Physical and genetic map of the Listeria monocytogenes EGD serotype 1/2a chromosome

Listeria monocytogenes is a facultative intracellular pathogen responsible for both invasive and non-invasive food-borne illness in animals and humans. In this study, macrorestriction analysis following pulsed-field gel electrophoresis was used to show that Listeria monocytogenes serovar 1/2a strain EGD has a single chromosome containing eight NotI fragments of 1100, 850, 365, 320, 275, 40, 30 and 20 kb in size and 11 AscI fragments of 860, 470, 410, 360, 320, 250, 110, 80, 50, 30 and 20 kb. The total genome therefore comprises 3000 +/- 50 kb. The creation of a physical and genetic map of the Listeria genome was achieved by generating NotI linking clones and their use in subsequent hybridisation analysis. Using isogenic mutants harbouring additional artificial NotI restriction sites, we were able to precisely map the positions of all currently known virulence genes on the chromosome.     

Differentiation of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by pulsed-field gel electrophoresis.

Clamped homogeneous electric field analysis of Listeria DNA with ApaI, AscI, SmaI, or NotI revealed species- and serotype-specific differences in genomic fingerprints. Clamped homogeneous electric field analysis may prove useful, therefore, in epidemiologic studies. Also, the summation of individually sized AscI fragments of genomic DNA from L. monocytogenes serotype 4b 101M and Scott A indicated genome lengths of 2,925 and 3,046 kb, respectively.     

Differentiation of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by pulsed-field gel electrophoresis.

Clamped homogeneous electric field analysis of Listeria DNA with ApaI, AscI, SmaI, or NotI revealed species- and serotype-specific differences in genomic fingerprints. Clamped homogeneous electric field analysis may prove useful, therefore, in epidemiologic studies. Also, the summation of individually sized AscI fragments of genomic DNA from L. monocytogenes serotype 4b 101M and Scott A indicated genome lengths of 2,925 and 3,046 kb, respectively.     

Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome.

A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI. The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests. The derived map is circular, and the genome size was estimated to be 1,623 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the map. Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome. In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis. The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed.     

Physical map of the Listeria monocytogenes chromosome.

The circular physical map of the pathogenic bacterium Listeria monocytogenes LO28 (serovar 1/2c) was established by using pulsed-field gel electrophoresis. The L. monocytogenes chromosome contains eight NotI fragments of 1,100, 940, 400, 335, 280, 45, 30, and 20 kb in size and eight Sse8387I fragments of 860, 680, 680, 370, 335, 130, 70, and 25 kb. Therefore, the total length of the genome is 3,150 kb. To order the NotI fragments on the chromosome, we used a strategy which can be of general use. We first cloned chromosomal HindIII or EcoRI fragments in pBR322. DNA extracted from the total libraries was digested by NotI and ligated to a NotI-kanamycin resistance cassette obtained by cutting Tn5 with NotI. After transformation in Escherichia coli, kanamycin-resistant clones originating from NotI-containing EcoRI or HindIII fragments were isolated. The two EcoRI-NotI or HindIII-NotI fragments of each recombinant plasmid were isolated and used as probes on Southern blot hybridizations to identify and link the corresponding NotI fragments. Seven NotI fragments were ordered in this way. The last junction was demonstrated by partial digest analysis. All L. monocytogenes genes identified so far as well as the six rRNA operons were localized on the NotI map. Regions homologous to genes from closely related bacteria were also detected and localized. Southern blot analysis of simple Sse8387I digests or double Sse8387I-NotI digests probed with the various NotI probes allowed us to align the Sse8387I fragments and localize the single SfiI site, resulting in the establishment of the first genetic and physical map of the L. monocytogenes chromosome.     

First Chromosomal Restriction Map of Actinobacillus pleuropneumoniae and Localization of Putative Virulence-Associated Genes

Combined physical and genetic maps of the genomes of Actinobacillus pleuropneumoniae AP76 (serotype 7 clinical isolate) and of A. pleuropneumoniae ATCC 27088 (serotype 1 reference strain) were constructed by using the restriction endonucleases ApaI, AscI, NotI, and SalI. The chromosome sizes as determined by the addition of estimated fragment sizes were 2.4 Mbp, and both maps had a resolution of approximately 100 kbp. The linkages between the ApaI, AscI, NotI, and SalI fragments and their relative positions were determined by (i) fragment excision and redigestion and (ii) partial digests of defined fragments and Southern blot using end-standing probes. The single SalI site within the chromosome of strain A. pleuropneumoniae AP76 was defined as position 1 of the map; for the map of A. pleuropneumoniae ATCC 27088, the corresponding SalI site was chosen. Putative virulence-associated genes (apx, omlA, sodA, tbpBA, ureC, and a repeat element) and housekeeping genes (glyA, metJ, recA, and rhoAP) were positioned on the physical maps and located on the ApaI and NotI fragments of A. pleuropneumoniae serotype reference strains.     

Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

A recombinant plasmid pool of the Saccharomyces diastaticus genome was constructed in plasmid YEp13 and used to transform a strain of Saccharomyces cerevisiae. Six transformants were obtained which expressed amylolytic activity. The plasmids each contained a 3.9-kilobase (kb) BamHI fragment, and all of these fragments were cloned in the same orientations and had identical restriction maps, which differed from the map of the STA1 gene (I. Yamashita and S. Fukui, Agric. Biol. Chem. 47:2689-2692, 1983). The glucoamylase activity exhibited by all S. cerevisiae transformants was approximately 100 times less than that of the donor strain. An even lower level of activity was obtained when the recombinant plasmid was introduced into Schizosaccharomyces pombe. No expression was observed in Escherichia coli. The 3.9-kb BamHI fragment hybridized to two sequences (4.4 and 3.9 kb) in BamHI-digested S. diastaticus DNA, regardless of which DEX (STA) gene S. diastaticus contained, and one sequence (3.9 kb) in BamHI-digested S. cerevisiae DNA. Tetrad analysis of crosses involving untransformed S. cerevisiae and S. diastaticus indicated that the 4.4-kb homologous sequence cosegregated with the glucoamylase activity, whereas the 3.9-kb fragment was present in each of the meiotic products. Poly(A)+ RNA fractions from vegetative and sporulating diploid cultures of S. cerevisiae and S. diastaticus were probed with the 3.9-kb BamHI fragment. Two RNA species, measuring 2.1 and 1.5 kb, were found in both the vegetative and sporulating cultures of S. diastaticus, whereas one 1.5-kb species was present only in the RNA from sporulating cultures of S. cerevisiae.     

A single gene encoding the fiber is responsible for variations in virulence in the fowl adenoviruses.

Intertypic recombinant fowl adenoviruses (FAVs) were generated to determine regions of the viral genome involved in virulence. Recombinants were produced with two serotype 8 FAVs, mildly virulent CFA 3 and hypervirulent CFA 40. Restriction endonuclease fragments from the genomes of the two FAVs were used to transfect primary chicken kidney cells. Virulence testing of these recombinants located the region responsible for differences in virulence to an 8.4-kb fragment of the genome located between kb 26.6 and 35.0. According to data available for a serotype 10 FAV that had been partially characterized in the laboratory, this segment of the genome contained three genes of known identity (100K, 33K, and pVIII) and a region between kb 31 and 35 with unknown coding potential (although this information subsequently became available for a serotype 1 FAV, CELO). Therefore, the region between kb 30.5 and 34.5 was sequenced. The results revealed that the unknown region encoded a fiber gene on the right strand and several small open reading frames of unknown identity on the left strand. Further recombinant viruses containing defined exchanges within the 4-kb fragment were constructed, and virulence testing of these viruses indicated that the fiber was responsible for differences in virulence for CFA 40 and CFA 3.     

DNA fragments from regions involved in surface antigen expression specifically identify Listeria monocytogenes serovar 4 and a subset thereof: cluster IIB (serotypes 4b, 4d, and 4e).

Listeria monocytogenes serotype 4b has frequently been implicated in sporadic as well as epidemic listeriosis. On the basis of pulsed-field fingerprinting, serotype 4b strains, along with strains of serotypes 4d and 4e, constitute one genomic cluster (IIB). We have identified two genomic regions essential for the expression of surface antigens which previously were shown to be specific to cluster IIB strains. A DNA probe of 1.1 kb derived from one of the regions (probe 1) hybridized only with strains of serotypes 4b, 4d, and 4e in Southern blots and dot blots. A different DNA probe of 0.3 kb (probe 2), derived from the other region, hybridized with all serovar 4 strains (serotypes 4b, 4a, 4c, 4d, and 4e). All other L. monocytogenes serotypes were negative with probe 1 or 2. Use of probe 1 in Southern blots of EcoRI-digested genomic DNA revealed a restriction fragment length polymorphism in serotype 4b strains, with the hybridizing EcoRI fragments being 4.5 kb (strains of the epidemic clone) and either 4.5 or 5.0 kb (all other serotype 4b strains). Although the probes hybridized with a special group of Listeria innocua strains which also expressed the surface antigens, the latter could be readily distinguished by the size of the hybridizing EcoRI fragment with probe 1 (ca. 2.2 kb). These data suggest that the combined use of these probes with L. monocytogenes can readily and specifically identify cluster IIB strains as well as the entire serovar 4 complex.     

