Herpes simplex virus type 1 oriL is not required for virus replication or for the establishment and reactivation of latent infection in mice.

During the course of experiments designed to isolate deletion mutants of herpes simplex virus type 1 in the gene encoding the major DNA-binding protein, ICP8, a mutant, d61, that grew efficiently in ICP8-expressing Vero cells but not in normal Vero cells was isolated (P. K. Orberg and P. A. Schaffer, J. Virol. 61:1136-1146, 1987). d61 was derived by cotransfection of ICP8-expressing Vero cells with infectious wild-type viral DNA and a plasmid, pDX, that contains an engineered 780-base-pair (bp) deletion in the ICP8 gene, as well as a spontaneous approximately 55-bp deletion in oriL. Gel electrophoresis and Southern blot analysis indicated that d61 DNA carried both deletions present in pDX. The ability of d61 to replicate despite the deletion in oriL suggested that a functional oriL is not essential for virus replication in vitro. Because d61 harbored two mutations, a second mutant, ts+7, with a deletion in oriL-associated sequences and an intact ICP8 gene was constructed. Both d61 and ts+7 replicated efficiently in their respective permissive host cells, although their yields were slightly lower than those of control viruses with intact oriL sequences. An in vitro test of origin function of isolated oriL sequences from wild-type virus and ts+7 showed that wild-type oriL, but not ts+7 oriL, was functional upon infection with helper virus. In an effort to determine the requirement for oriL in latency, ts+7 was compared with wild-type virus for its ability to establish, maintain, and be reactivated from latent infection in a murine eye model. The mutant was reactivated as efficiently as was wild-type virus from trigeminal ganglia after cocultivation with permissive cells, and each of the seven reactivated isolates was shown to carry the approximately 150-bp deletion characteristic of ts+7. These observations demonstrate that oriL is not required for virus replication in vitro or for the establishment and reactivation of latent infection in vivo.     

Analysis of genome plasticity in pathogenic and commensal Escherichia coli isolates by use of DNA arrays

Genomes of prokaryotes differ significantly in size and DNA composition. Escherichia coli is considered a model organism to analyze the processes involved in bacterial genome evolution, as the species comprises numerous pathogenic and commensal variants. Pathogenic and nonpathogenic E. coli strains differ in the presence and absence of additional DNA elements contributing to specific virulence traits and also in the presence and absence of additional genetic information. To analyze the genetic diversity of pathogenic and commensal E. coli isolates, a whole-genome approach was applied. Using DNA arrays, the presence of all translatable open reading frames (ORFs) of nonpathogenic E. coli K-12 strain MG1655 was investigated in 26 E. coli isolates, including various extraintestinal and intestinal pathogenic E. coli isolates, 3 pathogenicity island deletion mutants, and commensal and laboratory strains. Additionally, the presence of virulence-associated genes of E. coli was determined using a DNA "pathoarray" developed in our laboratory. The frequency and distributional pattern of genomic variations vary widely in different E. coli strains. Up to 10% of the E. coli K-12-specific ORFs were not detectable in the genomes of the different strains. DNA sequences described for extraintestinal or intestinal pathogenic E. coli are more frequently detectable in isolates of the same origin than in other pathotypes. Several genes coding for virulence or fitness factors are also present in commensal E. coli isolates. Based on these results, the conserved E. coli core genome is estimated to consist of at least 3,100 translatable ORFs. The absence of K-12-specific ORFs was detectable in all chromosomal regions. These data demonstrate the great genome heterogeneity and genetic diversity among E. coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes.     

Plasmodium falciparum: CD36 Dependent Cytoadherence or Rosetting of Infected Erythrocytes is Modulated by Knobs

A knobless (K-) line of the FCR-3 isolate of Plasmodium falciparum was obtained by gelatin flotation. Immunofluorescent staining and immunoblots indicated that both the K-line and the K+ (knobby) line from which it was derived contained similar forms of potentially adhesive modified band 3 protein. When the K+ and K-lines were assayed for their cytoadherent and rosetting abilities the K+ line showed a high level of CD36 dependent cytoadherence, whereas the K-line demonstrated a marked pH dependent increase in rosetting. Rosetting was inhibited by the addition of peptides based on band 3 motifs, suggesting that cytoadherence and rosetting involve the same adhesin but that the presence of knobs affects whether the adherent preference of the infected erythrocyte is uninfected red cells or endothelial/C32 amelanotic melanoma cells.     

