Isolation and phylogenetic analysis of cultivable manganese bacteria in sediments from the Arctic Ocean

Isolation, molecular identification and phylogenetic analysis were carried out to investigate the biodiversity of manganese bacteria in sediments which were collected from the Arctic Ocean during the 2nd Chinese Arctic Scientific Expedition. 21 and 19 species of cultivable strains were isolated from sediments at Stations P11 and S11, respectively, according to their distinct morphological character on the screening plate of manganese medium. Molecular identification and phylogenetic analysis showed that the cultivable manganese bacteria from Station P11 were basically composed of γ-Proteobacteria (γ subgroup of the Proteobacteria branch of the domain Bacteria) and Actinobacteria, which accounted for 86% and 14%, respectively. The isolates of γ-Proteobacteria mainly included Psychrobacter, Shewanella, Acinetobacter and Marinobacter, of which Psychrobacter was the major genus, which accounted for 67% of the γ-Proteobacteria. The cultivable manganese bacteria from Station S11 included α-Proteobacteria, γ-Proteobacteria and Flavobacteria of Bacteroides. The γ-Proteobacteria mainly included Shewanella, Marinomonas and Alteromonas. The majority of α-Proteobacteria was Sphingomonas. The phylogenetic analysis indicated that bacteria from sediments at Stations P11 and S11 had different cultivable manganese microbial communities. All tested strains had higher resistance to Mn2+, of which Marinomonas sp. S11-S-4 had the highest resistant ability.     

Correlation between numerical dynamics and reproductive behavior in Cypripedium lentiginosum

Isolation, molecular identification and phylogenetic analysis were carried out to investigate the biodiversity of manganese bacteria in sediments which were collected from the Arctic Ocean during the 2^nd Chinese Arctic Scientific Expedition. 21 and 19 species of cultivable strains were isolated from sediments at Stations P11 and S11, respectively, according to their distinct morphological character on the screening plate of manganese medium. Molecular identification and phylogenetic analysis showed that the cultivable manganese bacteria from Station P11 were basically composed of γ-Proteobacteria (γ subgroup of the Proteobacteria branch of the domain Bacteria) and Actinobacteria, which accounted for 86% and 14%, respectively. The isolates of γ-Proteobacteria mainly included Psychrobacter, Shewanella, Acinetobacter and Marinobacter, of which Psychrobacter was the major genus, which accounted for 67% of the γ-Proteobacteria. The cultivable manganese bacteria from Station S11 included α-Proteobacteria, γ-Proteobacteria and Flavobacteria of Bacteroides. The γ-Proteobacteria mainly included Shewanella, Marinomonas and Alteromonas. The majority of α-Proteobacteria was Sphingomonas. The phylogenetic analysis indicated that bacteria from sediments at Stations P11 and S11 had different cultivable manganese microbial communities. All tested strains had higher resistance to Mn²⁺, of which Marinomonas sp. S11-S-4 had the highest resistant ability.     

Isolation and phylogenetic analysis of cultivable manganese bacteria in sediments from the Arctic Ocean

Isolation, molecular identification and phylogenetic analysis were carried out to investigate the biodiversity of manganese bacteria in sediments which were collected from the Arctic Ocean during the 2^nd Chinese Arctic Scientific Expedition. 21 and 19 species of cultivable strains were isolated from sediments at Stations P11 and S11, respectively, according to their distinct morphological character on the screening plate of manganese medium. Molecular identification and phylogenetic analysis showed that the cultivable manganese bacteria from Station P11 were basically composed of γ-Proteobacteria (γ subgroup of the Proteobacteria branch of the domain Bacteria) and Actinobacteria, which accounted for 86% and 14%, respectively. The isolates of γ-Proteobacteria mainly included Psychrobacter, Shewanella, Acinetobacter and Marinobacter, of which Psychrobacter was the major genus, which accounted for 67% of the γ-Proteobacteria. The cultivable manganese bacteria from Station S11 included α-Proteobacteria, γ-Proteobacteria and Flavobacteria of Bacteroides. The γ-Proteobacteria mainly included Shewanella, Marinomonas and Alteromonas. The majority of α-Proteobacteria was Sphingomonas. The phylogenetic analysis indicated that bacteria from sediments at Stations P11 and S11 had different cultivable manganese microbial communities. All tested strains had higher resistance to Mn²⁺, of which Marinomonas sp. S11-S-4 had the highest resistant ability.     

