Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli

Summary A partial EcoRI fragment of Bacillus coagulans DNA cloned in an Escherichia coli K12 bacteriophage host-vector system was shown to direct the synthesis of a thermostable -amylase whose activity could be detected in situ on petri plates using the iodine staining method. A 3.31 kb EcoRI fragment containing the active gene with its own promoter was subcloned in pBR322; in the new clone, called pAMY2, the amylase was shown to accumulate in the periplasmic space. The molecular weight of the enzyme, confirmed by in vivo labelling of plasmid products in minicells, was estimated to be 60000.The restriction map of the plasmid was determined for five restriction enzymes and two new plasmids with smaller DNA inserts were constructed, both directing the synthesis of amylase; one of them with a 2.2 kb PstI insert was shown to be responsible for the synthesis of a fused -lactamase--amylase protein with amylase activity.     

Evidence for two separate Ca2+ pathways in smooth muscle plasmalemma

Summary The activation of rabbit aortic smooth muscle was studied by two most widely used vascular smooth muscle stimulants: -adrenoceptor activation by norepinephrine (NE) and high-K⁺ depolarization. This was studied by measurements of isometric contractions and net as well as unidirectional Ca²⁺ fluxes. These parameters showed markedly differential sensitivities towards two smooth muscle inhibitors used in this study: D 600 and amrinone. By choosing an appropriate concentration of D 600 or amrinone, Ca²⁺ uptake or Ca²⁺ influx induced by high K⁺ or NE could be selectively inhibited. Furthermore, by using unidirectional flux measurements it was demonstrated that Ca²⁺ influx stimulated by NE and high K⁺ were additive in nature. The data from the addivity experiment exclude the interpretation of a common Ca²⁺ pathway with two separate mechanisms for opening it. The data on three criteria employed in this study provide evidence for the existence of two independent Ca²⁺ pathways, one for each mode of activation, for Ca²⁺ influx known to be associated with these contractions.     

Typologie des Brackwassers

Zusammenfassung 1. Wenn das Venice System für die Klassifizierung der Brackwässer auf biologischen Unterschieden beruht, sollten die verschiedenen Klassen nicht in erster Stelle nach der Salinität, sondern nach den biologischen Erscheinungen definiert werden.2. Auf Grund der Zusammensetzung der Biozönosen ist es möglich, in Brackmeeren, Aestuaren und abgeschlossenen Brackwässern drei Untertypen zu unterscheiden, deren Grenzen mit dem mittleren Salzgehalt korreliert werden können, und welche gewöhnlich als oligohalin, mesohalin und polyhalin bezeichnet werden.3. Die entsprechenden Untertypen der drei verschiedenen Brackwassertypen sind aber nicht vergleichbar, da ihre Grenzen durch ganz verschiedene Faktoren bestimmt werden und nicht zusammenfallen; infolgedessen weisen ihre Biozönosen deutliche Differenzen auf.4. Die großen täglichen Salzgehaltsschwankungen in den Aestuaren formen eine effektive Barriere für viele euryhaline Meeresarten und fast alle Süßwasserarten.5. In den abgeschlossenen Brackwässern verursachen die oft großen jährlichen Salinitätsschwankungen das Fehlen stenohaliner Arten. Die Maximum- und Minimumsalzwerte entscheiden, welche euryhalinen Arten in einem bestimmten Wasser gut oder nicht existieren können, und zwar unabhängig vom Schwankungsmuster.6. Eine Einteilung des Brackwassers nach seiner räumlichen Kontinuität und der Periodizität der Salzgehaltsschwankungen ergibt ein typologisches System, in welchem folgende Typen erkannt wurden: a) Brackmeere; b) Flußmündungen in einem gezeitenlosen Meer; c) Aestuare; d) Schockbiotope; e) supralittorale Tümpel; f) lagunäre und abgeschlossene Brackwässer; g) Gezeitenzonen; h) Küstengrundwasser. Die verschiedenen Typen werden von speziellen Leitarten gekennzeichnet.7. Nur ein Teil dieser Brackwassertypen zeigt eine weitere Unterteilung, welche mit dem mittleren Salzgehalt korreliert werden kann.