Plasmodium falciparum: CD36 Dependent Cytoadherence or Rosetting of Infected Erythrocytes is Modulated by Knobs

A knobless (K-) line of the FCR-3 isolate of Plasmodium falciparum was obtained by gelatin flotation. Immunofluorescent staining and immunoblots indicated that both the K-line and the K+ (knobby) line from which it was derived contained similar forms of potentially adhesive modified band 3 protein. When the K+ and K-lines were assayed for their cytoadherent and rosetting abilities the K+ line showed a high level of CD36 dependent cytoadherence, whereas the K-line demonstrated a marked pH dependent increase in rosetting. Rosetting was inhibited by the addition of peptides based on band 3 motifs, suggesting that cytoadherence and rosetting involve the same adhesin but that the presence of knobs affects whether the adherent preference of the infected erythrocyte is uninfected red cells or endothelial/C32 amelanotic melanoma cells.     

Identification of the gene lon (capR) product as a 94-kilodalton polypeptide by cloning and deletion analysis.

A mutation in the lon (capR) gene of Escherichia coli K-12 effects several phenotypic alterations in the mutant cell, such as overproduction of capsular polysaccharide and sensitivity to ultraviolet or ionizing radiation. A previously cloned 9.2-megadalton (Md) EcoRI fragment contained the capR+ gene and specified two polypeptides, 94 kilodaltons (K) and 67K. To provide evidence that the 94K polypeptide is the capR+ gene product, we constructed a capR+ plasmid pJMC40, having a 2.0-Md EcoRI-PstI fragment which codes only for the 94K polypeptide. Plasmids pJMC22 and pJMC30, having deletions of 0.7 and 0.8 Md, respectively, from one end of the 2.0-Md fragment, were also constructed. Each codes for a shortened stable polypeptide (from the 94K). Neither plasmid can confer the capR+ phenotype to capR mutants, confirming that the unaltered 94K polypeptide is the capR+ gene product. Plasmids pJMC51 and pJMC52 each have a deletion of 0.7 Md from the other end of the 2.0-Md fragment, differing only in the orientation of the remaining 1.3-Md fragment with respect to the cloning vehicle. They are nonfunctional with respect to capR+ and do not code for a common polypeptide from the 1.3-Md fragment. These data indicate that the fragments in pJMC22 and pJMC30, which both code for shortened 94K polypeptides, contain the promoter-operator region of the capR gene. The deletion plasmids were also used to map chromosomal capR mutations.     

GrB deletion of the chorion locus of the silkmoth Bombyx mori. Localization of the left breakpoint and isolation of the deletion junction

In the silkmoth Bombyx mori, choriogenesis occurs through the developmentally controlled deposition of several related classes of chorion proteins onto the oocyte by surrounding follicular cells. In the GrB mutant strain, a distinctive family of proteins (Hc) normally expressed late in choriogenesis, as well as several proteins of middle development specificity, are missing due to the deletion of the corresponding genes from the chorion locus. In addition, a smaller set of proteins normally confined to mid-choriogenesis is found to be prolonged in expression in homozygote mutant but not heterozygote individuals. To elucidate the molecular organization of the chorion locus in the GrB genotype, we scanned a part of the wild-type locus represented by a chromosomal walk of 270,000 bases through library screening and genomic DNA hybridizations using a series of unique probes. A chromosomal clone, GrB4, whose sequences showed the expected characteristics of the deletion junction, was isolated from a partial EcoRI library of mutant genomic DNA. Through comparative hybridizations, mapping and sequencing, the precise location of one of the deletion breakpoints was identified on one of the clones mapping in the characterized part of the wild-type locus. Attempts to locate the other breakpoint in wild-type DNA and to extend the structural characterization past the deletion junction through chromosomal walking were unsuccessful, due to the apparent absence of these sequences from libraries of wild-type and mutant genomic DNA, respectively. Hybridizations of the deletion region on clone GrB4 to cDNA derived from follicular RNA indicate that no gene sequences are directly interrupted by the deletion, and reveal the presence of a gene sequence of unknown function 1000 to 5000 bases to the right of deletion junction.     

