Transgenic Zebrafish Expressing Chicken Lysozyme Show Resistance against Bacterial Diseases

We established a transgenic zebrafish strain expressing chicken lysozyme gene under the control of the Japanese flounder keratin gene promoter, and investigated its resistance to a pathogenic bacterial infection. To generate the lysozyme transgenic construct, Japanese flounder keratin promoter was linked to both the hen egg white (HEW) lyoszyme gene and green fluorescence protein (GFP) gene used as a selection marker for the transgenic strains, in a recombinant plasmid. The recombinant plasmid was microinjected into fertilized zebrafish eggs. In F2 transgenic zebrafish, GFP expression was strong in the epithelial tissues, liver and gill from the embryonic stage to the adult stage. The expressions of HEW lysozyme and GFP mRNA were confirmed in the liver and skin by RT-PCR. Western blot analysis showed that both HEW lysozyme and GFP were present in protein extracts from the liver of transgenic zebrafish, but not in protein extracts from the muscle. The lytic activity of protein extracts from the liver (assessed by a lysoplate assay using Micrococcus lysodeikticus as a substrate) was 1.75 times higher in F2 transgenic zebrafish than in the wild type. In a challenge experiment, 65% of the F2 transgenic fish survived an infection of Flavobacterium columnare and 60% survived an infection of Edwardsiella tarda, whereas 100% of the control fish were killed by both pathogens. However, the survival rates of the transgenic fish were not significantly higher when higher concentrations of bacteria were used.     

Two types of liver-specific F antigen are encoded by a locus located on chromosome 5 in mice

Two types of liver-specific F antigen in mice were distinguished by an immunoblotting technique after IEF of liver extracts. The IEF banding patterns consist of several bands whose pI vary from 7.57 to 8.15. One type of antigen (designated F2 antigen) showed a pattern that lacked some basic bands which are present in the pattern of the other type of antigen (designated F1 antigen). The latter type was found in AKR, CBA, C3H, DBA/2, and SM strains, while the former type was found in A, C57BL, and many other strains. Breeding experiments indicated that this variation is controlled by a single autosomal locus designated Laf (liver antigen F). Linkage analysis showed that the Laf locus is linked to the Pgm-1 locus on chromosome 5. The recombination frequency between these two loci is estimated to be 0.173 ± 0.037. The distribution of F1 and F2 antigen types among inbred strains is concordant with that of type 1 and type 2 F antigens, which have been previously distinguished by their immunogenic differences, i. e., whether alloimmunization with the liver extracts from a given strain of mice can produce the antibody to F antigen in certain strains of mice. It is suggested, therefore, that the Laf locus may encode an allogeneic moiety of F antigen molecules.Abbreviations used in this paper IEF isoelectric focusing - RI recombinant inbred - ICLAS International Council for Laboratory Animal Science     

Intimate genetic relationships and fungicide resistance in multiple strains of Aspergillus fumigatus isolated from a plant bulb

Fungal infections are increasingly dangerous because of environmentally dispersed resistance to antifungal drugs. Azoles are commonly used antifungal drugs, but they are also used as fungicides in agriculture, which may enable enrichment of azole-resistant strains of the human pathogen Aspergillus fumigatus in the environment. Understanding of environmental dissemination and enrichment of genetic variation associated with azole resistance in A. fumigatus is required to suppress resistant strains. Here, we focused on eight strains of azole-resistant A. fumigatus isolated from a single tulip bulb for sale in Japan. This set includes strains with TR₃₄/L98H/T289A/I364V/G448S and TR₄₆/Y121F/T289A/S363P/I364V/G448S mutations in the cyp51A gene, which showed higher tolerance to several azoles than strains harbouring TR₄₆/Y121F/T289A mutation. The strains were typed by microsatellite typing, single nucleotide polymorphism profiles, and mitochondrial and nuclear genome analyses. The strains grouped differently using each typing method, suggesting historical genetic recombination among the strains. Our data also revealed that some strains isolated from the tulip bulb showed tolerance to other classes of fungicides, such as QoI and carbendazim, followed by related amino acid alterations in the target proteins. Considering spatial–temporal factors, plant bulbs are an excellent environmental niche for fungal strains to encounter partners, and to obtain and spread resistance-associated mutations.     

