Drawing improves memory in patients with hippocampal damage.

The hippocampus plays a critical role in the formation of declarative memories, and hippocampal damage leads to significant impairments in new memory formation. Drawing can serve as a form of multi-modal encoding that improves declarative memory performance relative to other multimodal encoding strategies such as writing. We examined whether, and to what extent, patients with hippocampal damage could benefit from the mnemonic strategy of drawing. Three patients with focal hippocampal damage, and one patient with both hippocampal and cortical lesions, in addition to 22 age-, sex-, and education-matched controls, were shown a list of words one at a time during encoding and instructed to either draw a picture or repeatedly write each word for 40 s. Following a brief filled delay, free recall and recognition memory for words from both encoding trial types were assessed. Controls showed enhanced recall and recognition memory for words drawn versus those that were written, an effect that was even more pronounced in patients with focal hippocampal damage. By contrast, the patient with both hippocampal and cortical lesions showed no drawing-mediated boost in either recall or recognition memory. These findings demonstrate that drawing is an effective encoding strategy, likely accruing from the engagement of extra-hippocampal processes including the integration of cortical-based motor, visual, and semantic processing, enabling more elaborative encoding.

Factors affecting the electrophoretic mobility of the major outer membrane proteins of Escherichia coli in polyacrylamide gels.

The outer membrane proteins of Escherichia coli can be resolved by polyacrylamide gel electrophoresis in the presence of anionic detergents. Factors such as the choice of detergent and buffer system and the presence of urea in the separation gel are all shown to affect the charge and/or the configuration of the detergent-protein complexes and will affect the relative migration of these complexes to different extents. The procedures described in this paper may be of use in the determination of the relatedness of the proteins from the same or different strains. In addition, detailed examinations of the effects of these different parameters and the effect of changes in acrylamide concentrations may be useful in the detection of unusual characteristics which may indicate the presence of posttranslational modification.

In vivo effects of local anesthetics on the production of major outer membrane proteins by Escherichia coli.

Synthesis of a major outer membrane pore protein (the OmpF protein) by Escherichia coli K-12 was specifically and reversibly inhibited by low doses of procaine and other local anesthetics. The treated cells maintained the same total number of pores in their outer membrane by increased synthesis of the OmpC pore protein. Procaine also inhibited synthesis of the OmpF protein by Salmonella typhimurium and by E. coli B, although in the latter case, some OmpF protein was still detected in the outer membrane of treated cells. Experiments in which transcription was blocked by pretreatment with rifampicin indicated that procaine did not inhibit translation of the stable OmpF mRNA and that there was no pool of preformed OmpF and mRNA in cells grown in the presence of procaine. Procaine did not affect biosynthesis of the lipopolysaccharide core and did not inhibit the association of OmpF protein with the peptidoglycan. These results are discussed in terms of the known effects of procaine on membrane molecular packaging.

The conserved tetracysteine motif in the general secretory pathway component PulE is required for efficient pullulanase secretion.

The PulE component of the pullulanase secretion pathway, a typical main terminal branch of the general secretory pathway, has a tetracysteine motif (4Cys) that is also present in almost all of the many PulE homologues, including those involved in type-IV piliation and conjugal DNA transfer. The 4Cys resembles a zinc-binding motif found in other proteins such as adenylate kinases, which may be pertinent in view of the fact that PulE has a consensus ATP-binding motif and since at least one PulE homologue has been reported to have kinase activity. In PulE, the Cys residues of this motif form scrambled intra- and intermolecular disulfide bonds when cells are disrupted. Replacement of one or more Cys of this motif by Ser reduces PulE function, but at least two adjacent Cys must be replaced to prevent intramolecular disulfide bond formation.

Recent progress and future directions in studies of the main terminal branch of the general secretory pathway in Gram-negative bacteria--a review.

The main terminal branch (MTB) of the general secretory pathway is used by a wide variety of Gram- bacteria to transport exoproteins from the periplasm to the outside milieu. Recent work has led to the identification of the function of two of its 14 (or more) components: an enzyme with type-IV prepilin peptidase activity and a chaperone-like protein required for the insertion of another of the MTB components into the outer membrane. Despite these important discoveries, little tangible progress has been made towards identifying MTB components that determine secretion specificity (presumably by binding to cognate exoproteins) or which form the putative channel through which exoproteins are transported across the outer membrane. However, the idea that the single integral outer membrane component of the MTB could line the wall of this channel, and the intriguing possibility that other components of the MTB form a rudimentary type-IV pilus-like structure that might span the periplasm both deserve more careful examination. Although Escherichia coli K-12 does not normally secrete exoproteins, its chromosome contains an apparently complete set of genes coding for MTB components. At least two of these genes code for functional proteins, but the operon in which twelve of the genes are located does not appear to be expressed. We are currently searching for conditions which allow these genes to be expressed with the eventual aim of identifying the protein(s) that E. coli K-12 can secrete.