Common evolutionary origin of chromosomal beta-lactamase genes in enterobacteria

A 32P-labeled fragment of DNA, encoding the major part of the chromosomal ampC beta-lactamase gene of Escherichia coli K-12, was used as a hybridization probe for homologous DNA sequences in colonies of Neisseria gonorrhoeae, Pseudomonas aeruginosa, and different enterobacterial species. The ampC probe detected the presence of homologous DNA sequences in clinical isolates of E. coli, Shigella flexneri, Shigella sonnei, Klebsiella pneumoniae, Salmonella typhimurium, Serratia marcescens, and P. aeruginosa. No hybridization was found with N. gonorrhoeae colonies. In Southern blotting experiments the ampC probe hybridized to chromosomal DNA fragments of the same size in all enterobacterial species tested. However, the degree of hybridization differed with DNA from different species. DNA from the Shigella species strongly hybridized to the ampC probe. Furthermore, antibodies raised against purified E. coli K-12 ampC beta-lactamase precipitated beta-lactamases from the Shigella species, suggesting extensive sequence similarities between the ampC genes of these genera. The production of chromosomal beta-lactamase in S. sonnei increased with increasing growth rate similar to E. coli K-12. This growth rate response was abolished in two beta-lactamase-hyperproducing S. sonnei mutants, which thus seem similar to E. coli K-12 attenuator mutants. We propose that both the structure and regulation of the chromosomal beta-lactamase genes are very similar in E. coli and in S. sonnei.     

Characterization of the genetic element coding for lactose metabolism in Lactococcus lactis subsp. lactis KP3

The Lactococcus lactis subsp. lactis KP3 Lac genetic element was investigated. KP3 is a lactose-positive (Lac+) transconjugant which contains no detectable plasmid DNA. The KP3 Lac genetic element was self-transmissible (Tra+) and encoded a reduced bacteriophage sensitivity (Rbs+) phenotype. Matings of KP3 with a recombination-deficient (Rec-) recipient resulted in Lac+ transconjugants which were phenotypically indistinguishable from KP3 and contained a 96-MDa plasmid (pJS96). Phenotypic and physical analyses of pJS96 indicated that it was a deletion derivative of a putative pKB32::pJS88 Lac+ Tra+ cointegrate. pKB32 is the Lac plasmid and pJS88 is the Tra+ Rbs+ plasmid in L. lactis subsp. lactis 11007, the donor used in obtaining KP3. The results presented suggest that pJS96 is an episome, since it appeared to replicate both as a plasmid and as an integrated part of the chromosome. Conjugal transfer of chromosomal DNA mediated by pJS96 was not observed. Conjugal transfer of pJS96 resulted in Lac+ transconjugants containing plasmids ranging in size from 21 to 90 MDa. Only in Rec+ recipients were transconjugants isolated which appeared to contain pJS96 integrated into the host chromosome. Restriction analysis of several plasmids in the 21 to 90 MDa range suggested the deletions were due to intramolecular transposition of a transposable element on pJS96. This report suggests that a self-transmissible episome exists in KP3 and provides an explanation of how plasmids which vary in size yet encode similar phenotypes may be formed and disseminated.     

Increased resistance to ionizing and ultraviolet radiation in Escherichia coli JM83 is associated with a chromosomal rearrangement.

