Neiella litorisoli sp. nov., an alginate lyase: producing bacterium from South China Sea, and proposal of Echinimonadaceae fam. nov. in the order Alteromonadales.

A Gram-stain-negative, rod-shaped bacterium, designated HB171785T, was isolated from soil sample collected from Qishui Bay, Hainan, China. The strain grew optimally at pH 7-8, 37-40 °C and with NaCl 3-4%. The predominant isoprenoid quinone was found to be Q-8 and the major fatty acids were C16:0, C16:1 ω7c/C16:1 ω6c, C18:1 ω7c/C18:1 ω6c and C12:0 3OH. The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The size of the draft genome was 4.32 Mbp with G + C content 49.7%. Phylogenetic analysis of 16S rRNA gene sequence indicated that the closest phylogenetically related species were Neiella marina j221T, "Neiella holothuriorum" 126 and Echinimonas agarilytica KMM 6351T with the similarities of 98.2, 96.0 and 95.0%, respectively. The phylogenetic tree based on 16S rRNA gene and phylogenomic tree based on core genome showed that strain HB171785T clustered together with N. marina j221T, with the highest values of average nucleotide identity (82.9%) and digital DNA-DNA hybridization (25.4%). The combined phylogenetic relatedness, phenotypic and genotypic features supported the conclusion that strain HB171785T represents a novel species of the genus Neiella, for which the name Neiella litorisoli sp. nov. is proposed. The type strain is HB171785T (= MCCC 1K04625T = KCTC 82319T). In addition, Echinimonadaceae fam. nov. in the order Alteromonadales was proposed.

SNHG15 enhances cisplatin resistance in lung adenocarcinoma by affecting the DNA repair capacity of cancer cells.

BACKGROUND: Lung adenocarcinoma (LUAD) is a prevalent malignancy. SNHG15 has been demonstrated to be oncogenic in many kinds of cancers, however the mechanism of SNHG15 in LUAD cisplatin (DDP) resistance remains unclear. In this study, we demonstrated the effect of SNHG15 on DDP resistance in LUAD and its related mechanism. METHODS: Bioinformatics analysis was adopted to assess SNHG15 expression in LUAD tissues and predict the downstream genes of SNHG15. The binding relationship between SNHG15 and downstream regulatory genes was proved through RNA immunoprecipitation, chromatin immunoprecipitation and dual-luciferase reporter assays. Cell counting kit-8 assay was adopted to evaluate LUAD cell viability, and gene expression was determined by Western blot and quantitative real-time polymerase chain reaction. We then performed comet assay to assess DNA damage. Cell apoptosis was detected by Tunnel assay. Xenograft animal models were created to test the function of SNHG15 in vivo. RESULTS: SNHG15 was up-regulated in LUAD cells. Moreover, SNHG15 was also highly expressed in drug-resistant LUAD cells. Down-regulated SNHG15 strengthened the sensitivity of LUAD cells to DDP and induced DNA damage. SNHG15 could elevate ECE2 expression through binding with E2F1, and it could induce DDP resistance by modulating the E2F1/ECE2 axis. In vivo experiments verified that the SNHG15 could enhance DDP resistance in LUAD tissue. CONCLUSION: The results suggested that SNHG15 could up-regulate ECE2 expression by recruiting E2F1, thereby enhancing the DDP resistance of LUAD.

Paenibacillus sabuli sp. nov., isolated from the South China Sea.

A Gram-positive, rod-shaped, motile, spore-forming bacterium, designated strain IB182496T, was isolated from coastal sand of the South China Sea. The strain grew optimally at pH 7.0-9.0, 20-30 °C, and with NaCl 3.0-5.0 %. The predominant menaquinone was MK-7 and the major cellular fatty acids were anteiso-C15 : 0, iso-C16 : 0 and C16 : 0. The polar lipids in the cell wall included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unidentified phospholipids and one unidentified lipid. The comparison of 16S rRNA gene sequences indicated that strain IB182496T was most closely related to 'Paenibacillus sambharensis' SMB1 and Paenibacillus tarimensis SA-7-6T with similarities of 95.7 and 95.5 %, respectively. The whole-genome average nucleotide identity values between strain IB182496T and the two reference strains were 70.8 and 70.5%, and the digital DNA-DNA hybridization values were 18.7 and 18.0 %, respectively. Genomic analyses showed that strain IB182496T presented a genome of 6.22 Mbp with chromosomal G+C content of 60.3 %, and a total of 5261 genes were predicted. The combined phylogenetic relatedness, phenotypic and genotypic features supported the conclusion that strain IB182496T should be considered as representing a novel species of the genus Paenibacillus, for which we propose the name Paenibacillus sabuli sp. nov. with the type strain IB182496T (=MCCC 1K04627T=JCM 34216T).

