A novel mechanism by which silica defends grasses against herbivory.

BACKGROUND AND AIMS: Previous studies have shown that silica in grass leaves defends them against small herbivores, which avoid high-silica grasses and digest them less efficiently. This study tested the idea that silica can reduce digestibility by preventing the mechanical breakdown of chlorenchyma cells. METHODS: Both the percentage of total chlorophyll liberated from high- and low-silica grass leaves by mechanical grinding and the chlorophyll content of locust faeces were measured. KEY RESULTS: High-silica grasses released less chlorophyll after grinding and retained more after passing through the gut of locusts, showing that silica levels correlated with increased mechanical protection. CONCLUSIONS: These results suggest that silica may defend grasses at least in part by reducing mechanical breakdown of the leaf, and that mechanical protection of resources in chlorenchyma cells is a novel and potentially important mechanism by which silica protects grasses.

Temporal stability of the Learning Efficiency Test-II for adults.

This study examined the temporal stability of the Learning Efficiency Test-II with 101 undergraduate students over a mean test-retest time interval of 19 days. Temporal stability estimates were .79 for the Visual Modality factor score, .75 for the Auditory Modality factor score, and .80 for the Global Memory factor score. A repeated measures analysis of variance for these three factor scores indicated no significant mean differences from Test 1 to Test 2. Slightly lower retest correlations were obtained for each of the 12 subtests, with correlations ranging from .44 to .70. The findings indicate that some subtests are reliable to assess the memory processing of adults over time and also highlight the stability over 19 days of memory processes for intact learners. Other studies with older groups of persons are needed to examine the stability of scores.

The pI and pXI assembly proteins serve separate and essential roles in filamentous phage assembly.

Three non-capsid, phage-encoded proteins, pI, pIV and pXI, are required for assembly of the filamentous bacteriophage at the envelope of Escherichia coli. pIV forms the outer membrane component of the assembly site, and pI and pXI are predicted to form the cytoplasmic membrane component. pXI is the result of an in-frame internal translational initiation event in gene I and is identical with the carboxyl-terminal third of pI in amino acid sequence, membrane localization and topology. The two proteins share a cytoplasmic domain predicted to be an amphipathic helix, a transmembrane domain, and a periplasmic domain. By mutating the initiation site for pXI, a phage was made that produced only pI and was shown to absolutely require functional plasmid-encoded pXI for growth. Further mutational analysis was done to examine the functional determinants of the amphipathic helix and periplasmic domains of the pI and pXI proteins. The results show that the amphipathic helix region is very important for pI function but not for pXI function. Mutational analysis of the periplasmic domains of pI and pXI implies that these domains also perform separate functions, and suggests that the interaction between pI and pIV in the periplasm is critical for assembly. The results are discussed with regard to the separate roles that the pI and pXI proteins play in the overall process of phage assembly.

The major coat protein of filamentous bacteriophage f1 specifically pairs in the bacterial cytoplasmic membrane.

Filamentous bacteriophage are long, thin single-stranded DNA viruses that infect male strains of Escherichia coli without killing the host. Each phage contains approximately 2700 copies of the major coat protein, pVIII, which exists as a 5.2 kDa cytoplasmic membrane protein prior to incorporation into phage. Studies from a number of groups analyzing the behavior of wild-type and mutant pVIII in detergents suggested that pVIII might pair under these conditions. In order to test whether pVIII molecules pair in vivo in the cytoplasmic membrane, four plasmidencoded pVIII variants were constructed in which specific residues in the transmembrane region were mutated to cysteine in an attempt to stabilize any pair via disulfide bridges. Variants A35C and I39C were unable to complement phage with an amber mutation in gene VIII. The I39C variant was unable to be packaged into phage particles even though it was inserted into the membrane. In the case of A35C, the inability to complement was not due to a packaging defect because the variant protein could be packaged into phage in the presence of wild-type pVIII. Western blot analysis of cytoplasmic membrane samples revealed that the A35C variant formed stable disulfide dimers in vivo. Expression of A35C interfered with wild-type phage infection, indicating that the assembly machinery may recognize the disulfide dimers in some non-productive way. The results indicate that pVIII may specifically pair along a particular face in the cytoplasmic membrane prior to assembly; however, these pairs must be able to be separated in order for normal assembly to occur.