Effects of different molecular weight fractions of dissolved organic matter on the growth of bacteria,algae and protozoa from a highly humic lake

Effects of different molecular size fractions (< 1000 MW, < 10 000 MW, < 100 000 MW and <0.1 μm) of dissolved organic matter (DOM) on the growth of bacteria, algae and protozoa from a highly humic lake were investigated. DOM from catchment drainage water as well as from the lake consisted mostly (59–63%) of high molecular weight (HMW) compounds (> 10 000 MW). With excess inorganic nutrients, the growth rate and yield of bacteria were almost identical in all size fractions. However, in < 1000 MW fractions and with glucose added, a longer lag phase occurred. Without added nutrients both the growth rates and biomasses of bacteria decreased towards the smaller size fractions and the percentage of dissolved organic carbon (DOC) used during the experiment and the growth efficiency of bacteria were lower than with excess nutrients. The growth efficiency of bacteria was estimated to vary between 3–66% in different MW fractions, largely depending on the nutrient concentrations, but the highest growth efficiencies were observed in HMW fractions and with glucose. The growth of algae was clearly lowest in the < 1000 MW fraction. In dim light no net growth of algae could be found. In contrast, added nutrients substantially enhanced algal growth and in deionized water with glucose, algae achieved almost the same growth rate and biomass as in higher MW fractions of DOM. The results suggested that bacteria and some algae were favoured by DOM, but protozoans seemed to benefit only indirectly, through bacterial grazing. The utilization of DOM by bacteria and algae was strongly affected by the availability of phosphorus and nitrogen.     

桑粒肩天牛幼虫肠道菌群的研究

Intestinal flora of 47 Apriona germari(Hope) larvae,collected from fields,had been isolated and identified.The results showed that the predominant bacteria were Staphylococcus.Its viable count was 7.63±0.21,and the detection rate was 100%.Meanwhile,a strain of cellulose-utilizing bacterium was isolated from the fore-midgut fluid of A.germari larvae with the cellulose-congo red agar medium.The bacterium was tentatively identified as Cellulomonas.The detection rate of the cellulolytic bacterium was 23.40%,and…     

Preclinical transplantation and safety of HS/PCs expanded from human umbilical cord blood

AIM: To expand hematopoietic/progenitor stem cells (HS/PCs) from umbilical cord blood (UCB) and prepare the HS/PC product, and analyze preclinical transplantation and safety of HS/PC product. METHODS: Human bone marrow-derived mesenchymal stem cells (MSCs) were used as feeder cells to expand HS/PCs from UCB in a serum-free culture system. The proliferation potential of HS/PCs was analyzed. The expanded HS/PCs were suspended in the L-15 medium to prepare the HS/PC product. The contamination of bacteria, fungi and mycoplasmas, the infection of exogenous virus, the concentration of bacterial endotoxin, and the SCF residual in HS/PC product were determined. Finally, cells from the HS/PC product with or without bone marrow-derived mesenchymal stem cells (BM-MSCs) were transplanted into the irradiated NOD/SCID mice to determine the in vivo engraftment potential. RESULTS: After co-culture for 10 d, the total nuclear cells (TNCs) increased 125-fold, and CD34 + cells increased 43-fold. The granulocyte-macrophage colonyforming cells (GM-CFCs) and erythroid colony-forming cells (E-CFCs) increased 3.3and 4.7-fold respectively. The expanded cells were collected and prepared as the expanded product of HS/PCs by re-suspending cells in L-15 medium. For preclinical safety, the HS/PC product was analysed for contamination by bacteria, fungi and mycoplasmas, the bacterial endotoxin concentration and the SCF content. The results showed that the HS/PC product contained no bacteria, fungi or mycoplasmas. The bacterial endotoxin concentration was less than the detection limit of 6 EU/mL, and residual SCF was 75 pg/mL. Based on clinical safety, the HS/PC product was qualified for clinical transplantation. Finally, the HS/PC product was transplanted the irradiated mice where it resulted in rapid engraftment of hematopoietic cells. CONCLUSION: HSPC product prepared from UCB in the serum-free culture system with hMSCs as feeder cells should be clinically safe and effective for clinical transplantation.     

