attB site disruption in marine Actinomyces sp M048 via DNA transformation of a site-specific integration vector

An efficient conjugation method has been developed for the marine Actinomyces sp. isolate M048 to facilitate the genetic manipulation of the chandrananimycin biosynthesis gene cluster. A phi C31-derived integration vector pIJ8600 containing oriT and attP fragments was introduced into strain M048 by bi-parental conjugation from Escherichia coli ET12567 to strain M048. Transformation efficiency was (6.38 +/- 0.41) x 10(-5) exconjugants per recipient spore. Analysis of eight exconjugants showed that the plasmid pIJ8600 was stably integrated at a single chromosomal site (attB) of the Actinomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of antimicrobial activity analysis indicated that the insertion of plasmid pIJ8600 seemed to affect the biosynthesis of antibiotics that could strongly inhibit the growth of E. coli and Mucor miehei (Tu284). HPLC-MS analysis of the extracts indicated that disruption of the attB site resulted in the complete abolition of chandrananimycin A-C production, proving the identity of the gene cluster. Instead of chandrananimycins, two bafilomycins were produced through disruption of the attB site from the chromosomal DNA of marine Actinomyces sp. M048.

Transformation of accommodation space of the Cretaceous Qingshankou Formation, the Songliao Basin, NE China

Analysis of accommodation space variation during deposition of the Cretaceous Qingshankou Formation in the Songliao Basin, NE China, indicates that accommodation space changed both through time and across the basin as a seesaw movement. The mid-upper Qingshankou Formation is divided into three units. In each unit, changes of accommodation space differ in the southern and northern part of the basin. Increasing accommodation in the southern part is accompanied by a decrease in the northern part, and vice versa. Between the northern and southern basin, there was a neutral belt that is like a fulcrum, called the transformation belt here, where the accommodation did not change to any significant degree. We call this response 'accommodation transformation', whose characteristics are defined by tectonic subsidence analysis, palaeontological and sedimentary analyses. The accommodation increasing belt, decreasing belt, transformation belt and accommodation transformation boundary together constitute the accommodation transformation system. The recognition of accommodation transformation in the Songliao Basin provides a new insight into sequence stratigraphy and might be widely applicable.

Cross-Ocean distribution of Rhodobacterales bacteria as primary surface colonizers in temperate coastal marine waters

Bacterial surface colonization is a universal adaptation strategy in aquatic environments. However, neither the identities of early colonizers nor the temporal changes in surface assemblages are well understood. To determine the identities of the most common bacterial primary colonizers and to assess the succession process, if any, of the bacterial assemblages during early stages of surface colonization in coastal water of the West Pacific Ocean, nonnutritive inert materials (glass, Plexiglas, and polyvinyl chloride) were employed as test surfaces and incubated in seawater off the Qingdao coast in the spring of 2005 for 24 and 72 h. Phylogenetic analysis of the 16S rRNA gene sequences amplified from the recovered surface-colonizing microbiota indicated that diverse bacteria colonized the submerged surfaces. Multivariate statistical cluster analyses indicated that the succession of early surface-colonizing bacterial assemblages followed sequential steps on all types of test surfaces. The Rhodobacterales, especially the marine Roseobacter clade members, formed the most common and dominant primary surface-colonizing bacterial group. Our current data, along with previous studies of the Atlantic coast, indicate that the Rhodobacterales bacteria are the dominant and ubiquitous primary surface colonizers in temperate coastal waters of the world and that microbial surface colonization follows a succession sequence. A conceptual model is proposed based on these findings, which may have important implications for understanding the structure, dynamics, and function of marine biofilms and for developing strategies to harness or control surface-associated microbial communities.

Extracellular hydrolytic enzyme screening of culturable heterotrophic bacteria from deep-sea sediments of the Southern Okinawa Trough

The Southern Okinawa Trough is an area of focused sedimentation due to particulate matter export from the shelf of the East China Sea and the island of Taiwan. In order to understand the geomicrobiological characteristics of this unique sedimentary environment, bacterial cultivations were carried out for an 8.61 m CASQ core sediment sample. A total of 98 heterotrophic bacterial isolates were characterized based on 16S rRNA gene phylogenetic analysis. These isolates can be grouped into four bacterial divisions, including 13 genera and more than 20 species. Bacteria of the gamma-Proteobacteria lineage, especially those from the Halomonas ( 27 isolates) and Psychrobacter ( 20 isolates) groups, dominate in the culturable bacteria assemblage. They also have the broadest distribution along the depth of the sediment. More than 72.4% of the isolates showed extracellular hydrolytic enzyme activities, such as amylases, proteases, lipases and Dnases, and nearly 59.2% were cold-adapted exoenzyme-producers. Several Halomonas strains show almost all the tested hydrolases activities. The wide distribution of exoenzyme activities in the isolates may indicate their important ecological role of element biogeochemical cycling in the studied deep-sea sedimentary environment.