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Typology of brackish water

The Venice System for classification of brackish waters is based on one single factor, salinity, and consequently is not fit for a biological classification. I have amended the system by defining the different subdivisions according to their biological features (den Hartog 1960). It appears that the transition area between fresh and sea water usually shows a tripartition. In the different types of brackish water this tripartition is caused by different factors connected with salinity. In the estuaries the enormous daily salinity fluctuations exercise a too great pressure on the osmotic capacity of many species and thus form an effective barrier for them. In the Baltic Sea, where the salinity conditions are more or less stable, the salinity limit for many species is also the limit of their salinity tolerance. In lagoons and isolated brackish waters the minimum and maximum salinities are decisive for the absence or presence of several species. Although in all these waters the subdivision in an oligohaline, a mesohaline and a polyhaline subtype is evident, there is no reason to suppose that the ranges of the corresponding subtypes will coincide, the more so as their boundaries are determined by different environmental factors. In this paper, therefore, a typological system for the brackish waters is proposed, based on the continuity or discontinuity of the transition between fresh and sea water, and on the periodicity of the salinity fluctuations. In some of the eight recognized types a subdivision according to the amended Venice System is possible.

     

Floating stereospecific assignment revisited: Application to an 18 kDa protein and comparison with J-coupling data

We report a floating chirality procedure to treat nonstereospecifically assigned methylene orisopropyl groups in the calculation of protein structures from NMR data using restrainedmolecular dynamics and simulated annealing. The protocol makes use of two strategies toinduce the proper conformation of the prochiral centres: explicit atom swapping followingan evaluation of the NOE energy term, and atom floating by reducing the angle andimproper force constants that enforce a defined chirality at the prochiral centre. The individualcontributions of both approaches have been investigated. In addition, the effects of accuracyand precision of the interproton distance restraints were studied. The model system employedis the 18 kDa single-stranded DNA binding protein encoded by Pseudomonas bacteriophagePf3. Floating chirality was applied to all methylene and isopropyl groups that give rise to non-degenerate NMR signals, and the results for 34 of these groups were compared to J-couplingdata. We conclude that floating stereospecific assignment is a reliable tool in protein structurecalculation. Its use is beneficial because it allows the distance restraints to be extracteddirectly from the measured peak volumes without the need for averaging or addingpseudoatom corrections. As a result, the calculated structures are of a quality almostcomparable to that obtained with stereospecific assignments. As floating chirality furthermoreis the only approach treating prochiral centres that ensures a consistent assignment of the twoproton frequencies in a single structure, it seems to be preferable over using pseudoatoms or(R-6) averaging.     

Flooding resistance of Rumex species strongly depends on their response to ethylene: Rapid shoot elongation or foliar senescence

In excised Zea maus L. coleoptiles incubated in aerated media at high fresh weight per volume ratios, indole-3-acetic acid induces transient drops of extracellular pH. Based on the quantitative dependency of the response on the initial auxin concentration we developed a novel auxin bioassay, which allows reliable estimation of IAA concentrations between 10⁻⁸⁵ and 10⁻⁵M. Using the bioassay and complementary concentration measurements by IAA fluorescence we found the transient IAA-induced pH response paralleled by a decrease of auxin activity and concentration in the medium. This decline is rapid and starts immediately upon auxin addition, and insofar differs from the well known IAA degradation by epiphytic bacteria in long-term auxin tests. We conclude that the transient character of the auxin pH response is due to rapid IAA metabolism. The effect occurs under those experimental conditions that are necessary for reliable estimations of auxin-induced shifts of cell wall pH, which considerably complicates the interpretation of the results.     

