Constitutive production of the receptor-binding domain of the F18 fimbriae on the surface of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> using synthetic promoters

Nine synthetic constitutive promoters with different activities were produced for the surface display of the receptor-binding domain of the F18 fimbrial adhesin FedF in Lactococcus lactis. The promoters were synthesized from an oligonucleotide, designed to allow for randomization of bases between defined lactococcal promoter consensus regions. However, promoters with spontaneous modifications were selected for fedF expression, indicating that the consensus promoter was too strong. The amount of FedF on the surface of the best performing promoter clone was comparable to the amount of FedF produced by a L. lactis strain with an optimized NICE expression system. Stability of FedF production further suggested that an optimal constitutive expression level was attained.     

Adhesin receptors of human oral bacteria and modeling of putative adhesin-binding domains

Adherence by bacteria to a surface is critical to their survival in the human oral cavity. Many types of molecules are present in the saliva and serous exudates that form the acquired pellicle, a coating on the tooth surface, and serve as receptor molecules for adherent bacteria. The primary colonizing bacteria utilize adhesins to adhere to specific pellicle receptor molecules, then may adhere to other primary colonizers via adhesins, or may present receptor molecules to be utilized by secondary colonizing species. The most common primary colonizing bacteria are streptococci, and six streptococcal cell wall polysaccharide receptor molecules have been structurally characterized. A comparison of the putative adhesin disaccharide-binding regions of the six polysaccharides suggests three groups. A representative of each group was modeled in molecular dynamics simulations. In each case it was found that a loop formed between the galactofuranoseß (Galfß) and an oxygen of the nearest phosphate group on the reducing side of the Galfß, that this loop was stabilized by hydrogen bonds, and that within each loop resides the putative disaccharide-binding domain.Abbreviations used are as follows f furanose - Fuc d-fucose - Gal d-galactose - GalNAc N-Acetyl-d-Galactosamine - Glc d-glucose - Glyc glycerol - HPLC high-performance liquid chromatography - HPTLC high-performance thin-layer chromatography - HF hydrofluoric acid - LacCer lactosyl ceramide - NeuAc N-Acetyl neuraminic acid - NMR nuclear magnetic resonance - PRG proline-rich glycoproteins - PRP proline-rich proteins - p pyranose - Rha l-rhamnose - RMSD root-mean-square differences     

Relationship between phage resistance and emulsan production,interaction of phages with the cell-surface of Acinetobacter calcoaceticus RAG-1

The hydrocarbon-degrading strain Acinetobacter calcoaceticus RAG-1 produces an extracellular emulsifying agent capable of forming stable oil-in-water emulsions. The bioemulsifier, termed emulsan, is a polyanionic heteropolysaccharide (M.W. 10⁶) composed mainly of N-acyl D-galactosamine and an N-acyl hexosamine uronic acid. In order to probe the interaction of emulsan with the cell surface prior to its release into the growth medium, two new virulent bacteriophages for A. calcoaceticus RAG-1 were isolated from sewage and the properties of phage resistant mutants were studied. The two phages, ap-2 and ap-3, were differentiated on the basis of plaque morphology, electron microscopy and buoyant density. Isolated mutants of A. calcoaceticus RAG-1 which were resistant to one of the two phages retained sensitivity to the other phage. Resistance to phage ap-3 was accompanied by a severe drop in emulsan production. Independently isolated derivatives of A. calcoaceticus RAG-1 with a defect in emulsan production also turned out to be resistant towards phage ap-3. Antibodies prepared against purified emulsan specifically inhibited phage ap-3 adsorption to the cell surface of the parental strain.     

