Isolation of Surfactant-Resistant Bacteria from Natural, Surfactant-Rich Marine Habitats

Environmental remediation efforts often utilize either biodegradative microbes or surfactants, but not in combination. Coupling both strategies holds the potential to dramatically increase the rate and extent of remediation because surfactants can enhance the bioavailability of contaminants to microbes. However, many surfactants permeabilize bacterial cell membranes and are effective disinfectants. An important goal then is to find or genetically modify microorganisms that possess both desirable degradative capabilities and the ability to thrive in the presence of surfactants. The guts of some marine invertebrates, particularly deposit feeders, have previously been shown to contain high levels of biosurfactants. Our primary aim was to mine these natural, surfactant-rich habitats for surfactant-resistant bacteria. Relative to sediment porewaters, the gut contents of two polychaete deposit feeders, Nereis succinea and Amphitrite ornata, exhibited a significantly higher ratio of bacteria resistant to both cationic and anionic surfactants. In contrast, bacteria in the gut fluids of a holothuroid, Leptosynapta tenuis, showed surfactant susceptibility similar to that of bacteria from sediments. Analyses of 16S rRNA gene sequences revealed that the majority of surfactant-resistant isolates were previously undescribed species of the genus Vibrio or were of a group most closely related to Spongiobacter spp. We also tested a subset of resistant bacteria for the production of biosurfactants. The majority did produce biosurfactants, as demonstrated via the oil-spreading method, but in all cases, production was relatively weak under the culture conditions employed. Novel surfactant-resistant, biosurfactant-producing bacteria, and the habitats from which they were isolated, provide a new source pool for potential microorganisms to be exploited in the in situ bioremediation of marine sediments.     

Isolation and Characterization of Methanesulfonic Acid-Degrading Bacteria from the Marine Environment

Two methylotrophic bacterial strains, TR3 and PSCH4, capable of growth on methanesulfonic acid as the sole carbon source were isolated from the marine environment. Methanesulfonic acid metabolism in these strains was initiated by an inducible NADH-dependent monooxygenase, which cleaved methanesulfonic acid into formaldehyde and sulfite. The presence of hydroxypyruvate reductase and the absence of ribulose monophosphate-dependent hexulose monophosphate synthase indicated the presence of the serine pathway for formaldehyde assimilation. Cell suspensions of bacteria grown on methanesulfonic acid completely oxidized methanesulfonic acid to carbon dioxide and sulfite with a methanesulfonic acid/oxygen stoichiometry of 1.0:2.0. Oxygen electrode-substrate studies indicated the dissimilation of formaldehyde to formate and carbon dioxide for energy generation. Carbon dioxide was not fixed by ribulose bisphosphate carboxylase. It was shown that methanol is not an intermediate in methanesulfonic acid metabolism, although these strains grew on methanol and other one-carbon compounds, as well as a variety of heterotrophic carbon sources. These two novel marine facultative methylotrophs have the ability to mineralize methanesulfonic acid and may play a role in the cycling of global organic sulfur.     

北极海泥菌群的分离鉴定及生物学特性的初步研究

对从北极冰川海面下1500-4000m处的海泥样品中分离的8株冷适应细菌进行了生理特征和分子生物学研究。其中5株嗜冷菌、3株耐冷菌,利用16SrDNA通用引物对5株嗜冷菌基因组DNA进行扩增,测序得到其部分16SrDNA序列。经Blast调出与菌株16SrDNA同源的序列,按照Neighbor-Joining方法构建16SrDNA系统发育树。对8株细菌进行酶检测试验,结果表明其中有部分细菌产低温酶:N014产淀粉酶,R151产明胶酶,P371产纤维素酶。研究结果为进一步开发利用冷适应微生物产物提供参考依据。     

Method for the Isolation of Proteolytic Marine Bacteria

A two-layer plate consisting of a lower indicator layer of milk-agar and an upper nutrient agar layer was developed for isolating proteolytic marine bacteria.     

具有抑菌活性的海洋细菌的分离与鉴定

分离并筛选具有抑菌活性的海洋细菌对于开发和利用海洋微生物具有重要意义,该研究从6份海泥样品中共分离到78株海洋细菌,以6种细菌作为敏感指示菌,采用覆盖技术对分离菌株进行拮抗试验,17株海洋细菌具有抑菌活性,对其中2株具有较强抑菌活性的海洋细菌进行革兰氏染色,耐盐试验,运动性观察,过氧化氢酶测定,明胶液化试验,硫化氢产生试验,石蕊牛奶试验,糖类发酵,硝酸盐还原等特性分析,依据《伯杰氏细菌鉴定手册》进行分类鉴定,它们分别应归属为气单孢菌属（Aeromonas sp.）和假单孢菌属（Pseudomonas sp.）。     

