Population Genetic Analyses of Helicobacter pylori Isolates from Gambian Adults and Children

The gastric pathogen Helicobacter pylori is one of the most genetically diverse of bacterial species. Much of its diversity stems from frequent mutation and recombination, preferential transmission within families and local communities, and selection during persistent gastric mucosal infection. MLST of seven housekeeping genes had identified multiple distinct H. pylori populations, including three from Africa: hpNEAfrica, hpAfrica1 and hpAfrica2, which consists of three subpopulations (hspWAfrica, hspCAfrica and hspSAfrica). Most detailed H. pylori population analyses have used strains from non-African countries, despite Africa''s high importance in the emergence and evolution of humans and their pathogens. Our concatenated sequences from seven H. pylori housekeeping genes from 44 Gambian patients (MLST) identified 42 distinct sequence types (or haplotypes), and no clustering with age or disease. STRUCTURE analysis of the sequence data indicated that Gambian H. pylori strains belong to the hspWAfrica subpopulation of hpAfrica1, in accord with Gambia''s West African location. Despite Gambia''s history of invasion and colonisation by Europeans and North Africans during the last millennium, no traces of Ancestral Europe1 (AE1) population carried by those people were found. Instead, admixture of 17% from Ancestral Europe2 (AE2) was detected in Gambian strains; this population predominates in Nilo-Saharan speakers of North-East Africa, and might have been derived from admixture of hpNEAfrica strains these people carried when they migrated across the Sahara during the Holocene humid period 6,000–9,000 years ago. Alternatively, shared AE2 ancestry might have resulted from shared ancestral polymorphisms already present in the common ancestor of sister populations hpAfrica1 and hpNEAfrica.     

Helicobacter acinonychis: genetic and rodent infection studies of a Helicobacter pylori-like gastric pathogen of cheetahs and other big cats

Insights into bacterium-host interactions and genome evolution can emerge from comparisons among related species. Here we studied Helicobacter acinonychis (formerly H. acinonyx), a species closely related to the human gastric pathogen Helicobacter pylori. Two groups of strains were identified by randomly amplified polymorphic DNA fingerprinting and gene sequencing: one group from six cheetahs in a U.S. zoo and two lions in a European circus, and the other group from a tiger and a lion-tiger hybrid in the same circus. PCR and DNA sequencing showed that each strain lacked the cag pathogenicity island and contained a degenerate vacuolating cytotoxin (vacA) gene. Analyses of nine other genes (glmM, recA, hp519, glr, cysS, ppa, flaB, flaA, and atpA) revealed a approximately 2% base substitution difference, on average, between the two H. acinonychis groups and a approximately 8% difference between these genes and their homologs in H. pylori reference strains such as 26695. H. acinonychis derivatives that could chronically infect mice were selected and were found to be capable of persistent mixed infection with certain H. pylori strains. Several variants, due variously to recombination or new mutation, were found after 2 months of mixed infection. H. acinonychis ' modest genetic distance from H. pylori, its ability to infect mice, and its ability to coexist and recombine with certain H. pylori strains in vivo should be useful in studies of Helicobacter infection and virulence mechanisms and studies of genome evolution.     

Crystallographic and Electron Microscopic Analyses of a Bacterial Phytochrome Reveal Local and Global Rearrangements during Photoconversion

Phytochromes are multidomain photoswitches that drive light perception in plants and microorganisms by coupling photoreversible isomerization of their bilin chromophore to various signaling cascades. How changes in bilin conformation affect output by these photoreceptors remains poorly resolved and might include several species-specific routes. Here, we present detailed three-dimensional models of the photosensing module and a picture of an entire dimeric photoreceptor through structural analysis of the Deinococcus radiodurans phytochrome BphP assembled with biliverdin (BV). A 1.16-Å resolution crystal structure of the bilin-binding pocket in the dark-adapted red light-absorbing state illuminated the intricate network of bilin/protein/water interactions and confirmed the protonation and ZZZssa conformation of BV. Structural and spectroscopic comparisons with the photochemically compromised D207A mutant revealed that substitutions of Asp-207 allow inclusion of cyclic porphyrins in addition to BV. A crystal structure of the entire photosensing module showed a head-to-head, twisted dimeric arrangement with bowed helical spines and a hairpin protrusion connecting the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) and phytochrome-specific (PHY) domains. A key conserved hairpin feature is its anti-parallel, two β-strand stem, which we show by mutagenesis to be critical for BphP photochemistry. Comparisons of single particle electron microscopic images of the full-length BphP dimer in the red light-absorbing state and the photoactivated far-red light-absorbing state revealed a large scale reorientation of the PHY domain relative to the GAF domain, which alters the position of the downstream histidine kinase output module. Together, our data support a toggle model whereby bilin photoisomerization alters GAF/PHY domain interactions through conformational modification of the hairpin, which regulates signaling by impacting the relationship between sister output modules.     

