Kinetics of benzoate-induced loss of the TOL plasmid from Pseudomonas putida MT15 during growth in chemostat culture

Potassium-limited chemostat cultures of Pseudomonss putida MT15, grown on excess glucose, displayed approximately 100% plasmid loss after 60 generations of growth in the presence of 5 mM benzoate. The kinetics of plasmid loss indicated that plasmid-free cells displayed a growth rate advantage, which we attribute to selective inhibition of the growth of plasmid-containing cells by benzoate. However, stable, mixed populations of plasmid-free cells, deletants and plasmid-containing cells were selected during growth under glucose limitation in the presence of benzoate. This behaviour indicated that the plasmid-free cells in these cultures displayed a growth rate disadvantage and that their appearance was due entirely to benzoate-induced segregational instability of the plasmid.     

Isopropylbenzene catabolic pathway in Pseudomonas putida RE204: nucleotide sequence analysis of the ipb operon and neighboring DNA from pRE4

Pseudomonas putida RE204 employs a set of plasmid-specified enzymes in the catabolism of isopropylbenzene (cumene) and related alkylbenzenes. A 21,768 bp segment of the plasmid pRE4, whose sequence is discussed here, includes the ipb (isopropylbenzene catabolic) operon as well as associated genetic elements. The ipb operon, ipbAaAbAcAdBCEGFHD, encodes enzymes catalyzing the conversion of isopropylbenzene to isobutyrate, pyruvate, and acetyl-coenzyme A as well as an outer membrane protein (IpbH) of uncertain function. These gene products are 75 to 91% identical to those encoded by other isopropylbenzene catabolic operons and are somewhat less similar to analogous proteins of related pathways for the catabolism of mono-substituted benzenes. Upstream of ipbAa, ipbR encodes a positive regulatory protein which has about 56% identity to XylS regulatory proteins of TOL (xylene/toluate) catabolic plasmids. This similarity and that of the DNA sequence in the proposed ipb operator-promoter region (ipbOP) to the same region of the xyl meta operon (xylOmPm) suggest that, although the IpbR and XylS regulatory proteins recognize very different inducers, their interactions with DNA to activate gene expression are similar. Upstream of ipbR is an 1196 bp insertion sequence, IS1543, related to IS52 and IS1406. Separating ipbR from ipbAa are 3 additional tightly clustered IS elements. These are IS1544, related to IS1543, IS52, and other members of the IS5 family; IS1545, related to IS1240; and IS1546, related to IS1491. Encompassing the ipb catabolic genes and the other genetic elements and separated from each other by 18,492 bp, are two identical, directly repeated 1007 bp DNA segments. Homologous recombination between these segments appears to be responsible for the occasional deletion of the intervening DNA from pRE4.     

Mutational analysis of hydrophobic domain interactions in gamma B-crystallin from bovine eye lens.

gamma B-crystallin is a monomeric member of the beta gamma-superfamily of vertebrate eye lens proteins. It consists of two similar domains with all-beta Greek key topology associating about an approximate two-fold axis. At pH 2, with urea as the denaturant, the domains show independent equilibrium unfolding transitions, suggesting different intrinsic stabilities. Denaturation experiments using recombinant one- or two-domain proteins showed that the N-terminal domain on its own exhibits unaltered intrinsic stability but contributes significantly to the stability of its C-terminal partner. It has been suggested that docking of the domains is determined by a hydrophobic interface that includes phenylalanine at position 56 of the N-terminal domain. In order to test this hypothesis, F56 was substituted by site-directed mutagenesis in both complete gamma B-crystallin and its isolated N-terminal domain. All mutations destabilize the N-terminal domain to about the same extent but affect the C-terminal domain in a different way. Replacement by the small alanine side chain or the charged aspartic acid residue results in a significant destabilization of the C-terminal domain, whereas the more bulky tryptophan residue causes only a moderate decrease in stability. In the mutants F56A and F56D, equilibrium unfolding transitions obtained by circular dichroism and intrinsic fluorescence differ, suggesting a more complex denaturation behavior than the one observed for gamma B wild type. These results confirm how mutations in one crystallin domain can affect the stability of another when they occur at the interface. The results strongly suggest that size, hydrophobicity, and optimal packing of amino acids involved in these interactions are critical for the stability of gamma B-crystallin.     

The population genetics of sporophytic self-incompatibility in Senecio squalidus L. (Asteraceae): the number, frequency, and dominance interactions of S alleles across its British range

Sporophytic self-incompatibility (SSI) was studied in 11 British Senecio squalidus populations to quantify mating system variation and determine how its recent colonization of the United Kingdom has influenced its mating behavior. S allele number, frequency, and dominance interactions in populations were assessed using full diallels of controlled pollinations. A mean of 5.1 S alleles per population was observed, and no population contained more than six S alleles. Numbers of S alleles within populations of S. squalidus declined with increasing distance from the center of its introduction (Oxford). Cross-classification of S alleles allowed an estimate of approximately seven and no more than 11 S alleles for the entire British S. squalidus population. The low number of S alleles observed in British S. squalidus compared to other SI species is consistent with the population bottleneck associated with S. squalidus' introduction to the Oxford Botanic Garden and subsequent colonization of Britain. Extensive S allele dominance interactions were observed to be a feature of the S. squalidus SSI system and may represent an adaptive response to improve limited mate availability imposed by the presence of so few S alleles. Multilocus allozyme genotypes were also identified for individuals in all populations and geographic patterns of S locus and allozyme loci variation investigated. Less interpopulation structure was observed for the S locus than for allozyme diversity--a finding indicative of the effects of negative frequency-dependent selection at the S locus maintaining equal S phenotypes within populations and enhancing effective migration between populations.     

