Genetics and Regulation of Chitobiose Utilization in Borrelia burgdorferi

Borrelia burgdorferi spends a significant proportion of its life cycle within an ixodid tick, which has a cuticle containing chitin, a polymer of N-acetylglucosamine (GlcNAc). The B. burgdorferi celA, celB, and celC genes encode products homologous to transporters for cellobiose and chitobiose (the dimer subunit of chitin) in other bacteria, which could be useful for bacterial nutrient acquisition during growth within ticks. We found that chitobiose efficiently substituted for GlcNAc during bacterial growth in culture medium. We inactivated the celB gene, which encodes the putative membrane-spanning component of the transporter, and compared growth of the mutant in various media to that of its isogenic parent. The mutant was no longer able to utilize chitobiose, while neither the mutant nor the wild type can utilize cellobiose. We propose renaming the three genes chbA, chbB, and chbC, since they probably encode a chitobiose transporter. We also found that the chbC gene was regulated in response to growth temperature and during growth in medium lacking GlcNAc.     

紫外诱变菌降解菜籽饼粕中粗蛋白的实验研究

采用紫外照射法对原始菌株进行诱变和筛选,得到一株降解饼粕中粗蛋白能力较强的变异菌株UV—A,能使饼粕中粗蛋白的含量降到15．46％,是原始菌株的1．4倍。把该变异菌株接入到装有12．5kg菜籽饼粕的40L塑料箱内进行固体发酵,发现饼粕中粗蛋白的含量降解到8．9％。并通过正交设计试验确定变异菌株UV—A的培养条件为：NaCl5％,温度30℃,pH7．0,培养时间48h,其中温度对其影响最大。     

高效降解生活污水中COD的根际微生物的分离筛选

采用平板划线法从人工湿地的芦苇、美人蕉的根际土壤中分离出若干细菌、真菌、放线菌菌株,在实验室条件下检测了这些菌株对灭菌生活污水和自然生活污水COD的去除效果,结果表明4株细菌、1株放线菌、1株真菌对灭菌生活污水和自然生活污水COD均具有较高的去除率。4株细菌在降解灭菌生活污水COD的去除率在48 h后依次为75.4%、78.7%、83.5%、69.8%;其在降解未灭菌生活污水COD的去除率在48 h后依次为43.4%、47.8%、50.7%、36.8%;真菌对灭菌和未灭菌生活污水COD的去除率在48 h后分别为60.2%、41.3%;放线菌对灭菌和自然生活污水COD 48 h后的去除率分别为57.8%、46.4%。这几株高效降解COD的湿地根际微生物具有潜在的应用价值。     

Quantification of Frankia Strains and Other Root-Associated Bacteria in Pure Cultures and in the Rhizosphere of Axenic Seedlings by High-Performance Liquid Chromatography-Based Muramic Acid Assay

Application of a high-performance liquid chromatography-based muramic acid assay with precolumn fluorescence derivatization to quantification of root-associated bacteria was studied both in pure cultures and in the rhizosphere of axenic Festuca rubra seedlings. Quantities of muramic acid from acid-hydrolyzed cells of Frankia strains, Streptomyces griseoviridis, Enterobacter agglomerans, Klebsiella pneumoniae, Pseudomonas sp., and Bacillus polymyxa were mostly proportional to the respective cell protein and carbon quantities, but in some strains, culture age and particularly sporulation affected these ratios considerably. The muramic acid/cell protein ratio was generally 2 to 4 times higher in strains of the two actinomycete genera, Frankia and Streptomyces, than in the rest of the strains. Quantification of Frankia strains, S. griseoviridis, E. agglomerans, and Pseudomonas sp. was also attempted from the rhizosphere of F. rubra seedlings which had been inoculated with pure cultured bacteria and incubated briefly. It was possible to quantify Frankia cells by use of the muramic acid assay from both the root and the growth medium, whereas cells of the rest of the bacterial genera could only be detected in the medium. The detection limit for muramic acid was about 10 ng/ml hydrolysis volume, and from the Festuca rhizosphere, 28 to 63% of the muramic acid in the Frankia inoculum was recovered.     

Spatial Distribution of Bacterial Communities and Phenanthrene Degradation in the Rhizosphere of Lolium perenne L.

Rhizodegradation of organic pollutants, such as polycyclic aromatic hydrocarbons, is based on the effect of root-produced compounds, known as exudates. These exudates constitute an important and constant carbon source that selects microbial populations in the plant rhizosphere, modifying global as well as specific microbial activities. We conducted an experiment in two-compartment devices to show the selection of bacterial communities by root exudates and phenanthrene as a function of distance to roots. Using direct DNA extraction, PCR amplification, and thermal gradient gel electrophoresis screening, bacterial population profiles were analyzed in parallel to bacterial counts and quantification of phenanthrene biodegradation in three layers (0 to 3, 3 to 6, and 6 to 9 mm from root mat) of unplanted-polluted (phenanthrene), planted-polluted, and planted-unpolluted treatments. Bacterial community differed as a function of the distance to roots, in both the presence and the absence of phenanthrene. In the planted and polluted treatment, biodegradation rates showed a strong gradient with higher values near the roots. In the nonplanted treatment, bacterial communities were comparable in the three layers and phenanthrene biodegradation was high. Surprisingly, no biodegradation was detected in the section of planted polluted treatment farthest from the roots, where the bacterial community structure was similar to those of the nonplanted treatment. We conclude that root exudates and phenanthrene induce modifications of bacterial communities in polluted environments and spatially modify the activity of degrading bacteria.     

