Site-specific integration of the phage ΦCTX genome into the Pseudomonas aeruginosa chromosome: characterization of the functional integrase gene located close to and upstream of attP

The site-specific integration of the phage CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 by integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates.     

Stable integration and expression of heterologous genes in several lactobacilli using an integration vector constructed from the integrase and attP sequences of phage ΦAT3 isolated from Lactobacillus casei ATCC 393

An integration vector capable of stably integrating and maintaining in the chromosomes of several lactobacilli over hundreds of generations has been constructed. The major integration machinery used is based on the ΦAT3 integrase (int) and attP sequences determined previously. A novel core sequence located at the 3′ end of the tRNA^leu gene is identified in Lactobacillus fermentum ATCC 14931 as the integration target by the integration vector though most of such sequences found in other lactobacilli are similar to that determined previously. Due to the lack of an appropriate attB site in Lactococcus lactis MG1363, the integration vector is found to be unable to integrate into the chromosome of the strain. However, such integration can be successfully restored by cotransforming the integration vector with a replicative one harboring both attB and erythromycin resistance sequences into the strain. Furthermore, the integration vector constructed carries a promoter region of placT from the chromosome of Lactobacillus rhamnosus TCELL-1 which is used to express green fluorescence and luminance protein genes in the lactobacilli studied.     

Flagellin inhibits Myoviridae phage φCTX infection of Pseudomonas aeruginosa strain GuA18: purification and mapping of binding site

PhiCTX is a double-stranded DNA phage of the Myoviridae family that converts Pseudomonas aeruginosa into a cytotoxin producer. A 42-kDa phiCTX-inhibiting protein was purified from the outer membrane fraction of P. aeruginosa strain GuA18 by octyl-beta-glucoside extraction, DEAE-chromatography, and mono-Q HPLC. This protein had an isoelectric point of 5.4 and bound specifically [125I]-labeled phiCTX. The N-terminal amino acid sequence of six out of seven Lys-C fragments was highly similar (87%) to that of the entire of type-a flagellin of P. aeruginosa strain PAK. At a concentration of 14 nM, purified flagellin protein caused a 50% decrease in the phage titer after a 20-min incubation at 37 degrees C (PhI50). The presence of ethanol was necessary to reconstitute the inhibitory activity. In contrast, no ethanol treatment was necessary for the inhibitory activity of the sheared flagellin filaments from P. aeruginosa strain GuA18, which consists of the 42-kDa flagellin subunits and the synthesized 17-mer phage-binding-peptide NGSNSDSERTALNGEAK, representing flagellin residues 100-116 of P. aeruginosa strain PAK. The PhI50 was 10 nM and 200 nM, respectively. Antisera against the flagellin filament protein as well as against the 17-mer peptide neutralized phage infection. These results indicated that the amino acid region 100-116 of the flagellin subunit of strain GuA18 is involved in phiCTX binding. This region might play a role in phage attachment.     

Structural Insights into the Mechanism and Inhibition of the β-Hydroxydecanoyl-Acyl Carrier Protein Dehydratase from Pseudomonas aeruginosa

Fatty acid biosynthesis is an essential component of metabolism in both eukaryotes and prokaryotes. The fatty acid biosynthetic pathway of Gram-negative bacteria is an established therapeutic target. Two homologous enzymes FabA and FabZ catalyze a key step in fatty acid biosynthesis; both dehydrate hydroxyacyl fatty acids that are coupled via a phosphopantetheine to an acyl carrier protein (ACP). The resulting trans-2-enoyl-ACP is further polymerized in a processive manner. FabA, however, carries out a second reaction involving isomerization of trans-2-enoyl fatty acid to cis-3-enoyl fatty acid. We have solved the structure of Pseudomonas aeruginosa FabA with a substrate allowing detailed molecular insight into the interactions of the active site. This has allowed a detailed examination of the factors governing the second catalytic step. We have also determined the structure of FabA in complex with small molecules (so-called fragments). These small molecules occupy distinct regions of the active site and form the basis for a rational inhibitor design program.     

Presence of qacEΔ1 gene and susceptibility to a hospital biocide in clinical isolates of Pseudomonas aeruginosa resistant to antibiotics