Substrate flow in catalases deduced from the crystal structures of active site variants of HPII from Escherichia coli.

The active site of heme catalases is buried deep inside a structurally highly conserved homotetramer. Channels leading to the active site have been identified as potential routes for substrate flow and product release, although evidence in support of this model is limited. To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at approximately 2.0-A resolution. The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the heme distal pocket. In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the heme distal pocket filling the main channel. The differences in continuity of solvent molecules between the native and variant structures illustrate how sensitive the solvent matrix is to subtle changes in structure. It is hypothesized that the slightly larger H(2)O(2) passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent and peroxide. The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3-A resolution, revealing the existence of hydrogen peroxide binding sites both in the heme distal pocket and in the main channel. Unexpectedly, the largest changes in protein structure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departure from the 222-point group symmetry of the native enzyme. An active role for channels in the selective flow of substrates through the catalase molecule is proposed as an integral feature of the catalytic mechanism. The Asn201His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His-Tyr bond suggesting that the mechanistic pathways of the two reactions can be uncoupled.     

Base coupling in sequence-specific site recognition by the ETS domain of murine PU.1

The ETS domain of murine PU.1 tolerates a large number of DNA cognates bearing a central consensus 5'-GGAA-3' that is flanked by a diverse combination of bases on both sides. Previous attempts to define the sequence selectivity of this DNA binding domain by combinatorial methods have not successfully predicted observed patterns among in vivo promoter sequences in the genome, and have led to the hypothesis that energetic coupling occurs among the bases in the flanking sequences. To test this hypothesis, we determined, using thermodynamic cycles, the complex stabilities and base coupling energies of the PU.1 ETS domain for a set of 26 cognate variants (based on the lambdaB site of the Ig(lambda)2-4 enhancer, 5'-AATAAAAGGAAGTGAAACCAA-3') in which flanking sequences up to three bases upstream and/or two bases downstream of the core consensus are substituted. We observed that both cooperative and anticooperative coupling occurs commonly among the flanking sequences at all the positions investigated. This phenomenon extends at least three bases in the 5' side and is, at least on our experimental data, due exclusively to pairwise interactions between the flanking bases, and not changes in the local environment of the DNA groove floor. Energetic coupling also occurs between the flanking sides across the core consensus, suggesting long-range conformational effects along the DNA target and/or in the protein. Our data provide an energetic explanation for the pattern of flanking bases observed among in vivo promoter sequences and reconcile the apparent discrepancies raised by the combinatorial experiments. We also discuss the significance of base coupling in light of an indirect readout mechanism in ETS/DNA site recognition.     

Role of interchain alpha-helical hydrophobic interactions in Ca2+ affinity, formation, and stability of a two-site domain in troponin C.

We have previously shown that a 34-residue synthetic peptide representing the calcium-binding site III of troponin C formed a symmetric two-site dimer consisting of two helix-loop-helix motifs arranged in a head-to-tail fashion (Shaw, G.S., Hodges, R.S., & Sykes, B.D., 1990, Science 249, 280-283). In this study the hydrophobicities of the alpha-helices were altered by replacing L-98 and F-102 in the N-terminal region and/or I-121 and L-122 in the C-terminal region with alanine residues. Our results showed that substitution of hydrophobic residues either in the N- or C-terminal region have little effect on alpha-helix formation but resulted in a 100- and 300-fold decrease in Ca2+ affinity, respectively. Simultaneous substitution of both hydrophobes in the N- and C-terminal region resulted in a 1,000-fold decrease in Ca2+ affinity. Data from guanidine hydrochloride denaturation studies suggested that intermolecular interactions occur and that the less hydrophobic analogs had a lower overall conformational stability. These data support the contention that the hydrophobic residues are important in the formation of the two-site domain in troponin C, and this hydrophobic association stabilizes Ca2+ affinity.     

Intra-specific hybridization: generator of genetic diversification and heterosis in Andrographis paniculata Nees. A bridge from extinction to survival

Andrographis paniculata (AP) has been stated as a low-diverse, endangered and red-listed plant species. Self-pollinated mating system, being an introduced species and experiencing a bottleneck as well as over exploitation cause such a consequence. Inter and intra-specific hybridizations have been suggested as essential techniques for generating genetic diversity. To test the effect of intra-specific hybridization on diversification and heterosis of AP, seven accessions were outcrossed manually in all 21 possible combinations. Three types of markers including morphological, phytochemical and RAPD markers were employed to evaluate the mentioned hypothesis. The results revealed that hybridization acted as a powerful engine for diversification of AP as it caused heterotic expression of the studied traits, simultaneously. Initially, it seems that additive and non-additive gene effects both can be considered as the genetic basis of heterosis in AP for the investigated traits. Agronomic and morphological traits were differentiated from each other, while positive heterosis was recorded mainly for agronomic traits but not for the morphological traits. Intra-specific hybridization increased the genetic diversity in AP population. Nevertheless, a part of this variation could also be attributed to the negative heterosis. The current exploration demonstrated the first ever conducted manual intra-specific hybridization among AP accessions in a mass scale. However, the 17 RAPD primers produced a monomorph pattern, but perhaps increasing the number of markers can feature a new genetic profile in this plant.