Paenibacillus oceani sp. nov., isolated from surface seawater.

A Gram-stain-positive and motile bacterial strain, designated IB182363T, was isolated from surface seawater of the South China Sea. Cells grew at pH 5.0-9.5 (optimum, pH 7.0-8.0), 20-40 °C (optimum, 30 °C) and with 1-8 % (w/v) NaCl (optimum, 2-4 %). On the basis of 16S rRNA gene sequence analysis, strain IB182363T was affiliated to the genus Paenibacillus and the closest phylogenetically related species was Paenibacillus ginsengarvi DSM18677T with 96.9 % sequence similarity. The values of whole genome average nucleotide identity analysis and digital DNA-DNA hybridization between the isolate and the closely related type strains were less than 86.3 and 25.6 %, respectively. Chemotaxonomic analysis revealed that strain IB182363T possessed meso-diaminopimelic acid in the cell-wall peptidoglycan and contained menaquinone MK-7 as the predominant isoprenoid quinone. The major cellular fatty acids were anteiso-C15 : 0, C16 : 0 and iso-C16 : 0. The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified glycolipid, two unidentified aminolipids, two unidentified phospholipids and four unidentified aminophospholipids. The genomic DNA G+C content was 54.5 mol%. On the basis of the above results, strain IB182363T represents a novel species of the genus Paenibacillus, for which we propose the name Paenibacillus oceani sp. nov. with the type strain IB182363T (=MCCC 1K04630T=JCM 34214T).

Amaricoccus solimangrovi sp. nov., isolated from mangrove soil.

Strain HB172011T was isolated from mangrove soil sampled at the Bamenbay mangrove forest, PR China. Cells were easily recognized under the microscope as cocci that were usually arranged in distinctive tetrads. Results of phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate belongs to the genus Amaricoccus and has 95.6-96.3% 16S rRNA gene sequence similarities to the four Amaricoccus type strains. The strain was aerobic or facultatively anaerobic, Gram-stain-negative and non-motile. Cells were found to grow at 10-40 °C (optimum, 30 °C), pH 6.0-9.0 (optimum, pH 7.0) and with 0-9.0% (w/v) NaCl (optimum, 2-4%). Major fatty acids were feature 8 (C18:1 ω7c and/or C18:1 ω6c), C16:0, C19:0 cyclo ω8c and summed feature 2 (C16:1 iso I and/or C14:0-3 OH). Genome sequencing revealed a genome size of 4.87 Mbp and a DNA G+C content of 69.9 mol %. Based on these data, strain HB172011T represents a novel species of Amaricoccus, for which the name Amaricoccus solimangrovi sp. nov. is proposed. The type strain is HB172011T (=CGMCC 1.16728T=JCM 33334T).

Pontibacter mangrovi sp. nov., isolated from mangrove sediment.

A Gram-stain-negative, short-rod-shaped and pink-pigmented bacterial strain (HB172049T) was isolated from mangrove sediment. Cells grew at 10-45 °C (optimum, 30 °C), pH 6.0-9.0 (optimum, pH 7.0) and with 0.5-9.0 % (w/v) NaCl (optimum, 2-5 %). Analysis of the 16S rRNA gene sequence revealed that the isolate had highest sequence similarities to Pontibacter mucosus DSM 100162T (96.5 %) and Pontibacter korlensis X14-1T (96.5 %). The values of average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization between the isolate and its close neighbours were, respectively, less than 80.1, 81.7 and 23.2 %. Chemotaxonomic analysis indicated that the sole respiratory quinone was MK-7 and the predominant cellular fatty acids were summed feature 4 and iso-C15 : 0 (42.2 and 24.6 %, respectively). The major polar lipids consisted of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, one unidentified glycolipid, one unidentified phospholipid, one unidentified aminophospholipid and two unidentified polar lipids. The genomic DNA G+C content was 52.6 mol%. Based on polyphasic taxonomic characterization, it is proposed that strain HB172049T belongs to the genus Pontibacter and represents a novel species, for which the name Pontibacter mangrovi sp. nov. is proposed. The type strain is HB172049T (=CGMCC 1.16729T=JCM 33333T).