The TolQRA proteins are required for membrane insertion of the major capsid protein of the filamentous phage f1 during infection.

Infection of Escherichia coli by the filamentous bacteriophage f1 is initiated by interaction of the end of the phage particle containing the gene III protein with the tip of the F conjugative pilus. This is followed by the translocation of the phage DNA into the cytoplasm and the insertion of the major phage capsid protein, pVIII, into the cytoplasmic membrane. DNA transfer requires the chromosomally encoded TolA, TolQ, and TolR cytoplasmic membrane proteins. By using radiolabeled phages, it can be shown that no pVIII is inserted into the cytoplasmic membrane when the bacteria contain null mutations in tolQ, -R and -A. The rate of infection can be varied by using bacteria expressing various mutant TolA proteins. Analysis of the infection process in these strains demonstrates a direct correlation between the rate of infection and the incorporation of infecting bacteriophage pVIII into the cytoplasmic membrane.

Filamentous phage infection: required interactions with the TolA protein.

Infection of Escherichia coli by the filamentous phage f1 is initiated by binding of the phage to the tip of the F conjugative pilus via the gene III protein. Subsequent translocation of phage DNA requires the chromosomally encoded TolQ, TolR, and TolA proteins, after the pilus presumably has withdrawn, bringing the phage to the bacterial surface. Of these three proteins, TolA is proposed to span the periplasm, since it contains a long helical domain (domain II), which connects a cytoplasmic membrane anchor domain (domain I) to the carboxyl-terminal domain (domain III). By using a transducing phage, the requirement for TolA in an F+ strain was found to be absolute. The role of TolA domains II and III in the infective process was examined by analyzing the ability of various deletion mutants of tolA to facilitate infection. The C-terminal domain III was shown to be essential, whereas the polyglycine region separating domains I and II could be deleted with no effect. Deletion of helical domain II reduced the efficiency of infection, which could be restored to normal by retaining the C-terminal half of domain II. Soluble domain III, expressed in the periplasm but not in the cytoplasm or in the medium, interfered with infection of a tolA+ strain. The essential interaction of TolA domain III with phage via gene III protein appears to require interaction with a third component, either the pilus tip or a periplasmic entity.

The TolA protein interacts with colicin E1 differently than with other group A colicins.

The 421-residue protein TolA is required for the translocation of group A colicins (colicins E1, E2, E3, A, K, and N) across the cell envelope of Escherichia coli. Mutations in TolA can render cells tolerant to these colicins and cause hypersensitivity to detergents and certain antibiotics, as well as a tendency to leak periplasmic proteins. TolA contains a long alpha-helical domain which connects a membrane anchor to the C-terminal domain, which is required for colicin sensitivity. The functional role of the alpha-helical domain was tested by deletion of residues 56 to 169 (TolA delta1), 166 to 287 (TolA delta2), or 54 to 287 (TolA delta3) of the alpha-helical domain of TolA, which removed the N-terminal half, the C-terminal half, or nearly the entire alpha-helical domain of TolA, respectively. TolA and TolA deletion mutants were expressed from a plasmid in an E. coli strain producing no chromosomally encoded TolA. Cellular sensitivity to the detergent deoxycholate was increased for each deletion mutant, implying that more than half of the TolA alpha-helical domain is necessary for cell envelope stability. Removal of either the N- or C-terminal half of the alpha-helical domain resulted in a slight (ca. 5-fold) decrease in cytotoxicity of the TolA-dependent colicins A, E1, E3, and N compared to cells producing wild-type TolA when these mutants were expressed alone or with TolQ, -R, and -B. In cells containing TolA delta3, the cytotoxicity of colicins A and E3 was decreased by a factor of >3,000, and K+ efflux induced by colicins A and N was not detectable. In contrast, for colicin E1 action on TolA delta3 cells, there was little decrease in the cytotoxic activity (<5-fold) or the rate of K+ efflux, which was similar to that from wild-type cells. It was concluded that the mechanism(s) by which cellular uptake of colicin E1 is mediated by the TolA protein differs from that for colicins A, E3, and N. Possible explanations for the distinct interaction and unique translocation mechanism of colicin E1 are discussed.

