Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

On alternatives to selection-induced mutation in the Bgl operon of Escherichia coli

Selection-induced mutations are nonrandom mutations that occur as specific and direct responses to environmental challenge. Examples of selection-induced mutations have been reported both in bacteria and in yeast. I previously showed (Hall 1988) that excisions of the mobile genetic element IS150 from within bglF are selection induced and argued that they occurred because they were potentially advantageous under the selective conditions employed. Mittler and Lenski (Mittler and Lenski 1992) have argued that such excisions are not selection induced but that they occur randomly in nondividing cells. Here I provide further evidence that IS150 excisions are induced by selection and that the excisions are immediately, rather than only potentially, advantageous to the cell. I also provide evidence that excisions, which Mittler and Lenski claim occur randomly in saturated broth cultures, actually occur after samples from those cultures are plated onto selective medium.      

Oxygen permeation profile in lipid membranes: comparison with transmembrane polarity profile

Permeation of oxygen into membranes is relevant not only to physiological function, but also to depth determinations in membranes by site-directed spin labeling. Spin-lattice (T(1)) relaxation enhancements by air or molecular oxygen were determined for phosphatidylcholines spin labeled at positions (n = 4-14, 16) of the sn-2 chain in fluid membranes of dimyristoyl phosphatidylcholine, by using nonlinear continuous-wave electron paramagnetic resonance (EPR). Both progressive saturation and out-of-phase continuous-wave EPR measurements yield similar oxygen permeation profiles. With pure oxygen, the T(2)-relaxation enhancements determined from homogeneous linewidths of the linear EPR spectra are equal to the T(1)-relaxation enhancements determined by nonlinear EPR. This confirms that both relaxation enhancements occur by Heisenberg exchange, which requires direct contact between oxygen and spin label. Oxygen concentrates in the hydrophobic interior of phospholipid bilayer membranes with a sigmoidal permeation profile that is the inverse of the polarity profile established earlier for these spin-labeled lipids. The shape of the oxygen permeation profile in fluid lipid membranes is controlled partly by the penetration of water, via the transmembrane polarity profile. At the protein interface of the KcsA ion channel, the oxygen profile is more diffuse than that in fluid lipid bilayers.     

High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity

The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.      

Spin-labeled gramicidin a: channel formation and dissociation

Gramicidin A was studied by continuous wave electron spin resonance (CW-ESR) and by double-quantum coherence electron spin resonance (DQC-ESR) in several lipid membranes (using samples that were macroscopically aligned by isopotential spin-dry ultracentrifugation) and vesicles. As a reporter group, the nitroxide spin-label was attached at the C-terminus yielding the spin-labeled product (GAsl). ESR spectra of aligned membranes containing GAsl show strong orientation dependence. In DPPC and DSPC membranes at room temperature the spectral shape is consistent with high ordering, which, in conjunction with the observed high polarity of the environment of the nitroxide, is interpreted in terms of the nitroxide moiety being close to the membrane surface. In contrast, spectra of GAsl in DMPC membranes indicate deeper embedding and tilt of the NO group. The GAsl spectrum in the DPPC membrane at 35 degrees C (the gel to Pbeta phase transition) exhibits sharp changes, and above this temperature becomes similar to that of DMPC. The dipolar spectrum from DQC-ESR clearly indicates the presence of pairs in DMPC membranes. This is not the case for DPPC, rapidly frozen from the gel phase; however, there are hints of aggregation. The interspin distance in the pairs is 30.9 A, in good agreement with estimates for the head-to-head GAsl dimer (the channel-forming conformation), which matches the hydrophobic thickness of the DMPC bilayer. Both DPPC and DSPC, apparently as a result of hydrophobic mismatch between the dimer length and bilayer thickness, do not favor the channel formation in the gel phase. In the Pbeta and Lalpha phases of DPPC (above 35 degrees C) the channel dimer forms, as evidenced by the DQC-ESR dipolar spectrum after rapid freezing. It is associated with a lateral expansion of lipid molecules and a concomitant decrease in bilayer thickness, which reduces the hydrophobic mismatch. A comparison with studies of dimer formation by other physical techniques indicates the desirability of using low concentrations of GA (approximately 0.4-1 mol %) accessible to the ESR methods employed in the study, since this yields non-interacting dimer channels.     

Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae)

Individual plants of several Amelanchier taxa contain many polymorphic nucleotide sites in the internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA). This polymorphism is unusual because it is not recent in origin and thus has resisted homogenization by concerted evolution. Amelanchier ITS sequence polymorphism is hypothesized to be the result of gene flow between two major North American clades resolved by phylogenetic analysis of ITS sequences. Western North American species plus A. humilis and A. sanguinea of eastern North America form one clade (A), and the remaining eastern North American Amelanchier make up clade B. Five eastern North American taxa are polymorphic at many of the nucleotide sites where clades A and B have diverged and are thought to be of hybrid origin, with A. humilis or A. sanguinea as one parent and various members of clade B as the other parent. Morphological evidence suggests that A. humilis is one of the parents of one of the polymorphic taxa, a microspecies that we refer to informally as A. "erecta." Sequences of 21 cloned copies of the ITS1- 5.8S gene-ITS2 region from one A. "erecta" individual are identical to A. humilis sequence or to the clade B consensus sequence, or they are apparent recombinants of A. humilis and clade B ITS repeats. Amelanchier "erecta" and another polymorphic taxon are suspected to be relatively old because both grow several hundred kilometers beyond the range of one of their parents. ITS sequence polymorphisms have apparently persisted in these two taxa perhaps because of polyploidy and/or agamospermy (asexual seed production), which are prevalent in the genus.      

Arabidopsis At5g39790 encodes a chloroplast-localized,carbohydrate-binding,coiled-coil domain-containing putative scaffold protein

Background  

Starch accumulation and degradation in chloroplasts is accomplished by a suite of over 30 enzymes. Recent work has emphasized the importance of multi-protein complexes amongst the metabolic enzymes, and the action of associated non-enzymatic regulatory proteins. Arabidopsis At5g39790 encodes a protein of unknown function whose sequence was previously demonstrated to contain a putative carbohydrate-binding domain.     

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.     

The asymmetric function of Dph1–Dph2 heterodimer in diphthamide biosynthesis

JBIC Journal of Biological Inorganic Chemistry - Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue found in archaeal and eukaryotic translation...     

Mate choice copying and conspecific cueing in Japanese quail, Coturnix coturnix japonica

In four experiments, we examined the effects on the affiliative preferences of 'focal' female Japanese quail given the opportunity to watch a conspecific male interact with a 'model' female. Experiments were conducted in three, 10-min phases: (1) a pretest, during which a 'focal' female chose between two males; (2) an observation phase, when each focal female watched the male she had spent less time near during the pretest (her 'nonpreferred' male) interact with a 'model' quail; and (3) a post-test, during which each focal female again chose between her nonpreferred and preferred males. Focal females increased their preferences for nonpreferred males after seeing them together with a model female (but not a model male), even if the nonpreferred male and model female were separated by an opaque barrier that prevented them from interacting. A focal female's preference for the end of the enclosure containing her nonpreferred male was not increased when she either watched him court a concealed model female or watched a model female that was being courted by him. Taken together, the present results suggest that a simple tendency for females to approach areas where they have previously seen a male and female quail, in preference to locations where they have seen only a male quail, can explain some of the effect of watching a nonpreferred male mate on a female's tendency to affiliate with him. However, focal females also showed enhanced preferences for nonpreferred males they had seen mating after we both moved those males and controlled for effects of transposition. Thus, processes akin to both 'mate choice copying' and 'conspecific cueing' remain viable explanations for the increase in a focal female quail's tendency to affiliate with a male she watched mate with another female. Copyright 1999 The Association for the Study of Animal Behaviour.