Biocides play an important role in healthcare-associated infection control by either minimizing or preventing microorganism dissemination. This study evaluated the susceptibility of Pseudomonas aeruginosa clinical isolates to a quaternary ammonium (QAC) disinfectant and antibiotics, and verified the presence of qacEΔ1, a determinant of resistance to QAC. The disinfectant test was the Association of Official Analytical Chemists Use-Dilution Test, and polymerase chain reaction was used to examine for qacEΔ1. The qacEΔ1 gene was detected in 48% of the isolates. Eighty-eight percent of the multiresistant isolates carried qacEΔ1 gene, while 35% of the non-multiresistant isolates was positive to this gene, and multiresistance well correlated with its presence. Among isolates tested for the disinfectant, 46% showed a reduced susceptibility to the disinfectant. qacEΔ1 gene was present in 70% of the susceptible isolates to the biocide, whereas 90% of the less susceptible strains harbored this gene. Reduced susceptibility to the disinfectant was independent of presence of qacEΔ1 suggesting that it does not play an important role in biocide resistance in P. aeruginosa. As far as we know, it is the first report confirming this fact and testing with disinfectant at its in-use concentration. The evidence of less susceptible strains than the reference bacterium used in disinfectant testing, and the high percentage of qacEΔ1 gene detected are of special concern and suggests continued investigation in laboratory and in situ, not only in healthcare settings, but also in all areas of biocide usage, including different micro-organisms and biocides.     

Extensive proteolysis of head and inner body proteins by a morphogenetic protease in the giant Pseudomonas aeruginosa phage φKZ

Encased within the 280 kb genome in the capsid of the giant myovirus φKZ is an unusual cylindrical proteinaceous 'inner body' of highly ordered structure. We present here mass spectrometry, bioinformatic and biochemical studies that reveal novel information about the φKZ head and the complex inner body. The identification of 39 cleavage sites in 19 φKZ head proteins indicates cleavage of many prohead proteins forms a major morphogenetic step in φKZ head maturation. The φKZ head protease, gp175, is newly identified here by a bioinformatics approach, as confirmed by a protein expression assay. Gp175 is distantly related to T4 gp21 and recognizes and cleaves head precursors at related but distinct S/A/G-X-E recognition sites. Within the φKZ head there are six high-copy-number proteins that are probable major components of the inner body. The molecular weights of five of these proteins are reduced 35-65% by cleavages making their mature form similar (26-31 kDa), while their precursors are dissimilar (36-88 kDa). Together the six abundant proteins sum to the estimated mass of the inner body (15-20 MDa). The identification of these proteins is important for future studies on the composition and function of the inner body.     

Assignment of the gene for carboxypeptidase A to human chromosome 7q22→qter and to mouse chromosome 6

Summary A rat cDNA probe for preprocarboxypeptidase A was used to follow the segregation of the human gene for carboxypeptidase A (CPA) in 49 human x mouse somatic cell hybrids using Southern filter hybridization techniques. CPA was assigned to human chromosome 7q22qter. Similarly, the probe was used to follow the segregation of the mouse gene for carboxypeptidase A (Cpa) in 19 mouse x Chinese hamster somatic cell hybrids. Cpa was assigned to mouse chromosome 6. The gene for carboxypeptidase A forms part of a syntenic group that is conserved in man and mouse.Preliminary chromosomal assignments of carboxypeptidase A in man and mouse have been made in abstract (Honey et al. 1983a, b)     

Molecular characterization of head and neck cancer: how close to personalized targeted therapy?

Molecular targeted therapy in head and neck squamous cell carcinoma (HNSCC) continues to make strides, and holds much promise. Cetuximab remains the sole US FDA-approved molecular targeted therapy available for HNSCC, though several new biologic agents targeting the epidermal growth factor receptor (EGFR) and other pathways are currently in the regulatory approval pipeline. While targeted therapies have the potential to be personalized, their current use in HNSCC is not personalized. This is illustrated for EGFR-targeted drugs, where EGFR as a molecular target has yet to be individualized for HNSCC. Future research needs to identify factors that correlate with response (or lack of one) and the underlying genotype-phenotype relationship that dictates this response. Comprehensive exploration of genetic and epigenetic landscapes in HNSCC is opening new frontiers to further enlighten and mechanistically inform newer as well as existing molecular targets, and to set a course for eventually translating these discoveries into therapies for patients. This opinion offers a snapshot of the evolution of molecular subtyping in HNSCC and its current clinical applicability, as well as new emergent paradigms with implications for controlling this disease in the future.     

Coping with inadvertent lysis of Escherichia coli cultures: Strains resistant to lysogeny and infection by the stealthy lysogenic phage Φ80

Phage Φ80 can infect Escherichia coli in a stealthy manner and persist by forming lysogens. Such Φ80 lysogens are fairly common and often go undetected unless the host is grown at temperatures below 37°C. Since low growth temperatures are required for growing temperature-sensitive mutants and often preferred for large-scale applications such as protein production, Φ80-resistant strains would be useful. We report the construction of E. coli strains that cannot be efficiently lysogenized or infected by bacteriophage Φ80. These strains contain combinations of deletions or mutations in the bacterial attachment site for Φ80 integration and/or deletions in the genes required for phage absorption to the host outer membrane. These strains should help contain and prevent Φ80 infection of E. coli cultures in a laboratory or industrial setting.     