人源CLP的表达、纯化及生化特性研究

目的：为进一步研究CLP（coactosin-likeprotein）与5’-脂氧合酶、肌动蛋白的相互作用机制及功能,开展CLP克隆表达、分离纯化研究,以得到高纯度的CLP,并对其生物化学特性进行分析测定。方法：从人的胎肝cDNA文库中经PCR扩增得到CLP基因,克隆到原核表达载体pGEX-6p-1中并获得高效表达,经过Glutathione Sepharose 4B亲和层析和Su-perdex 75分子筛纯化,得到高纯度的CLP;在此基础上进行SDS-PAGE、动态光散射和分析型超速离心等实验,并进一步分析实验结果。结果：CLP在溶液中主要以单体形式呈现;CLP的摩擦率为1．909,证实该蛋白质具有线性化趋势存在的可能性,且线性化程度较高。结论：实验结果揭示了CLP作为线性化蛋白质的可能性,为进一步搭建CLP和丝状肌动蛋白的作用模型奠定了一定的数据基础。     

Reproductive characteristics of female Bengal tigers,in Ranthambhore Tiger Reserve,India

Reproductive characteristics of tigers (Panthera tigris) are important to understand population viability. We studied the reproductive parameters of female Bengal tigers (P. t. tigris) in a dry, tropical, deciduous habitat in Ranthambhore Tiger Reserve (RTR), western India, from April 2005 to March 2010. We monitored tigers by direct observation and with cameras placed throughout their habitat. The potential breeding population included 13 adult females. The average age at first reproduction was 3.3 years; 34 cubs were born during the study period (6.2?±?0.82 per year). Sixty-six percent of the births occurred between October and December. Mean litter size was 2.26?±?0.52 (n?=?13, range?=?1–3). The sex ratio of 32 cubs was 1.29 M:1.00 F. The survival rate of cubs (<12 months) was 85 % (95 % CI?=?0.68–0.94), whereas that of juveniles (12–24 months), and subadults (24–36 months) was 79 % (95 % CI?=?0.61–0.91). All breeding females were >3 years old. Only 2 of the 13 females reproduced twice during the 5 years of the study. The birth interval was 33.4?±?3.7 months (range 24–65 months). The mean reproductive rate was 0.59?±?0.23 cubs/female/year. Our study indicates that tiger populations can grow rapidly if the habitat provides adequate protection, an adequate population of prey, and minimal to no poaching.     

Microbiological, Biochemical, and Electron Microscopic Characterization of a Pectinatus Strain

A spoilage organism isolated from turbid beer is described. The bacterium was gram negative, catalase negative, strictly anaerobic, and rod shaped, having flagella only on one side of the cell. The main metabolic product was propionic acid. In addition acetic, succinic, and lactic acids and acetoin were formed. Malonate inhibited the production of propionic acid by the strain studied and by both Pectinatus and Propionibacterium strains. The guanine-plus-cytosine content of deoxyribonucleic acid was 36 mol%. Differences between this strain and Pectinatus strains were 2 to 5 percentage points. Immunofluorescent staining and gel diffusion precipitin tests revealed that the antigenic structure differed from those of Pectinatus strains. The isolated organism can, despite some differences, be regarded as belonging to the genus Pectinatus.     