Transduction inMycobacterium phlei

Transduction, the transfer of genetic information from one bacterial host to another by bacteriophage, has been demonstrated inMycobacterium phlei. The ability to utilized-xylose as a sole carbon source could be transferred fromM. phlei strain SN109 (xyl ⁺,str-s) toM. phlei strain F89 (xyl ⁻,str-r) by a bacterially sterile, DNase-treated phage Bo2-lysate of donorM. phlei SN109. The marker was expressed within 15 minutes of phage infection, but the recipient bacteria had to be in early stationary phase for transduction to occur. Controls employed in these experiments were bacteria incubated with heat-killed phage, with phage plus antiphage serum, or with sterile broth in place of phage. Phage conversion, the appearance of a new character due solely to phage infection, could not account for the observed phenomenon since phage Bo2, which was not previously propagated on axyl ⁺ donor, failed to confer the ability to utilize xylose on axyl ⁻ repicient. High frequency transduction (HFT) also could be demonstrated by using phage lysates obtained fromxyl ⁺ transductants. This work was presented in part at the 71st Annual Meeting of the American Society for Microbiology, Minneapolis, 2–8 May, 1971. Submitted by the senior author in partial fulfillment of the requirement for the Ph.D. degree at the Department of Microbiology, Loyola University — Stritch School of Medicine, Maywood, Illinois.     

Identification and characterization of peptides that bind the PPIase domain of Parvulin17

Peptidyl‐prolyl cis‐trans isomerases (PPIases) are the enzymes that increase the rate of isomerization of the peptide bond N‐terminal to the proline substrate. Par14 and its isoform Par17 belong to the Parvulin family of PPIases. Par14 can bind AT‐rich double‐stranded DNA and was shown to be part of the pre‐ribosomal ribonucleoprotein (pre‐rRNP) complexes, where it functions as an RNA processing factor that is involved in ribosome biogenesis. Its longer isoform Par17 is expressed only in cells of hominids, where it is targeted to the mitochondria. To find binding partners (peptides or proteins) for Par17, we applied the phage display technology. We panned 7‐mer and 12‐mer peptide libraries against Par17. The consensus sequence XHSXVHØ, where X can be any amino acid and Ø is a hydrophobic amino acid, was enriched from both libraries. We demonstrate the binding of this motif to the PPIase domain of Par17 using phage ELISA and NMR spectroscopy. We propose that residues Met90, Val91, Phe94, Gln95, Glu96, and Ala98 of Par17 are involved in substrate recognition, and that the phage display‐selected motif XHSXVHØ can be recognized by Par17 PPIase domain in vivo. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

The acidophilous taxa of thefestuca varia group in the alps: New studies on taxonomy and phytosociology

The acidophilous taxa of theFestuca varia group appearing in the Alps (F. varia Haenke s.str.,F. scabriculmis (Hack.)K. Richt. subsp.scabriculmis, F. scabriculmis subsp.luedii Markgr.-Dann., andF. acuminata Gaudin) have been investigated with regard to their taxonomy, phytosociology and ecology. Morphometrical analyses of the different taxa confirm that the division of the species group is justified. Moreover three varieties ofFestuca varia s.str. can clearly be distinguished. Apart from the already knownF. varia var.varia and var.handel-mazzettii Krajina a newF. varia var.winnebachensis Wallossek etMarkgr.-Dann. is described. The ploidy levels of 30 samples from all taxa have been determined by means of flow cytometry.Festuca acuminata is diploid (2n=14), although this taxon occurs in ecologically different habitats within an altitudinal range between 200 and 3100 m.Festuca varia var.varia is tetraploid whereas the other varieties occur in hexaploid populations, which can be associated with inter- or post-glacial migration stages. Hexaploidy was also determined for both subspecies ofF. scabriculmis. The phylogenesis of these taxa by allopolyploidy seems possible, as they show a morphologically intermediate position betweenF. acuminata and the basiphilousF. quadriflora Honck. The spatial distribution and separation of the taxa is shown by means of precise area maps. As a result of a numerical classification of 534 relevés, the Alpine variegated fescue grasslands can be subdivided into six independent phytosociological associations of theFestucion variae: the East AlpinePulsatillo albae-Festucetum variae, Gentianello anisodontae-Festucetum variae ass. nov. andSeslerio-Festucetum variae ass. nov. as well as the West AlpineFestucetum scabriculmis, Euphorbio cyparissiae-Festucetum luedii ass. nov. andFestuco scabriculmis-Potentilletum valderiae. The mixture of basiphilous and acidophilous species in theSeslerio-Festucetum variae can be explained by soil heterogeneity with acid and alkaline conditions alternating within a very small space.Festuca acuminata can rather be regarded as a rock plant as it preferably occurs in associations of the allianceAndrosacion multiflorae.     