Isolation of High Molecular Weight DNA from Marine Sponge Bacteria for BAC Library Construction

Metagenomics is a powerful tool for mining the genetic repositories from environmental microorganisms. Bacteria associated with marine sponges (phylum Porifera) are rich sources of biologically active natural products. However, to date, few compounds are discovered from the sponge metagenomic libraries, and the main reason might be the difficulties in recovery of high molecular weight (HMW) DNA from sponge symbionts to construct large insert libraries. Here, we describe a method to recover HMW bacterial DNA from diverse sponges with high quality for bacterial artificial chromosome (BAC) library construction. Microorganisms concentrated from sponges by differential centrifugation were embedded in agarose plugs to lyse out the HMW DNA for recovery. DNA fragments over 436 kb size were recovered from three different types of sponges, Halichondria sp., Haliclona sp., and Xestospongia sp. To evaluate the recovered DNA quality, the diversity of bacterial DNA comprised in the HMW DNA derived from sponge Halichondria sp. was analyzed, and this HMW DNA sample was also cloned into a shuttle BAC vector between Escherichia coli and Streptomyces sp. The results showed that more than five types of bacterial DNA, i.e., Proteobacteria, Nitrospirae, Cyanobacteria, Planctomycetes, and unidentified bacteria, had been recovered by this method, and an average 100 kb size insert DNA in a constructed BAC library demonstrated that the recovered HMW DNA is suitable for metagenomic library construction.     

洱海沉积物中反硝化细菌的分离与反硝化作用研究

自洱海十个点位的沉积物中富集筛选出101株反硝化细菌并从中筛选出1株较强反硝化能力的细菌,命名为EH314。该细菌接触酶(过氧化氢酶)试验、产硫化氢试验和淀粉水解均为阳性,葡萄糖氧化发酵实验结果为氧化菌,产脂酶(Tween 80)试验结果为阴性;初步鉴定该菌为产碱杆菌属细菌;对细菌反硝化能力进行测定发现,菌株EH314能有效地降解水体中的硝酸盐且反硝化可在有氧条件下进行。     

Isolation and Characterization of a GDSL Esterase from the Metagenome of a Marine Sponge-associated Bacteria

Using a metagenome library constructed from a bacterial associated with a marine sponge Hyrtios erecta, we identified a novel esterase that belongs to the SGNH hydrolase superfamily of esterases. The substrate specificity of EstHE1 was determined using p-nitrophenyl (pNP) ester (C2: acetate, C4: butylate, C6: caproate, C12: laurate, C16: palmitate). EstHE1 exhibited activity against C2 (5.6 U/mg), C4 (5.1 U/mg), and C6 (2.8 U/mg) substrates. The optimal temperature for EstHE1 esterase activity of the pNP acetate substrate was 40°C, and EstHE1 retained 60% of its enzymatic activity in the 30–50°C range. This esterase showed moderate thermostability, retaining 58% of its activity even after preincubation for 12 h at 40°C. EstHE1 also maintained activity in high concentrations of NaCl, indicating that this esterase is salt-tolerant. Thus, EstHE1 has the thermal stability and salt tolerance necessary for use as an industrial enzyme.     

Isolation and Identification of Photosynthetic Bacteria (Rhodospirillaceae) from Antarctic Marine and Freshwater Sediments

Sediment samples obtained from three freshwater lakes and off-shore coastal marine waters on Signy Island, South Orkney Islands, Antarctica have been inoculated into selective enrichment media for purple non-sulphur bacteria (Rhodospirillaceae). From the freshwater sediments strains of Rhodopseudomonas sphaeroides (1), Rhodopseudomonas palustris (1), Rhodospirillum fulvum (1), Rhodospirillum molischanum (1) and Rhodomicrobium vannielii (3) have been isolated. The only purple non-sulphur bacteria obtained from Lake 10 (Amos lake) were strains of Rhodomicrobium vannielii which were able to tolerate hydrogen sulphide (up to 0.04% w/v) found in this lake. Growth of all the other isolates is inhibited by the presence of hydrogen sulphide. Marine sediments yielded strains of Rhodopseudomonas palustris and Rhodomicrobium vannielii . All the isolates grow optimally at temperatures between 25 and 30 °C. mean generation times vary between 8 and 10.7 h depending on species. There is no evidence of cold adaptation in any of the strains studied.     

