Evidence of interactions between Gp27 and Gp28 constituents of the central part of bacteriophage T4 baseplate.

The central part of the bacteriophage T4 baseplate consists of several proteins. However, for a number of the constituents the manner of incorporation are not convincingly established. Recently, we have presented evidence that gp28 is the structural component of the central part of the baseplate, which possesses a hydrophobic region and is membrane bound [Nieradko et al., 1998]. By utilizing extracts prepared from Escherichia coli cells that overexpressed genes 27 and 28 of phage T4, we proved that gp28 forms a complex with an another baseplate structural components: gp27. This complex was located in the membrane fraction. Its affinity to the inner membrane indicates that the identified complex may function as an initiator of the central hub assembly. It was subsequently established that these products interact in the ratio 1:1. We have also demonstrated that the particular components of the complex can be separated by action of SDS and to a lesser extent by Triton X-100.

Characteristics of gene 28 product, the constituent of the central part of bacteriophage T4 baseplate.

The phage TD gene 28 product has been partially characterized and its biological role has been examined. It was found to be a protein with a molecular size of 24 kDa which cosediments with the membrane fraction of the bacterial extracts and could only be washed out by a 0.2% sarcosyl solution. Other observations indicate that gp 28 has a majority of hydrophobic residues on its surface and forms a homotrimeric complex in the absence of other phage proteins. The product was finally identified as a baseplate structural component. Incubation of purified phage preparation in a buffer which contained active protein 28, did not affect the efficiency of the plating. However, incubation of the phage particles with specific antiserum was found to neutralize phage infectivity.

Cloning and expression of the genes coding for tube associated proteins of bacteriophage T4.

Genes 29, 48 and 54 of bacteriophage T4, coding for specific tube associated proteins, were cloned to the expression vector pT7-5. The molecular mass of the products of these genes was estimated to be 64, 39 and 36 kDa, respectively. The examined genes are cotranscribed with genes 51, 27 and 28 from the same DNA strand and a common late promoter sequence located downstream of gene 51.

Expression of genes 51, 27, 28 coding for proteins of the central part of bacteriophage T4 baseplate in the bacteriophage T7 promoter/RNA polymerase expression system.

A fragment of T4 DNA (XbaI-HindIII) comprising the genes 51, 27, 28, which encodes the central plug proteins was cloned into plasmid pT7-5 and p7-6 (T7 RNA polymerase expressing system). The examined genes were only overexpressed when the orientation of cloned DNA to promoter phi 10 was as follows: promoter phi 10 and genes 51, 27, 28. This was achieved when the fragment (XbaI-HindIII) was cloned into plasmid pT7-5. Gene 27 and 28 were overexpressed when the intact fragment (XbaI-HindIII) was used. The high rate of the synthesis of proteins 27 and/or 28 had a strong inhibitory effect on the level of synthesis of the product of gene 51. For the overexpression of gene 51 in this system a deletion derivate which was devoid of gene 28 and a larger fragment of gene 27 was prepared.

Expression of bacteriophage T4 minor baseplate proteins in the bacteriophage T7 promoter/RNA polymerase expression system.

The central part of bacteriophage T4 baseplate is built of several proteins which are present in only a few copies per phage particle. Only some of these minor baseplate components have been identified previously as distinct protein species by biochemical analysis. We have used the bacteriophage T7 RNA polymerase expression system to identify and overexpress the minor baseplate proteins. The products of genes 25, 26 and 51 were identified on the autoradiographs after selective labelling with [35]S methionine. The overexpression of gene 25 and 51 products was high enough to make possible undertaking their purification and studies of their properties.

Construction and characterization of hybrids containing genes coding for proteins of the central part of bacteriophage T4 baseplate.

The central part (hub or plug) of bacteriophage T4 baseplate consist of several proteins which are present in only few copies per phage particle. The presence of these minor baseplate components was inferred from the genetic data but only some of them were identified as distinct proteins species by biochemical analysis. We have constructed a number of plasmids containing segments of bacteriophage T4 genome coding for baseplate proteins. The following genes were cloned into expression vectors: 54, 48, 29, 28, 27, 51, 26 and 25. The presence of a particular gene product was confirmed by in vivo complementation test. On the basis of these results we could more precisely localize the position of a particular gene on T4 phage genetic map. The hybrids contain sets of genes which make aggregation impossible, so bacteria harbouring these plasmids are convenient starting point for the purification of baseplate proteins.

The construction of the hybrid plasmid containing genes of the central part of phage T4 baseplate and gene 29 product separation.

A fragment of E. coli bacteriophage T4 genome including the four genes (genes 51, 27, 28, 29) coding for the central plug proteins was cloned into plasmid pMCC17. The genes present on this fragment were expressed in E. coli in the absence of phage infection producing hub proteins, which could be identified on polyacrylamide gels. By applying affinity chromatography protein 29 was purified from extracts of E. coli transformed with this hybrid plasmid. The isolated protein had the ability to complement T4 29 amber mutants. The molecular weight of the purified protein was estimated as 75,000 to 85,000 depending on the composition of SDS-polyacrylamide gel used for the assay.

Escherichia coli dnaJ- and dnaK-gene products: synthesis in minicells and membrane-affinity.

Escherichia coli dnaJ- and dnaK-gene products have been identified in a system of minicells infected with the appropriate transducing lambda phages. The molecular weights of these polypeptides in dodecyl sulphate/acrylamide electrophoresis amounted to 39,000 and 77,000, respectively. Equilibrium sedimentation of minicell lysates in metrizamide density gradients has revealed that both these host proteins, indispensable for lambda DNA replication, are membrane-bound.

Expression of the replication region of phage lambda DNA cloned into pBR322 in E. coli minicells.

Replication region of bacteriophage lambda DNA was cloned into pBR322 plasmid by the use of two restriction enzymes--PstI and HindIII. The restriction analysis of four obtained plasmids revealed that lambda DNA was cloned in both orientations. Recombinant plasmids were transferred to the minicell-producing strain of E. coli and synthesis of the plasmid-mediated proteins was analysed by polyacrylamide-gel electrophoresis. All four recombinant plasmids produced lambda DNA replication proteins pO and pP as well as some proteins specific for pBR322. The orientation of cloned fragment did not affect the synthesis of lambda DNA replication proteins.