Comparative studies on the effect of vitamins on sporulation in Fusarium oxysporum schlecht. ex. Fr. and Fusarium moniliforme V. subglutinans Wr. & Rg.

On the basis of sporulation (total output of all the three spore forms taken together) and fungal mat production bothF. oxysporum Schlecht ex.Fr. andF. moniliforme v.subglutinans Wr. &Rg. are auxoheterotrophic for thiamine, biotin, inositol, riboflavin and pyridoxine. The first three vitamins are selective in accelerating macro-conidial production also inF. moniliforme, which otherwise shows decrease with advance in days of incubation.F. moniliforme is an auxo-autotroph for nicotinic acid, Ca-pantothenate and folic acid and auxoheterotroph for ascorbic acid. Auxoautotrophy for Ca-pantothenate, folic acid, l-ascorbic acid and p-aminobenzoic acid cannot be suggested forF. oxysporum. Whereas nicotinic acid is a depressent of sporulation inF. oxysporum, inF. moniliforme another vitamin p-aminobenzoic acid depresses sporulation. As the two species ofFusarium show differences in preference as well as inhibition to at least five of the vitamins studied and also varied trends of pH changes exists there is full justification for their separate taxonomic placements.     

Expression and purification of the mannose recognition domain of the FimH adhesin

Type 1 fimbriae have been shown to be specifically required for Escherichia coli colonisation and pathogenesis of the urinary tract. These structural organelles mediate specific adhesion to alpha-D-mannosides by virtue of the FimH adhesin. FimH is a two-domain protein in which the N-terminal domain contains the receptor-binding site and the C-terminal domain is required for organelle integration. To date, FimH has only been isolated as a complex with the system-specific chaperone FimC. Here we report that a functional form of the FimH receptor-binding domain can be readily isolated and characterised by replacing the C-terminal domain with a histidine tag.     

Crystal structure of the MrkD1P receptor binding domain of Klebsiella pneumoniae and identification of the human collagen V binding interface

Klebsiella species are members of the family enterobacteriaceae, opportunistic pathogens that are among the eight most prevalent infectious agents in hospitals. Among other virulence factors in Klebsiella, type 3 pili exhibit a unique binding pattern in the human kidney via interaction of two MrkD adhesion variants 1C1 and 1P to type IV and/or V collagen. However, very little is known about the nature of this recognition. Here we present the crystal structure of the plasmid born MrkD_1P receptor domain (MrkDrd). The structure reveals a jelly‐roll β‐barrel fold comprising 17 β‐strands very similar to the receptor domain of GafD, the tip adhesin from the F17 pilus that recognizes n ‐acetyl‐d ‐glucosamine (GlcNAc). Analysis of collagen V binding of different MrkD_1P mutants revealed that two regions were responsible for its binding: a pocket, that aligns approximately with the GlcNAc binding pocket of GafD involving residues R105 and Y155, and a transversally oriented patch that spans strands β2a, β9b and β6 including residues V49, T52, V91, R102 and I136. Taken together, these data provide structural and functional insights on MrkD_1P recognition of host cells, providing a tool for future development of rationally designed drugs with the prospect of blocking Klebsiella adhesion to collagen V.     