Pseudomonas aeruginosa outer membrane protein F produced inEscherichia coli retains vaccine efficacy

Pseudomonas aeruginosa outer membrane protein F was purified by extraction from polyacrylamide gels of cell envelope proteins of anEscherichia coli strain expressing the cloned gene for protein F. Antisera directed against protein F purified fromP. aeruginosa PAO1 reacted with thisE. coli strain by immunofluorescence assay and immunoblotting, whereas these antisera were nonreactive withE. coli strains lacking thePseudomonas protein F gene. The protein F purified from thisE. coli strain was used to immunize mice by intramuscular injection of 10 µg of protein F preparation on days 1 and 14, followed by burn and challenge of the mice on day 28. As compared with control mice immunized withE. coli K-12 lipopolysaccharide, immunization with theE. coli-derived protein F afforded significant protection against subsequent challenge with heterologous Fisher-Devlin immunotype 5 and 6 strains ofP. aeruginosa. Antisera from mice immunized with theE. coli-derived protein F reacted at bands corresponding to protein F and 2-mercaptoethanol-modified protein F upon immunoblotting against cell envelope proteins of the PAO1, immunotype 5, and immunotype 6 strains ofP. aeruginosa and theE. coli strain containing the cloned F gene, but failed to react at these sites in anE. coli strain lacking the F gene. These data demonstrate thatP. aeruginosa protein F produced inE. coli through genetic engineering techniques retains its vaccine efficacy in the complete absence of anyP. aeruginosa lipopolysaccharide.     

Molecular typing and occurrence of beta‐lactam resistance genes of Shigella sonnei strains isolated from 1983 to 2014 in the São Paulo state of Brazil

Shigella sonnei , which has generally been associated with dysentery in developed countries, has recently been emerging in developing countries. Specifically, in Brazil few published studies have that molecularly characterized this species. The aims of this study were to analyze the efficacy of typing using multiple‐locus variable‐number tandem‐repeat analysis (MLVA), study the phylogeny by multi‐locus sequence typing (MLST) and assess the presence of some beta‐lactam resistance genes in S. sonnei strains isolated from human diarrhoeic faeces in the São Paulo State in Brazil between 1983 and 2014. Seventy‐two such S. sonnei strains were typed by MLVA and grouped into two clusters. The discrimination index of MLVA was found to be 0.996. Twenty strains were typed by MLST as ST152. In addition, the bla _TEM gene was detected in eight (72.7%) of the 11 S. sonnei strains that had previously been shown to be resistant to β‐lactams. However, bla _{CTX‐M‐1group}, bla _{CTX‐M‐9group} and bla _SHV genes were not found. MLVA results suggested the existence of two prevalent subtypes in the S. sonnei strains studied, confirming previous results. Moreover, MLVA efficiently discriminated monomorphic S. sonnei species. Because the S. sonnei strains studied belonged to clonal complex 152 and all isolates were typed as ST152, MLST is not a suitable method for studying the population structure of S. sonnei . Although, the rates of β‐lactam resistance were not high in the present study, the frequency of bla _TEM may represent a risk for patients receiving antimicrobial treatment. Taken together, the results provide better molecular characterization of this globally clinically important pathogen.

     

Integrative suppression of dnaA(Ts) mutations mediated by plasmid F in Escherichia coli is a DnaA-dependent process

Summary The thermosensitivity of dnaA(Ts) mutations can be suppressed by integration of plasmid F (integrative suppression). In the light of the recent finding that F requires DnaA protein for both establishment and maintenance, integrative suppression of 11 dnaA(Ts) mutations by a mini-F, pML31, integrated near oriC was examined. The plating efficiency of integratively suppressed strains was dnaA(Ts) allele-dependent and medium-dependent. The initiation capability of suppressed dnaA(Ts) strains lacking the oriC site and their F^- counterparts was determined at various temperatures between 30°C and 42°C. The degree of integrative suppression measured by the initiation capability varied in a dnaA(Ts) allele-dependent manner. F-directed DNA replication was most affected by the dnaA(Ts) mutations mapping in the middle of the gene whereas oriC-dependent replication was most thermosensitive in strains carrying mutations mapping in the carboxy-terminal half of the gene. The results indicated that the integrative suppression by F plasmid is a DnaA-dependent process and suggested that the requirements for DnaA protein in the oriC-dependent replication and F replication processes are qualitatively different.     