Cells cope with radiation damage through several mechanisms: (1) increased DNA repair activity, (2) scavenging and inactivation of radiation-induced radical molecules, and (3) entry into a G0-like quiescent state. We have investigated a chromosomal rearrangement to elucidate further the molecular and genetic mechanisms underlying these phenomena. A mutant of Escherichia coli JM83 (phi 80dlacZ delta M15) was isolated that demonstrated significantly increased resistance to both ionizing and ultraviolet radiation. Surviving fractions of mutant and wild-type cells were measured following exposure to standardized doses of radiation. Increased radioresistance was directly related to a chromosomal alteration near the bacteriophage phi 80 attachment site (attB), as initially detected by the LacZ- phenotype of the isolate. Southern hybridization of chromosomal DNA from the mutant and wild-type E. coli JM83 strains indicated that a deletion had occurred. We propose that the deletion near the attB locus produces the radioresistant phenotype of the E. coli JM83 LacZ- mutant, perhaps through the alteration or inactivation of a gene or its controlling element(s).     

水稻条纹病毒中国分离物和日本分离物RNA2节段序列比较

测定了来源于我国水稻条纹叶枯病常年流行区的云南楚雄(CX)及病害暴发区的江苏洪泽(HZ)的水稻条纹病毒(RSV)2个分离物RNA2全长序列,其长度分别为3506bp和3514 bp.与已报道的日本T和O分离物RNA2序列进行比较的结果表明,这4个分离物可分为两组,其中,HZ、T和O分离物为一组,组内分离物之间,RNA2的毒义链(vRNA2)及RNA2的毒义互补链(vcRNA2)上的ORF的核苷酸一致性分别为97.2%～98.0%和96.8%～97.1%,5′端和3′端非编码区的序列则完全一致.但HZ分离物与T分离物的亲缘关系更为密切,其基因间隔区(IR)与T和O分离物的等长.另一组为我国CX分离物,组与组之间,vRNA2及vcRNA2上的ORF的核苷酸一致性分别为95.0%～95.7%和93.9%～94.4%.CX分离物的IR与HZ分离物相比缺失了一段8 nt的片段.5′端非编码区的序列完全一致,但3′末端非编码区有一个碱基的差异.这些结果表明,RSV在自然界的分子变异与其地理分布具有密切的关系.此外,非编码区序列的高度保守性暗示着它们在病毒基因转录和复制的调控方面具有重要的功能.本文还讨论了RSV的分子流行病学.     

Direction of chromosome rearrangements in Saccharomyces cerevisiae by use of his3 recombinational substrates.

We used the his3 recombinational substrates (his3 fragments) to direct large interchromosomal (translocations) and intrachromosomal (deletions and tandem duplications) rearrangements in the yeast Saccharomyces cerevisiae. In strains completely deleted for the wild-type HIS3 gene, his3 fragments, one containing a deletion of 5' amino acid coding sequences and the other containing a deletion of 3' amino acid coding sequences, were first placed at preselected sites by homologous recombination. His+ revertants that arose via spontaneous mitotic recombination between the two his3 fragments were selected. This strategy was used to direct rearrangements in both RAD52+ and rad52 mutant strains. Translocations occurred in the RAD52+ genetic background and were characterized by orthogonal field alternating gel electrophoresis of yeast chromosomal DNA and by standard genetic techniques. An unexpected translocation was also identified in which HIS3 sequences were amplified. Two types of tandem duplications of the GAL(7, 10, 1) locus were also directed, and one type was not observed in rad52 mutants. Recombination mechanisms are discussed to account for these differences.     

Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.

