A new phage-ty-ing scheme for strains ofProteus mirabilis andProteus vulgaris

The results of serological and genetic analyses of 21 phages ofProteus mirabilis andProteus vulgaris of the new typing set confirmed their classification into morphological types A1, A2, B1, and C1 and the familiesMyoviridœ, Siphoviridœ andPodoviridœ. Phages and antisera reacted and cross-reacted only within each family. In DNA-DNA hybridization, DNA fragments reacted only with³²P-labelled DNA probes of their own families. Serological and genetic analysis showed that some phages in the familiesMyoviridœ andPodoviridœ were so closely related that they could be considered identical. First part:Folia Microbiol. 39, 381–386 (1994).     

Influence of hydrogen acceptors on growth and energy production ofProteus mirabilis

The generation time ofP. mirabilis in defined and in complex medium is shorter in the presence of hydrogen acceptors than in their absence. In the presence of hydrogen acceptors the molar growth yield for glucose and the acetate production are strongly increased. From the molar growth yield and the acetate production Y_ATP in defined medium was calculated as 5.5 g/mole, whereas in complex medium a value of 12.6 g/mole was obtained. The molar growth yield, the acetate production, the amount of hydrogen acceptor reduced and Y_ATP were used to calculate P/2e⁻ratios for phosphorylation coupled to electron transfer to oxygen, nitrate and tetrathionate as respectively 2.80; 1.48 and 1.23 in defined medium. Under anaerobic conditions in the presence of nitrate or tetrathionate as hydrogen acceptor in complex medium a bend in the growth curve is observed. In the period of rapid growth the P/2e⁻ratio for nitrate reduction is of the same magnitude as that in defined medium, however much lower P/2e⁻ratios are found during the subsequent period of slow growth. The P/2e⁻ratios for tetrathionate reduction in complex medium for both growth periods are lower than those in defined medium. Most probably these results indicate that during this period growth and energy production are uncoupled. Under aerobic conditions in complex medium a constant Y_O value of 32.2 g/atom O is found during a short period of the growth curve. Afterwards when the cell density increases a steady decrease of Y_O is observed.     

Fatty acid composition of membranes of L-forms ofStreptococcus faecalis andProteus mirabilis cultured at different osmolalities

The fatty acid composition of membranes of L-forms ofStreptococcus faecalis andProteus mirabilis cultured at different osmolalities and in different osmotic stabilizers was examined.S. faecalis L-forms cultured with sucrose in the medium showed a decrease in the unsaturated fatty acid C₁₈₁ and an increase in the C₁₈ fatty acid and C₁₉ cyclopropane fatty acid. Fatty acid composition ofS. faecalis L-forms cultured in medium containing 1.8% NaCl was similar to the fatty acid composition of L-forms cultured in brain-heart infusion broth (BHI) without osmotic stabilizer and was between the composition of fatty acids of L-forms in BHI with sucrose and that in BHI without 0.5 M sucrose. InProteus mirabilis L-forms, there were differences between L-forms cultured with and without sucrose, but these differences were not comparable to the changes observed inS. faecalis L-forms.P. mirabilis L-forms cultured with and without NaCl in the medium had similar fatty acid compositions.     

Localization of enterobacterial common antigen: Proteus mirabilis and its various L-forms.

An investigation of Proteus mirabilis wild-type strains and their various derived L-forms shows that the enterobacterial common antigen (ECA) is localized in the outer membrane of the cell envelope of these strains. In strains where the outer membrane is lacking (stable protoplast L-forms) or where its amount is reduced (spheroplast UL19) no ECA or only reduced amounts of it are detected by serological tests or by ferritin-labeling techniques.     