Characterization, expression, and antimicrobial activity of histones from Japanese flounder Paralichthys olivaceus.

Histone proteins are not only structurally important for chromosomal DNA packaging but also involved in the regulation of gene expression and the immune response of host against pathogens. Japanese flounder (Paralichthys olivaceus) as one of the most important marine flatfish, suffered from widespread outbreaks of diseases, and its immunological functioning remained to be elucidated. In the present study, we reported the expression patterns of four histones (H1, H2A, H3, and H3.3) and functional characterization of the histone H3.3 from flounder. Quantitative real time RT-PCR (RT-qPCR) analysis showed that expression of the four histones occurred in multiple tissues, but their levels of expression were relatively high in immune organs, and inducible in response to pathogens infection. Infection with extracellular and intracellular bacterial pathogens and viral pathogen regulated the expression of histones in a manner that depended on tissue type, pathogen, and infection stage. Specifically, H1 expression was highly induced by intracellular viral pathogens; H2AX and H3 expressions were highly induced by intracellular bacterial pathogen; dissimilarly, H3.3 expression was slightly induced by extracellular bacterial pathogen, but was inhibited by intracellular bacterial and viral pathogens. To further investigate H3.3 function, recombinant H3.3 (rH3.3) was obtained, and in vitro experiments showed rH3.3 possessed the capability of binding to both Gram-negative and Gram-positive bacteria and inhibiting the growth of some target bacteria. Consistently, In vivo results showed that overexpression of H3.3 promoted the host defense against invading pathogenic microorganism and regulated the expressions of several cytokines. These results suggested that flounder histones exhibit different expression patterns in response to the infection of different microbial pathogens, and H3.3 serves as an immune-related protein and plays an important role in antimicrobial immunity of Japanese flounder. Taken together, this study is the first report about the expression profile of different histones upon different kind of pathogens and anti-infectious immunity of H3.3 in teleost, which offered new insights into the immunological function of histones in teleost.

HutZ is required for biofilm formation and contributes to the pathogenicity of Edwardsiella piscicida.

Edwardsiella piscicida is a severe fish pathogen. Haem utilization systems play an important role in bacterial adversity adaptation and pathogenicity. In this study, a speculative haem utilization protein, HutZEp, was characterized in E. piscicida. hutZEp is encoded with two other genes, hutW and hutX, in an operon that is similar to the haem utilization operon hutWXZ identified in V. cholerae. However, protein activity analysis showed that HutZEp is probably not related to hemin utilization. To explore the biological role of HutZEp, a markerless hutZEp in-frame mutant strain, TX01ΔhutZ, was constructed. Deletion of hutZEp did not significantly affect bacterial growth in normal medium, in iron-deficient conditions, or in the presence of haem but significantly retarded bacterial biofilm growth. The expression of known genes related to biofilm growth was not affected by hutZEp deletion, which indicated that HutZEp was probably a novel factor promoting biofilm formation in E. piscicida. Compared to the wild-type TX01, TX01ΔhutZ exhibited markedly compromised tolerance to acid stress and host serum stress. Pathogenicity analysis showed that inactivation of hutZEp significantly impaired the ability of E. piscicida to invade and reproduce in host cells and to infect host tissue. In contrast to TX01, TX01ΔhutZ was defective in blocking host macrophage activation. The expression of hutZEp was directly regulated by the ferric uptake regulator Fur. This study is the first functional characterization of HutZ in a fish pathogen, and these findings suggested that HutZEp is essential for E. piscicida biofilm formation and contributes to host infection.

Universal stress proteins contribute Edwardsiella piscicida adversity resistance and pathogenicity and promote blocking host immune response.

