Hans-Joachim Fritz

The genome sequence of the extreme thermophile Thermus thermophilus

Thermus thermophilus HB27 is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment in Japan. This organism has considerable biotechnological potential; many thermostable proteins isolated from members of the genus Thermus are indispensable in research and in industrial applications. We present here the complete genome sequence of T. thermophilus HB27, the first for the genus Thermus. The genome consists of a 1,894,877 base pair chromosome and a 232,605 base pair megaplasmid, designated pTT27. The 2,218 identified putative genes were compared to those of the closest relative sequenced so far, the mesophilic bacterium Deinococcus radiodurans. Both organisms share a similar set of proteins, although their genomes lack extensive synteny. Many new genes of potential interest for biotechnological applications were found in T. thermophilus HB27. Candidates include various proteases and key enzymes of other fundamental biological processes such as DNA replication, DNA repair and RNA maturation.

Oligonucleotide-directed construction of mutations via gapped duplex DNA

Publisher Summary This chapter discusses the oligonucleotide-directed construction of mutations via gapped duplex deoxyribo nucleic acid (DNA). Oligonucleotide-directed construction of mutations has become the method of choice to introduce predetermined structural changes into DNA. This chapter discusses the gapped duplex DNA approach to oligonucleotide-directed mutation construction. The key intermediate in this process is a partial DNA duplex of a recombinant M13 genome, gapped duplex DNA (gdDNA), which has only the target region of mutation construction exposed in single-stranded form, and which furthermore, carries distinguishable genetic markers in the two DNA strands in such a way that, a rigorous selection can be applied in favor of phage progeny arising from the shorter strand— that is, (the minus strand of the MI3 genome). Two alternative variants of the gdDNA method are applicable: the “fill-in,” which incorporates DNA polymerase/ DNA ligase reactions in vitro , and the simplified “mix-heat-transfect” protocol. It bypasses these enzymatic manipulations.

Gene Islands Integrated into tRNA(Gly) Genes Confer Genome Diversity on a Pseudomonas aeruginosa Clone

Intraclonal genome diversity of Pseudomonas aeruginosa was studied in one of the most diverse mosaic regions of the P. aeruginosa chromosome. The ca. 110-kb large hypervariable region located near the lipH gene in two members of the predominant P. aeruginosa clone C, strain C and strain SG17M, was sequenced. In both strains the region consists of an individual strain-specific gene island of 111 (strain C) or 106 (SG17M) open reading frames (ORFs) and of a 7-kb stretch of clone C-specific sequence of 9 ORFs. The gene islands are integrated into conserved tRNA(Gly) genes and have a bipartite structure. The first part adjacent to the tRNA gene consists of strain-specific ORFs encoding metabolic functions and transporters, the majority of which have homologs of known function in other eubacteria, such as hemophores, cytochrome c biosynthesis, or mercury resistance. The second part is made up mostly of ORFs of yet-unknown function. Forty-seven of these ORFs are mutual homologs with a pairwise amino acid sequence identity of 35 to 88% and are arranged in the same order in the two gene islands. We hypothesize that this novel type of gene island derives from mobile elements which, upon integration, endow the recipient with strain-specific metabolic properties, thus possibly conferring on it a selective advantage in its specific habitat.

Contribution of the intramolecular disulfide bridge to the folding stability of REIv, the variable domain of a human immunoglobulin κ light chain

BACKGROUND Immunoglobulin domains contain about 100 amino acid residues folded into two beta-sheets and stabilized in a sandwich by a conserved central disulfide bridge. Whether antibodies actually require disulfide bonds for stability has long been a matter of debate. The contribution made by the central disulfide bridge to the overall folding stability of the immunoglobulin REIv, the variable domain of a human kappa light chain, was investigated by introducing stabilizing amino acid replacements followed by removal of the disulfide bridge via chemical reduction or genetic substitution of the cysteine residues. RESULTS Nine REIv variants were constructed by methods of protein engineering that have folding stabilities elevated relative wild-type REIv by (up to) 16.0 kJ mol-1. Eight of these variants can be cooperatively refolded after unfolding and chemical reduction of the disulfide bridge-in contrast to wildtype REIv. The stabilizing effect of one of these residue replacements (T39K) was rationalized by determining the structure of the respective REIv variant at 1.7 A. The loss of folding stability caused by reduction of the intramolecular disulfide bond is on average 19 kJ mol-1. Removal of the disulfide bridge by genetic substitution of C23 for valine resulted in a stable immunoglobulin domain in the context of the stabilizing Y32H amino acid exchange; again, REIv-C23V/Y32H has 18 kJ mol-1 less folding stability than REIv-Y32H. The data are consistent with the notion that all variants studied have the same overall three-dimensional structure with the disulfide bridge opened or closed. CONCLUSIONS A comparison of the magnitude of the stabilizing effect exerted by the disulfide bond and the length of the mainchain loop framed by it suggests lowering of the entropy of the unfolded state as the sole source of the effect. Disulfide bonds are not necessary for proper folding of immunoglobulin variable domains and can be removed, provided the loss of folding stability is at least partly compensated by stabilizing amino acid exchanges.