BioGPS and GXD: mouse gene expression data—the benefits and challenges of data integration

Mouse gene expression data are complex and voluminous. To maximize the utility of these data, they must be made readily accessible through databases, and those resources need to place the expression data in the larger biological context. Here we describe two community resources that approach these problems in different but complementary ways: BioGPS and the Mouse Gene Expression Database (GXD). BioGPS connects its large and homogeneous microarray gene expression reference data sets via plugins with a heterogeneous collection of external gene centric resources, thus casting a wide but loose net. GXD acquires different types of expression data from many sources and integrates these data tightly with other types of data in the Mouse Genome Informatics (MGI) resource, with a strong emphasis on consistency checks and manual curation. We describe and contrast the “loose” and “tight” data integration strategies employed by BioGPS and GXD, respectively, and discuss the challenges and benefits of data integration. BioGPS is freely available at http://biogps.org. GXD is freely available through the MGI web site (www.informatics.jax.org) or directly at www.informatics.jax.org/expression.shtml.     

The EEC and the law of the sea: How close to one voice?

Abstract

The Member States of the European Economic Community (EEC) constituted one of the most important negotiating groups at the Third United Nations Conference on the Law of the Sea (UNCLOS III). The EEC is competent to deal with several matters that were included in the Draft Treaty on the Law of the Sea. When such matters were considered at the Conference, the President of the EEC Council, rather than the Commission, spoke on behalf of the group. Coordination meetings were held at expert level and by the heads of delegations. Agreement was reached on the principal issues before the Conference such as the Economic Zone, the Area, protection of the marine environment, and scientific research. The group was less successful in having its views accepted by the Conference, largely because the member's views were those of the highly industrialized states and emphasized navigational rights. The EEC participation clause was almost as important as substantive issues for the group, because it enabled the Community to become a party to the Treaty. EEC competences are contrasted with single state competences, and some of the possible consequences of less than total ratification of the Treaty by EEC Members are considered.     

Human β-glucuronidase: Assignment of the structural gene to chromosome 7 using somatic cell hybrids

-Glucuronidase (GUS) has become an important enzyme model for the genetic study of molecular disease, enzyme realization, and therapy, and for the biogenesis and function of the lysosome and lysosomal enzymes. The genetics of human -glucuronidase was investigated utilizing 188 primary man-mouse and man-Chinese hamster somatic cell hybrids segregating human chromosomes. Cell hybrids were derived from 16 different fusion experiments involving cells from ten different and unrelated individuals and six different rodent cell lines. The genetic relationship of GUS to 28 enzyme markers representing 19 linkage groups was determined, and chromosome studies on selected cell hybrids were performed. The evidence indicates that the -glucuronidase gene is assigned to chromosome 7 in man. Comparative linkage data in man and mouse indicate that the structural gene GUS is located in a region on chromosome 7 that has remained conserved during evolution. Involvement of other chromosomes whose genes may be important in the final expression of GUS was not observed. A tetrameric structure of human -glucuronidase was demonstrated by the formation of three heteropolymers migrating between the human and mouse molecular forms in chromosome 7 positive cell hybrids. Linkage of GUS to other lysosomal enzyme genes was investigated. -Hexosaminidase HEX _B) was assigned to chromosome 5; acid phosphatase₂ (ACP ₂) and esterase A₄ (ES-A ₄) were assigned to chromosome 11; HEX _A was not linked to GUS; and -galactosidase (-GAL) was localized on the X chromosome. These assignments are consistent with previous reports. Evidence was not obtained for a cluster of lysosomal enzyme structural genes. In demonstrating that GUS was not assigned to chromosome 9 utilizing an X/9 translocation segregating in cell hybrids, the gene coding for human adenylate kinase₁ was confirmed to be located on chromosome 9.Supported by NIH Grants HD 05196, GM 20454, and GM 06321, by NSF Grant BMS 73-07072, and by HEW Maternal and Child Health Service, Project 417.     

Prothymosin α gene in humans: organization of its promoter region and localization to chromosome 2

A genomic clone encoding prothymosin (gene symbol: PTMA), a nuclear-targeted protein associated with cell proliferation, was isolated and the 5-regulatory region subcloned and sequenced. Because of previously reported discrepancies between several cDNA clones and a genomic clone for prothymosin , we determined the sequence of the first exon and of a 1.7-kb region 5 to the first exon. The sequence of the genomic clone reported here corresponds to the published cDNA sequences, suggesting that the previously noted discrepancies may be due to genetic polymorphism in this region. In addition, our sequence data extend the known 5-upstream sequence by an additional 1.5 kb allowing the identification of numerous, potential cis-acting regulatory sites. This 5-flanking cloned probe permitted us to localize the protothymosin gene to chromosome 2 in humans.