Complete amino acid sequence ofProteus mirabilis PR catalase. Occurrence of a methionine sulfone in the close proximity of the active site

The catalase ofProteus mirabilis PR, a peroxide-resistant (PR) mutant ofProteus mirabilis, binds strongly NADPH, which is a unique property among known bacterial catalases. The enzyme subunit consists of 484 amino acid residues for a mass of 55,647 daltons. The complete amino acid sequence was resolved through the combination of protein sequencing, mass spectrometry, and nucleotide sequencing of a PCR fragment. The sequence obtained was compared with that of other known catalases. Amino acids of the active site are all conserved as well as essential residues involved in NADPH binding. Among the amino acids interacting with the heme, a methionine sulfone was found at position 53, in place of a valine in most other catalases. The origin of oxidation of this methionine is unknown, but the presence of this modification could change iron accessibility by large substrates or inhibitors. This posttranslational modification was also demonstrated in the wild-typeP. mirabilis catalase.     

The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus mirabilis

The outer-membrane protein ShlB of Serratia marcescens activates and secretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA*) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins. Received: 17 February 1998 / Accepted: 30 June 1998     

Novel bacterial membrane surface display system using cell wall-less L-forms of Proteus mirabilis and Escherichia coli

We describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of Escherichia coli and Proteus mirabilis. The reporter protein, staphylokinase (Sak), was fused to transmembrane domains of integral membrane proteins from E. coli (lactose permease LacY, preprotein translocase SecY) and P. mirabilis (curved cell morphology protein CcmA). Both L-form strains overexpressed fusion proteins in amounts of 1 to 100 microg ml(-1), with higher expression for those with homologous anchor motifs. Various experimental approaches, e.g., cell fractionation, Percoll gradient purification, and solubilization of the CM, demonstrated that the fusion proteins are tightly bound to the CM and do not form aggregates. Trypsin digestion, as well as electron microscopy of immunogold-labeled replicas, confirmed that the protein was localized on the outside surface. The displayed Sak showed functional activity, indicating correct folding. This membrane surface display system features endotoxin-poor organisms and can provide a novel platform for numerous applications.     

Secondary structure prediction: combination of three different methods

A combination of three complementary secondary structure prediction methods is presented. The methods used are the GOR III method, the Homologue method and a new method, the bit pattern method, which is based on hydrophilic/hydrophobic residue patterns. For this purpose a hydropathy scale was developed and is presented here. The combination algorithm (Combine method) was designed to take the best results of each method and use their differences in order to improve the prediction. The combination yields 65.5% correctly predicted residues in three states: alpha-helix (H), beta-strand (E) and aperiodic structure (C) which is an improvement ranging from 2.5 to 6.5% compared with the individual methods when tested with a 67-polypeptide chain database. Seventy-five per cent of the regular secondary structure (H and E) runs are correctly located and beta-sheet runs are much better located by the Combine method in comparison to the other methods.     

Microbial community structure along an altitude gradient in three different localities

The microbial community structure along an altitude gradient was investigated in different localities, in Kalasi lake, Urumqi river and Sangong river, Xingjiang (China). The mean numbers of DAPI (4',6-diamidino-2-phenylindole)-stained cells were lower in Kalasi lake than that in Urumqi river and Sangong river; these differences were attributed to increasing environmental harshness including lower soil organic carbon and nitrogen content, more acidic pH and lower annual temperature. In each locality, the numbers of bacteria and archaea measured with two fluorescence-labeled 16S rRNA oligonucleotide probes (EUB338 and ARCH915) were higher in a coniferous forest and lower in desert vegetation. A significant and positive relationship was found between microbial and soil organic carbon and total nitrogen along the altitudinal gradient, indicating that plant communities and soil nutrients influence the soil microbial structure. The results show that the microbial population in higher latitudinal site was fewer than lower latitudinal one, soil microorganisms were positively correlated to soil organic carbon and total nitrogen, and plant communities had an obviously impact on soil microbes.     

Envelope formation and structure in the Euglenoid genus Trachelomonas

Ultrastructural details of envelope formation are described comparatively for several species of the genus Trachelomonas. The first-formed envelope component in T. oblonga var. punctata Prings. is an external “skin”. Next, a fibrillar layer is produced between the skin and the cell surface. Ferric hydroxide and manganese compounds are precipitated on this inner layer to form a thick envelope, species-specific in size, shape, ornamentation and collar characteristics. The original skin is then lost.