We have characterized Neurospora crassa transformants obtained with plasmid pJR2, which consists of the Neurospora glutamate dehydrogenase (am) gene cloned in pUC8 and an am132 host strain which contains a deletion encompassing the cloned fragment. Every one of 33 transformants tested showed extreme meiotic instability: less than 1 or 2% am+ progeny were obtained in initial or successive backcrosses between am+ transformants and am132 or in intercrosses between am+ progeny. Furthermore, am+ progeny from backcrosses gave a high proportion of auxotrophic (am) mitotic segregants during vegetative growth. These results indicate that the am+ character is not stably integrated into chromosomal DNA in any of the transformants tested. Nuclear DNAs from six transformants were analyzed by Southern hybridization. All six transformants contained sequences homologous to pJR2. Four showed restriction fragments expected for unmodified pJR2, but most showed additional bands. Southern blots of undigested DNAs showed that the plasmid sequences are present predominantly in high-molecular-weight form (larger than 20 kilobases). Southern blots showed that auxotrophic (am) progeny from a backcross to am132 had lost restriction bands corresponding to free plasmid but retained additional bands, apparently integrated into chromosomal DNA in a nonfunctional manner. Considered together, these results are most reasonably interpreted as follows: recombinant plasmids containing the am+ gene can replicate autonomously in N. crassa, the free plasmids are present in oligomeric or modified form or both, and plasmid sequences also integrate at multiple sites in the deletion host but in a nonfunctional manner. An alternate interpretation--that tandem repeats of the plasmid are integrated into chromosomal DNA but eliminated during meiosis--cannot be completely excluded. However, stable integration of the am gene can be obtained under a variety of other conditions, viz., using the am gene cloned in a phage lambda vector (J. A. Kinsey and J. A. Rambosek, Mol. Cell. Biol. 4:117-122, 1984), using derivatives of pJR2, or using pJR2 to transform a frameshift mutant.     

The occurrence of duplicate lysyl-tRNA synthetase gene homologs in Escherichia coli and other procaryotes.

The lysyl-tRNA synthetase (LysRS) system of Escherichia coli K-12 consists of two genes, lysS, which is constitutive, and lysU, which is inducible. It is of importance to know how extensively the two-gene LysRS system is distributed in procaryotes, in particular, among members of the family Enterobacteriaceae. To this end, the enterics E. coli K-12 and B; E. coli reference collection (ECOR) isolates EC2, EC49, EC65, and EC68; Shigella flexneri; Salmonella typhimurium; Klebsiella pneumoniae; Enterobacter aerogenes; Serratia marcescens; and Proteus vulgaris and the nonenterics Pseudomonas aeruginosa and Bacillus megaterium were grown in AC broth to a pH of 5.5 or less or cultured in SABO medium at pH 5.0. These growth conditions are known to induce LysRS activity (LysU synthesis) in E. coli K-12. Significant induction of LysRS activity (twofold or better) was observed in the E. coli strains, the ECOR isolates, S. flexneri, K. pneumoniae, and E. aerogenes. To demonstrate an association between LysRS induction and two distinct LysRS genes, Southern blotting was performed with a probe representing an 871-bp fragment amplified from an internal portion of the coding region of the lysU gene. In initial experiments, chromosomal DNA from E. coli K-12 strain MC4100 (lysS+ lysU+) was double digested with either BamHI and HindIII or BamHI and SalI, producing hybridizable fragments of 12.4 and 4.2 kb and 6.6 and 5.2 kb, respectively. Subjecting the chromosomal DNA of E. coli K-12 strain GNB10181 (lysS+ delta lysU) to the same regimen established that the larger fragment from each digestion contained the lysU gene. The results of Southern blot analysis of the other bacterial strains revealed that two hybridizable fragments were obtained from all of the E. coli and ECOR collection strains examined and S. flexneri, K. pneumoniae, and E. aerogenes. Only one lysU homolog was found with S. typhimurium and S. marcescens, and none was obtained with P. vulgaris. A single hybridizable band was found with both P. aeruginose and B, megaterium. These results show that the dual-gene LysRS system is not confined to E. coli K-12 and indicate that it may have first appeared in the genus Enterobacter.     