DNA polymorphism of the C2 and factor B genes

Factor B and the second component of complement (C2) in man are encoded within the major histocompatibility complex by single loci that are less than 1 kb apart. A 2.3 kb factor B-specific cDNA probe has been used to examine, by Southern blot analysis, the genomic DNA of individuals typed for C2 and factor B by protein electrophoresis. We have identified a restriction fragment length polymorphism using the endonuclease Taq I, which subdivides haplotypes carrying both the common variant of C2 (C2C) and the fast (F) variant of factor B. This DNA polymorphism has been mapped to lie in the C2 gene and represents a new genetic marker not defined by protein electrophoresis. This polymorphism may serve as a useful marker in the genetic analysis of diseases that are related to the major histocompatibility complex.     

Evaluation of M307 of <Emphasis Type="Italic">FUT1</Emphasis> gene as a genetic marker for disease resistance breeding of sutai pigs

Alpha (1,2) fucosyltransferase (FUT1) gene has been identified as a candidate gene for controlling the expression of the receptor for ETEC F18. The genetic variations in the position of M307 nucleotide in open reading frame of FUT1 have been proposed as a marker for selecting ETEC F18 resistant pigs. The polymorphisms of M307 in FUT1 of breeding base group for ETEC F18 resistance of Sutai pigs (Duroc × Meishan) was detected and their correlations to some immune indexes, growth and development ability, carcass traits and meat quality were also analyzed, which aimed to investigate feasibility of further breeding for diseases resistance based on M307 of FUT1 for Sutai pigs. After digested by Hin6 I, M307 of FUT1 gene could be divided into three kinds of genotypes, AA, AG, and GG. The frequencies were 0.235, 0.609, and 0.156, respectively. The results indicated that Sutai pigs with the AA genotype in M307 of FUT1 gene not only have relatively strong general disease resistance ability in piglets, but also have higher growth and development ability and stable carcass traits and meat quality. It is entirely feasible to raise the new strains of Sutai pigs resistant to Escherichia coli F18 based on genetic marker of the M307 position in FUT1gene.     

Characterisation of the nitrile hydratase gene clusters of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> strains AJ270 and AJ300 and <Emphasis Type="Italic">Microbacterium</Emphasis> sp. AJ115 indicates horizontal gene transfer and reveals an insertion of IS1166

The nitrile metabolising strains AJ270, AJ300 and AJ115 were isolated from the same location. The strains have very similar nitrile metabolising profiles. Sequencing of the 16S rRNA gene indicates that strains AJ270 and AJ300 are novel strains of Rhodococcus erythropolis while strain AJ115 is a novel Microbacterium strain very closely related to Microbacterium oxydans and Microbacterium liquefaciens. Analysis of the structure of the nitrile hydratase/amidase gene clusters in the three strains indicates that this region is identical in these strains and that this structure is different to other nitrile hydratase/amidase gene clusters. The major difference seen is the insertion of a complete copy of the insertion sequence IS1166 in the nhr2 gene. This copy of IS1166 generates a 10 bp direct duplication at the point of insertion and has one ORF encoding a protein of 434 amino acids, with 98% homology to the transposase of IS666 from Mycobacterium avium. A gene oxd, encoding aldoxime dehydratase is found upstream of the nitrile hydratase gene cluster and an open reading frame encoding a protein with homology to GlnQ type ABC transporters is found downstream of the nitrile hydratase/amidase genes. The identity of the nitrile hydratase/amidase gene clusters in the three strains suggests horizontal gene transfer of this region. Analysis of the strains for both linear and circular plasmids indicates that both are present in the strains but hybridisation studies indicate that the nitrile hydratase/amidase gene cluster is chromosomally located. The nitrile hydratase/amidase enzymes of strain AJ270 are inducible with acetonitrile or acetamide. Interestingly although a number of Fe-type nitrile hydratases have been shown to be photosensitive, the enzyme from strain AJ270 is not.     

