Characterization of the brewery spoilage bacterium Obesumbacterium proteus by automated ribotyping and development of PCR methods for its biotype 1

AIMS: To study the ability of automated ribotyping to characterize Obesumbacterium proteus and Hafnia alvei, to design primers and to evaluate standard end-point and real-time PCR for the detection of O. proteus biotype 1 in beer and in brewers's yeast-containing samples. METHODS AND RESULTS: Automated ribotyping was carried out using the standard method with EcoRI and PvuII. The digestions with both enzymes clearly differentiated O. proteus biotypes 1 and 2 and H. alvei. PCR primers were designed according to the 16S rRNA gene sequence of the O. proteus type strain. Two primer sets (Obs137-Obs558 and Obs137-Obs617) detected O. proteus biotype 1 and H. alvei but not O. proteus biotype 2 or other tested beer spoilage bacteria (40 species) in the end-point and real-time PCR, indicating their high specificity. The detection limit for O. proteus was 160-1600 CFU 100 ml(-1) beer in the end-point PCR reactions and < or =160 CFU 100 ml(-1) beer in the real-time PCR reactions. More cells (from 16 to 3200) were needed for detection in the presence of brewer's yeast cells. CONCLUSIONS: Automated ribotyping is a useful tool to characterize and identify O. proteus and H. alvei isolates. The designed primers are suitable for the rapid detection of O. proteus biotype 1 and H. alvei in brewery samples by PCR. Significance and IMPACT OF THE STUDY: Automated ribotyping and PCR could improve microbiological quality control in breweries by facilitating the detection, identification and tracing of spoilage bacteria.     

临床分离的变形杆菌中整合子分型研究

目的了解临床分离变形杆菌中1、2类整合子的流行现状及其耐药性。方法PCR扩增146株变形杆菌中1、2类整合酶基因,M-H肉汤稀释法检测146株变形杆菌的药敏情况。结果1类整合子检出率为36.9%(54/146),2类整合子检出率为38.4%(56/146),同时携带1、2类整合子的检出率为18.5%(27/146)。结论1、2类整合子在变形杆菌感染临床分离株中所占比例已相当高,1、2类整合子与变形杆菌的多重耐药具有相关性。     

Defense function of pigment granules in the ciliate Blepharisma japonicum against two predatory protists, Amoeba proteus (Rhizopodea) and Climacostomum virens (Ciliata)

The defense function of pigment granules in the red ciliate Blepharisma japonicum against two predatory protists, Amoeba proteus and Climacostomum virens, was investigated by (1) comparing normally-pigmented and albino mutant cells of B. japonicum as the prey of these predators and (2) comparing resistance of the predators to blepharismin, the toxic pigment contained in the pigment granules of B. japonicum. Normally pigmented cells which contained more blepharismin than albino cells were less vulnerable to A. proteus than albino cells, but not to C. virens. C. virens was more resistant than A. proteus to the lethal effect of blepharismin. The results indicate that pigment granules of B. japonicum function as defense organelles against A. proteus but not against C. virens and suggest that successful defense against a predator depends on the susceptibility of the predator to blepharismin.     

Rapid detection of Obesumbacterium proteus from yeast and wort using polymerase chain reaction

AIMS: To design and evaluate PCR primers for the rapid detection of Obesumbacterium proteus. METHODS AND RESULTS: The 16S rDNA from a wild-type Obesumbacterium proteus biotype II isolate was sequenced and the resultant data used to produce specific primers for this organism. These primers discriminated between biotype I (nonbrewery) and biotype II isolates. In addition, the primers were able to detect Obesumbacterium proteus in wort and in yeast slurries in the presence of competitive bacteria. The primers were tested against a range of other beer spoilage bacteria for any cross-reactions. None were detected. CONCLUSIONS:Obesumbacterium proteus primers can detect this contaminant without generating cross-reactions to related species. SIGNIFICANCE AND IMPACT OF THE STUDY: The primers generated in this study can now be used for PCR detection assays that will contribute to early detection of this important process contaminant.     