Molecular and Genetic Analyses of the B Type Surface Protein Gene from Paramecium Tetraurelia

The gene encoding the B type variable surface protein from Paramecium tetraurelia stock 51 has been cloned and sequenced. The 7,182 nucleotide open reading frame contains no introns and encodes a cysteine-rich protein that has a periodic structure including three nearly perfect tandem repeats in the central region. Interestingly, the B gene is located near a macronuclear telomere as was shown previously for two other paramecium surface protein genes. In this paper, we characterize four independent mutants with complete macronuclear deletions of the B gene. Previous analysis of different macronuclear deletion mutants of the A surface protein gene demonstrated two types of inheritance: typical Mendelian segregation (as illustrated by d12) and cytoplasmic inheritance (shown by d48). F(1) analysis of four B(-) mutants crossed with wild-type cells reveals heterozygous F(1) cell lines derived from both parental cytoplasms contain approximately the same copy number of the B gene, as expected for a recessive Mendelian mutation. Analysis of F(2) progeny from three of these four B(-) mutant crosses indicates that one of the three exhibits a Mendelian 1:1 segregation ratio of B(+) and B(-) cell lines. The other two show a preponderance of B(+) cells, but this is not correlated with the parental cytoplasmic type. In addition to having a large number of B(+) individuals, the d12.144, A(-), B(-) mutant produced some F(2) progeny that stably maintain less than normal macronuclear amounts of the A gene and/or the B gene.     

Electron Microscopic Study of a Slime Layer

Slime layers are being studied in our laboratories in an attempt to understand their functions in the control of pollution in natural streams. A method for fixing, staining, and embedding microorganisms in the intact slime has been developed. In this method, epoxy resin discs are placed in a holder and are introduced into a simulated stream. After various periods of time the discs are punched out of the holder into the fixative. The disc with the attached slime is fixed, stained (4% osmium tetroxide plus ruthenium red), dehydrated, and embedded in epoxy resin so that thin sections can be cut through the vertical plane of the slime mass. Such thin sections permit detailed examination of the attached layer, the surface-slime interface, the spatial relationships between cells in the vertical slime structure, and the strands of extracellular material between and around cells. No special attachment structures were noted as the cells appeared to be attached to the surface by extracellular material alone. This material was observed in strands and netlike forms between cells which are positioned 1 to 4 mum apart in the slime.     

Electron Microscopic Analysis of Circumsporozoite Protein Trail Formation by Gliding Malaria Sporozoites

Immunoelectron microscopic techniques were utilized to characterize the morphology of circumsporozoite protein-containing trails deposited on various substrates by gliding Plasmodium berghei and Plasmodium falciparum sporozoites. The basic components of the trails are beadlike particles, 25 to 90 nm in diameter, which are devoid of unit membrane and have an electronlucent center. Trails were captured on formvar-covered grids coated with anticircumsporozoite protein monoclonal antibodies and compared with trails produced on uncoated formvar; the results suggest that material containing circumsporozoite protein forms the matrix within which the particles are embedded. The trails exhibit morphological features similar to those displayed by circumsporozoite precipitation reactions; of note is the demonstration of sheaths of circumsporozoite protein-containing material that emanate from sporozoites prior to their gliding. The sheaths narrow into accumulations of electron-dense material, which eventually taper to form typical trails. The structural manifestation of sheaths and other morphological details of the formed trails enables us to correlate sporozoite behavior during trail formation with the motile actions of gliding sporozoites observed by light microscopy.     

Cloning, Expression, Purification and Toxicity Evaluation of Helicobacter pylori Outer Inflammatory Protein A

The Helicobacter pylori outer membrane proteins play an important role in pathogenesis; the outer inflammatory protein A (OipA) is one of these proteins which play the main role in the development of inflammation. In this study, purification of recombinant H. pylori OipA was performed by Ni–NTA affinity chromatography. Gastric carcinoma epithelial cells (AGS cell) were treated by different concentrations of recombinant OipA for various lengths of time and cell viability was evaluated by the viability assay. Statistical analysis showed that OipA had toxic effects on AGS cells in a concentration of 500 ng/ml after 24 and 48 h, and this toxic dose was 256 ng/ml after 72 h. OipA had direct toxic effects on gastric epithelial cells and the toxicity was observed to depend on time and dose of H. pylori exposure. Attachment of H. pylori to gastric epithelial cells is a key part in the pathogenesis and enables H. pylori to damage the epithelial cells with OipA.     

Identification of a Novel Penicillin-Binding Protein from Helicobacter pylori

The Helicobacter pylori genome encodes four penicillin-binding proteins (PBPs). PBPs 1, 2, and 3 exhibit similarities to known PBPs. The sequence of PBP 4 is unique in that it displays a novel combination of two highly conserved PBP motifs and an absence of a third motif. Expression of PBP 4, but not PBP 1, 2, or 3, is significantly increased during mid- to late-log-phase growth.     