Organization of the lux genes of photobacterium phosphoreum

The lux genes from Photobacterium phosphoreum (NCMB844) have been cloned into Escherichia coli in a plasmid containing the T7-bacteriophage promoter. By specific expression in vivo under the T7 promoter, five structural genes (luxA-E) coding for the fatty acid reductase and luciferase polypeptides were identified as well as a new gene, designated as luxF, which codes for a 26kDa polypeptide. This new gene is located between luxB and luxE and thus disrupts the structural gene order of luxCDABE found in the Vibrio genus. The luxF gene and the protein it codes for have recently been identified in other Photobacterium species and so appears to be widely distributed within this genus. Nucleotide sequencing of the luxF gene has shown it to code for a protein homologous to the luciferase subunits, coded by the luxA and luxB genes. Although this gene is not necessary for light emission in all luminescent bacteria, it must play an essential role in the biochemistry, physiology, or ecology of the luminescent system in species of the Photobacterium genus.     

The gene for ribosomal protein L25 (rplY) maps at 47.3 min near nalA in Escherichia coli K-12.

Summary A temperature sensitive mutant, termed JE1306, derived from Escherichia coli strain PA3092 was found to have an alteration in the ribosomal protein L25. Crosses with various Hfr strains and transductions with P1kc phage have revealed that the mutation maps at 47.3 min between nalA and fpk, in a region where no ribosomal protein gene has so far been located. The gene affected by this mutation is most probably the structural gene for protein L25 (rplY), because a strain heteromerozygous for the region shows both wild type and mutant forms of protein L25.     

A serum protein polymorphism determinant on chromosome 9 of Mus musculus

Summary Genetic polymorphism for a previously undescribed serum protein has been found among inbred strains of Mus musculus. The new serum protein locus, gene symbol Sep-1, has been located on Chromosome 9, gene order Lap-1-Sep-1-Mpi-1-d-Mod-1, by utilizing information obtained from 52 recombinant inbred strains together with standard genetic backcrosses. The strain distribution pattern for this locus, supernatant malic enzyme, and transferrin, also on Chromosome 9, are given for 67 inbred strains. Because the genotype of SEP-1 can be determined for individual mice without killing them, Sep-1 is a very useful gene in linkage studies and experimental biology.     

Fluorescent reference strains of bacteria by chromosomal integration of a modified green fluorescent protein gene

Fluorescent reference strains of bacteria carrying a stable chromosomally integrated single copy of the gfp gene have been developed. A modified version of the gfp gene has been generated by mutagenesis and expressed under the control of the bacteriophage lambda promoter P_L. A cassette comprising bacteriophage Mu transposon arms flanking the modified gfp gene and regulatory regions was irreversibly integrated as an in-vitro-assembled transposition complex into the genomes of Escherichia coli and Salmonella spp. The modified green fluorescent protein (GFP) protein retained the fluorescence excitation and emission wavelengths of wild-type GFP. However, it fluoresced more brightly in E. coli and Salmonella compared to wild-type GFP, presumably due to improved protein maturation. Fluorescent E. coli and Salmonella strains carrying the gfp gene cassette were easily differentiated from their respective non-fluorescent parental strains on various growth media by visualization under UV light. The bacterial strains produced by this method remained viable and stably fluorescent when incorporated into a matrix for delivery of exact numbers of viable bacterial cells for use as quality control agents in microbiological procedures.     

Regulation of expression of mouseC4 andSlp genes by non-H-2-linked genes

C4 (the fourth complement component) and Slp (sexlimited protein) are two homologous plasma proteins encoded by genes in theS-region of theH-2 gene complex. We studied the genetic factors influencing the plasma levels of these proteins and their mRNA levels in liver. Considerable differences in both protein and mRNA levels were found between mouse strains carrying the sameS-region allele on different genetic backgrounds, indicating a pretranslational effect of non-H-2-linked genes on the expression of the twoS-region genes. The expression of Slp is androgen-dependent in the strains tested. However, testosterone treatment cannot increase the low levels of Slp caused by non-H-2-linked regulatory genes. In mice with Slp-negativeS-region alleles we found liver mRNA hybridizing with Slp-specific oligonucleotides, indicating expression of theSlp gene in Slp-negative strains. Our data demonstrate the complexity of the regulation of theC4 andSlp genes and pave the way for the analysis of the regulatory factors involved.     

