Extending Phenomenological Crystal-Field Methods to C_{1} Point-Group Symmetry: Characterization of the Optically Excited Hyperfine Structure of

We show that crystal-field calculations for C_{1} point-group symmetry are possible, and that such calculations can be performed with sufficient accuracy to have substantial utility for rare-earth based quantum information applications. In particular, we perform crystal-field fitting for a C_{1}-symmetry site in ^{167}Er^{3+}:Y_{2}SiO_{5}. The calculation simultaneously includes site-selective spectroscopic data up to 20 000 cm^{-1}, rotational Zeeman data, and ground- and excited-state hyperfine structure determined from high-resolution Raman-heterodyne spectroscopy on the 1.5 µm telecom transition. We achieve an agreement of better than 50 MHz for assigned hyperfine transitions. The success of this analysis opens the possibility of systematically evaluating the coherence properties, as well as transition energies and intensities, of any rare-earth ion doped into Y_{2}SiO_{5}.

The formation of spores in biofilms of Anoxybacillus flavithermus.

AIMS: To examine the rate and the extent of spore formation in Anoxybacillus flavithermus biofilms and to test the effect of one key variable - temperature - on spore formation. METHODS AND RESULTS: A continuous flow laboratory reactor was used to grow biofilms of the typical dairy thermophile A. flavithermus (strain CM) in skim milk. The reactor was inoculated with either a washed culture or a spore suspension of A. flavithermus CM, and was run over an 8.5 h period at three different temperatures of 48, 55 and 60 degrees C. Change in impedance was used to determine the cell numbers in the milk and on the surface of the stainless steel reactor tubes. The biofilm developed at all three temperatures within 6-8 h. Spores formed at 55 and 60 degrees C and amounted to approx. 10-50% of the biofilm. No spores formed at 48 degrees C. CONCLUSIONS: The results suggest that both biofilm formation and spore formation of A. flavithermus can occur very rapidly and simultaneously. In addition, temperature variation has a considerable effect on the formation of spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This information will provide direction for developing improved ways in which to manipulate conditions in milk powder manufacturing plants to control biofilms and spores of A. flavithermus.

Contribution of receptor editing to the antibody repertoire.

Receptor editing, clonal deletion, and anergy are the mechanisms by which B cells maintain tolerance to self antigens. To determine the extent to which receptor editing shapes the normal antibody repertoire, we generated an immunoglobulin kappa polymorphism that facilitates the detection of editing of immunoglobulin light chains in vivo. We found that B cells are targeted for editing during a 2-hour delay in development at the pre-BII cell stage, and that about 25% of all antibody molecules are produced by gene replacement. These results suggest that receptor editing represents a major force in shaping the antibody repertoire.

Filamentous phage are released from the bacterial membrane by a two-step mechanism involving a short C-terminal fragment of pIII.

Filamentous phage assemble at the membrane of infected cells. The phage filament is released from the membrane at the end of assembly, after four to five copies of the minor proteins, pIII and pVI, have been added to the end of the virion. In the absence of pIII or pVI, phage filaments are not released, but remain associated with the cells. The C-terminal portion of pIII, termed the "C" domain, is required for the release of stable virions. With the use of pIII C-terminal fragments of increasing size, termination of assembly can be divided into various steps. An 83-residue fragment leads to the incorporation of pVI into the assembling phage, but does not release it from the membrane. A slightly longer fragment (93 residues) is sufficient to release the particle into the culture supernatant. However, these released particles are unstable in the detergent, sarkosyl, which does not disrupt wild-type phage. A fragment of >121 residues is needed for the particle to become detergent resistant. Thus, the C-domain can be divided into two subdomains: C2, sufficient for release, and C1, required for virion stability.A model for termination of phage assembly is proposed in which pIII and pVI dock to the membrane-associated filament and form a pre- termination complex. Then, a conformational change involving the C2 domain of pIII disrupts the hydrophobic interactions with the inner membrane, releasing the phage from the cells. The pIII-mediated release of phage from the membranes points to one possible mechanism for excision of membrane-anchored protein complexes from lipid bilayers.

A phagemid vector using the E. coli phage shock promoter facilitates phage display of toxic proteins.