Fine-scale vertical distribution of bacteria in the East Pacific deep-sea sediments determined via 16S rRNA gene T-RFLP and clone library analyses

Although the deep-sea sediments harbor diverse and novel bacteria with important ecological and environmental functions, a comprehensive view of their community characteristics is still lacking, considering the vast area and volume of the deep-sea sedimentary environments. Sediment bacteria vertical distribution and community structure were studied of the E272 site in the East Pacific Ocean with the molecular methods of 16S rRNA gene T-RFLP (terminal restriction fragment length polymorphism) and clone library analyses. Layered distribution of the bacterial assemblages was detected by both methods, indicating that the shallow sediments (40 cm in depth) harbored a diverse and distinct bacterial composition with fine-scale spatial heterogeneity. Substantial bacterial diversity was detected and nine major bacterial lineages were obtained, including Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Nitrospirae, Planctomycetes, Proteobacteria, and the candidate divisions OP8 and TM6. Three subdivisions of the Proteobacteria presented in our libraries, including the alpha-, gamma- and delta-Proteobacteria. Most of our sequences have low similarity with known bacterial 16S rRNA genes, indicating that these sequences may represent as-yet-uncultivated novel bacteria. Most of our sequences were related to the GenBank nearest neighboring sequences retrieved from marine sediments, especially from deep-sea methane seep, gas hydrate or mud volcano environments. Several sequences were related to the sequences recovered from the deep-sea hydrothermal vent or basalt glasses-bearing sediments, indicating that our deep-sea sampling site might be influenced to certain degree by the nearby hydrothermal field of the East Pacific Rise at 13A degrees N.

Molecular characterizations of chloramphenicol- and oxytetracycline-resistant bacteria and resistance genes in mariculture waters of China

In order to gain an understanding of the diversity and distribution of antimicrobial-resistant bacteria and their resistance genes in maricultural environments, multidrug-resistant bacteria were screened for the rearing waters from a mariculture farm of China. Both abalone Haliotis discus hannai and turbot Scophthalmus maximus rearing waters were populated with abundant chloramphenicol-resistant bacteria. These bacteria were also multidrug resistant, with Vibrio splendidus and Vibrio tasmaniensis being the most predominant species. The chloramphenicol-resistance gene cat II, cat IV or floR could be detected in most of the multidrug-resistant isolates, and the oxytetracycline-resistance gene tet(B), tet(D), tet(E) or tet(M) could also be detected for most of the isolates. Coexistence of chloramphenicol- and oxytetracycline-resistance genes partially explains the molecular mechanism of multidrug resistance in the studied maricultural environments. Comparative studies with different antimicrobial agents as the starting isolation reagents may help detect a wider diversity of the antimicrobial-resistant bacteria and their resistance genes. (C) 2009 Elsevier Ltd. All rights reserved.

Diversity of Both the Cultivable Protease-Producing Bacteria and Their Extracellular Proteases in the Sediments of the South China Sea

Protease-producing bacteria are known to play an important role in degrading sedimentary particular organic nitrogen, and yet, their diversity and extracellular proteases remain largely unknown. In this paper, the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China Sea was investigated. The richness of the cultivable protease-producing bacteria reached 10(6) cells/g in all sediment samples. Analysis of the 16S rRNA gene sequences revealed that the predominant cultivated protease-producing bacteria are Gammaproteobacteria affiliated with the genera Pseudoalteromonas, Alteromonas, Marinobacter, Idiomarina, Halomonas, Vibrio, Shewanella, Pseudomonas, and Rheinheimera, with Alteromonas (34.6%) and Pseudoalteromonas (28.2%) as the predominant groups. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria are serine proteases or metalloproteases. Moreover, these proteases have different hydrolytic ability to different proteins, reflecting they may belong to different kinds of serine proteases or metalloproteases. To our knowledge, this study represents the first report of the diversity of bacterial proteases in deep-sea sediments.