N-terminal myristoylation-dependent masking of neutralizing epitopes in the preS1 attachment site of hepatitis B virus

BACKGROUNDS & AIMS The N-terminally myristoylated preS1 domain of the large hepatitis B surface protein (LHBs) mediates specific attachment of hepatitis B virus (HBV) to hepatocytes. Its B-cell epitopes leading to neutralization of infectivity are not yet characterized. METHODS We inserted C- and N-terminal preS1 peptides into the most immunogenic region of HBV core particles, therewith immunized Balb/c mice and determined binding properties and neutralization potential of resulting antibodies in vitro. RESULTS The particles with preS1 inserts were highly immunogenic and the corresponding anti-preS antibodies strongly bound to HBV particles from chronic carriers infected with different HBV genotypes A-F. However, antibodies binding to the C-terminal part of preS1 did not neutralize HBV infectivity for susceptible hepatocyte cultures. In contrast, antibodies elicited by the complete N-terminal attachment site of preS1(2-48) strongly neutralized with an IC50<3μg/ml of total immunoglobulin. Interestingly, antibodies against the very N-terminal part of preS1(1-21) could not neutralize infectivity although this sequence contains the most conserved and essential part of the attachment site. These antibodies reacted well with non-myristoylated preS1 peptides but only weakly with myristoylated preS1 peptides, natural HBsAg or HBV. CONCLUSIONS N-terminal myristic acid obviously favors a topology of LHBs that makes the most essential part of the preS1 attachment site inaccessible for neutralizing antibodies, whereas antibodies to neighbouring sequences neutralized very well. Thus, addition of the preS1(2-48) peptide in a highly immunogenic form to the current hepatitis B vaccine may improve protective immunity and reduce selection of escape mutations.

Substrate preferences of Vsr DNA mismatch endonuclease and their consequences for the evolution of the Escherichia coli K-12 genome.

The substrate spectrum of Vsr DNA mismatch endonuclease of Escherichia coli K-12 was investigated using fluorescence-labelled oligonucleotide substrates and a DNA sequencer for detection and quantification of substrates and reaction products. Fourteen substrates were found to be processed by the enzyme, which differ in one or two positions from the canonical pentanucleotide sequence CTA/TGG (T mismatched to G). Relative second-order rate constants of these substrates were determined in groups of four by multiple substrate kinetics and compared to the underresentation of the corresponding pentanucleotides in the E. coli K-12 genome. The high quality of correlation further establishes active mutagenesis by VSP repair as a significant driving force of the evolution of the E. coli K-12 genome and provides clues to its possible selective value.

The Methanothermobacter thermautotrophicus ExoIII homologue Mth212 is a DNA uridine endonuclease

The genome of Methanothermobacter thermautotrophicus, as a hitherto unique case, is apparently devoid of genes coding for general uracil DNA glycosylases, the universal mediators of base excision repair following hydrolytic deamination of DNA cytosine residues. We have now identified protein Mth212, a member of the ExoIII family of nucleases, as a possible initiator of DNA uracil repair in this organism. This enzyme, in addition to bearing all the enzymological hallmarks of an ExoIII homologue, is a DNA uridine endonuclease (U-endo) that nicks double-stranded DNA at the 5′-side of a 2′-d-uridine residue, irrespective of the nature of the opposing nucleotide. This type of activity has not been described before; it is absent from the ExoIII homologues of Escherichia coli, Homo sapiens and Methanosarcina mazei, all of which are equipped with uracil DNA repair glycosylases. The U-endo activity of Mth212 is served by the same catalytic center as its AP-endo activity.

X-ray crystallography reveals stringent conservation of protein fold after removal of the only disulfide bridge from a stabilized immunoglobulin variable domain

