[Application of reporter strains for new antibiotic screening].

Screening for new antibiotics remains an important area of biology and medical science. Indispensable for this type of research is early identification of antibiotic mechanism of action. Preferentially, it should be studied quickly and cost-effectively, on the stage of primary screening. In this review we describe an application of reporter strains for rapid classification of antibiotics by its target, without prior purification of an active compound and determination of chemical structure.

[Common features of antibacterial compounds: an analysis of 104 compounds library].

Antibacterial compounds are one of the essential classes of clinically important drugs. High throughput screening allowed revealing potential antibiotics active towards any molecular target in bacterial cell. We used a library of 9820 organic compounds with highly diversified structures to screen for antibacterial activity. As the result of automated screening, 103 compounds were found to possess antibacterial activity against Escherichia coli. The properties of these compounds were compared with those of initial library. Non-linear Kohonen mapping was used to analyze the differences between non-active molecules from initial library, identified antibacterial hits and compounds with reported antibacterial activity. It was found that identified antibacterial compounds are located in the separated area of chemical space. It can be therefore suggested that these molecules belong to novel classes of antibacterial compounds and could be studied further.

Gene transfer to the rhesus monkey brain using SV40-derived vectors is durable and safe.

Gene transfer to central nervous system (CNS) has been approached using various vectors. Recombinant SV40-derived vectors (rSV40s) transduce human neurons and microglia effectively in vitro and in rodent brains in vivo, so we tested rSV40s gene transfer to rhesus monkey CNS in vivo, to characterize the distribution, duration and safety of such gene delivery. We used rSV40s carrying HIV-1 RevM10 with a carboxyl-terminal AU1 epitope tag as a marker, and others with the antioxidant enzymes, Cu/Zn superoxide dismutase and glutathione peroxidase. Vectors were injected stereotaxically into the caudate nucleus. Transgene expression was studied at 1 and 6 months by immunostaining serial brain sections. After intraparenchymal administration, numerous transgene-expressing cells were seen, with a longitudinal extent of 20 mm. In neurons and, more rarely, microglial cells, transgene expression remained strong throughout the 6-month study period. Astrocytes and oligodendroglia were not transduced. No evidence of inflammation or tissue damage was observed. SV40-derived vectors may thus be useful for long-term gene expression in the monkey brain and, potentially, in the human brain.

Engineering trimeric fibrous proteins based on bacteriophage T4 adhesins.

The adsorption specificity of bacteriophage T4 is determined by genes 12 and 37, encoding the short tail-fibers (STF) and the distal part of the long tail-fibers (LTF), respectively. Both are trimeric proteins with rod domains made up of similar tandem quasi-repeats, approximately 40 amino acids long. Their assembly requires the viral chaperones gp57A and gp38. Here we report that fusing fragments of gp12 and gp37 to another trimeric T4 fibrous protein, fibritin, facilitates correct assembly, thereby by-passing the chaperone requirement. Fibritin is an alpha-helical coiled coil protein whose C-terminal part (fibritin E, comprising the last 120 residues) has recently been solved to atomic resolution. Gp12 fragments of 109 and 70 amino acids, corresponding to three and two quasi-repeats respectively, were fused to the C-terminus of fibritin E. A similar chimera was designed for the last 63 residues of gp37, which contain four copies of the pentapeptide Gly-X-His-X-His and assume a narrow rigid structure in the LTF distal tip. Expressed from plasmids, all three chimeras form soluble trimers that are resistant to dissociation by SDS and digestion by trypsin, indicative of correct folding and oligomerization.

Properties of recombinant bacteriophage T4 tail sheath protein and its deletion fragments.

A vector for expression of recombinant bacteriophage T4 tail sheath protein (gp18) under control of phage T7 promoter in Escherichia coli cells has been constructed. The entire length recombinant gp18 (659 amino acids) polymerizes in vivo into extended polysheaths. To study gp18 folding mechanisms, six vectors for expression of deletion mutants have been constructed. Three proteins--1N, 2N, and 3N--contain, respectively, 268, 316, and 372 amino acids of the gp18 N-tail region. The other three fragments--1C, 2C, and 3C--contain, respectively, 455, 356, and 288 amino acids of the gp18 C-tail. The fragments 1N, 2N, 1C, 2C, and 3C form insoluble aggregates during expression. However, fragment 3N accumulates in soluble form in the cellular cytoplasm and does not form polymeric structures; this has allowed an effective purification method to be developed for it. The interaction of monoclonal antibodies against recombinant gp18 with protein fragments and with phage sheath before and after contraction has been studied. The fragment 3N seems to be a stable domain of native phage sheath gp18.

Chaperones in bacteriophage T4 assembly.

