Active and inactive state structures of unliganded Lactobacillus casei allosteric L‐lactate dehydrogenase

Lactobacillus casei L ‐lactate dehydrogenase (LCLDH) is activated through the homotropic and heterotropic activation effects of pyruvate and fructose 1,6‐bisphosphate (FBP), respectively, and exhibits unusually high pH‐dependence in the allosteric effects of these ligands. The active (R) and inactive (T) state structures of unliganded LCLDH were determined at 2.5 and 2.6 Å resolution, respectively. In the catalytic site, the structural rearrangements are concerned mostly in switching of the orientation of Arg171 through the flexible intersubunit contact at the Q‐axis subunit interface. The distorted orientation of Arg171 in the T state is stabilized by a unique intra‐helix salt bridge between Arg171 and Glu178, which is in striking contrast to the multiple intersubunit salt bridges in Lactobacillus pentosus nonallosteric L ‐lactate dehydrogenase. In the backbone structure, major structural rearrangements of LCLDH are focused in two mobile regions of the catalytic domain. The two regions form an intersubunit linkage through contact at the P‐axis subunit interface involving Arg185, replacement of which with Gln severely decreases the homotropic and hetertropic activation effects on the enzyme. These two regions form another intersubunit linkage in the Q‐axis related dimer through the rigid NAD‐binding domain, and thus constitute a pivotal frame of the intersubunit linkage for the allosteric motion, which is coupled with the concerted structural change of the four subunits in a tetramer, and of the binding sites for pyruvate and FBP. The unique intersubunit salt bridges, which are observed only in the R state structure, are likely involved in the pH‐dependent allosteric equilibrium. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Medium optimization and structural characterization of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3

In this study, response surface methodology was employed to optimize the medium compositions for the production of exopolysaccharides (EPS) from endophytic bacterium Paenibacillus polymyxa EJS-3. Firstly, fractional factorial design was applied to evaluate the effects of different components in the medium. It was found that sucrose, yeast extract and CaCl₂ influenced significantly the production of EPS. Then, steepest ascent method and central composite design were used to optimize the concentrations of the three variables. As results, the optimal medium compositions were determined as following (g/L): sucrose 188.2, yeast extract 25.8, K₂HPO₄ 5 and CaCl₂ 0.34, with a corresponding yield of 35.26 g/L. In addition, both polysaccharide fractions (EPS-1 and EPS-2) from crude EPS were mainly composed of (2 → 6)-linked β-d-fructofuranosyl residues backbone with (2 → 1)-linked branches based on their structural characterization by FT-IR spectroscopy, methylation analysis and ¹³C NMR spectroscopy.     

海洋菌W11产中温淀粉酶的酶学特性

本研究从威海文登海域筛选获得一株产淀粉酶海洋菌W11,初步鉴定为弧菌,并探讨pH、温度、无机离子对淀粉酶活性和稳定性的影响及该酶底物浓度效应和Km值。结果表明:在pH7.5左右酶活性最高,pH在4.0～7.5范围内体现较强的稳定性。最适酶解温度为55℃,酶液在60℃以下有较好的热稳定性;Ba2+、Mn2+对淀粉酶有激活作用,而Cu2+、Mg2+、Zn2+则抑制淀粉酶活性,表观Km值为0.973mg/mL。海洋菌W11所产的中温淀粉酶保存温度范围较广、适应pH作用的范围广及稳定性较强,将有着广泛的应用潜力。     

An ice-binding protein from an Antarctic sea ice bacterium

An Antarctic sea ice bacterium of the Gram-negative genus Colwellia, strain SLW05, produces an extracellular substance that changes the morphology of growing ice. The active substance was identified as a approximately 25-kDa protein that was purified through its affinity for ice. The full gene sequence was determined and was found to encode a 253-amino acid protein with a calculated molecular mass of 26,350 Da. The predicted amino acid sequence is similar to predicted sequences of ice-binding proteins recently found in two species of sea ice diatoms and a species of snow mold. A recombinant ice-binding protein showed ice-binding activity and ice recrystallization inhibition activity. The protein is much smaller than bacterial ice-nucleating proteins and antifreeze proteins that have been previously described. The function of the protein is unknown but it may act as an ice recrystallization inhibitor to protect membranes in the frozen state.     

Detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> by novel chromosomal polymerase chain reaction primers

A new sensitive and specific method for the detection of Erwinia amylovora was developed. The method is based on the detection of a chromosomal DNA sequence specific for this bacterial species and enables detection of E. amylovora pathogenic strains, including recent isolates that lack plasmid pEA29 and thus cannot be detected by the previously popular PCR methods based on the detection of this plasmid. A species-specific random amplified polymorphic DNA (RAPD) marker was identified, cloned, and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. The E. amylovora specific sequence, 1269 bp long, was amplified in polymerase chain reaction and detected with electrophoresis in agarose gel stained with ethidium bromide. Amplification with other bacterial species did not produce any PCR product detectable by electrophoresis. Matching of the E. amylovora specific sequence to chromosomal DNA was confirmed by computer analysis of the E. amylovora genome. A consistent sensitivity limit of the method was 3 CFU/reaction, and in some cases it was possible to detect 0.6 CFU/reaction. Due to its high sensitivity and specificity, our method of E. amylovora detection is currently the most reliable, taking into account that the reliability of PCR methods based on plasmid pEA29 has been compromised by the isolation of pathogenic E. amylovora strains that lack this plasmid.     

海洋细菌E18菌株的生物学特性及其蓝紫色素稳定性的研究

从海泥中分离获得一株海洋细菌Ｅ18菌株,发现其具有稳定产生蓝紫色素的特性。对该菌的形态特征、培养特征及生理生化特征进行了研究。对该菌的分子鉴定结果表明,该菌为假交替单胞菌属细菌。萃取该菌的色素,并试验光、紫外线、热、pH、氧化剂及还原剂对该色素的影响,结果表明：该色素的最大吸收峰为579nm,紫外光对其稳定性无影响,但自然光对其有一定的消色作用。60℃～80℃温度范围对色素有一定的增色作用,而90℃以上高温可使色素消色。色素在pH3～9区域内稳定。色素对还原剂Na₂SO₃较为稳定, 而高浓度的氧化剂H₂O₂可使色素改变颜色。     

三株光合细菌的毒性试验研究

以昆明小白鼠为实验动物,对类球红细菌Z1、沼泽红假单胞菌Z2及光合细菌分离株C2采用急性毒性试验、骨髓细胞微核试验和精子畸形试验进行安全性评定。结果表明,急性毒性试验:实验组小白鼠的一般状况、器官含水量、器官系数及血常规检查,与对照组差异均无显著性(P>0.05);骨髓细胞微核试验和精子畸形试验实验组与阴性对照组(生理盐水)差异均无显著性(P>0.05),与阳性对照组(环磷酰胺40 mg/kg)比较,骨髓细胞微核试验差异有极显著性(P<0.01),精子畸形率试验差异有显著性(P<0.05)。以上结果说明,三株光合细菌均不具有毒性。     

Biosynthesis of antibiotic prodiginines in the marine bacterium Hahella chejuensis KCTC 2396

AIMS: Hahella chejuensis KCTC 2396 produces red pigments, showing antibacterial and algicidal activities. The main red-coloured metabolite of the pigments was identified as antibiotic prodigiosin. With the expectation that the red pigments are a mixture of a series of close relatives, the aim of the present study is to detect new antibiotic prodigiosin analogues and to analyse the biosynthetic pattern for prodiginines in KCTC 2396. METHODS AND RESULTS: Except prodigiosin, the other constituents in the red pigments were confirmed as well-known dipyrrolyldipyrromethene prodigiosin, norprodigiosin, and undecylprodiginine. Additionally, four new prodigiosin analogues, each of which was distinguished from prodigiosin (C(5)), according to differences in alkyl chain length (C(3)-C(7)), were detected in small quantities by liquid chromatography mass spectrometry/mass spectrometry spectroscopy. Owing to the presence of a cytotoxic methoxy group, it is expected that all the new prodigiosin analogues are bioactive. CONCLUSIONS: Four characterized prodiginines, including prodigiosin and four new prodigiosin analogues are produced in different ratio in KCTC 2396. All of the prodiginines possess a common linear tripyrrolyl structure and a cytotoxic methoxy group. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows for the first time that KCTC 2396 is able to produce antibiotic prodigiosin, undecylprodiginine and new prodigiosin analogues in a mixture of pigments. It is also shown that KCTC 2396 possesses a novel system for the simultaneous production of multiple prodiginines in a single micro-organism.