A similar sequence of events occurs during envelope formation in other species, but differences exist in the details of the process and in the texture and complexity of construction of the mature envelopes. T. volvocina Ehrenb. has a fibrillar inner layer and granular precipitation, but produces a complex three-layered envelope with a honeycombed central layer. This is compared with the superficially similar three-layered envelope of an unnamed species from a wild collection, and subtle differences in ultrastructural patterning are discussed. T. pertyi Prings. forms a two-layered spiny envelope, less highly organised than that of T. volvocina. The spiny and porous envelopes of T. hispida (Perty) Stein em. Defl., T. echinata Singh and T. lefevrei Defl. are quite different in texture, being composed of flaky material woven into a spongy matrix. The presence of large amounts of iron in the mature envelopes of all the species under study was confirmed by microanalysis in the electron microscope using X-ray spectroscopy.

Details of envelope ultrastructure are discussed in relation to taxonomy, cell metabolism, the problem of why Trachelomonas cells form envelopes when those of other euglenoid genera do not, and the problem of cell control of species-specificity of envelope characters. Electron microscopy has added considerably to our knowledge of the structural details of trachelomonad envelope formation, but it has been less successful in helping to elucidate some of the enigmas associated with this phenomenon.     

Capsule structure of Proteus mirabilis (ATCC 49565).

Proteus mirabilis 2573 (ATCC 49565) produces an acidic capsular polysaccharide which was shown from glycose analysis, carboxyl reduction, methylation, periodate oxidation, and the application of one dimensional and two-dimensional high-resolution nuclear magnetic resonance techniques to be a high-molecular-weight polymer of branched trisaccharide units composed of 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine), 2-acetamido-2,6-dideoxy-L-galactose (N-acetyl-L-fucosamine), and D-glucuronic acid, having the structure: [formula: see text] P. mirabilis 2573 also produces an O:6 serotype lipopolysaccharide in which the O-chain component has the same structure as the homologous capsular polysaccharide. This is the first report of a defined capsular polysaccharide in this bacterial genus.     

Studies on the structure of isolated chromatin in three different solvents.

Properties of calf thymus chromatin, prepared by mild procedures, have been studied in various solvents. In 0.2 mM EDTA s-values ranged from 20 to 30 S and intrinsic viscosities from 5 to 24 dl/g. Dialysis against 0.15 M NaCl or 0.2 mM MgCl2 changed these values to 80 to 100 S and 0.2 to 5 dl/g, respectively, indicating an essentially more compact structure. In 0.2 mM EDTA X-ray scattering yielded a cross section diameter of 9 nm, which is associated with the tertiary structure of chromatin fiber (M/L = 21200 Dalton/nm). By dialysis against 0.15 M NaCl or 0.2 mM MgCl2 part of the material spontaneously formed quarterny structures (cross section diameters 25-29 nm). The rest of the material with cross section diameters less than 9 nm is supposed to be more strongly sheared tertiary structure which seems to be unable to form quarterny structure due to artificial conformational changes.     

云南三个地区野生猕猴的种群结构分析

通过齿式及牙齿磨损情况判断年龄及称量体重的方法,对2004 年至2010 年间从云南景东县、镇沅县、宁蒗县三个地区捕获的23 群共670 只野生猕猴进行了种群年龄组成、性别比例及体重差异的调查。结果表明:(1)景东县、镇沅县、宁蒗县三地猕猴种群雄雌性别比分别为1∶ 1. 21、1∶ 1. 55、1∶ 1. 52; (2) 三个地区猕猴种群年龄结构稳定,幼年组、青年组和中壮年组的个体数量占整个种群数量的80% 以上,处于发展阶段;(3)三个地区猕猴种群体重到了中壮年后均出现雄性明显高于雌性(P < 0. 01)的性二型现象,同时,发现宁蒗县猕猴种群体重明显高于其他两个地区(P < 0. 01)。以上三个地区野生猕猴种群均处于较高生育高峰,呈现出发展壮大的趋势。本文为云南省猕猴资源的保护与合理开发利用提供一定的依据,同时也为了解云南省野生猕猴群体结构及生长发育规律,建立人工繁育的不同地域(种、亚种)猕猴种质特性数据库和进一步研究提供相应的基础数据支持。