Dominant chloramphenicol-resistant bacteria and resistance genes in coastal marine waters of Jiaozhou Bay, China

Studies of abundance, diversity and distribution of antibiotic-resistant bacteria and their resistance determinants are necessary for effective prevention and control of antibiotic resistance and its dissemination, critically important for public health and environment management. In order to gain an understanding of the persistence of resistance in the absence of a specific antibiotic selective pressure, microbiological surveys were carried out to investigate chloramphenicol-resistant bacteria and the chloramphenicol acetyltransferase resistance genes in Jiaozhou Bay after chloramphenicol was banned since 1999 in China. About 0.15-6.70% cultivable bacteria were chloramphenicol resistant, and the highest abundances occurred mainly in the areas near river mouths or sewage processing plants. For the dominant resistant isolates, 14 genera and 25 species were identified, mostly being indigenous estuarine or marine bacteria. Antibiotic-resistant potential human or marine animal pathogens, such as Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Shewanella algae, were also identified. For the molecular resistance determinants, the cat I and cat III genes could be detected in some of the resistant strains, and they might have the same origins as those from clinical strains as determined via gene sequence analysis. Further investigation about the biological, environmental and anthropogenic mechanisms and their interactions that may contribute to the persistence of antibiotic-resistance in coastal marine waters in the absence of specific antibiotic selective pressure is necessary for tackling this complicated environmental issue.

Transformation field method for calculating the effective properties of isotropic graded composites having arbitrary shapes

A method of transformation field is developed to estimate the effective properties of graded composites whose inclusions have arbitrary shapes and gradient profiles by means of a periodic cell model. The boundary-value problem of graded composites having arbitrary inclusion shapes is solved by introducing the transformation field into the inclusion region. As an example, the effective dielectric response of isotropic graded composites having arbitrary shapes and gradient profiles is handled by the transformation field method (TFM). Moreover, TFM results are validated by the exact solutions of isotropic graded spherical inclusions having a power-law profile and good agreement is obtained in the dilute limit. Furthermore, it is found that the inclusion shapes and the parameters of the gradient profiles can have profound effect on the effective properties of composite systems at high concentration of inclusions.

The responsive expression of heat shock protein 22 gene in zhikong scallop Chlamys farreri against a bacterial challenge

HSP22 is a member of a small HSP subfamily contributing to the growth, transformation and apoptosis of the cell as well as acting as a molecular chaperone. In the present study, CfHSP22 cDNA was cloned from Chlamys farreri by the rapid amplification of cDNA ends technique. The full-length cDNA of CfHSP22 was of 1279 bp, consisting of a 5'-terminal untranslated region (5'UTR) of 122 bp, a 3'UTR of 581 bp with a canonical polyadenylation signal sequence AATAAA and a poly( A) tail, and an open reading frame of 576 bp encoding a polypeptide with a molecular mass of 22.21 kDa and a predicted isoelectric point of 9.69. There was an alpha-crystallin domain, a hallmark of the sHSP subfamily, in the C-terminus, and the deduced amino acid sequence of CfHSP22 showed high similarity to previously identified HSP22s. CfHSP22 was constitutively expressed in the haemocyte, muscle, kidney, gonad, gill, heart and hepatopancreas, and the expression level in the hepatopancreas was higher than that in the other tissues. CfHSP22 transcription was up-regulated and reached a maximal level at 12 h after the bacterial challenge, and then declined progressively to the original level at 48 h. These results suggested that CfHSP22 perhaps play a critical role in response to the bacterial challenge in haemocytes of scallop C. farreri.

Isolation and identification of bacteria associated with the surfaces of several algal species

We conducted this study to assess the diversity of bacteria associated with the surfaces of algae based on 16S rDNA sequence analyses. Twelve strains of bacteria were obtained from the surfaces of the following four species of algae: Gracilaria textorii, Ulva pertusa, Laminaria japonica, and Polysiphonia urceolata. The isolated strains of bacteria can be divided into two groups: Halomonas and Vibrio, in physiology, biochemical characteristics and 16S rDNA sequence analyses. The phylogenetic tree constructed based on 16S rDNA sequences of the isolates shows four obvious clusters, Halomonas venusta, Vibrio tasmaniensis, Vibrio lentus, and Vibrio splendidus. Isolates from the surface of P. urceolata are more abundant and diverse, of which strains P9 and P28 have a 16S rDNA sequence very similar (97.5%-99.8%) to that of V. splendidus. On the contrary, the isolates from the surfaces of G textorii, U. pertusa and L. japonica are quite simple and distribute on different branches of the phylogenetic tree. In overall, the results of this study indicate that the genetic relationships among the isolates are quite close and display a certain level of host species specificity, and alga-associated bacteria species are algal species specific.