大熊猫初生胎儿及幼仔组织器官的病理学研究

To elucidate the cause of prenatal and early postnatal death in giant panda, pathological studies have been carried out on paraffin-fixed tissue sections from two fetuses and four cubs. The fetuses appeared to have classical atrophic changes in lung and hemorrhage in multiple organs, whereas the cubs showed purulent inflmnmation in various organs, most profound in lung and umbilicus. Localized infusion of bacteria and neutrophils were identified in the focus. Acute enteritis and hepatitis were also observed, as well as purulent encephalitis in one case. Variable degrees of congestion, hemorrhage, denaturalization and putrescence were evident in heart, liver, spleen, kidney, intestines, and lymph nodes. The results indicated that the fetuses died from suffocation whereas the cubs died from infection.     

Community composition and spatial variation of bacteria in the sediments of a eutrophic fresh water urban lake， East Lake， Wuhan， China北大核心CSCD

［Objective］ Sediment bacteria are the important biological factors for remediating of eutrophic environments. To enrich our understanding of the bacteria communities in eutrophic urban lake sediments for better environment protection and pollution control in urban lake eco-systems, we resolved the composition of bacteria communities and their spatial variation in the sediments of a middle-size eutrophic urban lake, East Lake. ［Methods］We used 16S rRNA gene RFLP and sequencing methods to generate the phylogeny information of the bacteria community, used principal coordinates analysis （PCoA） and canonical correspondence analysis （CCA） methods to resolve the relationship between East Lake and other lakes, and the relationship between environmental factors and the bacteria communities. ［Results］ Sediments inhabited 13 phyla and 2 unclassified clusters. PCoA further revealed that the bacteria communities in three sub-lakes of East Lake sediments were closely related to the communities in similar eutropic lake environments, and divergent from the hypereutrophic sub-lake Miao Lake, which was also found to inhabit a relative abundant amount of Thermogymnomonas-type archaea. CCA further revealed that the distribution of bacteria was closely correlated with the carbon, nitrogen and phosphate contents in the sediments. ［Conclusion］The environment factors regulated the bacteria community composition and distribution. The results of this study providereference to the research, protection and pollution control on urban lake eco-systems.     

16S rDNA-based phylogeny of non-symbiotic bacteria of Entorno-pathogenic nematodes from infected insect cadavers

Using 16S rDNA gene sequencing technique, three different species of non-symbiotic bacteria of entomopatho-genic nematodes (EPNs) (Steinernema sp.and Heterorhabditis sp.) were isolated and identified from infected insect cadavers(Galleria mellonella larvae) after 48-hour post infections.Sequence similarity analysis revealed that the strains SRK3, SRK4 and SRK5 belong to Ochrobactrum cytisi,Schineria larvae and Ochrobactrum anthropi,respectively.The isolates O.anthropi and S.larvae were found to be associated with Heterorhabditis indica strains BDU-17 and Yer-136,respectively,whereas O.cytisi was associated with Steinernema siamkayai strain BDU-87. Phenotypically, temporal EPN bacteria were fairly related to symbiotic EPN bacteria (Photorhabdus and Xenorhabdus genera). The strains SRK3 and SRK5 were phylogeographically similar to several non-symbionts and contaminated EPN bacteria isolated in Germany(LMG3311T) and China (X-14),while the strain SRK4 was identical to the isolates of S.larvae (L1/57,L1/58, L1/68 and L2/11) from Wohlfahrtia magnifica in Hungary.The result was further confirmed by RNA secondary structure and minimum energy calculations of aligned sequences.This study suggested that the non-symbionts of these nematodes are phylogeographically diverged in some extent due to phase variation.Therefore,these strains are not host-dependent, but environment-specific isolates.     

Extraction of Magnetosome from Acidthiobacillus ferrooxidans

In physiological property and growing environment aspects, there are some similarities betweenAcidthiobacillus ferrooxidans（ATF） and magnetotactic bacteria. ATF shows magnetotacxis under the microscope. Themagnetosome can be extracted from ATF by using the extracting method of magnetosome from magnetotactic bacteria.The membrane of ATF was crushed by ultrasonic wave and the magnetosomes in the ATF which mainly contains Fethrough chemical detection were attracted by using magnetite. After purifing with sucrose density gradient centrifugationand washing by PBS, the magnetosomes can be seen clearly through a transmission electron microscope. The resultsindicate there are a small amount of intracellular magnetosomes which made the ATF be magnetotactic under the applied magnetic field. It is the first time to find that ATF was magnetotactic. The magnetotaxis of ATF can be utilizedand isolated by the magnetotactic properties to gain the high - performance ATF strains of mineral leaching, whichhave high activity and different magnetism.     

