), or tet(A). As no chloramphenicol-related antibiotic was ever used, coselection of the cat genes by other antibiotics, especially oxytetracycline, might be the cause of the high incidence of cat genes in the mariculture farm studied.