Phage display is a powerful tool with which to adapt the specificity of protease inhibitors. To this end, a library of variants of the potato protease inhibitor PI2 was introduced in a canonical phagemid vector. Although PI2 is a natural trypsin inhibitor, we were unable to select trypsin-binding variants from the library. Instead, only mutants carrying deletions or amber stop codons were found. Bacteria carrying these mutations had a much faster growth rate than those carrying the wt PI2-encoding gene, even when the promoter was repressed. To overcome these problems, two new phagemid vectors for g3-mediated phage display were constructed. The first vector has a lower plasmid copy number, as compared to the canonical vector. Bacteria harboring this new vector are much less affected by the presence of the PI2-g3 fusion gene, which appears from a markedly reduced growth retardation. A second vector was equipped with the promoter of the Escherichia coli psp operon, instead of the lac promoter, to control the PI2-g3 gene fusion expression. The psp promoter is induced upon helper phage infection. A phagemid vector with this promoter controlling a PI2-g3 gene fusion did not affect the viability of the host. Furthermore, both new vectors were shown to produce phage particles that display the inhibitor protein and were therefore considered suitable for phage display. The inhibitor library was introduced in both new vectors. Trypsin-binding phages with inhibitory sequences were selected, instead of sequences with stop codons or deletions. This demonstrates the usefulness of these new vectors for phage display of proteins that affect the viability of E. coli.

In vivo and in vitro activities of the Escherichia coli sigma54 transcription activator, PspF, and its DNA-binding mutant, PspFDeltaHTH.

Transcription of the phage-shock protein (psp) operon in Escherichia coli is driven by a sigma54 promoter, stimulated by integration host factor and dependent on an upstream, cis-acting sequence and an activator protein, PspF. PspF belongs to the enhancer binding protein family but lacks an N-terminal regulatory domain. Purified PspF is not modified and has an ATPase activity that is increased twofold in the presence of DNA carrying the psp cis-acting sequence. Purified mutant His-tagged PspF that lacks the C-terminal DNA-binding motif has a DNA-independent ATPase activity when present at 30-fold the concentration of the wild-type protein. Both proteins oligomerize in solution in an ATP and DNA-independent manner. The wild-type activator protein, but not the DNA-binding mutant, binds specifically to the cis-acting sequence. Analysis of the sequence protected by PspF demonstrates the presence of two upstream binding sites within the sequence, UAS I and UAS II, which together constitute the psp enhancer. Protection at low protein concentrations is more pronounced and more extensive on a supercoiled DNA than on a linear template. Full expression of the psp operon upon hyperosmotic shock depends on wild-type PspF, but only partially requires the presence of the psp enhancer.

Roles of pIII in filamentous phage assembly.

Filamentous phage protein III (pIII), located at one end of the phage, is required for infectivity and stability of the particle. Cells infected with phage from which gene III has been completely deleted produce particles that are not released into the medium but stay associated at the surface. These particles are much longer than normal phage. They can be released by subsequent expression of pIII. Viewed with the electron microscope, cells infected with gene III deletion phage are decorated with structures that resemble extremely long pili. Surprisingly, such cells are viable and can form colonies. The pIII deficiency can be complemented in trans, but there is a threshold concentration below which assembly does not occur. Above this threshold, pIII is used very efficiently and is incorporated into infectious but longer than unit length phage. As the concentration of pIII is increased, the number of infectious particles increases, and their average length decreases.pIII stabilizes pVI, a second phage protein found at the pIII end of the particle. In the absence of pIII, degradation of pVI is very rapid. pIII is thus not only required for infectivity and particle stability, but to terminate assembly and release the phage from its assembly site.

Filamentous phage infection-mediated gene expression: construction and propagation of the gIII deletion mutant helper phage R408d3.

We describe the use of transcriptional fusions to the phage shock protein (psp) promoter. These fusions are expressed only when cells are infected by filamentous phage. In an application, the psp promoter was fused to the protein coding part of filamentous phage gene III (gIII). Protein III (pIII) is needed to complement mutant f1 phage containing a deletion of gIII, but its synthesis also renders cells resistant to infection. By inducing pIII production from psp-gIII only in the cells that are already infected with phage, it was possible to obtain plaques from phage in which gIII had been completely deleted. gIII was deleted from two helper phages: R408 and VCSM13, which were then propagated on cells containing the psp-gIII fusion. These two phages were tested for use in a phage display method that requires generation of noninfectious, phagemid-containing virion-like particles. Both helpers worked, but R408d3 was superior to VCSM13d3, because it generated about 1800-times fewer background infectious particles.