Nutritional Behavior,Morphogenesis Cycle and Sediment Consolidation Capabilities of the Calcareous Bacteria Derived from Coastal Marine Sediments

Samples from stones and sediments of a coastal site in the Bay of Bengal (Indian Ocean) yielded as many as 39 new bacterial isolates capable of precipitating calcium carbonate (CaCO₃). Molecular identification revealed that these bacteria belonged predominantly to the phyla Firmicutes and Proteobacteria. Culture studies showed that nitrogen sources controlled the metabolic pathway of crystal precipitation, which was restricted to three reaction pathways, namely the deamination of amino acids, ureolytic nitrate reduction and dissimilatory nitrate reduction. The sequence of crystal morphogenesis clearly showed that bacterial precipitation of CaCO₃ led to predominantly spherical structures with time. The present investigation provides the first demonstration of the bacterial contribution and mechanisms involved in the calcareous consolidation of stones and sediments by bacteria in the marine environment.     

Properties of Alginate Lyases from Marine Bacteria

Alginate lyases (EC 4.2.2.3) from two marine bacteria were isolated and partially characterized. A cell-bound lyase from isolate A3 had a molecular weight of approximately 100,000 and cleaved mannuronate blocks, apparently in an exo manner. A lyase recovered from the culture medium of isolate W3 was soluble in saturated ammonium sulfate, cleaved guluronate blocks, apparently in an endo manner, and had a molecular weight of 35,000. The thiobarbiturate test and urea-polyacrylamide gel electrophoresis were used to determine substrate specificity and mode of substrate cleavage by the enzymes.     

Isolation and Analysis of Bacteria with Antimicrobial Activities from the Marine Sponge Haliclona simulans Collected from Irish Waters

Samples of the marine sponge Haliclona simulans were collected from Irish coastal waters, and bacteria were isolated from these samples. Phylogenetic analyses of the cultured isolates showed that four different bacterial phyla were represented; Bacteriodetes, Actinobacteria, Proteobacteria, and Firmicutes. The sponge bacterial isolates were assayed for the production of antimicrobial substances, and biological activities against Gram-positive and Gram-negative bacteria and fungi were demonstrated, with 50% of isolates showing antimicrobial activity against at least one of the test strains. Further testing showed that the antimicrobial activities extended to the important pathogens Pseudomonas aeruginosa, Clostridium difficile, multi-drug-resistant Staphylococcus aureus, and pathogenic yeast strains. The Actinomycetes were numerically the most abundant producers of antimicrobial activities, although activities were also noted from Bacilli and Pseudovibrio isolates. Surveys for the presence of potential antibiotic encoding polyketide synthase and nonribosomal peptide synthetase genes also revealed that genes for the biosynthesis of these secondary metabolites were present in most bacterial phyla but were particularly prevalent among the Actinobacteria and Proteobacteria. This study demonstrates that the culturable fraction of bacteria from the sponge H. simulans is diverse and appears to possess much potential as a source for the discovery of new medically relevant biological active agents.     

Bioactive Compounds from Marine Bacteria and Fungi

Marine bacteria and fungi are of considerable importance as new promising sources of a huge number of biologically active products. Some of these marine species live in a stressful habitat, under cold, lightless and high pressure conditions. Surprisingly, a large number of species with high diversity survive under such conditions and produce fascinating and structurally complex natural products. Up till now, only a small number of microorganisms have been investigated for bioactive metabolites, yet a huge number of active substances with some of them featuring unique structural skeletons have been isolated. This review covers new biologically active natural products published recently (2007–09) and highlights the chemical potential of marine microorganisms, with focus on bioactive products as well as on their mechanisms of action.     

Isolation and Characterization of Two Groups of Novel Marine Bacteria Producing Violacein

Thirteen strains of novel marine bacteria producing a purple pigment were isolated from the Pacific coast of Japan. They were divided into two groups based on their 16S ribosomal RNA gene sequences, and both groups of bacteria belonged to the genus Pseudoalteromonas. The UV-visible spectrum of the pigment was identical to those of violacein, a pigment produced by several species of bacteria including Chromobacterium violaceum, an opportunistic pathogen. Further analysis of the chemical structure of the pigment by mass spectroscopy and nuclear magnetic resonance spectroscopy showed that the pigment was violacein. The high purity of violacein in the crude extract enabled us to employ simple and nonpolluting procedures to purify the pigment. Isolated bacteria may be useful as a C. violaceum substitute for the safe production of violacein.     

Isolation of Marine Bacteria Capable of Producing Specific Lyases for Alginate Degradation

Alginate lyases (EC 4.2.2.3) were isolated from cultures of several marine bacterial isolates. The lyases were induced by native alginate and had activity toward both the mannuronic acid and the guluronic acid blocks of the alginate polymer. The guluronic acid-specific lyase was recovered from the medium, whereas the mannuronic acid-specific lyase was retained with the bacteria.     