Degradation of Polycyclic Aromatic Hydrocarbons in the Rhizosphere of Festuca arundinacea and Associated Microbial Community Changes

A phytoremediation growth chamber study was conducted to evaluate the contribution of soil microbial diversity to the contaminant degradation. Target contaminant removal from soil was assessed by monitoring concentrations of polycyclic aromatic hydrocarbons (PAHs), along with changes in the bacterial community structure over a time period of 10 months in the presence of tall fescue (Festuca arundinacea). Enhanced degradation of PAHs was observed in rhizosphere soil, with a maximum reduction in pyrene at a rate 36% higher than that noted for the unvegetated control. The dissipation of < 4-ring PAHs, 4-ring PAHs, and > 4-ring PAHs in unvegetated soil was 70%, 54%, and 49% respectively, whereas a higher dissipation rate was observed in tall fescue treated soil of 78%, 68%, and 61% at the end of the study. Microbial enumeration results showed greater total bacterial numbers and PAH-degrading bacteria in rhizosphere soil when compared to unvegetated soil. The results from the terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that there was a shift in the rhizosphere bacterial community during the phytoremediation process.     

Enhanced Hydrocarbons Degradation in the Rhizosphere of Mangrove Plants by a Halophilic Bacillus Subtilis Subtilis Strain

Sulaibikhat Embayment is a severely contaminated coastline in the State of Kuwait. The contaminating pollutants include hydrocarbons, heavy metals, and suspended particles. The objective of this study is to assess the ability of mangroves planted in the Sulaibikhat Embayment to enhance hydrocarbons degradation by the activities of rhizospheric hydrocarbon degrading bacteria (HDB). Accordingly, samples were collected from the rhizosphere of selected mangrove plants and from sediments in the same location but away from mangrove marshes. The samples were analyzed chemically and microbiologically before being enriched with a mixture of hydrocarbon compounds (HC) to isolate HDB.

A number of halophilic HDB were isolated from mangroves rhizosphere and the surrounding sediments such as Pseudomonas balearica, Microbacterium barkeri and Gordonia soli. On the other hand, Bacillus velezensis and Bacillus subtilis subtilis were both isolated only from mangroves rhizosphere. Among the isolated HDB, Bacillus subtilis subtilis was distinguished with its high degradation rates of the tested HC including poly aromatic hydrocarbons. According to our knowledge, this is the first Bacillus subtilis HC-degrading strain that was isolated from Kuwait Bay and from mangroves rhizosphere.     

无饲培养细柄半网菌Hemitrichia calyculata

报道了细柄半网菌在无饲培养条件下的完整生活史过程,描述了孢子萌发、游动胞、黏变形体、原质团及孢囊形成过程中的主要发育特点。结果表明：孢子预处理可以提高萌发率;游动胞鱼形,具有一根较短的鞭毛;黏变形体前缘具有较厚的透明外质,滑动运动方式。原质团属于“发达的中间类型”原质团。     

Quantitative Growth of Naegleria in Axenic Culture

A strain of Naegleria gruberi, isolated from a Vero cell culture and designated TS-1, was axenically cultivated in monolayer and mass aerating suspension culture. Cultural conditions for constant growth parameters and high-exponential cell densities were defined. Serum or other supplemented fractions were found essential in both Trypticase-yeast extract-glucose (TYG) and Casitone (CAS)-based media. Monolayer cultures grown in the CAS medium required lower levels of serum to reach maximum stationary densities of amoebae than cultures grown in the TYG medium. Heat-killed (121 C, 10 min) whole cell and cell lysate bacterial fractions were capable of replacing the serum in both the TYG and CAS media. Heat-killed bacterial fractions provided the same levels of growth as attained with serum in TYG medium, whereas the bacterial lysate supported only minimal growth in the same medium. In the CAS medium, both bacterial fractions resulted in the same level of growth which was equal to that obtained in reduced serum content. Strain TS-1 was established in suspension culture with the CAS medium used in monolayer culture. The addition of sheep red blood cells (RBC) or RBC lysate greatly enhanced growth responses. Further modifications resulted in a final medium for suspension culture consisting of Casitone-yeast extract-glucose-vitamin base, supplemented with serum and RBC lysate. This medium supported growth with a mean generation time of 9 h at 30 C and a stationary phase yield of greater than 5 x 10(6) amoebae per ml.     

Axenic culture of Leishmania amastigotes

One of the future goals in Leishmania research will be to reproduce the entire life cycle oxenically, in vitro. In this article, Paul Bates reviews recent progress in the axenic culture of amastigotes and addresses some of the remaining problems associated with culture methods for both amostigote and promostigote forms.     