On remaining cytoplasmic.

The published literature contains a number of examples of normally non-cytoplasmic proteins whose transport out of the cytoplasm is not completely abolished by drastic alterations to their routing signals (signal sequences, etc). Furthermore, there are numerous examples of cytoplasmic proteins that can be routed to and across plasma or organelle membranes by fusing them to routing signals. These 2 sets of observations lead to a re-evaluation of the reliability and accuracy of protein routing and to consideration of the consequences of the errors which might occur.

Pullulanase: model protein substrate for the general secretory pathway of gram-negative bacteria.

Pullulanase of Klebsiella oxytoca is one of a wide variety of extracellular proteins that are secreted by Gram-negative bacteria by the complex main terminal branch (MTB) of the general secretory pathway. The roles of some of the 14 components of the MTB are now becoming clear. In this review it is proposed that most of these proteins form a complex, the secretion, that spans the cell envelope to control the opening and closing of channel in the outer membrane. Progress toward the goal of testing this model is reviewed.

The complete general secretory pathway in gram-negative bacteria.

The unifying feature of all proteins that are transported out of the cytoplasm of gram-negative bacteria by the general secretory pathway (GSP) is the presence of a long stretch of predominantly hydrophobic amino acids, the signal sequence. The interaction between signal sequence-bearing proteins and the cytoplasmic membrane may be a spontaneous event driven by the electrochemical energy potential across the cytoplasmic membrane, leading to membrane integration. The translocation of large, hydrophilic polypeptide segments to the periplasmic side of this membrane almost always requires at least six different proteins encoded by the sec genes and is dependent on both ATP hydrolysis and the electrochemical energy potential. Signal peptidases process precursors with a single, amino-terminal signal sequence, allowing them to be released into the periplasm, where they may remain or whence they may be inserted into the outer membrane. Selected proteins may also be transported across this membrane for assembly into cell surface appendages or for release into the extracellular medium. Many bacteria secrete a variety of structurally different proteins by a common pathway, referred to here as the main terminal branch of the GSP. This recently discovered branch pathway comprises at least 14 gene products. Other, simpler terminal branches of the GSP are also used by gram-negative bacteria to secrete a more limited range of extracellular proteins.

The immunity and lysis genes of ColN plasmid pCHAP4.

Nucleotide sequencing of part of the plasmid pCHAP4, which encodes the ca. 42,000 Da putative poreforming colicin N, confirmed previous results indicating that the colicin N immunity gene (cni) and the colicin release or lysis gene (cnl) are located immediately downstream from the colicin N structural gene (cna) in the order cna-cni-cnl. The cni gene is transcribed in the opposite direction to cna and probably encodes an Mr 15239 Da protein. The putative immunity protein was detected among the [35S]methionine-labelled proteins produced by minicells carrying cni cloned under lac promoter control, and when the gene was subcloned into expression vectors under the control of a bacteriophage T7 promoter. Deletion of the region immediately upstream from cni completely abolished colicin N immunity, presumably because the natural promoter had been deleted. cnl is in the same operon as cna, and encodes a typical Col plasmid pro-lysis protein comprising a signal peptide and a 34 residue mature polypeptide with high homology to all but one of the other known Col lysis proteins, including the fatty acylated amino-terminal cysteine residue which was specifically labelled with 3H-palmitate. Cell fractionation studies indicated that the cnl gene product was located predominantly in the outer membrane.

Nucleotide sequencing of the structural gene for colicin N reveals homology between the catalytic, C-terminal domains of colicins A and N.