幽门螺杆菌热休克蛋白A基因的克隆、表达及免疫原性研究

幽门螺杆菌的感染可诱发人体产生胃炎和消化性溃疡,其组成成分热休克蛋白Ａ（ＨｓｐＡ）可刺激机体产生保护性的免疫反应。用ＰＣＲ方法从幽门螺杆菌的染色体ＤＮＡ上扩增出ＨｓｐＡ基因片段,将其插入原核表达载体ｐＥＴ２２ｂ（＋）中,并在ＢＬ２１（ＤＥ３）大肠杆菌表达。经测序ＨｓｐＡ基因片段有３５４ｂｐ组成,可编码１１８个氨基酸残基的多肽。ＳＤＳＰＡＧＥ和免疫印迹分析检测发现,ＨｓｐＡ基因表达的蛋白质分子量约为１５ｋＤ,并证实该重组蛋白质可以被幽门螺杆菌感染阳性患者的血清所识别,同时将其免疫小鼠可刺激机体产生抗该重组蛋白质的抗体。ＨｓｐＡ有可能作为一种有效的蛋白质疫苗用于幽门螺杆菌感染的预防和治疗。     

幽门螺杆菌Lpp20融合蛋白的表达、标签切除及鉴定

目的:利用GST融合基因表达系统表达Lpp20-GST融合蛋白,并利用凝血酶切除GST标签.方法:IPTG诱导重组质粒Lpp20/pGEX-4T -1在大肠杆菌BL21 (DE3)中表达,菌体经反复冻融、溶菌酶裂解及超声破菌后,发现Lpp20-GST融合蛋白以部分可溶性的形式表达.采用GST蛋白纯化系统对其纯化,得到Lpp20- GST融合蛋白,再用凝血酶进行GST标签的切除,所得产物进行Western Blot鉴定.结果:高效表达出Lpp20-GST融合蛋白的相对分子质量约4.5kDa,凝血酶成功切除了GST标签,Western Blot证实Lpp20蛋白能被鼠抗Lpp20单克隆抗体识别.结论:成功表达和纯化了重组Lpp20蛋白,为深入研究Lpp20的功能奠定了基础.     

Spherical Parasporal Inclusions of the Lepidoptera-Specific and Coleoptera-Specific Bacillus thuringiensis Strains: A Comparative Electron Microscopic Study

Four Lepidoptera-specific Bacillus thuringiensis strains that belong to the four H serogroups (serovars sumiyoshiensis, fukuokaensis, darmstadiensis, and japonensis) and a Coleoptera (Scarabaeidae)-specific strain belonging to serovar japonensis were examined for comparative ultrastructure of spherical parasporal inclusions. The prominent feature of the inclusions of the Lepidoptera-specific strains was the existence of thick, highly electron-dense envelopes surrounding a homogeneous protein matrix. The envelopes were 15.0–66.7 nm thick and consisted of 5–12 layers of membrane. This is also the case with inclusions of a Coleoptera-specific strain. The ultrastructure of inclusions from the five strains was in marked contrast to that of the bipyramidal parasporal inclusions produced by a Lepidoptera-specific serovar sotto strain. Received: 26 July 1999 / Accepted: 30 August 1999     

Electron Microscopic Analysis of a Spherical Mitochondrial Structure

Mitochondria undergo dynamic structural alterations to meet changing needs and to maintain homeostasis. We report here a novel mitochondrial structure. Conventional transmission electron microscopic examination of murine embryonic fibroblasts treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, found that more than half of the mitochondria presented a ring-shaped or C-shaped morphology. Many of these mitochondria seemed to have engulfed various cytosolic components. Serial sections through individual mitochondria indicated that they formed a ball-like structure with an internal lumen surrounded by the membranes and containing cytosolic materials. Notably, the lumen was connected to the external cytoplasm through a small opening. Electron tomographic reconstruction of the mitochondrial spheroids demonstrated the membrane topology and confirmed the vesicular configuration of this mitochondrial structure. The outside periphery and the lumen were defined by the outer membranes, which were lined with the inner membranes. Matrix and cristae were retained but distributed unevenly with less being kept near the luminal opening. Mitochondrial spheroids seem to form in response to oxidative mitochondrial damage independently of mitophagy. The structural features of the mitochondrial spheroids thus represent a novel mitochondrial dynamics.     

Sequence Divergence and Conservation in Genomes of Helicobacter cetorum Strains from a Dolphin and a Whale

Background and Objectives

Strains of Helicobacter cetorum have been cultured from several marine mammals and have been found to be closely related in 16 S rDNA sequence to the human gastric pathogen H. pylori, but their genomes were not characterized further.