Hepatic Canalicular Membrane Transport of Bile Salt in C57L/J and AKR/J Mice: Implications for Cholesterol Gallstone Formation

C57L/J (gallstone-susceptible) and AKR/J (gallstone-resistant) mice have been utilized for quantitative trait loci (QTL) analysis to identify the Lith 1 locus for cholelithiasis. Abcb11 encodes for the liver canalicular membrane bile salt export pump (BSEP), which maps to this QTL and is a candidate gene for Lith 1. We investigated the transmembrane transport of taurocholate in canalicular liver membrane vesicles isolated from these murine strains. Canalicular liver plasma membranes (cLPM) and RNA were isolated from C57L/J and AKR/J mice livers, and were utilized for Northern and Western blot analysis and functional ³H-taurocholate uptake studies. ATP-dependent ³H-taurocholate uptake was significantly higher in AKR/J, compared to C57L/J mice. V _max was 127 vs. 42 pmol TC/mg/s in the murine strains, respectively, while K _m was unchanged. In contrast, gene and protein expression of hepatic Abcb11 was increased three-fold in C57L/J, compared to AKR/J mice. Thus, Abcb11 bile salt transport activity per unit protein was reduced nine-fold in the C57L/J, compared to AKR/J mice. In contrast, canalicular membrane cholesterol:phospholipid content was also significantly higher in the C57L/J mice. We conclude that gallstone-susceptible C57L/J mice demonstrate increased gene and canalicular membrane expression of Abcb11, however, taurocholate transport is functionally diminished. The latter may be due to the increased cholesterol membrane content of the cLPM in C57L/J mice. These findings may be important for the pathogenesis of gallstone formation.     

On the variability of the types ofC. diphtheriae

Conclusion From this investigation it is evident that most strains ofC. diphtheriae can be easily typed; with the typing of some strains difficulties have been encountered. By close examination of the properties some of these can be typed after some delay, superisolation being indispensable.The use of insoluble starch is essential in the fermentation test. All bacteria which differ in one or more respects from the typical ones must be labelled as atypical. This also concerns gravis strains which do not ferment starch and glycogen. If the bacteria are thus typed erroneous typing can be minimized, though faults will be inevitable.The typical as well as the atypical bacteria have great stability. For the moment we wish to consider the types as fixates rather than as subspecies. Notwithstanding the observation of changesin vitro in some cases, the types appear to be sufficiently stable for epidemiological investigation.     

DNA sequence analysis of point mutations in traA, the F pilin gene, reveal two domains involved in F-specific bacteriophage attachment

Summary Six missense point mutations in traA (WPFL43,44,45,46,47 and 51), the gene encoding F pilin in the transfer region of the F plasmid, have been characterized for their effect on the transfer ability, bacteriophage (R17, QB and fl) sensitivity and levels of piliation expressed by the plasmid. The sequence analysis of the first five of these mutations revealed two domains in the F pilin subunit exposed on the surface of the F pilus which mediate phage attachment. These two domains include residues 14–17 (approximately) and the last few residues at the carboxy-terminus of the pilin protein. One of these mutants had a pleiotropic affect on pilus function and was thought to have affected pilus assembly. The sixthe point mutant (WPFL51), previously thought to be in traA, was complemented by chimeric plasmids carrying the traG gene of the F transfer region, which may be involved in the acetylation of the pilin subunit. A traA nonsense mutant (JCFL1) carried an amber mutation near the amino-terminus which is well suppressed in SuI⁺ (supD) and SuIII⁺ (supF) strains. Neither the antigenicity of the pilin nor the efficiency of plating of F-specific bacteriophages were affected when this plasmid was harbored by either suppressor strain. A second amber mutant (JCFL25) which is not suppressible, carried its mutation in the codon for the single tryptophan in F pilin, suggesting that this residue is important in subunit interactions during pilus assembly. Two other point mutants (JCFL32 and 44) carried missense mutations in the leader sequence (positions 9 and 13) which affected the number of pili per cell presumably by altering the processing of propilin to pilin.