Volatile Fatty Acids and Hydrogen as Substrates for Sulfate-Reducing Bacteria in Anaerobic Marine Sediment

The addition of 20 mM MoO₄²⁻ (molybdate) to a reduced marine sediment completely inhibited the SO₄²⁻ reduction activity by about 50 nmol g⁻¹ h⁻¹ (wet sediment). Acetate accumulated at a constant rate of about 25 nmol g⁻¹ h⁻¹ immediately after MoO₄²⁻ addition and gave a measure of the preceding utilization rate of acetate by the SO₄²⁻-reducing bacteria. Similarly, propionate and butyrate (including isobutyrate) accumulated at constant rates of 3 to 7 and 2 to 4 nmol g⁻¹ h⁻¹, respectively. The rate of H₂ accumulation was variable, and a range of 0 to 16 nmol g⁻¹ h⁻¹ was recorded. An immediate increase of the methanogenic activity by 2 to 3 nmol g⁻¹ h⁻¹ was apparently due to a release of the competition for H₂ by the absence of SO₄²⁻ reduction. If propionate and butyrate were completely oxidized by the SO₄²⁻-reducing bacteria, the stoichiometry of the reactions would indicate that H₂, acetate, propionate, and butyrate account for 5 to 10, 40 to 50, 10 to 20, and 10 to 20%, respectively, of the electron donors for the SO₄²⁻-reducing bacteria. If the oxidations were incomplete, however, the contributions by propionate and butyrate would only be 5 to 10% each, and the acetate could account for as much as two-thirds of the SO₄²⁻ reduction. The presence of MoO₄²⁻ seemed not to affect the fermentative and methanogenic activities; an MoO₄²⁻ inhibition technique seems promising in the search for the natural substrates of SO₄²⁻ reduction in sediments.     

Isolation of Marine Bacteria by In Situ Culture on Media-Supplemented Polyurethane Foam

Polyurethane foam (PUF) supplemented with various agar media was used in situ to trap marine bacteria and it consequently provided a substrate on which they could be cultivated while exposed to natural seawater in the coral reef area. The bacterial population on the PUF blocks was analyzed by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. Changing the composition of the cultivation medium in the PUF blocks and selecting different sampling sites resulted in different bacteria being detected on the PUF blocks. For example, iron-utilizing (IU) bacteria, siderophore-producing (SP) bacteria, and petroleum-degrading (PD) bacteria were isolated from PUF blocks and it was discovered that IU and SP contained iron and PD contained hydrocarbon. This method opens up the possibility for isolating novel and useful marine bacteria.     

Screening and Isolation of PHB-Producing Bacteria in a Polluted Marine Microbial Mat

The characteristics of microbial mats within the waste stream from a seafood cannery were compared to a microbial community at a pristine site near a sandy beach at Puerto San Carlos, Baja California Sur, Mexico. Isolation of poly-beta-hydroxybutyrate (PHB)-producing bacteria, recognition of brightly refractile cytoplasmatic inclusions, lipophilic stains with Sudan Black and Nile Red, and chemical extraction of PHB were used as a culture-dependent strategy for the detection of PHB-producing bacteria. The culture-independent approach included denaturing gradient gel electrophoresis of phylotypes of 16S rRNA of microbial communities from environmental samples. Significant differences in community structure were found among the polluted and pristine sites. These differences were correlated with the physicochemical characteristics of the seawater column. At the polluted site, the seawater was rich in nutrients (ammonia, phosphates, and organic matter), compared to the pristine location. Partial sequencing of 16S rDNA of cultures of bacteria producing PHB included Bacillus and Staphylococcus at both sites; Paracoccus and Micrococcus were found only at the polluted site and Rhodococcus and Methylobacterium were found only at the pristine site. Bands of the sequences of 16S rDNA from both field samples in the denaturing gradient gel electrophoresis (DGGE) analyses affiliated closely only with bacterial sequences of cultures of Bacillus and Staphylococcus. High concentrations of organic and inorganic nutrients at the polluted site had a clear effect on the composition and diversity of the microbial community compared to the unpolluted site.     

Construction of Plasmid Vectors for Screening Replicons from Gram-Positive Bacteria and Their Use as Shuttle Cloning Vectors

Plasmids play a central role in engineering recombinant bacteria because they are the primary vehicles used to manipulate targeted sequences. In some cases, bacteria of interest are poorly provided with suitable tools for these molecular or genetic manipulations. In this context, we constructed from two shuttle cloning vectors, pUCB2871 and pUCB2872, the basic vectors pUCB30 and pUCB31, which could represent suitable tools to isolate replicons from Gram-positive bacteria. These plasmid vectors are characterized by the following after-features: (a) the pUC origin of replication is unable to replicate in Gram-positive bacteria; (b) an erythromycin-resistance encoding gene that is functional in both Gram-negative and -positive bacteria; (c) the pUC19 multiple cloning site (MCS) within the lacZα reporter gene; and (4) an additional multiple cloning site (MCS). Cloning replicons from Gram-positive bacteria in this additional MCS would allow the derivative vectors to function directly as shuttle cloning vectors.