Vectors that facilitate the replacement of transcriptional lacZ fusions in Streptococcus mutans and Bacillus subtilis with fusions to gfp or gusA

Plasmid vectors have been constructed for Streptococcus mutans and Bacillus subtilis that make possible rapid replacement of the widely used reporter gene lacZ (encoding beta-galactosidase) with either gfp (encoding green fluorescent protein) or gusA (encoding beta-glucuronidase). The lacZ-->gfp replacement vectors greatly facilitate the analysis of the spatial location of gene expression in biofilms of S. mutans and in sporulating B. subtilis. The lacZ-->gusA replacement vectors facilitate the comparison of two promoters within the same organism. A vector is also described that enables gusA to be replaced with gfp in B. subtilis.     

Molecular phylogeny of the springhare, Pedetes capensis, based on mitochondrial DNA sequences

The phylogenetic position of the Pedetidae, represented by a single species Pedetes capensis, is controversial, reflecting in part the retention of both Hystricomorphous and Sciurognathous characteristics in this rodent. In an attempt to clarify the species evolutionary relationships, mtDNA gene sequences from 10 rodent species (representing seven families) were analyzed using phenetic, parsimony, and maximum-likelihood methods of phylogenetic inference; the rabbit, Oryctolagus cuniculus (Order Lagomorpha), and cow, Bos taurus (Order Artiodactyla), were used as outgroups. Investigation of 714 base pairs of the protein-coding cytochrome b gene indicate strong base bias at the third codon position with significant rate heterogeneity evident between the three structural domains of this gene. Similar analyses conducted on 816 base pairs of the 12S rRNA gene revealed a transversion bias in the loop sections of all taxa. The cytochrome b gene sequences proved useful in resolving associations between closely related species but failed to produce consistent tree topologies at the family level. In contrast, phylogenetic analysis of the 12S rRNA gene resulted in strong support for the clustering of Pedetidae/Heteromyidae/Geomyidae and Muridae in one clade to the exclusion of the Hystricidae/Thryonomyidae and Sciuridae, a finding which is concordant with studies of rodent fetal membranes as well as reproductive and other anatomical features.      

A bridged Mo(VI) complex containing six and five coordinate Mo: Synthesis, 95Mo N.m.r. spectroscopy and X-ray crystal structure of Mo2O5(C17H17N2O)2

The kinetics of the reaction between Carcinus maenas hemocyanin and cyanide has been studied at various KCN concentrations and a different temperatures (21° and 4°C) by following the decrease of the copper-peroxide absorption band, centered at 337 nm, of the copper still bound to the protein and the intrinsic fluorescence changes as functions of time. In all conditions used, the absorption band completely disappears and KCN concentration affects only the rate of the process. The reaction is kinetically homogeneous indicating no site-site interaction. The apparent rate constant increases with the square of cyanide concentration and the inverse of O₂ concentration. The copper still bound decreases at a rate slower than the 337 nm absorption and the process is not kinetically homogeneous. The fluorescence of the protein increases after an induction period showing an inflection point at about 50% of the total effect. A kinetic model has been proposed on the assumption that the two metal ions are removed sequentially from the active site. The experimental data are in agreement with the theoretical equations derived from the model. The equilibrium constants for the formation of the complex between the first and the second copper ion with cyanide and the rate constants of their decomposition have been calculated. The rate-limiting process for the removal of the second copper ion is the formation of the complex with cyanide.     

Analysis by fluorescence microscopy of the development of compartment-specific gene expression during sporulation of Bacillus subtilis.

The use of a fluorogenic substrate, 5-octanoylaminofluorescein-di-beta-D-galactopyranoside, for beta-galactosidase has made it possible to visualize enzyme activity in individual cells of sporulating populations of Bacillus subtilis by fluorescence microscopy. lacZ fusions to different sporulation-associated genes have been used to investigate the cell compartmentalization of gene expression during sporulation. A strain with a lacZ fusion to sspA, a gene which is transcribed by E-sigma G at a late stage of sporulation, displayed predominantly compartment-specific fluorescence. Expression of the early-expressed spoIIA locus, which includes the structural gene for sigma F, was seen not to be compartmentalized. Populations of strains with lacZ fusions to gpr and dacF, genes which are transcribed by E-sigma F at intermediate stages of sporulation, included some organisms showing uncompartmentalized fluorescence and others showing compartment-specific fluorescence; the proportion showing compartment-specific fluorescence increased in samples taken later in sporulation. Several possible explanations of the results obtained with gpr and dacF are considered. A plausible interpretation is that sigma F activity is initially not compartmentalized and becomes compartmentalized as sporulation progresses. The progression to compartmentalization does not require the activities of the sporulation-specific factor sigma E or sigma G but may require some product of sigma F activity.     

