Restriction Enzyme Digestion Profiles Research Articles

Molecular techniques for the assessment of Cr (VI) reduction by Bacillus thuringiensis

Effluent pollution with Cr (VI) is a worldwide environmental problem. In the Pasto River (southeastern, Colombia), previous studies reported contamination with this metal at points near tanneries. To establish the role of Bacillus thuringiensis in Cr (VI) reduction in water from Pasto River, experiments were carried out with untreated Pasto River water (treatment 1), sterile Pasto River water inoculated with B. thuringiensis (treatment 2), and unsterilized Pasto River water inoculated with B. thuringiensis (treatment 3). All experiments were conducted in bioreactors with a controlled temperature of 20 °C and constant agitation for 156 h. Samples of 20 mL were taken every 12 h from each treatment to track Cr (VI) reduction levels and to confirm microorganism identity via molecular methods involving denaturing gradient gel electrophoresis (DGGE), restriction enzyme digestion profiles (RFLP), and bioinformatic analysis. Cr (VI) reduction was higher in treatment 3 (99:42 %) as opposed to treatment 2 (76:12 %) and treatment 1 (74:46 %). The molecular identity of B. thuringiensis was determined via sequencing of the 16SrRNA gene, and RFLP assessments in all three treatments revealed B. thuringiensis profiles. Since B. thuringiensis was present in all three treatments trough time, Cr (VI) reduction can be attributed to this bacterium.

Prevalent and persistent Escherichia coli O157:H7 strains on farms are selected by bovine passage

Escherichia coli O157:H7 is a human pathogen capable of causing hemorrhagic colitis and in some cases hemolytic uremic syndrome. Cattle are an asymptomatic carrier and a major reservoir of this pathogen that can be transmitted by contaminated foods like beef products and vegetables. To further understand persistence in cattle and on farms, a total of 1716 samples over a two-year period were collected from a Wisconsin dairy farm (Farm R) and 91 were positive for the presence of E. coli O157:H7. Seventy-six of 1373 (4.8%) fecal samples and 10/190 (5.3%) water samples were positive. Genotyping of the 341 E. coli O157 isolates by pulsed-field gel electrophoresis showed nine different restriction enzyme digestion profile (REDP) types, seven of which were 93–98% similar (comprised of serotype O157:H7 isolates) and two that were dissimilar (serotype O157:H-isolates). The REDP 31 strain dominated and was isolated from 59 fecal and 9 water samples; 75% of the positive samples (68/91) contained this strain. Growth studies of representative strains from each the REDP groups in Luria broth at 25 and 39°C found no significant differences between the strains. In LB supplemented with bile salts (3, 6, and 9%; 39°C, 48h), the REDP 30 strain had a longer lag phase and achieved a lower maximum density than the other strains in the presence of 6 and 9% bile salts. Likewise, the survival of the strains in low-pH conditions (HCl, pH 2.0 and acetic acid, pH 3.0) were similar except for the REDP 30 strain which was significantly less acid tolerant at pH 2.0. A screening for differences in carbohydrate utilization found that the dominant strain (REDP 31) utilized the most carbon sources and was the only strain that oxidized amygdalin, citraconic acid, α-ketoglutarate, and γ-cyclodextrin. The inoculation of Holstein calves with a three-strain mixture (REDP 30, 31, and 36 strains) found the REDP 31 strain (FRIK 2455) dominated in fecal and rectal swab samples throughout the durations of shedding. These results suggested that carbohydrate utilization and host factors encountered during animal passage select for persistent and predominant strains on farms.

Characterization of 4 T1-like lytic bacteriophages that lyse Shiga-toxin Escherichia coli O157:H7

Bacteriophages are associated with reduced fecal shedding of Shiga-toxin-producing Escherichia coli O157:H7 (STEC O157:H7) in cattle. Four phages exhibiting activity against 12 of 14 STEC O157:H7 strains, representing 11 common phage types, were isolated. Phages did not lyse non-O157 E. coli, with 11 of the 12 STEC strains exhibiting extreme susceptibility (average multiplicity of infection (MOI) = 0.0003-0.0007). All phages had icosahedral heads with tapered, noncontractile tails, a morphology indicative of T1-like Siphoviridae. Genome size of all phages was ∼44 kb, but EcoRІ or HindIII digestion profiles differed among phages. Based on restriction enzyme digestion profiles, phages AHP24, AHS24, and AHP42 were more related (66.7%-82.4%) to each other than to AKS96, while AHP24 and AHS24, isolated from the same feedlot pen, exhibited the highest identity (88.9%-92.3%). Phages AHP24 and AHS24 exhibited the broadest host range and strongest lytic activity against STEC O157:H7, making them strong candidates for biocontrol of this bacterium in cattle.