Molecular Cloning of a Preprohormone from Hydra magnipapillata Containing Multiple Copies of Hydra-LWamide (Leu-Trp-NH2) Neuropeptides: Evidence for Processing at Ser and Asn Residues

Abstract: The simple, freshwater polyp Hydra is often used as a model to study development in cnidarians. Recently, a neuropeptide, 2, has been isolated from sea anemones that induces metamorphosis in a hydroid planula larva to become a polyp. Here, we have cloned a preprohormone from Hydra magnipapillata containing 11 (eight different) immature neuropeptide sequences that are structurally related to the metamorphosis-inducing neuropeptide from sea anemones. During the final phase of our cloning experiments, another research team independently isolated and sequenced five of the neuropeptides originally found on the preprohormone. Comparison of these mature neuropeptide structures with the immature neuropeptide sequences on the preprohormone shows that most immature neuropeptide sequences are preceded by Ser or Asn residues, indicating that these residues must be novel processing sites. Thus, the structure of the Hydra prepro-hormone confirms our earlier findings that cnidarian pre-prohormones contain unusual or novel processing sites. Nearly all neuropeptide copies located on the Hydra preprohormone will give rise to mature neuropeptides with a C-terminal Gly-Leu-Trp-NH₂ sequence (the most frequent one being Gly-Pro-Pro-Pro-Gly-Leu-Trp-NH₂; Hydra-LWamide I; three copies). Based on their structural similarities with the metamorphosis-inducing neuropeptide from sea anemones, the mature peptides derived from the Hydra-LWamide preprohormone are potential candidates for being developmentally active neurohormones in Hydra .     

Thermodynamics of melting of the circular dumbbell d〈pCGC-TT-GCG-TT〉

The conformational behavior of DNA minihairpin loops is sensitive to the directionality of the base pair that closes the loop. Especially tailored circular dumbbells, consisting of a stem of three Watson–Crick base pairs capped on each side with a minihairpin loop, serve as excellent model compounds by means of which deeper insight is gained into the relative stability and melting properties of hairpin loops that differ only in directionality of the closing pair: C-G vs G-C. For this reason the thermodynamic properties of the circular DNA decamers 5′-d〈pCGC-TT-GCG-TT〉-3′( I ) and reference compounds 5′-d〈pGGC-TT-GCC-TT≤-3′( II ) and 5′-d(GCG-TC-CGC)-3′( III ) are studied by means of nmr spectroscopy. Molecules I and II adopt dumbbell structures closed on both sides by a two-membered hairpin hop. At low temperature I consists of a mixture of two slowly exchanging forms, denoted L2L2 and L2L4 . The low-temperature L2L2 form is the fully intact minihairpin structure with three Watson–Crick C-G base pairs. The high-temperature form, L2L4 ,contains a partially disrupted closing G-C base pair in the 5′-GTTC-3′ loop, with the cytosine base placed in a syn orientation. The opposite 5′-CTTG-3′ loop remains stable. A study of the noncircular hairpin structure III shows similar conformational behavior for the 5′-GTTC-3′ loop as found in I a syn orientation for C(6) and two slowly exchanging imino proton signals for G(3). The melting point T_m of II was estimated to lie above 365 K. The T_m value of the duplex stem and the 5′-CTTG-3′ loop of the L2L4 form ofIis 352 ± 2 K. The ΔH° is calculated as ?89 ± 10 kJ/mol. The T_m value determined for the individual residues of the 5′-GTTC-3′ loop lies 4°–11° lower. The enthalpy ΔH° of melting the thymine residues in the 5′-GTTC-3′ loop is calculated to be -61± 7 kJ/mol. Thermodynamic data of the equilibrium between the slowly exchanging two- and four-membered loop conformers of I reveal an upper limit for ΔH° of +30 kJ/mol in going from a two-memberedto a four-membered loop, in agreement with the enthalpy difference of +28 k.j/mol between the two loops at the T_m midpoint. For hairpin III the upper limit for ΔH° going from a two-membered to a four-membered loop amounts to ±21 kJ/mol. The mutual exchange rate between the L2 and L4 form in III is estimated as 13.6 s^?1. Our results clearly suggest that small four-way DNA junctions(model for immobilized Holliday junctions) can be designed that consist of a single DNA strandthat features -CTTG-caps on three of the four arms of the junction. © 1995 John Wiley & Sons, Inc.     