Reduced heat resistance of mutant spores after cloning and mutagenesis of the Bacillus subtilis gene encoding penicillin-binding protein 5.

Part of the gene encoding penicillin-binding protein 5 from Bacillus subtilis 168 was cloned in Escherichia coli with a synthetic oligonucleotide as a hybridization probe. The gene was designated dacA by analogy with E. coli. The nucleotide sequence was determined, and the predicted molecular mass was 45,594 daltons (412 amino acids). A comparison of the predicted amino acid sequence with that of the E. coli penicillin-binding protein 5 indicated that these enzymes showed about 25% identity. The B. subtilis dacA gene was mutated by integration of a plasmid into the structural gene by homologous recombination. A comparison of the mutant and control strains revealed that (i) the mutant lacked detectable penicillin-binding protein 5, (ii) the D-alanine carboxypeptidase activity of membranes isolated from the mutant was only 5% of that measured in membranes from the control strain, (iii) the mutant cells showed apparently normal morphology only during exponential growth, and after the end of exponential phase the cells became progressively shorter, (iv) the mutant sporulated normally except that the forespore occupied about two-thirds of the mother cell cytoplasm and, during its development, migrated towards the center of the mother cell, and (v) purified mutant spores were 10-fold less heat resistant but possessed normal refractility and morphology. Preliminary chemical analysis indicated that the structure of the cortex of the mutant was different.     

The Bacillus subtilis spoIIA locus is expressed at two times during sporulation

Abstract The Bacillus subtilis spoIIA and spoIIAB genes were fused to the Escherichia coli lacZ gene on a novel integrational plasmid vector. The constructs were integrated into the B. subtilis chromosome, and used to show that the spoIIA locus was expressed at two times during sporulation.     

The division during bacterial sporulation is symmetrically located in Sporosarcina ureae

Immunofluorescence microscopy was used to visualize the FtsZ band that marks the site of septation in Sporosarcina ureae . Image analysis indicated that the vegetative division was symmetrically located with respect to the ends of the cells. Fusions of lacZ to the sporulation loci, spoIIA and cotE , of Bacillus subtilis were introduced into S . ureae by mobilization of plasmids containing the fusions from Escherichia coli . The fusions showed similar patterns of sporulation-associated expression in S . ureae to those observed in B . subtilis . Formation of β-galactosidase encoded by the spoIIA–lacZ fusion made it possible to identify early sporulating cells by immunofluorescence microscopy. Analysis of the position of FtsZ bands in cells expressing spoIIA–lacZ indicated that the location of sporulation division was symmetrical with respect to the ends of the cells, in sharp contrast to the asymmetrical location of septation in sporulating Bacilli . It is inferred that asymmetry of location of the sporulation division is not essential for the compartmentalization of gene expression that follows the division.     

The NorM MATE Transporter from N. gonorrhoeae: Insights into Drug and Ion Binding from Atomistic Molecular Dynamics Simulations

The multidrug and toxic compound extrusion transporters extrude a wide variety of substrates out of both mammalian and bacterial cells via the electrochemical gradient of protons and cations across the membrane. The substrates transported by these proteins include toxic metabolites and antimicrobial drugs. These proteins contribute to multidrug resistance in both mammalian and bacterial cells and are therefore extremely important from a biomedical perspective. Although specific residues of the protein are known to be responsible for the extrusion of solutes, mechanistic details and indeed structures of all the conformational states remain elusive. Here, we report the first, to our knowledge, simulation study of the recently resolved x-ray structure of the multidrug and toxic compound extrusion transporter, NorM from Neisseria gonorrhoeae (NorM_NG). Multiple, atomistic simulations of the unbound and bound forms of NorM in a phospholipid lipid bilayer allow us to identify the nature of the drug-protein/ion-protein interactions, and secondly determine how these interactions contribute to the conformational rearrangements of the protein. In particular, we identify the molecular rearrangements that occur to enable the Na⁺ ion to enter the cation-binding cavity even in the presence of a bound drug molecule. These include side chain flipping of a key residue, GLU-261 from pointing toward the central cavity to pointing toward the cation binding side when bound to a Na⁺ ion. Our simulations also provide support for cation binding in the drug-bound and apo states of NorM_NG.