Dynamic disulfide scanning of the membrane-inserting Pf3 coat protein reveals multiple YidC substrate contacts

The membrane insertase YidC inserts newly synthesized proteins into the plasma membrane. While defects in YidC homologs in animals and plants cause diseases, YidC in bacteria is essential for life. Membrane insertion and assembly of ATP synthase and respiratory complexes is catalyzed by YidC. To investigate how YidC interacts with membrane-inserting proteins, we generated single cysteine mutants in YidC and in the model substrate Pf3 coat protein. The single cysteine mutants were expressed and analyzed for disulfide formation during 30 s of synthesis. The results show that the substrate contacts different YidC residues in four of the six transmembrane regions. The residues are located either in the region of the inner leaflet, in the center, as well as in the periplasmic leaflet, consistent with the hypothesis that YidC presents a hydrophobic platform for inserting membrane proteins. In a YidC mutant where most of the contacting residues were mutated to serines, YidC function was severely disturbed and no longer active in a complementation test, suggesting that the residues are important for function. In addition, a Pf3 mutant with a defect in membrane insertion was deficient to contact the periplasmic residues of YidC.     

Identification of dynamic structural motifs involved in peptidoglycan glycosyltransfer

We have determined the structure of a new form of the bifunctional peptidoglycan glycosyltransferase (GT)/transpeptidase penicillin-binding protein 2 from the pathogen Staphylococcus aureus. We observe several previously unstructured regions of the GT substrate-binding pockets, including a π-bulge in the outer helix that may be responsible for the conformational flexibility of active-site motifs required for transfer of product to the donor binding site during processive rounds of peptidoglycan polymerization. The identification of a β-hairpin in the usually unstructured region of the fold shares local structural homology to that of an exomuramidase, heightening comparisons between this biosynthetic enzyme and lytic peptidoglycan transglycosylases. This new form also shows remarkable interdomain flexibility, causing the linker region of the fold to project into the GT active site. This self-interaction may have significant consequences for the regulation of polymerization activity. The derived information is used to build a catalytic model of both donor and acceptor glycolipid substrates.     

A novel site-specific recombination system derived from bacteriophage phiMR11

We report identification of a novel site-specific DNA recombination system that functions in both in vivo and in vitro, derived from lysogenic Staphylococcus aureus phage phiMR11. In silico analysis of the phiMR11 genome indicated orf1 as a putative integrase gene. Phage and bacterial attachment sites (attP and attB, respectively) and attachment junctions were determined and their nucleotide sequences decoded. Sequences of attP and attB were mostly different to each other except for a two bp common core that was the crossover point. We found several inverted repeats adjacent to the core sequence of attP as potential protein binding sites. The precise and efficient integration properties of phiMR11 integrase were shown on attP and attB in Escherichia coli and the minimum size of attP was found to be 34bp. In in vitro assays using crude or purified integrase, only buffer and substrate DNAs were required for the recombination reaction, indicating that other bacterially encoded factors are not essential for activity.     

Cloning of genes for bacteriophage T5 stable RNAs

One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage λ XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo ³²P-labeled T5 4–5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI¹440 fragment contains the gene for tRNA 10 (tRNA^Asp), the EcoRI/HindIII¹220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNA^His), and the EcoRI/HindIII¹960 fragment contains only a part of the gene for tRNA 9 (tRNA^Gln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNA^Gln-tRNA^His-RNA III-RNA I-…-tRNA^Asp.     

Cloning and antigenic expression of two genes from Chlamydia psittaci avian strain

Abstract: Genes from Chlamydia psittaci P-1041 were cloned into the Bam HI site of pUC19 and were transformed to host Escherichia coli JM109. Two recombinant plasmids that expressed protein antigens of Chlamydia were isolated. The sizes of the DNA fragments were 1350 and 1710 bp, and encoded for polypeptides of M _r 25 and 42 kilodaltons (kDa), respectively. The 25-kDa protein had cross-reactivity with antisera to ten C. psittaci strains and two C. trachomatis strains, whereas the 42-kDa protein reacted only with homologous antiserum to the C. psittaci P-1041 strain. Furthermore, in Southern hybridization analysis these two fragments as probes hybridized with DNA of ten C. psittaci strains and four C. trachomatis strains. These results indicated that the two fragments shared a DNA sequence common to the chlamydial genus.     

Editorial

Circular double-stranded phage M13 DNA-containing gaps of defined size and location were obtained in preparative amounts by in vitro hybridization of plus- and minus-strands derived from different phage strains.     

Fluorescence study of secondary structure of DNA within bacteriophage lambda

Bromoacetaldehyde (BAA) was used to study the secondary structure of DNA in lambda-phage particles. It was determined that about 1% of the adenines in the intraphage lambda-DNA reacts readily with BAA, thus, they are placed in DNA sites with disturbed complementary interactions. These adenines are close to the tryptophan residues of the phage protein. Fluorescence emission of epsilon A in the intraphage DNA is dramatically quenched. This, apparently, indicates the interaction between epsilon A and Trp- and/or Tyr- and/or Met-residues of phage protein.     

Interactions between the soil-borne bacteriophage Fo-l and Pseudomonas spp. on sugarbeet roots

The large plaquing (24 mm²) soilborne bacteriophage, Fo-l, did not affect the colonization ability on sugarbeet roots of its host, fluorescent Pseudomonas sp. strain B26. Phage Fo-l did not increase in numbers on sugarbeet roots when seeds were coated with less than 10⁶ CFU (colony forming units) of B26 and when less than 300 PFU (plaque forming units) of phage Fo-l was added per g of soil (dry weight). Above these threshold values, phage replication occurred and up to 2 × 10⁶ PFU per root system could be recovered.