Characterization of the tol-pal and cyd region of Escherichia coli K-12: transcript analysis and identification of two new proteins encoded by the cyd operon.

Sequence analysis showed that the cyd operon is immediately upstream of the tol-pal region. Northern (RNA) blot analysis demonstrated that the transcript for the cyd operon terminates just before the promoter for transcription of the tol genes. The cyd transcript contains cydA cydB followed by two open reading frames: orfC, encoding a 37-residue peptide, and orfD, encoding a 97-residue peptide. Both OrfC and OrfD are synthesized in minicells.

Characterization of the tol-pal region of Escherichia coli K-12: translational control of tolR expression by TolQ and identification of a new open reading frame downstream of pal encoding a periplasmic protein.

The TolQ, TolR, TolA, TolB, and Pal proteins appear to function in maintaining the integrity of the outer membrane, as well as facilitating the uptake of the group A colicins and the DNA of the infecting filamentous bacteriophages. Sequence data showed that these genes are clustered in a 6-kb segment of DNA with the gene order orf1 tolQ tolR tolA tolB pal orf2 (a newly identified open reading frame encoding a 29-kD9 protein). Like those containing orf1, bacteria containing an insertion mutation in this gene showed no obvious phenotype. Analysis of beta-galactosidase activity from fusion constructs in which the lac operon was fused to various genes in the cluster showed that the genes in this region constitute two separate operons: orf1 tolQRA and tolB pal orf2. In the orf1 tolQRA operon, translation of MR was dependent on translation of the upstream tolQ region. Consistent with this result, no functional ribosome-binding site for TolR synthesis was detected.

Membrane insertion characteristics of the various transmembrane domains of the Escherichia coli TolQ protein.

The Escherichia coli TolQ protein is a 230-amino acid integral cytoplasmic membrane protein required for the import of group A colicins, for infection by the filamentous phage, and for maintenance of the integrity of the bacterial envelope. TolQ is a polytopic protein with three membrane-spanning regions. The first membrane-spanning region has a 19-residue periplasmic NH2-terminal tail, while the second and third membrane-spanning segments are separated by a short 17-amino acid periplasmic loop. To study the membrane assembly of TolQ, fusions of different membrane-spanning regions were examined for their ability to insert in the absence of functional SecA or the membrane potential. Fusions containing the first membrane-spanning region plus the adjacent cytoplasmic domain and a construct containing the "hairpin loop," formed by the second and third membrane-spanning regions, insert in the absence of functional SecA. The fusion containing the second and third membrane-spanning regions required the membrane potential for insertion while the first membrane-spanning region was able to insert even in the absence of a membrane potential. Taken together, these results suggest that insertion of intact TolQ is not dependent on the Sec system, but does require the membrane potential.

The products of gene I and the overlapping in-frame gene XI are required for filamentous phage assembly.

The class I filamentous bacteriophage are non-lytic single-stranded DNA phage, which are assembled at the cell envelope as they are extruded from the Gram-negative bacteria, Escherichia coli. The process requires the products of the phage genes I and IV, which reside in the inner and outer membrane, respectively, and are not present in the mature phage particle. Gene I encodes two proteins, the full length 348-residue pI and a smaller pI*, which this report shows is the result of an internal translation initiation event at methionine codon 241. Both pI and pI* are shown to be required for phage assembly. Therefore, pI* can be considered the product of an additional phage gene, XI, which is a separate in-frame gene that overlaps gene I. Both proteins contain a 13-residue region adjacent to the cytoplasmic face of the inner membrane that probably exists as a positively charged amphiphilic helix. Although this region is not required for membrane insertion of pI and pI*, it is shown to be required for phage assembly. Oligonucleotide-directed mutagenesis of this region, which removes positive charges or alters the hydrophobic face of the putative helix, renders pI and pI* unable to function in phage assembly. This region of pI and pI* is highly homologous in structure to the carboxyl-terminal 11 amino acids of pVIII, the main coat protein, which also reside adjacent to the cytoplasmic face of the inner membrane.