Reversible phase variation in the phnE gene, which is required for phosphonate metabolism in Escherichia coli K-12

It is known that Escherichia coli K-12 is cryptic (Phn-) for utilization of methyl phosphonate (MePn) and that Phn+ variants can be selected for growth on MePn as the sole P source. Variants arise from deletion via a possible slip strand mechanism of one of three direct 8-bp repeat sequences in phnE, which restores function to a component of a putative ABC type transporter. Here we show that Phn+ variants are present at the surprisingly high frequency of >10(-2) in K-12 strains. Amplified-fragment length polymorphism analysis was used to monitor instability in phnE in various strains growing under different conditions. This revealed that, once selection for growth on MePn is removed, Phn+ revertants reappear and accumulate at high levels through reinsertion of the 8-bp repeat element sequence. It appears that, in K-12, phnE contains a high-frequency reversible gene switch, producing phase variation which either allows ("on" form) or blocks ("off" form) MePn utilization. The switch can also block usage of other metabolizable alkyl phosphonates, including the naturally occurring 2-aminoethylphosphonate. All K-12 strains, obtained from collections, appear in the "off" form even when bearing mutations in mutS, mutD, or dnaQ which are known to enhance slip strand events between repetitive sequences. The ability to inactivate the phnE gene appears to be unique to K-12 strains since the B strain is naturally Phn+ and lacks the inactivating 8-bp insertion in phnE, as do important pathogenic strains for which genome sequences are known and also strains isolated recently from environmental sources.     

Characterisation of Toxoplasma gondii isolates using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the non-coding Toxoplasma gondii (TGR)-gene sequences

The Toxoplasma gondii (TGR) genes constitute a family of non-coding sequences, three of which have been previously described as possible tools for typing of Toxoplasma gondii isolates. We obtained new isolates of T. gondii from domestic and wild animals, and used these to evaluate the possibility of using TGR gene variants as markers to distinguish among T. gondii isolates from different animals and different geographical sources.Based on the band patterns obtained by restriction fragment length polymorphism (RFLP) analysis of the polymerase chain reaction (PCR) amplified TGR sequences, the T. gondii isolates could be separated into seven groups. Sequencing the amplified products showed that at least 20 TGR sequences not hitherto described had been found, demonstrating that the TGR gene family comprises a large number of different yet highly homologous sequences. Each isolate had its own unique TGR sequence. The TGR gene family therefore seems a promising target for typing individual T. gondii isolates and for studying the genetic distance between two isolates, which can be used for tracing routes of infection.     

Cloning and analysis of duplicated rfbM and rfbK genes involved in the formation of GDP-mannose in Escherichia coli O9:K30 and participation of rfb genes in the synthesis of the group I K30 capsular polysaccharide.

The rfbO9 gene cluster, which is responsible for the synthesis of the lipopolysaccharide O9 antigen, was cloned from Escherichia coli O9:K30. The gnd gene, encoding 6-phosphogluconate dehydrogenase, was identified adjacent to the rfbO9 cluster, and by DNA sequence analysis the gene order gnd-rfbM-rfbK was established. This order differs from that described for other members of the family Enterobacteriaceae. Nucleotide sequence analysis was used to identify the rfbK and rfbM genes, encoding phosphomannomutase and GDP-mannose pyrophosphorylase, respectively. In members of the family Enterobacteriaceae, these enzymes act sequentially to form GDP-mannose, which serves as the activated sugar nucleotide precursor for mannose residues in cell surface polysaccharides. In the E. coli O9:K30 strain, a duplicated rfbM2-rfbK2 region was detected approximately 3 kbp downstream of rfbM1-rfbK1 and adjacent to the remaining genes of the rfbO9 cluster. The rfbM isogenes differed in upstream flanking DNA but were otherwise highly conserved. In contrast, the rfbK isogenes differed in downstream flanking DNA and in 3'-terminal regions, resulting in slight differences in the sizes of the predicted RfbK proteins. RfbMO9 and RfbKO9 are most closely related to CpsB and CpsG, respectively. These are isozymes of GDP-mannose pyrophosphorylase and phosphomannomutase, respectively, which are thought to be involved in the biosynthesis of the slime polysaccharide colanic acid in E. coli K-12 and Salmonella enterica serovar Typhimurium. An E. coli O-:K30 mutant, strain CWG44, lacks rfbM2-rfbK2 and has adjacent essential rfbO9 sequences deleted. The remaining chromosomal genes are therefore sufficient for GDP-mannose formation and K30 capsular polysaccharide synthesis. A mutant of E. coli CWG44, strain CWG152, was found to lack GDP-mannose pyrophosphorylase and lost the ability to synthesize K30 capsular polysaccharide. Wild-type capsular polysaccharide could be restored in CWG152, by transformation with plasmids containing either rfbM1 or rfbM2. Introduction of a complete rfbO9 gene cluster into CWG152 restored synthesis of both O9 and K30 polysaccharides. Consequently, rfbM is sufficient for the biosynthesis of GDP-mannose for both O antigen and capsular polysaccharide E. coli O9:K30. Analysis of a collection of serotype O8 and O9 isolates by Southern hybridization and PCR amplification experiments demonstrated extensive polymorphism in the rfbM-rfbK region.     