An 1800 bp fragment of DNA from a natural ColN plasmid (pCHAP4) encompassing the colicin N structural gene (cna) and its regulatory region was subjected to nucleotide sequencing and deletion analysis. The region of DNA immediately upstream from cna contains two tandemly-arranged and overlapping potential LexA binding sites (SOS boxes), in line with the previous demonstration that cna expression is repressed by LexA protein. Deletion of the LexA binding site allowed efficient transcription of cna from an upstream lacZ promoter, whereas its presence reduced lacZ-promoted cna expression to varying extents depending on the proximity of lacZp and the SOS boxes. The molecular weight of colicin N, as deduced from the nucleotide sequence, is 41,696, which is close to the experimentally determined molecular weight of 39,000. Colicin N has a glycine-rich amino terminus similar to that found in many other colicins. Part of the glycine-rich domain of colicin N could be replaced by an unrelated sequence devoid of glycine residues without affecting either colicin release or activity. The carboxy-terminal half of colicin N exhibits significant homology to the C-terminus of colicin A. The latter colicin forms pores in the cytoplasmic membrane of Escherichia coli, thereby depolarizing the membrane and causing cell death. The C-terminus of colicin A is endowed with this catalytic activity. Although colicin N was previously found to cause lysis of Escherichia coli cells, a more detailed investigation revealed that it too depolarizes the Escherichia coli cytoplasmic membrane and that lysis is a secondary effect.

Processing and methylation of PuIG, a pilin-like component of the general secretory pathway of Klebsiella oxytoca.

The signal sequence of the Klebsiella oxytoca puIG gene product, which is required for extracellular secretion of the enzyme pullulanase, is similar in many respects to the corresponding segment of the precursors of type IV (me-Phe) pilins. The significance of this similarity is confirmed by the observation that the puIO gene product processes prePuIG at the consensus type IV prepilin peptidase cleavage site at the amino-terminal end of the PuIG signal sequence. Like most type IV pilins, processed PuIG was found to have a methylated amino-terminal phenylalanine residue. Site-directed mutagenesis was used to replace amino acids in prePuIG that correspond to residues shown by others to be essential for processing, methylation and assembly of type IV pilins. The glycine residue on the amino-terminal side of the prePuIG cleavage site is absolutely required for processing and for pullulanase secretion. The glutamate residue at position 11(+5) is also required for pullulanase secretion but not for processing or methylation. This result contrasts with that reported for corresponding variants of Pseudomonas aeruginosa type IV prepilin, which were processed but only inefficiently N-methylated. Cleavage of prePuIG and pullulanase secretion were both unaffected by replacement of the phenylalanine residue on the carboxy-terminal side of the cleavage site by leucine, isoleucine or valine, by a conservative substitution within the hydrophobic core of the prePuIG signal sequence, or by a glutamine to proline substitution within the processed segment. However, replacement of the same glutamine residue by arginine abolished secretion without affecting either processing or methylation.

Translocation of a folded protein across the outer membrane in Escherichia coli.

A mutation in the Escherichia coli dsbA gene (coding for a periplasmic disulfide oxidoreductase) reduces the rate of disulfide bond formation in the enzyme pullulanase and also reduces the rate at which the enzyme is secreted to the cell surface, as measured by protease accessibility. The enzyme did not become protease accessible when disulfide bond formation was completely prevented in the mutant strain by carboxymethylation. These results indicate that a disulfide bond may be required for, and certainly does not impede, the translocation of pullulanase across the outer membrane. Since it is unlikely that a disulfide bond could be formed and then reduced again in the periplasm, these results would appear to strengthen the argument that pullulanase polypeptides fold into or close to their final conformation before they are transported across the outer membrane. It is suggested that this might be a feature common to all proteins that are secreted by other Gram-negative bacteria by a pullulanase-like pathway.

A mutation in the dsbA gene coding for periplasmic disulfide oxidoreductase reduces transcription of the Escherichia coli ompF gene.

An insertion mutation in the Escherichia coli dsbA gene, coding for periplasmic disulfide oxidoreductase, dramatically reduces the level of OmpF porin protein in the cell envelope. Studies with chromosomal ompF-lacZ operon and gene fusions indicate that this is due to reduced ompF transcription. Identical effects resulted from growth in medium containing the reducing agent dithiothreitol, but the combined effects of the reducing agent and dsbA were no greater than the effects of each individually. The dsbA mutation did not prevent normal regulation of ompF transcription by the local anaesthetic procaine or by osmolarity. OmpF does not contain a cysteine residue, and the sole cysteine residue in the cytoplasmic membrane regulator of ompF transcription, EnvZ, is predicted to be located on the cytoplasmic side of the membrane, and is therefore unlikely to be involved in the effects of the dsbA mutation. The effects of the dsbA mutation and of the reducing agent on ompF transcription may be due to the failure to from an essential disulfide bond in an as yet unidentified envelope protein that affects ompF transcription.