Structural and Thermodynamic Characterization of Pre- and Postpolymerization States in the F4 Fimbrial Subunit FaeG

Enterotoxigenic Escherichia coli expressing F4 fimbriae are the major cause of porcine colibacillosis and are responsible for significant death and morbidity in neonatal and postweaned piglets. Via the chaperone-usher pathway, F4 fimbriae are assembled into thin, flexible polymers mainly composed of the single-domain adhesin FaeG. The F4 fimbrial system has been labeled eccentric because the F4 pilins show some features distinct from the features of pilins of other chaperone-usher-assembled structures. In particular, FaeG is much larger than other pilins (27  versus ∼ 17 kDa), grafting an additional carbohydrate binding domain on the common immunoglobulin-like core. Structural data of FaeG during different stages of the F4 fimbrial biogenesis process, combined with differential scanning calorimetry measurements, confirm the general principles of the donor strand complementation/exchange mechanisms taking place during pilus biogenesis via the chaperone-usher pathway.     

Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pIII

Protein III (pIII) of filamentous phage is required for both the beginning and the end of the phage life cycle. The infection starts by binding of the N-terminal N2 and N1 domains to the primary and secondary host receptors, F pilus and TolA protein, respectively, whereas the life cycle terminates by the C-terminal domain-mediated release of the membrane-anchored virion from the cell. It has been assumed that the role of the C-terminal domain of pIII in the infection is that of a tether for the receptor-binding domains N1N2 to the main body of the virion. In a poorly understood process that follows receptor binding, the virion disassembles as its protein(s) become integrated into the host inner membrane, resulting in the phage genome entry into the bacterial cytoplasm. To begin revealing the mechanism of this process, we showed that tethering the functional N1N2 receptor-binding domain to the virion via termination-incompetent C domain abolishes infection. This infection defect cannot be complemented by in trans supply of the functional C domain. Therefore, the C domain of pIII acts in concert with the receptor-binding domains to mediate the post receptor binding events in the infection. Based on these findings, we propose a model in which binding of the N1 domain to the periplasmic portion of TolA, the secondary receptor, triggers in cis a conformational change in the C domain, and that this change opens or unlocks the pIII end of the virion, allowing the entry phase of infection to proceed. To our knowledge, this is the first virus that uses the same protein domain both for the insertion into and release from the host membrane.     

Infection of transformable cells ofHaemophilus influenzae by bacteriophage and bacteriophage DNA

Summary A phage HP1, infecting transformable cells ofHaemophilus influenzae Rd, has been isolated. The general properties of the wild type and of a clear plaquemutantc1 employed for most of the experiments are described. Phage DNA is infective for transformableHaemophilus cells with an efficiency (plaqueforming units of the original phage recovered as DNA-infected cells) of up to 6×10^–3. The competence ofHaemophilus cells for infection with phage DNA parallels the competence for transformation with bacterial DNA.Both HP1 and thec1 mutant are able to lysogenize their host, and the lysogenic cells are readily induced by UV. Competent non-lysogenicHaemophilus cells can be infected by DNA of lysogenic cells, thereby giving rise to phage progeny. Thus, the phage genetic material can be introduced into competentHaemophilus cells in three different ways: injection from intact phage, and infection with either phage DNA or with bacterial DNA carrying the prophage.The UV inactivation curves for infectious phage DNA and for complete phages are similar, both indicating the occurrance of host-cell reactivation. Photoreactivationin vitro of infectious phage DNA takes place to about the same high extent as observed with bacterial transforming DNA.The usefulness of this system for investigating bacterial transformation and biological effects ofin vitro treatment of DNA is discussed.with the technical assistance ofSandra J. Antoine With 4 Figures in the TextPreliminary report presented at the 7th Annual Bacterial Transformation Meeting, Aspen, Colorado, June 17–19, 1963.Supported by a travel grant from the Deutsche Forschungsgemeinschaft.Supported by Research Carreer Development Award GM-K3-7500 and Research Grant RH 00221 from the U.S. Public Health Service.     

