Peter Model

Homologous recombination based modification in Esherichia coli and germline transmission in transgenic mice of a bacterial artificial chromsome

Escherichia coli-based artificial chromosomes have become important tools for physical mapping and sequencing in various genome projects. The lack of a general method to modify these large bacterial clones, however, has limited their utility in functional studies. We developed a simple method to modify bacterial artificial chromosomes directly in the recombination-deficient E. coli host strain by homologous recombination for in vivo studies. The IRES-LacZ marker gene was introduced into a 131 kb BAG containing the murine zinc finger gene, RU49. No rearrangements or deletions were detected in the modified BACs. Furthermore, transgenic mice were generated by pronuclear injection of the modified BAG, and germline transmission of the intact BAG has been obtained. Proper expression of the lacZ transgene in the brain has been observed, which could not be obtained with conventional transgenic constructs.

Sorting of protein a to the staphylococcal cell wall

The cell wall of gram-positive bacteria can be thought of as representing a unique cell compartment, which contains anchored surface proteins that require specific sorting signals. Some biologically important products are anchored in this way, including protein A and fibronectin binding protein of Staphylococcus aureus and streptococcal M protein. Studies of staphylococcal protein A and Escherichia coli alkaline phosphatase show that the signal both necessary and sufficient for cell wall anchoring consists of an LPXTGX motif, a C-terminal hydrophobic domain, and a charged tail. These sequence elements are conserved in many surface proteins from different gram-positive bacteria. We propose the existence of a hitherto undescribed sorting mechanism that positions proteins on the surface of gram-positive bacteria.

Cell wall sorting signals in surface proteins of gram-positive bacteria.

Staphylococcal protein A is anchored to the cell wall, a unique cellular compartment of Gram‐positive bacteria. The sorting signal sufficient for cell wall anchoring consists of an LPXTG motif, a C‐terminal hydrophobic domain and a charged tail. Homologous sequences are found in many surface proteins of Gram‐positive bacteria and we explored the universality of these sequences to serve as cell wall sorting signals. We show that several signals are able to anchor fusion proteins to the staphylococcal cell wall. Some signals do not sort effectively, but acquire sorting activity once the spacing between the LPXTG motif and the charged tail has been increased to span the same length as in protein A. Thus, signals for cell wall anchoring in Gram‐positive bacteria are as universal as signal (leader) sequences.

The Escherichia coli phage-shock-protein (psp) operon

The phage‐shock‐protein (psp) operon helps to ensure survival of Escherichia coli in late stationary phase at alkaline pH, and protects the cell against dissipation of its proton‐motive force against challenge. It is strongly induced by filamentous phage pIV and its bacterial homologues, and by mutant porins that don’t localize properly, as well as by a number of other stresses. Transcription of the operon is dependent on σ54 and a constitutively active, autogenously controlled activator. psp‐operon expression is controlled by one negatively and several positively acting regulators, none of which is a DNA‐binding protein. The major product of the operon, PspA, may also serve as a negative regulator of an unusual porin, OmpG.

An artificial anchor domain: hydrophobicity suffices to stop transfer

A hydrophobic sequence of 23 contiguous, uncharged residues anchors the coliphage f1 gene III protein (pIII) to the Escherichia coli cytoplasmic membrane; mutations removing this domain allow secretion of the protein to the periplasm. Multiple copies of an oligonucleotide encoding the hydrophobic repeat, Leu-Ala-Leu-Val, were introduced into genes for secreted forms of pIII. Artificial domains of 16 or more hydrophobic residues function to anchor the protein. Pronase protection experiments demonstrate that the new sequences act to halt transfer of the protein across the membrane, thus specifying a transmembrane topology. Relocating the hydrophobic domain within the polypeptide chain predictably alters the resultant protein/membrane topology. Repeats of a polar sequence were inserted with no effect on secretion. Furthermore, an unrelated hydrophobic sequence, uncovered by a gene III frameshift mutation, acts to anchor the protein. We conclude that function simply reflects hydrophobicity and not some more subtle feature of structure or sequence.

