Random mutagenesis of Schizosaccharomyces pombe SRP RNA: lethal and conditional lesions cluster in presumptive protein binding sites.

Signal recognition particle (SRP), a ribonucleoprotein composed of six polypeptides and one RNA subunit, serves as an adaptor between the cytoplasmic protein synthetic machinery and the translocation apparatus of the endoplasmic reticulum. To begin constructing a functional map of the 7SL RNA component of SRP, we extensively mutagenized the Schizosaccharomyces pombe SRP7 gene. Phenotypes are reported for fifty-two mutant alleles derived from random point mutagenesis, seven alleles created by site-directed mutagenesis to introduce restriction sites into the SRP7 gene, nine alleles designed to pinpoint conditional lesions, and three alleles with extra nucleotides inserted at position 84. Our data indicate that virtually all single nucleotide changes as well as many multiple substitutions in this highly structured RNA are phenotypically silent. Six lethal alleles and eleven which result in sensitivity to the combination of high temperature and elevated osmotic strength were identified. These mutations cluster in conserved regions which, in the mammalian RNA, are protected from nucleolytic agents by SRP proteins. The effects of mutations in the presumptive binding site for a fission yeast SRP 9/14 homolog indicate that both the identity of a conserved residue and the secondary structure within which it is embedded are functionally important. The phenotypes of mutations in Domain IV suggest particular residues as base-specific contacts for the fission yeast SRP54 protein. A single allele which confers temperature-sensitivity in the absence of osmotic perturbants was identified in this study; the growth properties of the mutant strain suggest that the encoded RNA is somewhat defective even at the permissive temperature, and is most likely unable to correctly assemble with SRP proteins at the nonpermissive temperature.     

Mobilization of closely related plasmids pUB110 and pBC16 by Bacillus plasmid pXO503 requires trans-acting open reading frame beta.

Genetic analysis of the closely related nonconjugative plasmids pUB110 and pBC16 has demonstrated that the open reading frame beta (ORF-beta) region in pUB110 and the corresponding homologous region in pBC16 are essential for mobilization of these plasmids by pLS20 or its derivatives. Deletions in this region or insertions that interrupted ORF-beta severely impaired or eliminated the mobilization of pUB110::pUC18 and pBC16::pUC18 hybrids. In contrast, a hybrid in which pUC18 was inserted into pBC16 at a point outside ORF-beta transferred at a frequency comparable to that of intact pUB110 or pBC16 (10(-4) transcipients per donor cell). The defect of most transfer-deficient (Mob-) hybrid plasmids could be complemented by an intact sister plasmid (i.e., pBC16 for pUB110::pUC18 Mob- hybrids). The inability to complement certain constructs suggested that the origin of transfer might be located in an area 5' to ORF-beta. Furthermore, cloning the region 5' to ORF-beta onto a nonmobilizable pC194::pUC18 construct resulted in a hybrid plasmid, pUCCoriTBC16, that could be mobilized with complementation. These results indicate that mobilization of pUB110 and pBC16 by conjugative helper plasmids requires ORF-beta in trans and at least one other region, including the RSA sequence, which presumably functions as an origin of transfer, in cis.     

Inhibition of substance P degradation in rat brain preparations by peptide hydroxamic acids

A peptidase activity of rat diencephalon membranes, which acts on the C-terminal hexapeptide sequence of substance P, was characterized using the radiolabeled substrate N alpha-[( 125I]iododesaminotyrosyl)-substance P (6-11)-hexapeptide. This activity presents certain characteristics similar to those of the substance-P-degrading enzyme purified from human brain by Lee et al. [Eur. J. Biochem. 114, 315-327 (1981)]. It is inhibited by metal chelators and some thiol reagents, but is insensitive to inhibitors of serine proteases and aminopeptidases. The activity is different from angiotensin-converting enzyme and enkephalinase, since it is not affected by specific inhibitors of these enzymes. Substance P and substance P C-terminal fragments longer than the pentapeptide inhibited the degradation of the radiolabeled substrate with inhibition constants around 200 microM. Short fragments of the substance P sequence, such as Boc-Phe-Phe-OMe and Boc-Phe-Phe-Gly-OEt, were also found to inhibit the degradation of the substrate. When the metal-chelating hydroxamic acid moiety was attached to the carboxyl terminus of these short peptides, potent inhibitors of the substance-P-degrading activity were obtained, with inhibition constants in the micromolar range. The most potent of these compounds, iododesaminotyrosyl-Phe-Phe-Gly-NHOH (IBH-Phe-Phe-Gly-NHOH), is a competitive inhibitor, with a Ki value of 1.9 microM. The degradation of substance P by rat diencephalon slices was inhibited to the same extent (40-50%) by IBH-Phe-Phe-Gly-NHOH (20 microM) and by phosphoramidon (1 microM). A combination of both reagents reduced the degradation rate by 75-80%, suggesting that both enkephalinase and the substance-P-degrading activity are involved in the metabolism of substance P in this preparation. IBH-Phe-Phe-Gly-NHOH seems to be quite specific for the latter enzyme, since at a high concentration (0.1 mM) it did not affect the degradation of the radiolabeled substrate by alpha-chymotrypsin, papain, or thermolysin.     