PrgB promotes aggregation,biofilm formation,and conjugation through DNA binding and compaction

Gram‐positive bacteria deploy type IV secretion systems (T4SSs) to facilitate horizontal gene transfer. The T4SSs of Gram‐positive bacteria rely on surface adhesins as opposed to conjugative pili to facilitate mating. Enterococcus faecalis PrgB is a surface adhesin that promotes mating pair formation and robust biofilm development in an extracellular DNA (eDNA) dependent manner. Here, we report the structure of the adhesin domain of PrgB. The adhesin domain binds and compacts DNA in vitro. In vivo PrgB deleted of its adhesin domain does not support cellular aggregation, biofilm development and conjugative DNA transfer. PrgB also binds lipoteichoic acid (LTA), which competes with DNA binding. We propose that PrgB binding and compaction of eDNA facilitates cell aggregation and plays an important role in establishment of early biofilms in mono‐ or polyspecies settings. Within these biofilms, PrgB mediates formation and stabilization of direct cell‐cell contacts through alternative binding of cell‐bound LTA, which in turn promotes establishment of productive mating junctions and efficient intra‐ or inter‐species T4SS‐mediated gene transfer.     

Adsorption of bacteriophage on bacterial surface and on the surface of Hg dropping electrode

Summary The influence of the ionic strength of the medium on the adsorption of bacteriophage T 2 to the surfaces of a mercury dropping electrode on one hand and ofbacteria E. coli B on the other hand was studied. The adsorption on the mercury surface was determined by measurement of the differential capacity of the electrode double layer, the adsorption to bacteria was estimated from the decrease of free phage particles in a bacterial suspension with time. The adsorption to the mercury electrode increases with increasing ionic strength of the medium, but adsorption to the surface of bacteria increases at first, has a maximum at concentrations between 0,1 to 0,5 M and decreases with further increase of ionic strength. The decrease of adsorption of phage to the bacterial surface is assumed to be caused by the blocking of specific sites on the bacterial surface by adsorbed ions which sterically prevent the adsorption of the phage. Such specific sites are not present on the electrode surface, therefore adsorption increases further with increasing ionic strength probably due to the neutralization of surface charges of the phage and of the electrode. The saturated surface-concentration of the phage _s was calculated from the dependence of the differential capacity on the concentration. It is concluded from _s value obtained that the phage particles are scattered with wide intervals on the electrode surface with a degree of coverage of approximately 140.Abbreviations used DNA deoxyribonucleic acid - N Avogadro number The authors wishes to express their gratitude to the late Prof.Ferdinand Hercík, director of the Institute of Biophysics, for the initiation of this work and stimulating interest. The authors are also indebted to Dr. J.Koudelka for his kind gift of phage T 2 sample and to Dr. M.Vízdalová for her valuable comments during preparation of this article.     

On the validity of the genusDactylococcopsis (Cyanophyceae)

Summary The genusDactylococcopsis Hansg. 1888 (Cyanophyceae) is based on the typeD. rupestris, which was later identified as a green algae. Most of the many species described later were also placed to other groups of algae. Several authors even doubted about the existence of the genus. As, however, some species of Cyanophyceae correspond to the original generic diagnosis, the name Dactylococcopsis Hansg. ex R. et F.Chod. 1925 has been proposed as a nomen conservandum, and a new type (D. smithii R. et F.Chod.) has been defined. Further speciesD. linearis Geitl. 1935 and D.Planctonica Teil. 1942 has been unambiguously described till now.     

Systematik,Verbreitung und Karyologie derFestuca violacea-Gruppe (Poaceae) im Ostalpenraum

The biosystematics of theFestuca violacea group (F. rubra subsp.violacea sensuHackel) in the Eastern Alps is studied.F. picturata Pils (=F. picta Kit. exSchultes nonJ. F. Gmelin) is confirmed as diploid, whereas the chromosome numbers forF. nitida (diploid),F. puccinellii (hexaploid), andF. norica (diploid, tetraploid and hexaploid) are reported for the first time. Details of morphology, leaf anatomy, and epidermal structures now allow a better separation of these species; new maps illustrate their distribution. Karyological, chorological, morphological, and anatomical data form the basis for a discussion of the phylogeny of theF. violacea group and its position withinF. rubra s. latiss.