Characterization and sequence of the Escherichia coli stress-induced psp operon☆

We describe a new Escherichia coli operon, the phage shock protein (psp) operon, which is induced in response to heat, ethanol, osmotic shock and infection by filamentous bacteriophages. The operon includes at least four genes: pspA, B, C and E. PspA associates with the inner membrane and has the heptad repeats characteristic of proteins that can form coiled coils. The operon encodes a factor that activates psp expression, and deletion analyses indicate that this protein is PspC; PspC is predicted to possess a leucine zipper, a motif present in many eukaryotic transcription factors. The pspE gene is expressed in response to stress as part of the operon, but is also transcribed from its own promoter under normal conditions. In vitro studies suggest that PspA and C are modified in vivo. Expression of the psp genes does not require the heat shock sigma factor, sigma32. The increased duration of psp induction in a sigma32 mutant suggests that a product (or products) of the heat shock response down-regulates expression of the operon.

Fine structure of a membrane anchor domain

We describe a detailed deletion analysis of the anchoring domain of a model membrane protein. Removal of the 23 contiguous uncharged amino acids from the carboxy terminus of the bacteriophage fl gene III protein (pIII) converts it from an integral membrane protein to a secreted periplasmic form. Deletions that remove six or fewer residues of the hydrophobic core result in no diminution of the proteins capacity to anchor in the membrane. Longer deletions into this hydrophobic domain gradually destablize the protein-membrane association. pIII derivatives with over half of the hydrophobic core deleted retain substantial residual anchor function. The basic residues, arginine and lysine, which provide a carboxy-terminal boundary for this domain, can be deleted without loss of anchoring capacity.

In vitro synthesis of bacteriophage f1 proteins.

Abstract Covalently closed, circular, double-stranded DNA isolated from cells infected with bacteriophage f1 has been used as a template for coupled transcription and translation in vitro . By using appropriate amber mutants of the phage, it has been possible to identify six gene-specific polypeptides. Three of these correspond to previously known proteins (products of genes III, V and VIII) , while three others had not been previously identified (products of genes I, II and IV ). No polypeptides could be identified as the products of genes VI and VII . The relative amounts of the various polypeptide products are highly sensitive to the concentration of magnesium ions, somewhat sensitive to the concentration of monovalent cations, but are not affected by the introduction of a limited number of single-strand breaks into the template. No differences could be detected between the profile of products made in extracts of phage f 1 -infected cells when compared with the profile of products made in extracts of uninfected cells. Suppression of the amber mutations by Su + tRNA is demonstrated. Additionally, however, Su + tRNA added to the in vitro system promotes the synthesis of an additional polypeptide not found in the presence of Su − tRNA. This phenomenon is independent of the genetic constitution of the template, and is postulated to result from the suppression of a weak terminator.

Binding of mammalian ribosomes to ms2 phage rna reveals an overlapping gene encoding a lysis function

The main binding site for mammalian ribosomes on the single-stranded RNA of bacteriophage MS2 is located nine tenths of the way through the coat protein gene. Translation initiated at an AUG triplet in the +1 frame yields a 75 amino acid polypeptide which terminates within the synthetase gene at a UAA codon, also in the +1 frame. Partial amino acid sequence analysis of the product synthesized in relatively large amounts by mammalian ribosomes confirms this assignment of the overlapping cistron. The same protein is made in an E. coli cell-free system, but only in very small amounts. Analysis of the translation products directed by RNA from op3, a UGA nonsense mutant of phage f2, identifies the overlapping cistron as a lysis gene. In this paper we show that the op3 mutation is a C yield U transition occurring in the second codon of the synthetase cistron, which explains the lowered production of phage replicase (as well as lack of lysis) upon op3 infection of nonpermissive cells. We discuss the properties of the overlapping gene in relation to its lysis function, recognition of the lysis initiator region by E. coli versus eucaryotic ribosomes and op3 as a ribosome binding site mutant for the f2 synthetase cistron.

The PspA protein of Escherichia coli is a negative regulator of sigma(54)-dependent transcription.

In Eubacteria, expression of genes transcribed by an RNA polymerase holoenzyme containing the alternate sigma factor sigma(54) is positively regulated by proteins belonging to the family of enhancer-binding proteins (EBPs). These proteins bind to upstream activation sequences and are required for the initiation of transcription at the sigma(54)-dependent promoters. They are typically inactive until modified in their N-terminal regulatory domain either by specific phosphorylation or by the binding of a small effector molecule. EBPs lacking this domain, such as the PspF activator of the sigma(54)-dependent pspA promoter, are constitutively active. We describe here the in vivo and in vitro properties of the PspA protein of Escherichia coli, which negatively regulates expression of the pspA promoter without binding DNA directly.