BACKGROUND Immunoglobulin domains owe a crucial fraction of their conformational stability to an invariant central disulfide bridge, the closure of which requires oxidation. Under the reducing conditions prevailing in cell cytoplasm, accumulation of soluble immunoglobulin is prohibited by its inability to acquire and maintain the native conformation. Previously, we have shown that disulfide-free immunoglobulins can be produced in Escherichia coli and purified from cytoplasmic extracts. RESULTS Immunoglobulin REIv is the variable domain of a human kappa light chain. The disulfide-free variant REIv-C23V/Y32H was crystallized and its structure analyzed by X-ray crystallography (2.8 A resolution). The conformation of the variant is nearly identical to that of the wild-type protein and the conformationally stabilized variant REIv-T39K. This constitutes the first crystal structure of an immunoglobulin fragment without a disulfide bridge. The lack of the disulfide bridge produces no obvious local change in structure (compared with the wild type), whereas the Y32H mutation allows the formation of an additional hydrogen bond. There is a further change in the structure that is seen in the dimer in which Tyr49 has flipped out of the dimer interface in the mutant. CONCLUSIONS Immunoglobulin derivatives without a central disulfide bridge but with stringently conserved wild-type conformation can be constructed in a practical two-step approach. First, the protein is endowed with additional folding stability by the introduction of one or more stabilizing amino acid exchanges; second, the disulfide bridge is destroyed by substitution of one of the two invariant cysteines. Such derivatives can be accumulated in soluble form in the cytoplasmic compartment of the E. coli cell. Higher protein yields and evolutionary refinement of catalytic antibodies by genetic complementation are among the possible advantages.

1.7 A structure of the stabilized REIv mutant T39K. Application of local NCS restraints.

The X-ray structure of the T39K mutant of the variable domain of a human immunoglobulin kappa light chain has been determined at room temperature to 1.7 A resolution with a conventional R factor of 0. 182. T39K crystallizes in the triclinic space group P1 [a = 35.4 (1), b = 40.1 (1), c = 43.1 (1) A, alpha = 66.9 (1), beta = 85.4 (1), gamma = 73.8 (1) degrees ]. The unit-cell contains two monomers, related by a non-crystallographic twofold axis. The use of a novel type of local non-crystallographic symmetry restraints on related isotropic displacement parameters and 1-4 distances as incorporated in the refinement program SHELXL improves the model and quality of the maps, but local differences between both monomers in areas subject to different packing contacts can still be observed. 12 overall anisotropic scaling parameters were refined. These may have compensated for the difficulties in accurately scaling single rotation axis image plate data from a triclinic crystal, because of the scarcity of common equivalent reflections. The final model has been used to perform a number of tests on anisotropic scaling, non-crystallographic symmetry, anisotropic refinement, determination of standard uncertainties and bulk solvent correction. It is remarkable that removal of the NCS restraints from the final model caused Rfree to increase. These tests clarify the strategies for optimum use of SHELXL for refinement at medium as opposed to atomic resolution.

ToxR co-operative interactions are not modulated by environmental conditions or periplasmic domain conformation

ToxR is a transmembrane regulatory protein that controls virulence gene expression in Vibrio cholerae. Previous experiments using λ repressor–ToxR chimeric proteins and a λ repressor‐controlled reporter system (OR1 PR–lacZY ) established that ToxR sequences can effectively dimerize the amino‐terminal domain of λ repressor in Escherichia coli. However, in E. coliToxR does not respond to environmental signals that control virulence gene expression in V. cholerae. Here, we report the results of experiments designed to test whether environmental signals that modulate virulence gene expression in V. cholerae also modulate a monomer to dimerization transition of λ–ToxR chimeras. When the OR1 PR–lacZY reporter fusion and chimeric proteins were transferred to V. cholerae, we unexpectedly found that λ–ToxR chimeras did not dimerize significantly. Interestingly, experiments evaluating the ability of λ–ToxR proteins to form tetramers in E. coli suggested that λ–ToxR dimers could act co‐operatively. Using a redesigned reporter system containing multiple λ operator sites (OR1 OR2 OR3 PR–lacZY ), we found that λ–ToxR could dimerize quite efficiently in V. cholerae. These data imply that multiple DNA binding sites might enhance the ability of ToxR to dimerize in V. cholerae and suggest that ToxR dimers might be capable of co‐operative interactions. However, we failed to correlate a monomer–dimer transition of the λ–ToxR chimeras with changes in virulence gene expression in response to environmental signals in V. cholerae. Finally, because of conflicting results in the literature, the importance of membrane localization of ToxR and dimerization of the ToxR periplasmic domain was re‐evaluated. This was accomplished by measuring the ability of various chimeric proteins to activate toxin gene expression in both E. coli and V. cholerae. These assays suggest that, in V. choleraedeletion of the transmembrane domain has a profound effect on ToxR activity, although it is not an absolute requirement when ToxR is dimerized by a heterologous domain. In addition, we noted differences in chimeric protein activity when expressed in E. coli and V. cholerae. A construct substituting the monomeric MalE domain for the periplasmic domain of ToxR was unable to activate a ctx::lacZ reporter fusion in E. coli. Although the addition of leucine zipper sequences to this construct resulted in enhanced activity of the chimera in E. coli, both chimeras were able to produce wild‐type levels of toxin in V. cholerae. These data support the notion that dimerization of ToxR stimulates its activity as a transcriptional activator in E. coli. In V. cholerae, however, we present data that do not demonstrate a correlation between dimerization of the periplasmic domain and ToxR activity.