Isolation and characterization of phages infecting Bacillus cereus

To isolate and characterize bacteriophages (phages) that infect the foodborne pathogen Bacillus cereus. Two phages were isolated from soil based on their ability to form plaques on four indicator hosts including Bacillus thuringiensis subsp. israelensis, and three isolates of B. cereus. The purified phages were characterized by morphology, host range, single-step growth curves and restriction enzyme digestion profiles. The phages appeared to be of the Myoviridae family based on their structure in electron micrographs. The phages lysed bacteria of several species, produced average burst sizes of 322 and 300 phages per infected cell, and both had genomes over 90 kb. The phages were chloroform-resistant and stable at 4°C. They reduced the concentration of B. cereus in mashed potatoes by >6 log(10) CFU ml(-1) within 24 h at room temperature, when applied at a high concentration. The relatively narrow host range within B. cereus might mean that these phages need to be used as part of a 'cocktail' of phages for biocontrol, but their efficacy for the control of their host in food was demonstrated. This is the first report of biocontrol by phages of B. cereus in food.

HideNseek, a post-genome approach to locate transgenes exemplified in Arabidopsis thaliana

Determination of transgene location is essential for investigating the effects of position on transgene expression levels and facilitates cloning of the resident gene affected by insertion. Currently used PCR-based approaches for determination of transgene location are relatively complicated and often fail when the transgene is duplicated, rearranged or fragmented. HideNseek is a new bioinformatics tool that allows computation of transgene locations, provided that a suitable genomic restriction enzyme digestion profile is available. Since the new approach is not based on the terminal sequences of the transgene insert, it is less sensitive to transgene duplication, rearrangement or fragmentation. HideNseek has been tested experimentally and by in silico simulation. The experimental example provided here shows that this simple approach is feasible, permitting rapid location of transgenes with little bench work. available on request from the authors. HideNseek input and output examples, experimental procedures and figures showing experimental results are provided as supplementary files: Supplementary material 1, 2, 3 and Supplementary figures (Figs 1 and 2), respectively. Supplementary data is available at Bioinformatics online.

Dairy Farm Reservoir of Listeria monocytogenes Sporadic and Epidemic Strains

Identifying the reservoirs of a pathogen is vital for control of sporadic disease and epidemics. Listeria monocytogenes is a zoonotic foodborne pathogen that is responsible for 28% of food-related deaths in the United States annually, as well as a major cause of massive product recalls worldwide. To examine the role of the dairy farm as a potential source or reservoir for L. monocytogenes subtypes shown to cause human listeriosis, we compared the pulsed-field gel electrophoresis (PFGE) restriction enzyme digestion profiles of L. monocytogenes dairy farm–associated strains (milk, environmental, and bovine) to human sporadic and epidemic disease strains. Twenty-three percent of human sporadic strains had PFGE patterns identical to that of farm isolate(s). Additionally, three farm environmental strains and one human sporadic strain had a PFGE pattern identical to a strain of L. monocytogenes responsible for the 1985 California epidemic. These data indicate that this epidemic strain continues to cause sporadic human illness and has a potential dairy farm as a reservoir.

Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome

Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV.