Universal stress proteins (Usps) exist ubiquitously in bacteria and other organisms. Usps play an important role in adaptation of bacteria to a variety of environmental stresses. There is increasing evidence that Usps facilitate pathogens to adapt host environment and are involved in pathogenicity. Edwardsiella piscicida (formerly included in E. tarda) is a severe fish pathogen and infects various important economic fish including tilapia (Oreochromis niloticus). In E. piscicida, a number of systems and factors that are involved in stress resistance and pathogenesis were identified. However, the function of Usps in E. piscicida is totally unknown. In this study, we examined the expressions of 13 usp genes in E. piscicida and found that most of these usp genes were up-regulated expression under high temperature, oxidative stress, acid stress, and host serum stress. Particularly, among these usp genes, usp13, exhibited dramatically high expression level upon several stress conditions. To investigate the biological role of usp13, a markerless usp13 in-frame mutant strain, TX01Δusp13, was constructed. Compared to the wild type TX01, TX01Δusp13 exhibited markedly compromised tolerance to high temperature, hydrogen peroxide, and low pH. Deletion of usp13 significantly retarded bacterial biofilm growth and decreased resistance against serum killing. Pathogenicity analysis showed that the inactivation of usp13 significantly impaired the ability of E. piscicida to invade into host cell and infect host tissue. Introduction of a trans-expressed usp13 gene restored the lost virulence of TX01Δusp13. In support of these results, host immune response induced by TX01 and TX01Δusp13 was examined, and the results showed reactive oxygen species (ROS) levels in TX01Δusp13-infected macrophages were significantly higher than those in TX01-infected cells. The expression level of several cytokines (IL-6, IL-8, IL-10, TNF-α, and CC2) in TX01Δusp13-infected fish was significantly higher than that in TX01-infected fish. These results suggested that the deletion of usp13 attenuated the ability of bacteria to overcome the host immune response to pathogen infection. Taken together, our study indicated Usp13 of E. piscicida was not only important participant in adversity resistance, but also was essential for E. piscicida pathogenicity and contributed to block host immune response to pathogen infection.

Major histocompatibility complex class I (MHC Iα) of Japanese flounder (Paralichthys olivaceus) plays a critical role in defense against intracellular pathogen infection.

The major histocompatibility complex (MHC) is a highly polymorphic region of the vertebrate genome that plays a critical role in initiating immune responses towards invading pathogens. It is well known that MHC I molecules play a central role in the immune response to viruses. However, rare literatures were reported the role of MHC I in the resistance to intracellular bacteria. Sequences of MHC Iα were identified in multiple teleost species, including Japanese flounder (Paralichthys olivaceus), however, the immunological function of MHC Iα remain largely unknown. In this study, we examined the expression profile and biological activity of an MHC Iα homologue, PoMHC Iα, from P. olivaceus. Structural analysis showed that PoMHC Iα possesses conserved structural characteristics of MHC Iα proteins, including MHC_I domain, IGc1 domain, transmembrane region. Expression of PoMHC Iα was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen infection. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoMHC Iα increased the capability of host cells to defense against intracellular pathogen Edwardsiella tarda infection and enhanced the expression of immune related genes. The knockdown of PoMHC Iα attenuated the ability of cells to eliminate E. tarda, which was sustained by the in vivo results that overexpression of PoMHC Iα promoted the host defense against invading E. tarda. Antigen uptake assay indicated PoMHC Iα participated in cells antigen presentation. Collectively, this study is the first report that MHC Iα plays an important role in immune defense against intracellular bacterial pathogen in teleost. Taken together, these findings add new insights into the biological function of teleost MHC Iα and emphasize the importance of MHC I gene products for the control of E. tarda infection.

Thioredoxin H (TrxH) contributes to adversity adaptation and pathogenicity of Edwardsiella piscicida.