Ability of the Listeria monocytogenes strain Scott A to cause systemic infection in mice infected by the intragastric route

Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involve Listeria monocytogenes serotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates of L. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection with L. monocytogenes Scott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strain L. monocytogenes EGD. Prior administration of sodium bicarbonate did not enhance the virulence of L. monocytogenes strain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains of L. monocytogenes (Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth of L. monocytogenes Scott A at temperatures of 12.5 to 37 degrees C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4b L. monocytogenes strains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.     

Human chromosome 17 NotI linking clones and their use in long-range restriction mapping of the Miller-Dieker chromosome region (MDCR) in 17p13.3

A NotI linking library constructed from flow-sorted human chromosome 17 material was screened to aid in construction of a long-range restriction map of the Miller-Dieker chromosome region (MDCR) in 17p13.3. A total of 66 clones were mapped to one of eight regions of chromosome 17 using a somatic cell hybrid panel, and 44/66 (67%) of these clones cross-hybridized to rodent DNA on Southern blots. Of these, 24 clones were tested and all mapped to mouse chromosome 11, the homolog of human chromosome 17. Four linking clones mapped to 17p13.3 and were used for pulsed-field gel electrophoresis studies along with six other anonymous probes previously mapped to this region. Clone L132 was found to be deleted in all Miller-Dieker patients tested (n = 15) and therefore lies within the critical region for this disorder. It detects two NotI fragments (180 and 320 kb), one of which (320 kb) was shared by YNZ22 and YNH37, two probes previously shown to be co-deleted in all patients with the Miller-Dieker syndrome (MDS). These results indicate that all MDS patients share a minimum deletion region of greater than 370 kb. Two other NotI clones, L53 and L125, mapped telomeric to the MDS critical region and share a 600-kb MluI fragment with each other and with YNZ22/YNH37. This provides a 930-kb MluI map that encompasses the distal boundary of the MDS critical region but does not include the proximal boundary. A total of over 2 Mbp is represented in the MluI fragments by probes in subband p13.3, a cytogenetic region estimated to be 3-4 Mbp.     

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay.

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.     

Physical genome map of the unicellular cyanobacterium Synechococcus sp. strain PCC 7002.

A physical restriction map of the genome of the cyanobacterium Synechococcus sp. strain PCC 7002 was assembled from AscI, NotI, SalI, and SfiI digests of intact genomic DNA separated on a contour-clamped homogeneous electric field pulsed-field gel electrophoresis system. An average genome size of 2.7 x 10(6) bp was calculated from 21 NotI, 37 SalI, or 27 SfiI fragments obtained by the digestions. The genomic map was assembled by using three different strategies: linking clone analysis, pulsed-field fragment hybridization, and individual clone hybridization to singly and doubly restriction-digested large DNA fragments. The relative positions of 21 genes or operons were determined, and these data suggest that the gene order is not highly conserved between Synechococcus sp. strain PCC 7002 and Anabaena sp. strain PCC 7120.     

A physical map of the human Y-chromosome short arm

A physical map of the Y-chromosome short arm was constructed using DNA probes p19B, Y-286/la5, pZFY, Y-280, and Y-227. These probes hybridize with four NotI fragments of 400 kb (p19B and Y-286/la5), 350 kb, 1.9 Mb, and 3.0 Mb, respectively. The restriction fragments were shown to be adjacent to each other by analysis of NotI partial digests, overlapping restriction fragments, and/or the detection of rearranged restriction fragments in a 46,XX male. The present map covers approximately 5.6 Mb of contiguous DNA of Yp. Previously, the size of the pseudoautosomal region was estimated to be 2.3 Mb, and a 5.3-Mb NotI fragment containing Y-specific repeated DNA was assigned to proximal Yp. These and the present data account for approximately 13 Mb and thus for most of the DNA content of the Y short arm.     

A restriction map of IncFIV plasmid R124

A physical and genetic map of the 125.7-kb IncFIV plasmid R124 was constructed using the restriction enzymes Sal1 and EcoR1. Two discrete regions involved in plasmid replication were identified on the plasmid genome. One region was located on a 4.66-kb segment of an EcoR1 fragment at map coordinates 73.87 to 78.53 kb. Another was located within an 8.05-kb segment of an EcoR1 fragment at map coordinates 113.40 to 121.45. This region was very unstable but, when ligated to the 3.21-kb EcoR1 fragment E13 located at map coordinates 18.83 to 22.06 kb, replication was stable. Thus, at least three regions of R124 widely separated around the genome are associated with plasmid replication and stable maintenance. Each of these three regions expressed incompatibility with R124. The Tc resistance gene of R124 was located on the contiguous EcoR1 fragments E8 and E12 located at map coordinates 100.49 to 113.40.     