Prevalence of Listeria monocytogenes Subtypes in Bulk Milk of the Pacific Northwest

The prevalence of Listeria monocytogenes in bulk milk from three Pacific Northwest states was assessed for 474 herds at three time points. For samples collected in November 2000 and June 2001, the L. monocytogenes prevalence levels were 4.9 and 7.0%, respectively. All isolates were subtyped by serotyping and by pulsed-field gel electrophoresis (PFGE). Forty-nine of the 55 isolates belonged to serogroup 1/2a, while 6 belonged to serogroup 4. Subtyping by PFGE revealed that isolates from 31 herds shared 10 patterns; there was a weak but significant association between PFGE subtype and geographical distance. Six herds were positive for L. monocytogenes at both time points. Of these six herds, four had indistinguishable PFGE patterns at both time points. Twenty-five of the 33 herds that were positive in June 2001 were sampled again in June 2002. L. monocytogenes was recovered from 17 of these 25 herds (68%), with the ApaI restriction enzyme digestion profiles (REDP) for 8 herds being identical to those of isolates recovered from these herds the previous year. The ApaI REDP for the bulk milk isolates were compared with those for isolates recovered from environmental and human samples that were collected by the Washington Department of Health (n = 23). Analysis of ApaI digestion profiles revealed that only two of the Washington Department of Health isolates had digestion profiles similar to those for isolates from bulk milk; however, further analysis with the use of a second enzyme (AscI) was capable of discriminating between isolates from the two sources. Thus, we found no direct REDP matches between bulk milk and clinical isolates.

CTX-M-14, a plasmid-mediated CTX-M type extended-spectrum beta-lactamase isolated from Escherichia coli.

Four Escherichia coli isolates harboring CTX-M-14, with a single Ala231-->Val substitution compared to CTX-M-9, had three different ribotypes. Cefotaxime resistance was plasmid encoded and conjugatively transferable. Three isolates had the same plasmid restriction enzyme digestion profile, suggesting clonal spread of a resistant plasmid. A high k(cat)/K(m) value for cefotaxime (20.3 microM(-1) s(-1)) but low values for ceftazidime and aztreonam (< 0.02 microM(-1) s(-1)) were observed in hydrolysis assays, indicating resistance to cefotaxime (MIC > or = 64 microg/ml) but susceptibility to ceftazidime (MIC < or = 2 microg/ml).

Phenotypic characterization of Listeria monocytogenes and Listeria innocua strains isolated from short-ripened cheeses

Listeria monocytogenes and Listeria innocua strains isolated from short-ripened cheeses and classified, respectively, into five (m1 to m5) and two (i1 and i2) REDP clusters (restriction enzyme digestion profiles by PFGE), were characterized on the basis of their carbohydrate fermentation profiles, growth in milk, haemolytic activity, sensitivity to sanitizing agents, tolerance to low pH, organic acids and NaCl, and resistance to pasteurization. All isolates fermented L -rhamnose and α-methyl- D -mannoside and most of them were lactose positive; melezitose was fermented by L. monocytogenes strains but not by L. innocua. The haemolytic activity of L. monocytogenes varied depending on the strain. The isolates were sensitive to all cleaning and disinfectant agents tested (acid, alkaline, chlorine and peroxide-containing agents), with the only exception of NaOH. Storage at refrigeration temperature enhanced the tolerance to NaCl of both species and the resistance of L. monocytogenes to the acid. Organic acids were more inhibitory than HCl and their effectiveness was greater at higher than at lower concentration and followed the order acetic>lactic>citric. M4, a group genetically related to certain pathogenic L. monocytogenes strains, was unable to ferment lactose although its growth in milk was not affected. The slightly reduced tolerance to NaCl of this group with respect to the others might have prevented a wider distribution in dairy environments. Although pasteurization was effective in most cases, two different L. monocytogenes strains isolated from the same cheese sample displayed a certain degree of thermotolerance; this resistance could have been induced by successive heat treatments of strains contaminating the dairy environment. All this data pointed to a relationship between several phenotypic characteristics and the ability of Listeria to colonize certain environments.

The non-collagenous dentin matrix proteins are involved in dentinogenesis imperfecta type II (DGI-II).