Thioredoxins (Trxs) play an important role in defending against oxidative stress and keeping disulfide bonding correct to maintain protein function. Edwardsiella piscicida, a severe fish pathogen, has been shown to encode several thioredoxins including TrxA, TrxC, and TrxH, but their biological roles remain unknown. In this study, we characterized TrxH of E. piscicida (named TrxHEp) and examined its expression and function. TrxHEp is composed of 125 residues and possesses typical thioredoxin H motifs. Expression of trxHEp was upregulated under conditions of oxidative stress, iron starvation, low pH, and during infection of host cells. trxHEp expression was also regulated by ferric uptake regulator (Fur), an important global regulatory of E. piscicida. Compared to the wild type TX01, a markerless trxHEp in-frame mutant strain TX01∆trxH exhibited markedly compromised tolerance of the pathogen to hydrogen peroxide, acid stress, and iron deficiency. Deletion of trxHEp significantly retarded bacterial biofilm growth and decreased resistance against serum killing. Pathogenicity analysis shows that the inactivation of trxHEp significantly impaired the ability of E. piscicida to invade host cells, reproduce in macrophages, and infect host tissues. Introduction of a trans-expressed trxH gene restored the lost virulence of TX01∆trxH. There is likely to be a complex relationship of functional complementation or expression regulation between TrxH and another two thioredoxins, TrxA and TrxC, of E. piscicida. This is the first functional report of TrxH in fish pathogens, and the findings suggest that TrxHEp is essential for coping with adverse circumstances and contributes to host infection of E. piscicida.

Granulocyte colony stimulating factor (GCSF) of Japanese flounder (Paralichthys olivaceus): Immunoregulatory property and anti-infectious function.

Granulocyte colony stimulating factor (GCSF) is a key regulator of neutrophil production, and plays a vital role in immune response of mammals and teleost against pathogen. Sequences of GCSF were identified in several teleost species, however, the function and activity of GCSF in teleost remain largely unknown. In this study, we examined the biological activity and the immunomodulatory property of a GCSF homologue, PoGCSF, from Japanese flounder (Paralichthys olivaceus). Structural analysis showed that PoGCSF possesses conserved structural characteristics of GCSF proteins, including a signal peptide and a typical IL-6 domain. The expression of PoGCSF was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoGCSF increased the capability of host cells to defense against pathogen infection and enhanced the expression of immune related genes. The knockdown of PoGCSF attenuated the ability of host cells to eliminate pathogenic bacteria. In vivo results showed that overexpression of PoGCSF promoted the host defense against invading pathogenic microorganism. Collectively, this study is the first report about the immunoregulatory property and anti-infectious immunity of GCSF in teleost. These findings suggested that PoGCSF serves as an immune-related cytokine and plays an important role in the immune defense system of Japanese flounder.

Global discovery of small RNAs in the fish pathogen Edwardsiella piscicida: key regulator of adversity and pathogenicity.

Recently, bacterial small RNA (sRNA) has been shown to be involved as a key regulator in stress responses. sRNAs of Edwardsiella piscicida, an important aquatic pathogen, are not well characterized to date. In this study, using RNA-seq technology, we globally found and identified sRNA candidates expressed from E. piscicida grown in normal LB medium, acid pressure, iron deficiency stress, and oxidation pressure. A total of 148 sRNAs were found, including 19 previously annotated sRNAs and 129 novel sRNA candidates by searching against the Rfam database. Compared in normal condition, the expression of 103 sRNAs (DEsRNA, differentially expressed sRNA) and 1615 mRNAs (DEmRNAs, differentially expressed mRNA) showed significant differences in three stress sample. Based on the prediction by IntaRNA and relational analysis between DEsRNAs and DEmRNAs, 103 DEsRNAs were predicted to regulate 769 target mRNAs. Pleiotropic function of target DEmRNAs indicated that sRNAs extensively participated in a variety of physiological processes, including response to adversity and pathogenicity, the latter was further confirmed by infection experiment. A large number transcription factors appeared in target genes of sRNAs, which suggested that sRNAs likely deeply interlaced within complex gene regulatory networks of E. piscicida. Moreover, 49 Hfq-associated sRNAs were also identified in this study. In summary, we globally discovered sRNAs for the first time in pathogenic bacteria of fish, and our findings indicated that sRNAs in E. piscicida have important roles in adaptation to environmental stress and pathogenicity. These results also provide clues for deciphering regulation mechanism of gene expression related to physiological response and pathogenicity.

Strepchazolins A and B: Two New Alkaloids from a Marine Streptomyces chartreusis NA02069.