Detailed analysis of the IL-5-IL-5R alpha interaction: characterization of crucial residues on the ligand and the receptor.

The receptor for interleukin-5 (IL-5) is composed of two different subunits. The IL-5 receptor alpha (IL-5R alpha) is required for ligand-specific binding while association with the beta-chain results in increased binding affinity. Murine IL-5 (mIL-5) has similar activity on human and murine cells, whereas human IL-5 (hIL-5) has marginal activity on murine cells. We found that the combined substitution of K84 and N108 on hIL-5 by their respective murine counterpart yields a molecule which is as potent as mIL-5 for growth stimulation of a murine cell line. Since the unidirectional species specificity is due only to the interaction with the IL-5R alpha subunit, we have used chimeric IL-5R alpha molecules to define regions of hIL-5R alpha involved in species-specific hIL-5 ligand binding. We found that this property is largely determined by the NH2-terminal module of hIL-5R alpha, and detailed analysis defined D56 and to a lesser extent E58 as important for binding. Moreover, two additional residues, D55 and Y57, were identified by alanine scanning mutagenesis within the same region. Based on the observed homology between the NH2-terminal module and the membrane proximal (WSXWS-containing) module of hIL-5R alpha we located this stretch of four amino acid residues (D55, D56, Y57 and E58) in the loop region that connects the C and D beta-strands on the proposed tertiary structure of the NH2-terminal module.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational analysis of the Yersinia enterocolitica virC operon: characterization of yscE, F, G, I, J, K required for Yop secretion and yscH encoding YopR

Pathogenic yersiniae secrete the Yop anti-host proteins using a type-III secretion pathway. The components of the secretion machinery are encoded by three loci on the pYV plasmid: virA, virB, and virC . In this paper we describe the characterization of eight non-polar mutants of the virC locus, constructed by allelic exchange. The yscE, FG, I, J and K mutants were defective in Yop secretion and independent of Ca²⁺ (Cl) for their growth at 37°C. Substitution of the 12 N-terminal amino-acid residues of YscF impaired secretion of YopB and YopD only and led also to a Cl phenotype. The culture supernatant of the yscH mutant contained all the Yops except the 18 kDa YopR. Complementation experiments and an immunoblot analysis confirmed that YopR is encoded by the yscH gene. The LD₅₀ for the mouse of the yscH mutant was 10-fold higher than that of the parental strain indicating that YopR is involved in pathogenesis. The phenotype of the yscM mutant was similar to that of the wild-type strain. However, overproduction of YscM from a multicopy plasmid in wild-type Yersinia enterocolitica prevented Yop secretion and synthesis. A hybrid YopH—LacZ' protein, encoded by a gene transcribed from the lac promoter, was secreted by a strain overexpressing YscM, showing that the secretion machinery was still functional. These results indicate that YscM plays a role in the feedback inhibition of Yop synthesis when secretion is compromised by acting as a negative regulator of Yop synthesis.     

Trehalose induces antagonism towards Pythium debaryanum in Pseudomonas fluorescens ATCC 17400.

Pseudomonas fluorescens ATCC 17400 shows in vitro activity against Pythium debaryanum under conditions of iron limitation. A lacZ reporter gene introduced by transposon mutagenesis into the P. fluorescens ATCC 17400 trehalase gene (treA) was induced by a factor released by the phytopathogen Pythium debaryanum. The induction of the lacZ gene was lost upon treatment of the Pythium supernatant with commercial trehalase. A trehalose concentration as low as 1 microM could induce the expression of treA. The mutation did not affect the wild-type potential for fungus antagonism but drastically decreased the osmotolerance of the mutant in liquid culture and suppressed the ability of P. fluorescens ATCC 17400 to utilize trehalose as a carbon source. A subsequent transposon insertion in treP, one of the trehalose phosphotransferase genes upstream of treA, silenced the lacZ gene. This double mutant restricted fungal growth only under conditions of high osmolarity, which probably results in internal trehalose accumulation. These data confirm the role of the disaccharide trehalose in osmotolerance, and they indicate its additional role as an initiator of or a signal for fungal antagonism.