Cultivation of Pseudocohnilembus persalinus in Axenic Medium

SYNOPSIS. Bacteria can be eliminated from protozoan cultures by adding a bacterium supplanting those difficult to destroy, if the added bacterium can be easily eliminated. Pseudocohnilembus persalinus was cultivated axenically in a simple medium containing peptones, nucleic acid extract, and vitamins. Growth was stimulated by saturated or unsaturated fatty acids.     

Growth of the Peritrich Opercularia coarctata in Axenic Culture*

A synthetic medium for Opercularia coarctata was developed that contains 20 amino acids, 10 vitamins, an 8-component balanced salt solution, Fe₂(SO₄)₃·(NH₄)₂SO₄·24H₂O, Tween 80, stigmasterol, a 7-component nucleic acid mixture, phenol red as an indicator, and 2,500 U.S.P. units/ml penicillin to maintain sterility. This medium supported axenic survival for 96 hr. Multiple supplements of thioctic acid, niacin, niacinamide, inositol, PABA, oleic acid, and Fe(NO₃)₂·9H₂O instead of Fe₂(SO₄)₃·(NH₄)₂SO₄·24H₂O coverted the survival medium into a growth medium, which permitted 36–45 days continuous cultivation of populations in excess of 4 × 10³ cells/3.0 ml final volume. Five generations were produced during the 48 hr logarithmic growth period. Serial transfers at 72 hr and during periods of greatest cell density produced a maximum of 8 generations 96 hr after initiation but the medium failed to sustain growth through more than 6 serial transfers. Extension of this investigation to formulating a minimal axenic medium is discussed.     

Rhizosphere micro-organisms in relation to the apple replant problem

Summary One of the factors giving rise to soil sickness in apple orchards is the rhizosphere microflora. The composition of the microbial coenosis in the rhizosphere changes with increasing age of the apple trees. An increase in the counts of micromycetes and actinomycetes and a decrease in bacterial counts was found in agreement with the decreasing pH of the rhizosphere soil. The presence of fluorescent pseudomonads in the rhizosphere of old apple trees was rare, but the planting of apple seedling into sick soil induced their proliferation. The relative proportion of individual genera of micromycetes changed according to the tree age; fungi of the genus Mucor were more often found in the rhizosphere of younger trees than in that of older ones while fungi of the genus Penicillium had an opposite trend. Biological tests showed that Penicillium fungi form the majority of the phytotoxic microflora. The amount of phytotoxic micromycetes was higher in ‘sick’ soil as compared with control soil in which apple trees had not been grown for at least 15 years. Higher numbers of phytotoxic micromycetes were isolated also from the rhizosphere of apple seedlings grown in ‘sick’ soil as compared with those growing in control soil. An increase in the amount of phytotoxic micromycetes in apple tree rhizosphere could be induced by mere addition of 5% (w/w) ‘sick’ soil to the soil in which apple trees were grown for the first time. By adding sterilized ‘sick’ soil, the amount of phytotoxic micromycetes in the apple seedling rhizosphere was not affected. Increased numbers of phytotoxic micromycetes affected negatively the growth of apple trees and the morphology of apple tree roots. This demonstrated the possibility of transfer of a factor participating in the etiology of soil sickness in apple orchards.     

Dauer Larvae of Caenorhabditis briggsae in Axenic Culture

The free-living hermaphroditic nematode, Caenorhabditis briggsae, enters a dauer stage under certain conditions in axenic culture. Dauer larvae differ from directly-developing third-stage larvae in internal structure, size at time of second molt, morphology of second and third cuticles, separation zone of cuticular caps, and survival at 4 C and 37 C, temperatures fatal to other stages. Males, which occur rarely in liquid medium, may mature under conditions which cause most of the hermaphrodites to go into the dauer stage, resulting in a culture with increased male-to-hermaphrodite ratio.     

Chitobiose utilization in Borrelia burgdorferi is dually regulated by RpoD and RpoS

Background  

Borrelia burgdorferi has limited biosynthetic capabilities and must scavenge N-acetylglucosamine (GlcNAc), an essential component of the microbial cell wall, from the surrounding environment. Spirochetes cultured in the absence of free GlcNAc exhibit biphasic growth; however, addition of chitobiose (a dimer of GlcNAc) substitutes for free GlcNAc resulting in a single exponential phase. We evaluated the effect of RpoS and RpoN, the only alternative sigma factors in B. burgdorferi, on biphasic growth and chitobiose utilization in the absence of free GlcNAc. In addition, we investigated the source of GlcNAc in the second exponential phase.     

Axenic Culture of Myxamoebae of the Myxomycete Physarum polycephalum

Myxamoebae of the acellular slime mold Physarum polycephalum have been cultured axenically in a soluble medium. The growth medium contains bovine serum albumin, embryo extracts, liver infusion broth, peptone, and glucose. Cell densities ranging from 3 x 10(6) to 5 x 10(6) cells/ml have been obtained with this medium. To date, myxamoebae have been serially transferred more than 100 times without deleterious effect.