L-Form Induction, Morphology, and Development in Two Related Strains of Erysipelothrix rhusiopathiae

Two related strains of Erysipelothrix rhusiopathiae, one the parent and the other an L-form revertant, were studied for their propensity or ability to produce L-forms under the influence of penicillin. The parent strain produced L-forms in nutrient solid media in an osmolarity range between 0.85 and 5.0% NaCl concentration whereas the revertant strain did so between 0.5 and 3.0% NaCl concentration. When various hyperosmolar media were tried without penicillin, recovery of L-forms from the revertant strain was optimal at a salt concentration of 2.0%, whereas the parent strain occasionally produced a few L-forms on 3.0% salt medium only. The process of penicillin-induced transformation from bacteria to L-form followed an unusual morphological sequence, beginning with beading of the bacterial body, followed by disintegration into granules from which the L-form colony derived. No large bodies were seen during the initial process of L-form induction, but they evolved later from the original granules and had the potential to reproduce L-type growth. The spontaneous development of L-forms in hyperosmolar media had a different morphological sequence starting with elongation of the bacteria into filaments which later developed polar and central dilatations from which granules and L-type growth developed. The differences in biological behavior between these related bacterial strains suggest that the revertant strain developed new properties, probably of genetic origin. Consequently, the assumption that L-forms revert to the "parent" bacteria may not always be justified. It can be made only after the biological properties of the parent and the revertant organisms have been properly identified.     

伤寒杆菌和甲型副伤寒杆菌的稳定L型外膜蛋白和染色体DNA酶切研究

非高渗培养基传代培养的伤寒杆菌和甲型副伤寒杆菌的稳定L型丧失了主要外膜蛋白、特异性表面抗原和染色体DNA部分片段,保留了沙门氏菌共同的内部抗原和形成L型独特的表面抗原。提示伤寒杆菌和甲型副伤寒杆菌的稳定L型多种特性的丧失同其基因或主要外膜蛋白的缺失有关。     

Lipid composition of Staphylococcus aureus and its derived L-forms.

Two strains of Staphylococcus aureus (Newman and Tazaki) and their derived L-forms were cultured in serum-containing broth and the differences in their lipid compositions were analyzed. Cardiolipin accounted for more than 50% of the total phospholipid phosphorus in L-forms, but for less than 25% in parent bacteria. The cardiolipin content of L-forms was very high through all growth phases, although it increased gradually as growth proceeded. Significant amounts of cholesterol and its esters were present in parent strains and L-forms, all of which incorporated serum cholesterol into the cell membrane. On the other hand, they could be detected in the L-forms but not in the parent strains when they were cultured in serum-free broth. To examine the ability of L-forms to synthesize cholesterol, the cholesterol content of L-forms cultured in serum-free broth was compared with that of the medium. The results indicated that staphylococcal L-forms could synthesize cholesterol and its esters. These differences in lipid composition suggested that modification of membrane lipids may occur as an adaptational change in response to the disappearance of the cell wall.     

Biological properties of L-forms and their parent bacteria

L-forms of Staphylococcus aureus, Streptococcus faecalis, Listeria monocytogenes and Salmonella typhy and their parent bacteria were examined for biological properties and compared with their parental forms. Some L-forms differed from their parent bacteria and required a longer incubation period.     

A novel method for differentiating L-form bacteria from their parental form using the Hucker Gram staining technique

L-forms of Enterococcus faecium, Bacillus subtilis and Pseudomonas syringae pv. phaseolicola were differentiated from their parent, cell-walled forms by a modified Gram staining technique. The addition of glutaraldehyde to the culture medium fixed the cells to prevent lysis of the L-forms. The cell-walled forms exhibited typical Gram staining reactions whereas the L-forms remained red due to the counterstain. L-forms were easily differentiated from cell-walled forms by their size and morphology which was made more obvious by the staining procedure. This is a very rapid and easy technique which distinguishes L-form bacteria from cell-walled organisms.     