Protein folding in the cell is controlled at the levels of translation and post-translational modification, depends on a number of conserved proteins known as chaperones, and is catalyzed by specific enzymes, such as protein disulfide isomerase and peptidyl prolyl cis-trans isomerase. The chaperones stabilize folding intermediates and participate in assembly and disaggregation of supramolecular structures. Bacteriophage T4 is an especially convenient system for studying of protein folding mechanisms, since its genome encodes several virus-specific chaperones. In this review, the chaperones of phage T4 that take part in capsid formation (gp31 and gp40) and in folding and assembly of virion tail fibers (gp38, gp57A) have been considered. Protein encoded by gene 31 completely substitutes co-chaperonin GroES of the host cell in folding of the major capsid protein, gp23, aided by chaperonin GroEL. The product of gene 40, which is homologous to analogs of eukaryotic GroEL and peptidyl prolyl cis-trans isomerase, participates in assembly of gp20 while the formation of procapsid connector. The chaperone encoded by gene 57A is essential for folding and oligomerization of both long and short phage tail fibers. gp38, together with gp57A, participates in the formation of the distal part of the long fibers. This protein seems to represent a principally new group of chaperones that change steric structure of folded polypeptide. One phage chaperone, fibritin, encoded by gene wac (whiskers antigen control) and taking part in assembly the subunits of the long tail fibers is a constituent of the virion. Fibritin is a convenient model for studying mechanisms of folding and oligomerization of fibrous proteins due to its labile triple-stranded alpha-helical coiled-coil structure.

Evolution of T4-related phages.

Much progress has been made in understanding T-even phage biology in the last 50 years. We now know the entire sequence of T4, encoding nearly 300 genes, only 69 of which have been shown to be essential under standard laboratory conditions; no specific function is yet known for about 140 of them. The origin of most phage genes is unclear, and only 42 genes in T4 have significant similarity to anything currently included in GenBank. Comparative analysis of related phages is now being used to gain insight into both the evolutionary origins and interrelationships of these phage genes, and the functions of their protein products. The genomes of phages isolated from Tbilisi hospitals, Long Island sewage plants, the Denver zoo, and Khabarovsk show basic similarity. However, these phages show substantial insertions and deletions in a number of regions relative to each other, and closer investigation of specific sequences often reveals much more complex relationships. There are only a few cases in T4-related phages in which there is evidence for evolution through DNA duplication. These include the fibrous products of genes 12, 34, and 37; head proteins gp23 and gp24; and the Alt enzyme and its downstream neighbors. T4 also contains 13 apparent relatives of group I and group II intron homing endonucleases. Distal portions of the tail fibers of various T-even phages contain segments closely related to tail-fiber regions of other DNA coliphages, such as Mu, P1, P2, and lambda. Horizontal gene transfer clearly emerges as a major factor in the evolution of at least the tail-fiber regions, where site-specific recombination probably is involved in the exchange of host-range determinants.

A proposed structure of bacteriophage T4 gene product 22--a major prohead scaffolding core protein.

Gene 22 of bacteriophage T4 encodes a major prohead scaffolding core protein of 269 amino acid residues. From its nucleotide sequence the gene product (gp) 22 has a predicted Mr of 29.9 and a pI of 4.3. The protein is rich in charged residues (glutamic acid and lysine) and contains low amounts of proline and glycine and no cysteine residues. We suggest that gp22 undergoes limited proteolytic processing which eliminates the short C-terminal piece from the molecule during the early steps of prohead assembly. Most amino acid residues of the gp22 polypeptide chain (80%) have an alpha-helical conformation and form seven peculiar alpha-helices. A model suggesting the spatial organization of gp22 is presented. Three long alpha-helices numbered 1 (1A and 1B), 3, and 5 (5A and 5B) are packed in an antiparallel fashion along the major axis of the road-shaped molecule. Two rather short alpha-helices (2 and 4) are located at the distal and proximal ends of the protein molecule, respectively. Helix number 2, which is a proteolytic fragment of gp22 found in mature T4 heads, is packed with helices 1A and 3, similar to a novel element of supersecondary structure, the alpha alpha-corner. Helix number 4 probably interacts with the gp20 connector of the prohead. The implications of the structure of the gp22 molecule for the assembly of the prohead core are discussed.

Cascade of overlapping late genes in bacteriophage T4.

The DNA sequences of genes 9, 10, 11, 12, and wac, which encode the structural proteins in the bacteriophage T4 base plate, were determined. These genes form a single operon which is transcribed in a clockwise direction from a single late promoter in the TATAAATA region located upstream of gene 9 at position -10. A feature of the operon is an overlap between the termination codon of each upstream gene and the initiation codon of its downstream gene. With the exception of gene 10, the open reading frames encode proteins which have a calculated molecular mass close to that obtained experimentally. The reading frame of gene 10 encodes a polypeptide with a calculated molecular mass of 66.2 kDa, which is at least 22 kDa less than that in the phage particle. Thus the mature protein encoded by gene 10 is possibly a product of the fusion of two adjacent phage genes. The hybrid protein may be formed by a frameshift during the translation of messenger RNA at the end of gene 9 or gene 10.