Biological activity of a red-tide alga - A-tamarense under co-cultured condition with bacteria

The relationship between Alexandrium tamarense (Lebour) Balech, one of red-tide alga, and two strains of marine bacteria, Bacillius megaterium(S-7) and B. halmapulus(S-10) isolated from Xiamen Western Sea, was investigated by evaluating the growth state of A. tamarense and the variation of P-glucosidase activity in co-culture system. The results showed the growth and multiplication of the alga were related with the concentration, genus speciality of the bacteria, and growth stage of the alga itself. The growth of A. tamarense was obviously inhibited by S7 and S, at high concentration. Either inhibition or promotion contributed much more clearly in earlier than in later stage of the growth of the alga. Furthermore, there was a roughly similar variation trend of the activity of extra-cellular enzyme, beta-glucosidase, in the water of the separately co-cultured bacteria S-7 and S-10 with the alga. The beta-glucosidase activity (beta-GlcA) rapidly increased during the later algal growth accompanying the increase of the lysis of the alga cells. The obvious inhibition of A. tamarense by marine bacteria at high concentration and evident increase of beta-GlcA in co-colture system would help us in better understanding the relationship between red-tide alga and bacteria, and also enlightened us the possible use of bacteria in the bio-control of red-tide.

Marine bacteria associated with marine macroorganisms: the potential antimicrobial resources

In order to explore marine microorganisms with medical potential, marine bacteria were isolated from seawater, sediment, marine invertebrates and seaweeds collected from different coastal areas of the China Sea. The antimicrobial activities of these bacteria were investigated. Ethyl acetate extracts of marine bacterial fermentation were screened for antimicrobial activities using the method of agar diffusion. The results showed that 42 strains of the isolates have antimicrobial activity. The proportion of active bacteria associated with marine invertebrates (20%) and seaweeds (11%) is higher than that isolated from seawater (7%) and sediment (5%). The active marine bacteria were assigned to the genera Alteromonas, Pseudomonas, Bacillus and Flavobacterium. The TLC autobiographic overlay assay implied that the antimicrobial metabolites produced by four strains with wide antimicrobial spectrum were different. Due to a competitive role for space and nutrient, the marine bacteria associated with marine macroorganisms (invertebrates and seaweeds) could produce more antibiotic substances. These marine bacteria were expected to be potential resources of natural antibiotic products.

Toxin-screening and identification of bacteria isolated from highly toxic marine gastropod Nassarius semiplicatus

Bacteria isolated from a highly toxic sample of gastropod Nassarius semiplicatus in Lianyungang, Jiangsu Province in July 2007, were studied to probe into the relationship between bacteria and toxicity of nassariid gastropod. The toxicity of the gastropod sample was 2 x 10(2) mouse unit (MU) Per gram Of tissue (wet weight). High concentration of tetrodotoxin (TTX) and its analogues (TTXs) were found in the digestive gland and muscle of the gastropod, using high performance liquid chromatography coupled with mass chromatography (LC-MS). Bacterial strains isolated from the digestive gland were cultured and screened for TTX with a competitive ELISA method. Tetrodotoxin was detected in a proportion of bacterial strains, but the toxin content was low. Partial 16S ribosomal DNA (rDNA) of the TTX-producing strains was then sequenced and compared with those published in the GenBank to tentatively identify the toxic strains. It was found that most of the toxic strains were closely affiliated with genus Vibrio, and the others were related to genus Shewanella, Marinomonas, Tenacibaculum and Aeromonas. These findings suggest that tetrodotoxin-producing bacteria might play an important role in tetrodotoxin accumulation/production in N. semiplicatus. (C) 2008 Elsevier Ltd. All rights reserved.

Spatial variability in biomass and production of heterotrophic bacteria in the East China Sea and the Yellow Sea

The distributions of heterotrophic bacterial abundance and production were investigated in the East China Sea and the Yellow Sea during the autumn of 2000 and spring of 2001. Bacterial abundance varied in the range 3.2-15.7 (averaging 5.7) x 10(5) and 2.3-13.6 (averaging 6.2) x 10(5) cells cm(-3) in the spring and autumn, respectively. During autumn, bacterial production (BP) (0.27-7.77 mg C m(-3) day(-1)) was on average 3 fold that in spring (0.001-2.04 mg C m(-3) day(-1)). Bacterial average turnover rate (ratio of bacterial production:bacterial biomass, mu=0.21 day(-1)) in autumn was 3 times as high as in spring (0.07 day(-1)). The ratio of integrated bacterial biomass to integrated phytoplankton biomass in the euphotic zone ranged from 4 to 101% (averaging 35%) in spring and 24 to 556% (averaging 121%) in autumn. The results indicate that the distributions of heterotrophic bacteria were controlled generally by temperature in spring and additionally by substrate supply in autumn. (C) 2010 Elsevier Ltd. All rights reserved.