The chemical composition of the membranes of protoplasts and L-forms of Staphylococcus

Membrane fractions were prepared from Staphylococcus aureus H and 100 after dissolution of the cell walls by a lytic enzyme from Streptomyces griseus. Membranes were also prepared from the L-forms derived from the same strains. The membranes were analysed for protein, lipid, carbohydrate and RNA contents, and the fatty acid composition of the lipids was determined. A branched-chain saturated C(15) acid was the major component in all samples, and the correspondence between L-forms and parent bacteria was fairly close. The lipids were separated into non-polar-lipid, glycolipid and phospholipid fractions; the L-forms contained a little more neutral lipid and much more glycolipid than the parent bacteria. In all membranes the glycolipid, which accounted for all the carbohydrate present, was a diglucosyl diglyceride. The major phospholipids of the protoplast membranes were phosphatidylglycerol and some lipoamino acids (lysine and a little alanine). On the other hand, diphosphatidylglycerol was the chief phospholipid found in L-form membranes.     

Growth and adhesion of Enterococcus faecium L-forms

Abstract Comparisons of growth and surface colonisation of Enterococcus faecium L-forms and their cell-walled forms were undertaken to produce information about their ability to form sessile cells. The growth of L-forms in liquid culture was slower than that of the parent. This was reflected in their longer lag phase and slower specific growth rates: 0.16 h⁻¹ for the L-form and 0.81 h⁻¹ for the parent. Although E. faecium L-forms attached to a silastic rubber surface, the attached population density was 10–100-fold less than that of the parent. Confluent biofilms on the silastic surfaces were not observed for either bacterial form. Comparison of the attachment of E. faecium L-form and parent may provide important information on how bacteria overcome host defence mechanisms and antibiotic treatment.     

Induction, growth and antibiotic production of Streptomyces viridifaciens L-form bacteria

AIMS: To induce, cultivate and investigate the characteristics of L-form bacteria derived from the filamentous actinomycete Streptomyces viridifaciens. METHODS AND RESULTS: L-forms were induced in a liquid medium supplemented with lysozyme and penicillin. A stable culture which no longer required inducing agents but could still revert, was obtained by the twelfth subculture. The specific growth rate of stable L-forms was faster (0.751) than unstable L-forms (0.361). After the exponential growth phase, the cell diameter continued to increase, as did the percentage of vacuoles. Morphologically, the L-forms appeared as spherical bodies with no signs of differentiation and were sensitive to osmotic stress, indicating removal of the cell wall. The L-forms produced secondary metabolites although much lower levels of antibiotic were assayed in the L-forms compared with the cell walled forms. CONCLUSION: Stable L-form bacteria were induced from S. viridifaciens and their growth characterized. The L-forms produced secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Stable Streptomyces L-forms were induced and have potential as biocontrol agents.     

Polyacrylamide Gel Identification of Bacterial L-Forms and Mycoplasma Species of Human Origin

Polyacrylamide gel electrophoretic patterns of acidified phenol extracts prepared from whole cells can be used for the identification of bacterial L-forms and Mycoplasma species of human origin. Ten human Mycoplasma serotypes and eight L-forms belonging to five different genera were studied. The gel patterns were sufficiently distinct and reproducible that it was possible not only to identify L-forms at the genus level (group with streptococci) and different Mycoplasma serotypes but also to differentiate between the two of them. The parentage of L-forms of Streptobacillus moniliformis L1, Listeria monocytogenes, Streptococcus MG, and Staphylococcus aureus Smith strain was established by relating their gel patterns directly to parent bacteria. It was found that an L-form designated S. moniliformis An (ATCC 14220) was actually an L-form of Proteus. In addition, it was shown electrophoretically that no relationship existed between the Streptococcus MG L-form and M. pneumoniae. The applicability of this method as a diagnostic and taxonomic tool for the differentiation of L-forms and mycoplasmas is discussed.     

L-forms ofHaemophilus influenzae: Growth and reversion as influenced by a strain ofNeisseria perflava

Factors excreted into the medium by various bacteria and fungi exert a significant effect on the growth and revertibility of bacterial L-forms. Studies with L-forms ofHaemophilus influenzae have been facilitated by a strain ofNeisseria perflava, which not only promotes the growth of the L-forms on media of normal osmolarity (horse blood agar), but also enhances the revertibility of the L-forms to the parent bacterium. L-forms derived fromN. perflava also exhibit this growth-enhacing property, an effect that does not appear to be related to X and V factors. Preliminary characterization of the active component produced byNeisseria perflava indicates that it is present in broth filtrate, diffusible through agar, heat labile, and of large molecular size.