     

Protein characterization of lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages isolated from cheese wheys

Proteins of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages were studied using antibody inhibition assay and immunoblotting. Antisera were prepared against four representative L. lactis ssp. lactis and L. lactis ssp. cremoris phages (D59-1, F4-1, G72-1, and I37-1), which were selected from 17 isolates, derived from commercial cheese wheys. The reactivities of the four antisera with 13 other phage isolates were tested. Among these isolates, two phage groups having distinct serological properties were found. Group I reacted with the antisera against phages D59-1/F4-1 and Group II reacted with the antisera against phages G72-1/I37-1. Strongly lytic phages, capable of lysing phage-resistant host strains, were found to share protein similarities with the phage protein group I, and phages isolated from phage-sensitive host strains belonged to the phage protein group II. Furthermore, group I was composed of all prolate and some isometric phages, whereas group II was composed solely of the isometric phages. Thus, the two serologically distinct phage groups were not correlated with the two morphological groups, prolate and isometric. Proteins of the four phages were further characterized by immunoblotting and silver staining. A 22.5-kDa antigenic polypeptide of phage I37-1, and three polypeptides of 65, 37, 21 kDa in phage F4-1 were responsible for the cross-reactivities in group II and group I, respectively. Correspondence to: R. A. Ledford     

Comparison between Enterotoxigenic Escherichia coli Strains Expressing “F42,” F41 and K99 Colonization Factors

Two enterotoxigenic Escherichia coli (ETEC) strains (coded 567/7 and 103) isolated from piglets with neonatal diarrhea were described as producers of a new adhesin (F42). With the use of molecular biology and immunology techniques such as DNA hybridization with probes for F41 and K99 genes and Western-blotting of the superficial proteins of these strains and standard E. coli strains carrying genes for F41 and K99 adhesins, it was demonstrated that this new adhesin either shares extensive genetic and immunological determinants with F41 adhesin or they are the same fimbriae.     

Structural and energetic basis of infection by the filamentous bacteriophage IKe

Filamentous phage use the two N‐terminal domains of their gene‐3‐proteins to initiate infection of Escherichia coli. One domain interacts with a pilus, and then the other domain binds to TolA at the cell surface. In phage fd, these two domains are tightly associated with each other, which renders the phage robust but non‐infectious, because the TolA binding site is inaccessible. Activation for infection requires partial unfolding, domain disassembly and prolyl isomerization. Phage IKe infects E. coli less efficiently than phage fd. Unlike in phage fd, the pilus‐ and TolA‐binding domains of phage IKe are independent of each other in stability and folding. The site for TolA binding is thus always accessible, but the affinity is very low. The structures of the two domains, analysed by X‐ray crystallography and by NMR spectroscopy, revealed a unique fold for the N‐pilus‐binding domain and a conserved fold for the TolA‐binding domain. The absence of an activation mechanism as in phage fd and the low affinity for TolA probably explain the low infectivity of phage IKe. They also explain why, in a previous co‐evolution experiment with a mixture of phage fd and phage IKe, all hybrid phage adopted the superior infection mechanism of phage fd.     

Drei neue Arten desFestuca ovina-Formenkreises (Poaceae) aus dem Osten Österreichs

WithinFestuca ovina agg. two hexaploid taxa:F. brevipila andF. carnuntina, and one tetraploid:F. eggleri, are described as new species.F. pseudovina Hack. exWiesb. is better treated asF. valesiaca Schleich. exGaudin subsp.parviflora (Hack.), comb. nova.

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Drei neue Arten desFestuca ovina-Formenkreises (Poaceae) aus dem Osten Österreichs