A decarboxylase encoded at the Cochliobolus heterostrophus translocation-associated Tox1B locus is required for polyketide (T-toxin) biosynthesis and high virulence on T-cytoplasm maize

Genes at two unlinked loci (Tox1A and Tox1B) are required for production of the polyketide T-toxin by Cochliobolus heterostrophus race T, a pathogenic fungus that requires T-toxin for high virulence to maize with T-cytoplasm. Previous work indicated that Tox1A encodes a polyketide synthase (PKS1) required for T-toxin biosynthesis and for high virulence. To identify genes at Tox1B, a wild-type race T cDNA library was screened for genes missing in the genome of a Tox1B deletion mutant. The library was probed, first with a 415-kb NotI restriction fragment from the genome of the Tox1B mutant, then with the corresponding 560-kb fragment from the genome of wild type. Two genes, DEC1 (similar to acetoacetate decarboxylase-encoding genes) and RED1 (similar to genes encoding members of the medium-chain dehydrogenase/reductase superfamily), were recovered. Targeted disruption of DEC1 drastically reduced both T-toxin production and virulence of race T to T-cytoplasm maize, whereas specific inactivation of RED1 had no apparent effect on T-toxin production (as determined by bioassay) or on virulence. DEC1 and RED1 map within 1.5 kb of each other on Tox1B chromosome 6;12 and are unique to the genome of race T, an observation consistent with the hypothesis that these genes were acquired by C. heterostrophus via a horizontal transfer event.     

A BlnI restriction map of the Salmonella typhimurium LT2 genome.

BlnI or AvrII (5'-CCTAGG) sites are very rare in the Salmonella typhimurium LT2 genome. BlnI was used to construct a physical map which was correlated with the genetic map by using three methods. First, Tn10 carries BlnI sites, and the extra restriction sites produced by 34 genetically mapped Tn10 insertions were physically mapped by using pulsed-field gel electrophoresis. Second, six genetically mapped Mud-P22 prophage insertions were used to assign BlnI fragments. Integration of Mud-P22 introduces 30 kb of DNA that can easily be detected by a "shift up" in all but the largest BlnI fragments. Finally, induced Mud-P22 insertions package more than 100 kb of genomic DNA adjacent to one side of the insertion. Some of the smaller BlnI fragments were localized by hybridization to a dot blot array of 52 lysates from induced Mud-P22 insertions. Of the 10 BlnI sites mapped, 6 probably occur in or near the 16S rRNA genes at about 55, 71, 83, 86, 88.5, and 89.5 min. There is one BlnI site in the 90-kb pSLT plasmid. Two additional BlnI fragments of about 7 and 4 kb have not been localized. The size of the genome was estimated as 4.78 Mb (+/- 0.1 Mb) excluding pSLT but including prophages Fels-1 and Fels-2. One BlnI fragment that maps between 55 and 59 min showed a 40-kb reduction in size in a strain cured of the approximately 40-kb Fels-2 prophage.     

Physical and functional map of an amikacin-resistance plasmid isolated from a multiresistant strain of Serratia marcescens

pTK159, a multiresistance 40-kilobase (kb) plasmid, was isolated from a clinical strain of Serratia marcescens. pTK159 was nonconjugative and carried determinants for resistance to amikacin, streptomycin/spectinomycin, sulfamethoxazole and ampicillin. A physical and functional map of pTK159 was constructed. By cloning various fragments of pTK159 in pACYC184 or pBR322, genes for resistance to amikacin, streptomycin/spectinomycin, and sulfamethoxazole were found to be located on a 2.0-kb BamHI-HindIII fragment, a 1.4-kb HindIII fragment and a 2.1-kb HindIII fragment, respectively. The map of pTK159 was compared with published maps of amikacin-resistance determinants and transposons.     

Cloning of the listeriolysin O gene and development of specific gene probes for Listeria monocytogenes

A clone containing 3.1 kb of Listeria DNA was selected from a gene library of Listeria monocytogenes Scott A strain. The Escherichia coli clone produced hemolysin on sheep blood agar and in sonicated extracts but very little in the culture supernatant. This 3.1-kb DNA fragment and a 650-bp HindIII fragment located within the listeriolysin gene were used as probes in a colony hybridization assay. Both probes were specific for L. monocytogenes and did not hybridize with any other Listeria strains at high stringency. Two synthetic probes, one from the 650-bp HindIII fragment and one from the carboxy-terminal region of the protein, were also specific for L. monocytogenes.