A comparison of the performance of cognitively disabled children on the WISC-R and WISC-III.

This study compared differences in performance on the WISC-III and on the WISC-R. Sixty-one students with learning impairments, due for reevaluation of their special education placement, were administered the WISC-III as part of a psychological assessment battery. Results indicate a mean difference between WISC-R and WISC-III FSIQ of 7.95 points, which is similar to WISC/WISC-R comparisons. Substantial differences that averaged 9.21 points were found between WISC-R and WISC-III PIQ means. These findings suggest that for a special education sample an average decrease of at least 8 points can be expected on the WISC-III Full Scale IQ. Caution should be exercised when one is considering changes in educational classification or interpreting qualitative differences in performance on WISC-III scores compared with WISC-R scores.

Adjustment issues with adult children of alcoholics.

The current study assessed locus of control, general level of life satisfaction, and self-reported grade-point averages among adults who had experienced either alcoholism within the family of origin, traumatic life events other than alcoholism, or who indicated neither problem during their childhood. Results indicated that both the adult children of alcoholics (ACOA) and traumatic experience (TE) groups had lower life satisfaction scores than the control group. Significantly lower levels of locus of control also were found for the ACOA group in comparison to the control group. There were no significant differences among the three groups based on self-reported GPAs. Results of the current study support the concept that family dysfunction during childhood can influence negatively later life experiences and adjustment.

Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity.

TolQ is a 230-amino-acid protein required to maintain the integrity of the bacterial envelope and to facilitate the import of both filamentous bacteriophage and group A colicins. Cellular fractionation experiments showed TolQ to be localized to the cytoplasmic membrane. Bacteria expressing a series of TolQ-beta-galactosidase and TolQ-alkaline phosphatase fusion proteins were analyzed for the appropriate enzyme activity, membrane location, and sensitivity to exogenously added protease. The results are consistent with TolQ being an integral cytoplasmic membrane protein with three membrane-spanning regions. The amino-terminal 19 residues as well as a small loop in the 155 to 170 residue region appear exposed in the periplasm, while the carboxy terminus and a large loop after the first transmembrane region are cytoplasmic. Amino-terminal sequence analysis of TolQ purified from the membrane revealed the presence of the initiating formyl methionine group, suggesting a rapid translocation of the amino-terminal region across the cytoplasmic membrane. Analysis of various tolQ mutant strains suggests that the third transmembrane region as well as parts of the large cytoplasmic loop are necessary for activity.

Membrane topology of the Escherichia coli TolR protein required for cell envelope integrity.

TolR is a 142-amino-acid protein required for the import of colicins and bacteriophage and for maintenance of cell envelope integrity. The topology of TolR in the inner membrane was analyzed by two methods. First, bacteria expressing a series of TolR-beta-galactosidase, TolR-alkaline phosphatase, and TolR-beta-lactamase fusions were assayed for the appropriate enzymatic activity. Second, the accessibility of TolR to proteinase K was determined in permeabilized cells and everted vesicles with an antibody elicited against the carboxyl-terminal 70% of TolR. The results are consistent with TolR spanning the inner membrane once via residues 23 to 43 and with the carboxyl-terminal moiety being exposed to the periplasm. Quantitative studies with the anti-TolR antibody indicated the presence of 2 x 10(3) to 3 x 10(3) TolR molecules per cell.

The filamentous bacteriophage assembly proteins require the bacterial SecA protein for correct localization to the membrane.

The noncapsid assembly proteins pI and pI of the filamentous bacteriophage f1 are inserted into the inner membrane of Escherichia coli via an internal signal sequence. Inhibition of the activity of SecA with low concentrations of sodium azide results in rapid accumulation of pI and pI proteins in the cytoplasm. However, both proteins are inserted into the membrane under the same conditions when synthesized in bacteria containing a secA azide resistance mutation. The other noncapsid assembly protein, pIV, is an outer membrane protein synthesized with a cleavable signal sequence. Wild-type bacteria accumulate the precursor to pIV when protein synthesis is in the presence of low concentrations of sodium azide. These results suggest that the f1 bacteriophage assembly proteins require SecA and consequently the bacterial Sec system to reach their proper membrane location.