Multimers of the precursor of a type IV pilin-like component of the general secretory pathway are unrelated to pili.

Both the mature and precursor forms of PulG, a type IV pilin-like component of the general secretory pathway of Klebsiella oxytoca, can be chemically cross-linked into multimers similar to those obtained by cross-linking the components of type IV pili. To explore the possibility that the PulG precursor could form a pilus-like structure, the PulG sequence was altered in a variety of ways, including (i) replacement of the characteristic hydrophobic region, which is required for the assembly of type IV pilins by the MalE signal peptide, or (ii) fusion of beta-lactamase (beta laM) to the C-terminus. Neither of these changes affected multimerization. PulG precursor could be post-translationally processed by prepilin peptidase (PulO), indicating that the N-terminus of prePulG remains on the cytoplasmic side of the cytoplasmic membrane where it is accessible to the catalytic site of this enzyme. Finally, precursor and mature forms of PulG could be efficiently cross-linked in a mixed dimer, indicating that at least a subpopulation of the two forms of the protein are probably located in clusters in the cytoplasmic membrane. These results provide further evidence that the cross-linked multimers of the precursor form of PulG are unrelated to type IV pilus-like structures. It is still unclear whether a subpopulation of processed PulG can be assembled into a rudimentary pilus-like structure.

Obligatory coupling of colicin release and lysis in mitomycin-treated Col+ Escherichia coli.

Previous work has shown that Escherichia coli K12 strains carrying the small, high copy number ColE2-P9 plasmid produce large amounts of colicin and then lyse and release colicin when grown in broth culture containing mitomycin C. Strains carrying the larger, low copy number ColIa-CA53 plasmid produced much less colicin and did not lyse or discharge more than 15% of their colicin when grown under the same conditions. Naturally-occurring Col+ strains and E. coli K12 derivatives carrying different Col plasmids could be classified either as ColE2+-like or ColIa+-like according to whether or not they produced large amounts of colicin and lysed and discharged colicin when grown in the presence of mitomycin, and also by the size and presumed copy number of the Col plasmid they carried. Strains carrying multiple copies of the cloned colicin Ia structural gene produced large amounts of colicin but did not lyse or release colicin when grown in the presence of mitomycin. This result rules out the possibility that high level accumulation of colicin is sufficient to cause lysis. Conditions were sought under which colicin Ia could be released from the producing cells. It was found that mitomycin-treated cultures of strains carrying both ColE2 and ColIa plasmids released both colicins when they lysed, although colicin Ia release occurred later than colicin E2 release. It was also noted that colicin Ia-laden cells released their colicin when diluted into fresh culture medium.

Colicin E4-CT9 is proteolytically degraded after discharge from producing cells in liquid cultures.

Colicin E4 was produced in very large amounts when Escherichia coli K12 strain W3110 pColE4-CT9 was grown in the presence of 0.5 microgram mitomycin C ml-1. The colicin was discharged from the producing cells when they lysed and was then degraded by a protease located on the surface of the producing cells. Colicins and proteolytic fragments derived from them could be concentrated from spent culture medium by filtration through Millipore membrane filters. Colicins are probably retained by these filters by hydrophobic interactions since binding was unaffected by changes in ionic conditions but was completely inhibited in the presence of ionic or non-ionic detergents.

Autoinduced synthesis of colicin E2.

Escherichia coli K-12 cells carrying the high copy number plasmid ColE2-P9 and a sfiA-lacZ gene fusion exhibit abnormally high levels of SOS-regulated phi sfiA-lacZ expression. Increased sfiA-lacZ expression is caused by the action of colicin E2, which is a DNase, rather than by the presence of multiple copies of a binding site for LexA protein, the repressor for the sfiA and colicin E2 genes. Expression of sfiA-lacZ was reduced to normal levels if the ColE2+ strain lacked the outer membrane colicin E2 receptor protein (BtuB) or if they carried an increased number of colicin E2 immunity genes. The results suggest that cultures of ColE2+ strains contain a small number of cells which produce colicin which can then enter other, non-producing cells in the culture and cause sufficient damage to the DNA to induce the SOS system. The levels of colicin E2 immunity in the producing cells is presumably sufficient to prevent extensive lethal effects of the colicin, but insufficient to prevent limited endonuclease activity. An important consequence of this phenomenon is that the DNase action of colicin E2 can stimulate its own production.