Two new alkaloids, strepchazolins A (1) and B (2), together with a previously reported compound, streptazolin (3), were isolated from a marine actinomycete, Streptomyces chartreusis NA02069, collected in the Coast of Hainan Island, China. The structures of new compounds were determined by extensive NMR, mass spectroscopic and X-ray crystallographic analysis, as well as modified Mosher's method. Compound 1 showed weak anti-Bacillus subtilis activity with the MIC value of 64.0 µM, and weak inhibitory activity against acetylcholinesterase (AChE) in vitro with IC50 value of 50.6 µM, while its diastereoisomer, Compound 2, is almost inactive.

Pseudonocardia nematodicida sp. nov., isolated from mangrove sediment in Hainan, China.

Two aerobic, Gram-stain positive actinobacterial strains with nematicidal activity, designated HA11164(T) and HA12591, were isolated from mangrove sediments in Hainan, China. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strains HA11164(T) and HA12591 belong to the genus Pseudonocardia and are closely related to Pseudonocardia carboxydivorans (with the similarities of 98.30 and 98.24 %, respectively), Pseudonocardia alni (98.23 and 98.16 %, respectively) and Pseudonocardia antimicrobica (98.10 and 98.03 %, respectively). The major polar lipids of the strain HA11164(T), as a representative strain of the two strains, were found to consist of phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, five unidentified glycolipids and four unidentified polar lipids. The predominant menaquinone of strain HA11164(T) was identified as MK-8 (H4), and the major fatty acids were identified as iso-C16:0, C17:1 ω10, C16:0 and C16:1 ω9. The G+C content of strain HA11164(T) was determined to be 74.9 mol%. The DNA-DNA relatedness values between strains HA11164(T) and P. alni, Pseudonocardia tropica, Pseudonocardia antarctica, P. carboxydivorans and Pseudonocardia parietis were 58.3, 56.2, 50.0, 57.1 and 46.0 %, respectively. Based on the results of this polyphasic study, strains HA11164(T) and HA12591 are considered to represent a novel species of the genus Pseudonocardia, for which the name Pseudonocardia nematodicida sp. nov. is proposed. The type strain is HA11164(T) (=CGMCC 4.7118(T) = DSM 45940(T)).

Nocardiopsis mangrovei sp. nov., isolated from mangrove sediment.

Two Gram-positive actinobacterial strains, designated HA11166(T) and HA12420, were isolated from mangrove sediments in Hainan, China. The bacterial cells grew with 0-9 % (w/v) NaCl, at 15-40 °C and pH 5.0-10.0, with the optimum growth at 1 % NaCl, 30-37 °C and pH 7.0. The organisms had a range of chemical and morphological properties consistent with their classification in the genus Nocardiopsis. Phylogenetic analysis of the 16S rRNA gene sequences indicated that strains HA11166(T) and HA12420 can be affiliated to the genus Nocardiopsis and most closely related to Nocardiopsis trehalosi VKM Ac-942(T) (with the similarity of 97.2 and 97.5 %, respectively). The value of DNA-DNA relatedness between type strain HA11166(T), selected as the representative strain, and N. trehalosi VKM Ac-942(T) was 38.8 %. The DNA G+C content of strain HA11166(T) was 73.7 %. On the basis of these phenotypic and genotypic data, strains HA11166(T) and HA12420 are proposed to represent a novel species of the genus Nocardiopsis, for which the name Nocardiopsis mangrovei sp. nov. is proposed. The type strain is HA11166(T) (=CGMCC 4.7119(T)=DSM 46665(T)).

Gracilibacillus marinus sp. nov., isolated from the northern South China Sea.

Two gram-positive, aerobic, spore-forming, rod-shaped bacteria, designated HB09003(T) and HB12160, were isolated from seawater and sediment in the northern South China Sea, respectively. Cells were found to be motile by means of peritrichous flagella. The strains were found to grow with 0-15 % (w/v) NaCl, at 10-45 °C and pH 5.0-10.7, with an optimum of 3 % NaCl, 28 °C and pH 8.5, respectively. The predominant isoprenoid quinone of strain HB09003(T), selected as the representative strain, was identified as MK-7. This strain was found to possess anteiso-C15:0, iso-C15:0, anteiso-C17:0 and C16:0 as the major fatty acids. The G+C contents of strain HB09003(T) and HB12160 were determined to be 34.1 and 34.3 mol%, respectively. Analysis of the 16S rRNA gene sequences of the two strains showed an affiliation with the genus Gracilibacillus, with Gracilibacillus kekensis CGMCC 1.10681(T) (similarity of 97.4, 98.0 %, respectively) and Gracilibacillus ureilyticus CGMCC 1.7727(T) (similarity of 97.1, 97.8 %, respectively) as their closest relatives. The DNA-DNA hybridization values between strain HB09003(T) and the two type strains were 42.2 and 54.1 %, respectively. On the basis of phenotypic and genotypic data, strain HB09003(T) and HB12160 are proposed to represent a novel species of the genus Gracilibacillus, for which the name Gracilibacillus marinus sp. nov. is proposed. The type strain is HB09003(T) (=CGMCC 1.10343(T) = DSM 23372(T)).

