Identification of receptor binding sites by competitive peptide mapping: phages T1, T5, and phi 80 and colicin M bind to the gating loop of FhuA.

Previously we proposed a transmembrane model of the FhuA receptor protein in the outer membrane of Escherichia coli. Removal of the largest loop at the cell surface converted the FhuA transport protein into an open channel and rendered cells resistant to the FhuA-specific phages T1, T5, and phi 80 and to colicin M. In the present study we employed acetylated hexapeptide amides covering the entire surface loop to investigate binding of the phages and of colicin M. Competitive peptide mapping proved to be a powerful technique to uncover three ligand binding sites within a region of 34 amino acid residues. Hexapeptides derived from three specific regions of the surface loop inhibited infection of cells by the phages and killing by colicin M. Two of these regions were common among all four FhuA ligands. Electron microscopy of phage T5 revealed that one inhibitory peptide triggered a strong conformational change leading to the release of DNA from the phage head. These results suggest that the FhuA gating loop is the target for specific binding of phages T1, T5, and phi 80 and colicin M.     

A mixed-ligand iron-sulfur cluster (C556SPaB or C565SPsaB) in the Fx-binding site leads to a decreased quantum efficiency of electron transfer in photosystem I.

The proposed structure of Photosystem I depicts two cysteines on the PsaA polypeptide and two cysteines on the PsaB polypeptide in a symmetrical environment, each providing ligands for the interpolypeptide Fx cluster. We studied the role of Fx in electron transfer by substituting serine for cysteine (C565SPsaB and C556SPsaB), thereby introducing the first example of a genetically engineered, mixed-ligand [4Fe-4S] cluster into a protein. Optical kinetic spectroscopy shows that after a single-turnover flash at 298 K, the contribution of A1- (lifetime of 10 microseconds, 40% of total and lifetime of 100 microseconds, 20% of total) and Fx- (lifetime of 500-800 microseconds, 10-15% of total) to the overall P700+ back reaction have increased in C565SPsaB and C556SPsaB at the expense of the back reaction from [FA/FB]-. The electron paramagnetic resonance spectrum of Fx shows g-values of 2.04, 1.94, and 1.81 in both mutants and a similarly decreased amount of FA and FB reduced at 15 K after a single-turnover flash. These results indicate that the mixed-ligand (3 cysteines, 1 serine) Fx cluster is an inefficient electron carrier, but that a small leak through Fx still permits FA and FB to be reduced quantitatively when the samples are frozen during continuous illumination. The data confirm that Fx is a necessary intermediate in the electron transfer pathway from A1 to FA and FB in Photosystem I.     

Interspecific interactions in the marine rotifer microcosm

Copepods and protozoans often co-exist in marinerotifer mass cultures. Interspecific interactionbetween the rotifer Brachionus rotundiformisTschugunoff and eight other zooplankton species,namely Brachionus plicatilis O. F. Müller (rotifer),Diaphanosoma celebensis Stingelin (cladoceran),Tigriopus japonicus Mori, Acartia sp. (copepod),Euplotes sp., Vorticella sp., an unidentifiedprotozoan species (P1 strain) (protozoan) and Artemiasp. (anostracan) at two developmental stages (nauplii –0.95 mm, 0 days old; adults – 3.3 mm, 19 days old) wereinvestigated in the laboratory.There was no contaminating species that contributedto an increase in rotifer population growth during theexperiments. Four types of interspecific interactionswere seen between B. rotundiformis and otherco-existing zooplankton species. These include effectson population growth: (1) both species declined, (2) onespecies is promoted while the other is not influenced,(3) one species is declined while the other is notinfluenced and (4) one species is promoted while theother declined. The first type was exhibited by B. rotundiformis vs B. plicatilis, B. rotundiformis vsD. celebensis and B. rotundiformis vs Artemia sp. The second type was exhibited by B. rotundiformis vsVorticella sp. and the third type by B. rotundiformisvs Euplotes sp. and B. rotundiformis vs T. japonicus. The fourth type was exhibited by B. rotundiformis vs Acartia sp. and B. rotundiformis vs P1 strain.     

Genetic variation of herpesvirus saimiri subgroup A transforming protein and its association with cellular src.