Proteus syndrome associated with liver involvement: case report

We report a patient with proteus syndrome who has epidermal nevus, right-sided asymmetric growth of extremities, pelvis, vertebrae and hemimegalencephaly. This patient also had enlargement of the liver which is not reported before in the proteus syndrome.     

Photophobic and phototactic responses of Amoeba proteus in KCN and SHAM solutions

The effects of the metabolic inhibitors KCN and SHAM on phototaxis and photophobic response in Amoeba proteus have been studied. Both drugs neither change amoebae photophobic response nor the phototactic reaction. The results indicate clearly that the negative phototactic orientation is not impaired by impediment of respiration thus it is not directly coupled to the differences in energy production in different parts of A. proteus body.     

Enumeration of Obesumbacterium proteus in brewery yeasts and characterization of isolated strains using Biolog GN microplates and protein fingerprinting

Of a range of media tested for enumeration of Obesumbacterium proteus in brewers' yeast, Universal Beer agar and Wallerstein Laboratories Differential medium were most effective. MacConkey agar (several types) and Membrane Lauryl Sulphate agar were least effective. Other media (Wort agar, YM agar) were of intermediate efficacy. Nine O. proteus strains from commercial yeast samples were characterized using the API 20E test kit, the Biolog GN microplate (BGNM) and by SDS-PAGE of their total soluble proteins. Both the BGNM and SDS-PAGE techniques allowed the strains to be differentiated from one another: the API 20E kit did not. All strains isolated from UK breweries belonged to O. proteus biogroup II. Four of these strains displayed a branching cell morphology not hitherto described in any member of the Enterobacteriaceae.     

Is actin involved in the nuclear division in Amoeba proteus?

During semi-open mitosis of Amoeba proteus the nuclear envelope is not dispersed and nucleus divides by fission. The presence of actin layer close to nuclear envelope was demonstrated in interphase and telophase nuclei of that amoeba stained with rhodamine labelled phalloidin. In telophase, an accumulation of actin arises in the space between the future daughter nuclei. It appears to be comparable with the contractile ring of dividing cells. This suggests that actin associated with the nuclear envelope of Amoeba proteus may be involved in final separation of the daughter nuclei, forming a constriction ring at the middle of dividing nucleus.     

Presence of aquaporin and V-ATPase on the contractile vacuole of Amoeba proteus.

BACKGROUND INFORMATION: The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (contractile vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe (2004) Cell Struct. Funct. 29, 85-90]. RESULTS: In the present study, we cloned an AQP gene from A. proteus [ApAQP (A. proteus AQP)] that encodes a 295-amino-acid protein. The protein has six putative TMs (transmembrane domains) and two NPA (Asn-Pro-Ala) motifs, which are conserved among various AQPs and are thought to be involved in the formation of water channels that span the lipid bilayer. Using Xenopus oocytes, we have demonstrated that the ApAQP protein product can function as a water channel. Immunofluorescence microscopy with anti-ApAQP antibody revealed that ApAQP is detected on the CV membrane and on the vesicles around the CV. The presence of V-ATPase (vacuolar H+-ATPase) on the vesicle membrane around the CV was also detected. CONCLUSIONS: Our data on ApAQP allow us to provide the first informed explanation of the high water permeability of the CV membrane in amoeba. Moreover, the results suggest that vesicles possessing V-ATPase are involved in generating an osmotic gradient. Based on our findings, we propose a new hypothesis for the mechanism of CV function.     

Response to light-shade difference in anucleate and polynucleate specimens of Amoeba proteus

The enucleated specimens of Amoeba proteus, the anucleate fragments, and the polynucleate individuals which all are capable of cortical contraction but not of locomotion, may be reactivated by the light-shade difference established across their body. Individual cells or fragments migrate toward the shade. The motory polarity and coordinated movement disappear immediately after cessation of the stimulus. The results are interpreted according to the earlier hypothesis that the necessary to maintain the motory polarity of amoebae. It is suggested that the anucleate and polynucleate specimens are incapable of coordinated movements when non-stimulated, because of a deficiency or an excess, respectively, of the regulatory relaxing factor secreted by the nucleus of Amoeba proteus.     