Methods

The genomes of H. cetorum strains from a dolphin and a whale were sequenced completely using 454 technology and PCR and capillary sequencing.

Results

These genomes are 1.8 and 1.95 mb in size, some 7–26% larger than H. pylori genomes, and differ markedly from one another in gene content, and sequences and arrangements of shared genes. However, each strain is more related overall to H. pylori and its descendant H. acinonychis than to other known species. These H. cetorum strains lack cag pathogenicity islands, but contain novel alleles of the virulence-associated vacuolating cytotoxin (vacA) gene. Of particular note are (i) an extra triplet of vacA genes with ≤50% protein-level identity to each other in the 5′ two-thirds of the gene needed for host factor interaction; (ii) divergent sets of outer membrane protein genes; (iii) several metabolic genes distinct from those of H. pylori; (iv) genes for an iron-cofactored urease related to those of Helicobacter species from terrestrial carnivores, in addition to genes for a nickel co-factored urease; and (v) members of the slr multigene family, some of which modulate host responses to infection and improve Helicobacter growth with mammalian cells.

Conclusions

Our genome sequence data provide a glimpse into the novelty and great genetic diversity of marine helicobacters. These data should aid further analyses of microbial genome diversity and evolution and infection and disease mechanisms in vast and often fragile ocean ecosystems.     

Electron Microscopic Immunocytochemical Localization of Myelin Proteolipid Protein and Myelin Basic Protein to Oligodendrocytes in Rat Brain During Myelination

Electron microscopic immunocytochemical studies were carried out to localize myelin basic protein and myelin proteolipid protein during the active period of myelination in the developing rat brain using antisera to purified rat brain myelin proteolipid protein and large basic protein. The anti-large basic protein serum was shown by the immunoblot technique to cross-react with all five forms of basic protein present in the myelin of 8-day-old rat brain. Basic protein was localized diffusely in oligodendrocytes and their processes at very early stages in myelination. The immunostaining for basic protein was not specifically associated with any subcellular structures or organelles. The ultrastructural localization of basic protein suggests that it may be involved in fusion of the cytoplasmic faces of the oligodendrocyte processes during compaction of myelin. Immunoreactivity in the oligodendrocyte and myelin due to proteolipid protein appeared at a later stage of myelination than did that due to basic protein. Staining for proteolipid protein in the oligodendrocyte was restricted to the membranes of the rough endoplasmic reticulum, the Golgi apparatus, and apparent Golgi vesicles. The early, uncompacted periaxonal wrappings of oligodendrocyte processes were well stained with antiserum to large basic protein whereas staining for proteolipid protein was visible only after the compaction of myelin sheaths had begun. Our evidence indicates that basic protein and proteolipid protein are processed differently by the oligodendrocytes with regard to their subcellular localization and their time of appearance in the developing myelin sheath.     

Supramolecular Assembly of Fucoxanthin-Chlorophyll-Protein Complexes Isolated from a Brown Alga, Petalonia fascia. Electron Microscopic Studies

Using a mild detergent, octyl sucrose, light-harvesting fucoxanthin-Chla/c-protein complexes of a brown alga, Petalonia fascia, wereisolated in the form of supramolecular assemblies. Negativelystained images of these assemblies (FCPAs) were extremely uniformin size and shape. Each was discoidal in shape, being 11.2 nmin diameter and 10.2 nm in height, with a small pit at the centerof disc. From the sedimentation rate (S_{20, w} = 21.6) and theobserved dimensions, the molecular mass (Mr) of FCPA was calculatedas about 697?10³, and each FCPA was deduced to contain 128 moleculesof Chl a 27 of Chl c, 69 of fucoxanthin and 8 of violaxanthin.Fresh FCPA showed highly efficient transfer of excitation energyfrom fucoxanthin to Chl a but the energetic coupling was disruptedon storage with accompanying distortion of fine structures.Given the occurrence of similar supramolecular assemblies offucoxanthin-chlorophyll a/c-protein complexes in another brownalga, Dictyota dichotoma [Katoh et al. (1989) Biochim. Biophys.Acta 976: 233], the molecular assemblies of fucoxanthin-Chla/c-protein complexes is assumed to be common to the light harvestingsystems in all brown algae. (Received December 28, 1989; Accepted March 5, 1990)