Escherichia coli O157:H7 Strain Origin,Lineage, and Shiga Toxin 2 Expression Affect Colonization of Cattle

Enterohemorrhagic Escherichia coli O157:H7 has evolved into an important human pathogen with cattle as the main reservoir. The recent discovery of E. coli O157:H7-induced pathologies in challenged cattle has suggested that previously discounted bacterial virulence factors may contribute to the colonization of cattle. The objective of the present study was to examine the impact of lineage type, cytotoxin activity, and cytotoxin expression on the amount of E. coli O157:H7 colonization of cattle tissue and cells in vitro. Using selected bovine- and human-origin strains, we determined that lineage type predicted the amount of E. coli O157:H7 strain colonization: lineage I > intermediate lineages > lineage II. All E. coli O157:H7 strain colonization was dose dependent, with threshold colonization at 10³ to 10⁵ CFU and maximum colonization at 10⁷ CFU. We also determined that an as-yet-unknown factor of strain origin was the most dominant predictor of the amount of strain colonization in vitro. The amount of E. coli O157:H7 colonization was also influenced by strain cytotoxin activity and the inclusion of cytotoxins from lineage I or intermediate lineage strains increased colonization of a lineage II strain. There was a higher level of expression of the Shiga toxin 1 gene (stx₁) in human-origin strains than in bovine-origin strains. In addition, lineage I strains expressed higher levels of the Shiga toxin 2 gene (stx₂). The present study supports a role for strain origin, lineage type, cytotoxin activity, and stx₂ expression in modulating the amount of E. coli O157:H7 colonization of cattle.Enterohemorrhagic Escherichia coli O157:H7 is a bacterium that causes serious human disease outbreaks through the consumption of contaminated food or water (39). Mature cattle are considered the primary reservoir for E. coli O157:H7 and historically were reported to have no symptoms or pathologies (17, 23, 38); this was attributed both to a lack of receptors for a critical E. coli O157:H7 virulence factor, Shiga toxin 1 (Stx1 [29]), and to a differential expression of type III protein secretion system effector molecules such as EspA, EspD, and Iha (25, 30) in cattle compared to humans. In 2008, it was established for the first time that E. coli O157:H7 causes mild to severe intestinal pathology in persistent shedding cattle (5, 26) and that the secreted cytotoxins enhanced E. coli O157:H7 colonization of intestinal tissues of cattle (6). This suggested that cattle were susceptible to E. coli O157:H7 infection and that previously discounted virulence factors could influence the amount of colonization in cattle.Three distinct E. coli O157:H7 lineages have been identified based on the lineage specific polymorphism assay (LSPA-6) that suggests both the evolutionary history of the strain and their propensity to be present among animals, the environment, and clinical human isolates (21, 22, 24, 33, 40, 42). Typically, two predominant lineages have been described, lineages I and II (22, 40) and, more recently, intermediate lineages that have characteristics of lineage I and/or II have been reported at higher frequency among cattle (34). Although all E. coli O157:H7 lineages have been isolated from feedlot cattle, the predominant recovery of lineage I from clinical human illnesses suggests that this particular lineage type has unique expression patterns that may contribute to its preferential colonization of humans. There is some evidence to suggest that lineage I strains do not express certain virulence factors in bovine hosts, whereas other factors such as cytotoxins are expressed equally irrespective of host (30). One virulence factor associated with all lineages is the bacterium''s ability to form intimate attaching-and-effacing lesions or colonization sites in the ilea of susceptible animals (28). The amount of colonization is enhanced by the expression of Shiga toxin 2 (Stx2) through both an increase in the expression of alternative non-TIR (translocated intimin receptor) colonization sites (31) and toxicity to the absorptive epithelial cells (32). In cattle, attaching-and-effacing lesions are also formed (5), and Stx2 increases colonization but is not cytotoxic to epithelial cells from the jejuna and descending colons of cattle (4). Differential expression of stx₂ among E. coli O157:H7 lineages is also linked to the increased pathogenicity of lineage I strains in humans (25), and this may affect cattle similarly. Together, this information suggests that at least some similar virulence factors affecting E. coli O157:H7 colonization in humans also function in cattle.In order to gain a better understanding of the factors modulating E. coli O157:H7 colonization in cattle, we compared the ability of lineage I, lineage II, and intermediate lineages isolated from human sources to colonize the jejunum tissue and a colonic cell line from cattle. We hypothesized that the bovine colonic cell line could be used as a model system to reflect E. coli O157:H7 colonization of tissue. To confirm the value of this model, the role of strain origin in colonization of cattle was examined. In order to understand the differences in colonization associated with lineage and strain origins, we assessed cytotoxin expression, secreted cytotoxin activity, and cytotoxin-induced changes in E. coli O157:H7 colonization. Given the known lack of Stx1 activity in cattle, we examined the effects of LSPA-6 genotype, strain origin (human versus bovine), and cytotoxin activity on E. coli O157:H7 colonization of cattle.     