Intraspecific polymorphism of glucose-6-phosphate dehydrogenase in Amoeba proteus

Seven Amoeba proteus strains of different origin were studied using a polyacrylamide gel electrophoresis micromethod in the presence of NADP+ in cathode buffer. The strains examined differ in glucose-6-phosphate dehydrogenase (G6PD) zymograms: the majority of them (A, L, F, Da and Bk) display 3 bands, whereas strains B and C show 4 and 2 bands, respectively. All but strain C have a common band of high relative mobility. Strain B shows two fast migrating bands instead of a single band typical of strains A, L, F, Da and Bk. The results obtained testify to a certain intraspecific polymorphism of electrophoretic G6PD patterns in A. proteus. These difference between strains may be used as genetic markers in the nuclear transfer experiments.     

Biochemical and Molecular Characterization of Obesumbacterium proteus, a Common Contaminant of Brewing Yeasts

We have evaluated the effectiveness of API 20E, Biolog testing, plasmid profiling, ribotyping, and enteric repetitive intergenic consensus (ERIC)-PCR to characterize, classify, and differentiate nine bacterial isolates of the common brewery contaminant Obesumbacterium proteus. Of the five typing techniques, Biolog testing, plasmid profiling, and ERIC-PCR provided the most differentiation, and API 20E testing and ribotyping were relatively indiscriminate. The molecular biology approach of ERIC-PCR offered the ideal combination of speed, simplicity, and discrimination in this study. Overall, the results are supportive of the view that O. proteus can be subdivided into two biogroups, biogroup 1, which has considerable biochemical and genetic homology to Hafnia alvei, and biogroup 2, which is relatively heterogeneous.     

Mechanics and control of the cytoskeleton in Amoeba proteus.

Many models of the cytoskeletal motility of Amoeba proteus can be formulated in terms of the theory of reactive interpenetrating flow (Dembo and Harlow, 1986). We have devised numerical methodology for testing such models against the phenomenon of steady axisymmetric fountain flow. The simplest workable scheme revealed by such tests (the minimal model) is the main preoccupation of this study. All parameters of the minimal model are determined from available data. Using these parameters the model quantitatively accounts for the self assembly of the cytoskeleton of A. proteus: for the formation and detailed morphology of the endoplasmic channel, the ectoplasmic tube, the uropod, the plasma gel sheet, and the hyaline cap. The model accounts for the kinematics of the cytoskeleton: the detailed velocity field of the forward flow of the endoplasm, the contraction of the ectoplasmic tube, and the inversion of the flow in the fountain zone. The model also gives a satisfactory account of measurements of pressure gradients, measurements of heat dissipation, and measurements of the output of useful work by amoeba. Finally, the model suggests a very promising (but still hypothetical) continuum formulation of the free boundary problem of amoeboid motion. by balancing normal forces on the plasma membrane as closely as possible, the minimal model is able to predict the turgor pressure and surface tension of A. proteus. Several dynamical factors are crucial to the success of the minimal model and are likely to be general features of cytoskeletal mechanics and control in amoeboid cells. These are: a constitutive law for the viscosity of the contractile network that includes an automatic process of gelation as the network density gets large; a very vigorous cycle of network polymerization and depolymerization (in the case of A. proteus, the time constant for this reaction is approximately 12 s); control of network contractility by a diffusible factor (probably calcium ion); and control of the adhesive interaction between the cytoskeleton and the inner surface of the plasma membrane.     

Ca2+-binding modulator protein in protozoa and myxomycete.

Ca2+-binding protein with the properties of brain modulator protein of 3,5-cyclic nucleotide phosphodiesterase was identified in Physarum polycephalum plasmodia and in Euglena gracilis and Amoeba proteus cells by urea polyacrylamide gel electrophoresis and activation of cyclic nucleotide phosphodiesterase and of myosin light chain kinase.     