The membrane domain of a bacteriophage assembly protein. Transmembrane-directed proteolysis of a membrane-spanning fusion protein.

A tripartite fusion construct encoding the amino-terminal half of EcoRI endonuclease followed by amino acids 217-299 of the filamentous bacteriophage gene I protein (pI) attached to the enzymatic portion of alkaline phosphatase results in the production of two proteins. The larger protein, pIf, is the complete tripartite fusion protein while the smaller protein, pIf*, results from internal initiation of translation at pI methionine 241. Both pIf and pIf* span the Escherichia coli inner membrane via a 20-amino-acid hydrophobic stretch of pI with their amino termini in the cytoplasm and their carboxyl-terminal alkaline phosphatase domains in the periplasm. The alkaline phosphatase moiety of approximately 70% of pIf is released into the periplasm by in vivo proteolysis, but only about 10% of pIf* is cleaved. Neither DegP, OmpT, nor protease III are responsible for the cleavage in vivo, and leader peptidase is unable to cleave the fusion protein in vitro. Deletion and substitution analyses demonstrate that the degree of periplasmic cleavage depends on the sequence of the cytoplasmic domain of the fusion proteins. Possible mechanisms for this transmembrane-directed cleavage event are compared to proposed models for signal transduction.

The membrane domain of a bacteriophage assembly protein. Membrane insertion and growth inhibition.

The gene I protein (pI) of the f1 filamentous bacteriophage is a non-capsid protein that is required for the assembly of the bacteriophage. It spans the Escherichia coli inner membrane once with its amino terminus in the cytoplasm and its carboxyl-terminal portion in the periplasm. The presence of moderate amounts of this protein in the membrane results in rapid inhibition of cell growth, probably from a loss of membrane potential. Previous observations defined a 55-amino-acid sequence within pI required for its membrane insertion which includes a 20-residue hydrophobic stretch preceded by a 13-residue positively charged amphiphilic helix. To define the minimal sequence required for membrane translocation and for growth inhibition, a deletion analysis was performed on a tripartite fusion construct containing the 55-residue pI sequence flanked upstream by the amino-terminal portion of EcoRI endonuclease and downstream by the enzymatic portion of alkaline phosphatase. Only the 20-residue hydrophobic stretch immediately preceded by 1 arginine residue is required for membrane insertion of the fusion proteins. This region also sufficed to inhibit cell growth provided it contained protein domains exposed in both the cytoplasm and periplasm. It was not possible to separate the domains required for membrane insertion and cell growth inhibition. No requirement for the positively charged amphiphilic helix was detected either for membrane insertion or growth inhibition, suggesting that it plays a role in phage assembly and not membrane insertion.

Role of the carboxyl-terminal domain of TolA in protein import and integrity of the outer membrane.

The TolA protein is involved in maintaining the integrity of the outer membrane of Escherichia coli, as mutations in tolA cause the bacteria to become hypersensitive to detergents and certain antibiotics and to leak periplasmic proteins into the medium. This protein also is required for the group A colicins to exert their effects and for many of the filamentous single-stranded bacteriophage to infect the bacterial cell. TolA is a three-domain protein, with the amino-terminal domain anchoring it to the inner membrane. The helical second domain is proposed to span the periplasmic space to allow the carboxyl-terminal third domain to interact with the outer membrane. A plasmid that allowed the synthesis and transport of the carboxyl-terminal third domain into the periplasmic space was constructed. The presence of an excess of this domain in the periplasm of a wild-type cell resulted in an increased sensitivity to deoxycholate, the release of periplasmic alkaline phosphatase and RNase into the medium, and an increased tolerance to colicins E1, E2, E3, and A. There was no effect on the cells' response to colicin D, which depends on TonB instead of TolA for its action. The presence of the free carboxyl-terminal domain of TolA in the periplasm in a tolA null mutation did not restore the wild-type phenotype, suggesting that this domain must be part of the intact TolA molecule to perform its function. Our results are consistent with a model in which the carboxyl-terminal domain of TolA interacts with components in the periplasm or on the inner surface of the outer membrane to function in maintaining the integrity of this membrane.