Biochemical characterization of a truncated penta-EF-hand Ca2+ binding protein from maize

Plants possess multiple genes encoding calcium sensor proteins that are members of the penta-EF-hand (PEF) family. Characterized PEF proteins such as ALG-2 (apoptosis-linked gene 2 product) and the calpain small subunit function in diverse cellular processes in a calcium-dependent manner by interacting with their target proteins at either their N-terminal extension or Ca2+ binding domains. We have identified a previously unreported class of PEF proteins in plants that are notable because they do not possess the hydrophobic amino acid rich N-terminal extension that is typical of these PEF proteins. We demonstrate that the maize PEF protein without the N-terminal extension has the characteristics of known PEF proteins; the protein binds calcium in the 100 nM range and, as a result of calcium binding, displays an increase in hydrophobicity. Characterization of the truncated maize PEF protein provides insights into the role of the N-terminal extension in PEF protein signaling. In the context of the current model of how PEF proteins are activated by calcium binding, these results demonstrate that this distinctive class of PEF proteins could function as calcium sensor proteins in plants even in the absence of the N-terminal extension.     

Investigation of Mannheimia haemolytica bacteriophages relative to host diversity

Aims

This study aimed to characterize the impact of lytic and temperate bacteriophages on the genetic and phenotypic diversity of Mannheimia haemolytica from feedlot cattle.

Methods and Results

Strictly lytic phages were not detected from bovine nasopharyngeal (n = 689) or water trough (n = 30) samples, but Myoviridae‐ or Siphoviridae‐like phages were induced from 54 of 72 M. haemolytica strains by mitomycin C, occasionally from the same strain. Phages with similar restriction fragment length polymorphism profiles (RFLP ≥70% relatedness) shared common host serotypes 1 or 2 (P < 0·000 1). Likewise, phages with similar RFLP tended to occur in genetically related host bacteria (70–79% similarity). Host range assays showed that seven phages from host serotypes 1, 2 and 6 lysed representative strains of serotypes 1, 2 or 8. The genome of vB_MhM_1152AP from serotype 6 was found to be collinear with P2‐like phage φMhaA1‐PHL101.

Conclusions

Prophages are a significant component of the genome of M. haemolytica and contribute significantly to host diversity. Further characterization of the role of prophage in virulence and persistence of M. haemolytica in cattle could provide insight into approaches to control this potential respiratory pathogen.

Significance and Impact of the Study

This study demonstrated that prophages are widespread within the genome of M. haemolytica isolates and emphasized the challenge of isolating lytic phage as a therapeutic against this pathogen.     

High catalytic efficiency and resistance to denaturing in bacterial Rho GTPase-activating proteins

Several major bacterial pathogens use the type III secretion system (TTSS) to deliver virulence factors into host cells. Bacterial Rho GTPase activating proteins (RhoGAPs) comprise a remarkable family of type III secreted toxins that modulate cytoskeletal dynamics and manipulate cellular signaling pathways. We show that the RhoGAP activity of Salmonella SptP and Pseudomonas ExoS toxins is resistant to variations in the concentration of NaCl or MgCl(2), unlike the known salt dependant nature of the activity of some eukaryotic GAPs such as p190, RanGAP and p120GAP. Furthermore, SptP-GAP and ExoS-GAP display full activity after treatment at 80°C or with 6 m urea, which suggests that these protein domains are capable of spontaneous folding into an active state following denaturing such as what might occur upon transit through the TTSS needle. We determined the catalytic activity of bacterial GAPs for Rac1, CDC42 and RhoA GTPases and found that ExoS, in addition to Yersinia YopE and Aeromonas AexT toxins, display higher catalytic efficiencies for Rac1 and CDC42 than the known eukaryotic GAPs, making them the most catalytically efficient RhoGAPs known. This study expands our knowledge of the mechanism of action of GAPs and of the ways bacteria mimic host activities and promote catalysis of eukaryotic signaling proteins.