Nucleotide sequence, mutational analysis, transcriptional start site, and product analysis of nov, the gene which affects Escherichia coli K-12 resistance to the gyrase inhibitor novobiocin.

In a previous study, we demonstrated the existence of a gene locus, nov, which affects resistance of Escherichia coli K-12 to the gyrase inhibitor novobiocin and, to a lesser degree, coumermycin (J. Rakonjac, M. Milic, D. Ajdic, D. Santos, R. Ivanisevic, and D. J. Savic, Mol. Microbiol. 6:1547-1553, 1992). In the present study, sequencing of the nov gene locus revealed one open reading frame that encodes a protein of 54,574 Da, a value. found to be in correspondence with the size of the Nov protein identified in an in vitro translation system. We also located the 5' end of the nov transcript 8 bp downstream from a classical sigma70 promoter. Transcription of the gene is in the counterclockwise direction on the E. coli chromosome. Transposon mutagenesis of nov followed by complementation analyses and replacement of chromosomal alleles with mutated nov confirmed our previous assumption that the nov gene exists in two allelic forms and that the Novr gene is an active allele while the Novs gene is an inactive form. After comparing nucleotide sequences flanking the nov gene with existing data, we conclude that the gene order in this region of the E. coli K-12 map is att phi 80-open reading frame of unknown function-kch (potassium channel protein)-nov-opp. Finally, the possible identity of the nov gene with cls, the gene that codes for cardiolipin synthase, is also discussed.

DNA sequence of the serum opacity factor of group A streptococci: identification of a fibronectin-binding repeat domain.

The serum opacity factor (SOF) is a group A streptococcal protein that induces opacity of mammalian serum. The serum opacity factor 22 gene (sof22) from an M type 22 strain was cloned from an EMBL4 library by screening for plaques exhibiting serum opacity activity. DNA sequencing yielded an open reading frame of 3,075 bp. Its deduced amino acid sequence predicts a protein of 1,025 residues with a molecular weight of 112,735, a size that approximates that of the SOF22 protein isolated from both the original streptococcal strain and Escherichia coli harboring the cloned sof22 gene. The molecule is composed of three domains: an N-terminal domain responsible for the opacity reaction (opacity domain), a repeat domain with fibronectin-binding (Fn-binding) activity, and a C-terminal cell attachment domain. The C-terminal end of SOF22 is characterized by a hexameric LPXTGX motif, an adjacent hydrophobic region, and a charged C terminus, which are the hallmarks of cell-bound surface proteins found on nearly all gram-positive bacteria. Immediately upstream of this cell anchor region, SOF22 contains four tandem repeat sequence blocks, flanked by prolinerich segments. The repeats share up to 50% identity with a repeated motif found in other group A streptococcal Fn-binding proteins and exhibit Fn-binding activity, as shown by subcloning experiments. According to deletion analysis, the opacity domain is confined to the region N terminal to the repeat segment. Thus, SOF22 is unique among the known Fn-binding proteins from gram-positive bacteria in containing an independent module with a defined function in its N-terminal portion. Southern blot analysis with a probe from this N-terminal region indicates that the opacity domain of SOF varies extensively among different SOF-producing M types.

nov: a new genetic locus that affects the response of Escherichia coli K-12 to novobiocin.

We have identified a new gene locus (nov) affecting the resistance of Escherichia coli K-12 to novobiocin. The gene also affects, although to a lesser extent, tolerance to another gyrase inhibitor coumermycin. Transductional and complementation analysis show that nov is located between att phi 80 and the osmZ (hns) genes at minute 27 of the E. coli K-12 genetic map. In standard laboratory strains of E. coli K-12 nov exists at least in two allelic forms.

cysB and cysE mutants of Escherichia coli K12 show increased resistance to novobiocin.

Mutations in the cysB and cysE genes of Escherichia coli K12 cause an increase in resistance to the gyrase inhibitor novobiocin but not to coumermycin, acriflavine and rifampicin. This unusual relationship was also observed among spontaneous novobiocin resistant (Novr) mutants: 10% of Novr mutants isolated on rich (LA) plates with novobiocin could not grow on minimal plates, and among those approximately half were cysB or cysE mutants. Further analyses demonstrated that cysB and cysE negative alleles neither interfere with transport of novobiocin nor affect DNA supercoiling.