Cloning and expression of the Escherichia coli glgC gene from a mutant containing an ADPglucose pyrophosphorylase with altered allosteric properties.

A mutant strain of Escherichia coli K-12, designated 618, accumulates glycogen at a faster rate than wild-type strain 356. The mutation affects the ADPglucose pyrophosphorylase regulatory properties (N. Creuzat-Sigal, M. Latil-Damotte, J. Cattaneo, and J. Puig, p. 647-680, in R. Piras and H. G. Pontis, ed., Biochemistry of the Glycocide Linkage, 1972). The enzyme is less dependent on the activator, fructose 1,6 bis-phosphate for activity and is less sensitive to inhibition by the inhibitor, 5'-AMP. The structural gene, glgC, for this allosteric mutant enzyme was cloned into the bacterial plasmid pBR322 by inserting the chromosomal DNA at the PstI site. The glycogen biosynthetic genes were selected by cotransformation of the neighboring asd gene into an E. coli mutant also defective in branching enzyme (glgB) activity. Two recombinant plasmids, pEBL1 and pEBL3, that had PstI chromosomal DNA inserts containing glgC and glgB were isolated. Branching enzyme and ADPglucose pyrophosphorylase activities were increased 240- and 40-fold, respectively, in the asd glgB mutant, E. coli K-12 6281. The E. coli K-12 618 mutant glgC gene product was characterized after transformation of an E. coli B ADPglucose pyrophosphorylase mutant with the recombinant plasmid pEBL3. The kinetic properties of the cloned ADPglucose pyrophosphorylase were similar to those of the E. coli K-12 618 enzyme. The inserted DNA in pEBL1 was arranged in opposite orientation to that in pEBL3.     

Tn554 inserts in methicillin-resistant Staphylococcus aureus from Australia and England: comparison with an American methicillin-resistant group.

We have compared methicillin-resistant (Mcr) Staphylococcus aureus isolates from Australia, the UK and the USA with regard to chromosomal inserts of the macrolides-lincosamides-streptogramin B (MLS)-resistance transposon Tn554. The American isolates were known to have a distinctive Tn554 insert, designated insert 6, which was closely associated epidemiologically with the methicillin-resistance phenotype. Southern blots of DNA from Australian and London, UK Mcr isolates were hybridized with a range of probes related to Tn554. The isolates had similar or identical Tn554 inserts, and we consider them to be a single group, designated 'Australondon'. Australondon isolates were compared in detail with a deletion mutant, ANS62, that had lost the methicillin-resistance determinant mec, plus other resistance determinants resident in the mec region of the chromosome, and with an American Mcr isolate containing Tn554 insert 6. The Australondon isolates had three Tn554 inserts. Sequence analysis with the polymerase chain reaction showed that all of these inserts differed from classical Tn554 in that the 3'-terminal residues of the transposons were reverse complements of the usual GATGTA. One of the Australondon inserts, designated 6B, closely resembled Tn554 insert 6 in the sequence of its left flanking chromosomal DNA. This insert was found to abut the deletion from the mec region which results in strain ANS62. We infer that Tn554 insert 6B is part of the mec region of the chromosome in Australondon isolates, supporting the idea that insert 6 of the American isolates is also part of this chromosomal region.