[Seven kinds of new SCCmec type in Methicillin-resistant Staphyloccus aureus and their susceptibility to the antibiotics].

In order to explore the resistance and the staphylococcal chromosome cassette mec (SCCmec) types of Methicillin-resistant S. aureus (MRSA) in the area of Haikou, 686 strains of MRSA had been distinguished from 1174 strains of S. aureus using PBP2a testing. The resistance to the seven deputies of seven kinds antibiotics which in common use in clinic, including Oxacillin, Vancomycin, Doxycyclin, Amikacin, Erythromycin, Chloramphenicol, Ciprofloxacin, and SCCmec type of 58 strains had been tested using the K-B Agar diffuse, E-test and multiplex PCR strategy, and seven kinds of new SCCmec types were found in 17 strains. Their specialties of structure are: type-new3 possess four loci of A, F, H, M; New4 possess three loci of F, H, M; New5 possess three loci of D, B, M; New6 possess three loci of A, B, M; New7 possess four loci of H, E, C, M; New8 possess two loci of A, M; New9 possess three loci of A, C, M. All of them are different from the types reported. The strains carrying new SCC mec types are different from that carrying old SCC mec types in the epidemical distribution and resistance to the antibiotics: they were mostly isolated from the out-patients and have high level and wider range of resistance to antibiotics and deserve to pay more attention.

[Two kinds of new Sccmec type in Methicillin-resistant Staphylococcus aureus and their susceptibility to the antibiotics].

In order to explore the resistance, epidemical distribution and the staphylococcal chromosome cassette mec (SCCmec) types of Methicillin-resistant S. aureus (MRSA) in the area of Haikou, the resistance and SCCmec type of MRSA isolated from clinical sample has been tested using the K-B Agar diffuse, E-test and multiplex PCR strategy, resulting that two kinds of new SCC mec types were found in them. Type-newl possess three loci of B, M, F. Locus M, which locate in mecA, is the indicator of mecA, and also used as the positive control. Locus B, which locate in upstream of mec A, is specific for type- II and F, which locate in downstream of mec A, is specific for type- iU ; and so, type-newl was combined from the type- II and - I . Type-new2 possess five loci of A, B, M, F, H. Locus A, which is locate in upstream of mecA, is specific for type- I and H, which is locate in downstream of mecA, is also specific for type- 1 ; and so, type-new2 was combined from the type- I , - II and - I . The strains carrying new SCC mec types are different from that carrying old SCC mec types in the epidemical distribution and resistance to the antibiotics. They can be usually isolated from the out-patients and have high level and wider range of resistance to antibiotics. It must be paid more attention to them.

[Archaeal diversity associated with sponge Pachychalina sp. analyzed by culture-independent approach].

With culture-independent approach, microbial total DNA was directly extracted from Pachychalina sp. Using the total microbial DNA as template, archaeal 16S rDNAs were amplified by PCR with universal primers. Amplified products were cloned into T-vector and secondarily amplified by PCR. Then the secondarily amplified products were purified to be further characterized by termed ARDRA (amplified rDNA restriction analysis). According to the enzyme restriction mapping, the apparent difference among them were disclosed. Furthermore eight archaeal cloned partial sequences were acquired and built up a phylogenetic tree. In the phylogenetic tree, the eight archaea belonged to Methanogenium organophilum and Methanoplanus petrolearius, but the 16S rDNAs similarities among them and those archaea registered in RDP Database didn't excess to 90%. It means that they maybe represent some novel archaeal groups.