Isolation and partial characterization of two plasmid deoxyribonucleic acids from endosymbiotic bacteria of Amoeba proteus.

Obligatory endosymbiotic bacteria in a strain of Amoeba proteus were found to harbor two distinct species of plasmid, pHJ11 and pHJ12. Their molecular weights were 39 x 10(6) and 14 x 10(6), respectively.     

Heat shock proteins of freshwater protists and their involvement in adaptation to changes in the environmental salinity

Changes in the level of heat shock proteins (HSP) in cells of freshwater protists, amoebae Amoeba proteus and ciliates Paramecium jenningsi, in response to changes in the environmental salinity were investigated. Changes in salinity levels were considered as a stress factor. The immunoblotting method revealed a polypeptide antigen cross-reacting with antibodies against bovine HSP70 in total protein extracts of both intact cells and cells subjected to salinity stress. The same polypeptide antigen was revealed in A. proteus cells subjected to heat shock. Therefore, it may be supposed that the polypeptide revealed after salinity shock is a heat shock protein related to the vertebrate HSP70. Under the impact of stress factor, well acclimated protists mostly spend their own previously accumulated HSP70. A conclusion is made that freshwater protists, living under conditions of increased salinity, appear to be preadapted to changes in environmental factors.     

Dynamics of the cytoskeleton in Amoeba proteus. III. Influence of microinjected antibodies on the organization and function of the microfilament system

Affinity-purified antibodies against actin, myosin, alpha-actinin and vinculin cross-reacted with corresponding proteins from Amoeba proteus in immunoblotting experiments. Antibody staining of cells fixed during locomotion revealed different distribution patterns with a local concentration of anti-actin in the intermediate and of anti-myosin in the uroid region. Anti-alpha-actinin labeled a thin layer at the internal face of the plasma membrane, whereas anti-vinculin was distinctly concentrated at the base of advancing pseudopodia. Microinjection of different control solutions or antibodies against actin, myosin and alpha-actinin neither influenced the normal morphology and motile activity of amoebae nor changed the cellular distribution pattern of complementary antigens. However, antibodies against vinculin disorganized controlled locomotion and altered the spatial morphology of the microfilament system as well as the localization of the vinculin antigen thus pointing to a function of this protein in adhesion and locomotion of A. proteus. The results of the present paper show similarities to observations on mammalian tissue culture cells.     

Studies on changes in the nuclear helices of Amoeba proteus during the cell cycle.

The fine structure of the nuclei of synchronously growing cell population of Amoeba proteus was studied at I-h intervals during the interphase. This study showed that the nuclear helices undergo increases in their number at certain stages during interphase. These changes were found to correlate with ultrastructural changes occurring in the nucleoli.     

华南贵州早奥陶世红花园组的镰箭类牙形刺

本文描述了产自华南贵州下奥陶统红花园组(桐梓红花园标准剖面,沿河甘溪剖面和贵阳黄花冲剖面)的四个镰箭类牙形刺种:Drepanoistodus sp.cf.nowlani,Paltodus sp.cf.P.subaequalis,Paltodus sp.A和Paroisto-dus sp.。D.sp.cf.nowlani为红花园组常见种,但另三种则相对稀少。在红花园组标准剖面,Paltodus sp.cf.P.subaequalis最低出现于该组底部,证实此层位为晚特马豆克期。文中详细图示了产于瑞典下奥陶统的两个带化石种:Paltodus deltifer和Paroistodus proteus,以及瑞典镰箭刺属的三个常见种:Drepanoistodus forceps,D.basiovalis和D.sp.cf.D.suberectus。通过与瑞典材料的详细对比,我们认为华南以前关于P.proteus和P.deltifer两种的记述多有误,须重新厘定。     

Osmotic homeostasis in the freshwater ameba

Structural and physiological osmotic adaptations of freshwater protozoa are reviewed using Amoeba proteus as an example. A particular emphasis is given to the contractile vacuole. Recent results on the effects of exogenous ATP and vasopressin on the contractile vacuole are also presented.