Herpesvirus saimiri strain 11 of subgroup A contains a gene called the saimiri transformation-associated protein, STP, which is not required for viral replication but is required for in vitro immortalization and for the lymphoma-inducing capacity of the virus. To assess the effects of sequence variation on STP function, STP genes from six subgroup A isolates were cloned and sequenced. Sequence comparisons revealed extensive amino acid substitutions within the central region, but the acidic amino terminus and the hydrophobic carboxyl terminus were well conserved. Amino acid identities varied from 73 to 99% among all two-way comparisons. The highly conserved YAEV/I motif at amino acid residues 115 to 118 was preceded by negatively charged glutamic acid residues and thus matched very well the consensus sequence for binding to SH2 domains of src family kinases. The STPs of these subgroup A strains were shown to associate with cellular src and to be an in vitro substrate for src kinase. Mutational analysis of STP-A11 showed that binding to src kinase required the tyrosine residue at 115, showing that YAEV/I is a likely binding motif for src. Also, tyrosine phosphorylation of STP-A11 by src led to subsequent binding to lck and fyn in vitro. Thus, the association of STP with src is likely to be important for T-cell transformation by subgroup A strains of herpesvirus saimiri.     

A role for natural simian immunodeficiency virus and human immunodeficiency virus type 1 nef alleles in lymphocyte activation.

A T-lymphoid cell line termed 221 was derived from a rhesus monkey infected with herpesvirus saimiri. Growth of 221 cells was dependent on the addition of interleukin-2 (IL-2) to the culture medium. In the absence of IL-2, 221 cells arrested in G0-G1 but did not die. Simian immunodeficiency virus (SIV) replicated efficiently in IL-2-stimulated 221 cells whether or not the nef gene was present. In the absence of IL-2, nef-containing SIV replicated 8 to 100 times more efficiently in 221 cells than did the same virus lacking nef. nef-containing virus preferentially stimulated the production of IL-2 from 221 cells. HIV-1 nef and v-ras genes, but not the c-ras gene, were shown to substitute functionally for SIV nef when tested as recombinant viruses in this assay system. These results demonstrate a role for natural nef in causing lymphoid cell activation, and they provide a system for delineating the biochemical mechanisms responsible for this activation.     

Immunodominant CD4+ T-cell epitope within nonstructural protein 3 in acute hepatitis C virus infection.

In acute hepatitis C virus infection, 50 to 70% of patients develop chronic disease. Considering the low rate of spontaneous viral clearance during chronic hepatitis C infection, the first few months of interaction between the patient's immune system and the viral population seem to be crucial in determining the outcome of infection. We previously reported the association between a strong and sustained CD4+ T-cell response to nonstructural protein 3 (NS3) of the hepatitis C virus and a self-limited course of acute hepatitis C infection. In this study, we identify an immunodominant CD4+ T-cell epitope (amino acids 1248 to 1261) that was recognized by the majority (14 of 23) of NS3-specific CD4+ T-cell clones from four of five patients with acute hepatitis C infection. This epitope can be presented to CD4+ T cells by HLA-DR4, -DR11, -DR12, -DR13, and -DR16. HLA-binding studies revealed a high binding affinity for 10 of 13 common HLA-DR alleles. Two additional CD4+ T-cell epitopes, amino acids 1388 to 1407 and amino acids 1450 to 1469, showed a very narrow pattern of binding to individual HLA-DR alleles. Our data suggest that the NS3-specific CD4+ T-cell response in acute hepatitis C infection is dominated by a single, promiscuous peptide epitope which could become a promising candidate for the development of a CD4+ T-cell vaccine.     

Epitope mapping of antibodies directed against hypervariable region 1 in acute self-limiting and chronic infections due to hepatitis C virus.

Epitopes of hypervariable region 1 (HVR1) were mapped by enzyme-linked immunosorbent assay using follow-up sera of patients, all of whom were infected with the same isolate of hepatitis C virus (HCV). Our results suggest that (i) an early appearance (up to month 13 postinfection) of antibodies directed to the N terminus of HVR1 is associated with acute self-limiting infections of HCV and (ii) isolate-independent antibodies which are mainly directed to the C terminus of HVR1 seem to persist in chronically infected patients. The relevance of HVR1-specific antibodies for neutralization was evaluated by characterization of a rabbit serum.     

Combination of the tod and the tol pathways in redesigning a metabolic route of Pseudomonas putida for the mineralization of a benzene, toluene, and p-xylene mixture.

Construction of a hybrid strain which is capable of mineralizing components of a benzene, toluene, and p-xylene mixture simultaneously was attempted by redesigning the metabolic pathway of Pseudomonas putida. Genetic and biochemical analyses of the tod and the tol pathways revealed that dihydrodiols formed from benzene, toluene, and p-xylene by toluene dioxygenase in the tod pathway could be channeled into the tol pathway by the action of cis-p-toluate-dihydrodiol dehydrogenase, leading to complete mineralization of a benzene, toluene, and p-xylene mixture. Consequently, a hybrid strain was constructed by cloning todC1C2BA genes encoding toluene dioxygenase on RSF1010 and introducing the resulting plasmid into P. putida mt-2. The hybrid strain of P. putida TB105 was found to mineralize a benzene, toluene, and p-xylene mixture without accumulation of any metabolic intermediate.