Dentinogenesis Imperfecta type II (DGI-II) is a localized form of mesodermal dysplasia of the dentin affecting both the primary and permanent dentitions. This is an autosomal-dominant disease in which there is a disorder in dentin mineralization. Several studies have localized DGI-II to human chromosome 4 in the region 4q 12-21. Many ECM genes-such as OPN, DMP1, DMP2, DMP3 (DSPP), and BSP-have been mapped to the same locus. Biochemical studies indicated that dentin phosphophoryn (DMP2) might be a candidate gene in DGI-II. In this study, we have used histological and RFLP analyses of tissues from a DGI-II-affected patient, as compared with two normal controls, to determine if DMP1, 2, or 3 was linked to DGI-II. The histology of the affected tooth was very different in the DGI-II patient as compared with the normals. In particular, the dentinal tubules in the DGI-II patient were very irregular, which could be the result of perturbations in the process of dentin formation. Patient and control DNA samples were digested with EcoRI or PstI and Southern-hybridized with the DMP1, DMP2, and DMP3 cDNAs. Few differences in the restriction pattern were observed between affected and normal samples for DMP1 and DMP3-3' region (phosphophoryn-like sequences) probes. On the other hand, DMP2 showed a dramatic shift in the restriction pattern in DGI-II. This study suggests that the different restriction enzyme digestion profiles of the DNA from the DGI-II patient, as probed by DMP2, might be related to the defective mineralization of dentin in DGI-II.

Polymerase chain reaction and restriction endonuclease digestion for selected members of the "Mycoplasma mycoides cluster" and Mycoplasma putrefaciens.

A specific diagnostic method using the polymerase chain reaction, together with restriction endonuclease digestion patterns, was developed for members of the "Mycoplasma mycoides cluster" that normally occur in the United States (i.e., Mycoplasma mycoides subsp. mycoides Large Colony and Mycoplasma capricolum subsp. capricolum in addition to "cluster" mycoplasma, bovine serogroup 7, and Mycoplasma putrefaciens. The digestion of "cluster" polymerase chain reaction DNA (1,225 bp) amplification products with restriction enzymes AseI and SspI gave mycoplasma species-specific patterns for all strains of M. mycoides subsp. mycoides Large Colony, M. capricolum subsp. capricolum, and bovine group 7 tested. Moreover, we found a nonspecific amplification product for M. putrefaciens that occurred with the oligonucleotide primers used for the "M. mycoides cluster" reaction. However, the restriction endonuclease digestion patterns observed with the restriction enzymes AluI, AseI, and SspI for M. putrefaciens were different than the digestion patterns obtained for the other "cluster" mycoplasmas. This report confirms the usefulness of polymerase chain reaction DNA amplification allied with restriction enzyme digestion profile analysis for the rapid and specific identification of mycoplasmas belonging to the "M. mycoides cluster" and M. putrefaciens.

The recombination mediated by double-strand breaks in extrachromosomal DNA substrate carrying mouse immunoglobulin switch regions Sμ and Sγ2b

Recombination in mouse cells was analyzed using extrachromosomal DNA substrates carrying the mouse immunoglobulin switch regions Sμ and Sγ2b. Recombination was detected at a frequency of 10 −2–10 −3 in mouse fibroblasts and in pre-B cell lines, but at a low frequency in a scid fibroblast cell line. Restriction enzyme digestion profile revealed that most recombination occurred between the CMV promoter region, which neighbors the Sμ upstream region, and the Sγ2b region. However, frequency of direct recombination between the CMV promoter region and the Sy2b region was low as measured by the substrate-lacking SR region. Nucleotide sequence analysis showed that recombination occurred between several homologous base-pairs, and extranucleotides were frequently found at the recombination junctions. These results indicate that recombination took the form of the recombination mediated by double-strand breaks. Double-strand breaks likely occurred in the Sμ and/or Sγ2b region, and the ends joined.

Simple method for detection of Clostridium botulinum type A to F neurotoxin genes by ploymerase chain reaction.

A polymerase chain reaction (PCR)-based method was established to detect each type of neurotoxin genes of Clostridium botulinum types A to F by employing the oligonucleotide primer sets corresponding to special regions of the light chains of the neurotoxins. In this procedure, the PCR products were easily confirmed by restriction enzyme digestion profiles, and as little as 2.5 pg of template DNAs from toxigenic strains could be detected. The specific PCR products were obtained from toxigenic C. botulinum types A to F, a type E toxin-producing C. butyricum strain, and a type F toxin-producing C. baratii strain, but no PCR product was detected in nontoxigenic strains of C. botulinum and other clostridial species. The neurotoxin genes were also detected in food products of a seasoned dry salmon and a fermented fish (Izushi) which had caused type E outbreaks of botulism. Therefore, it is concluded that this PCR-based detection method can be used for the rapid diagnosis of botulism.

Urinary isolates of apramycin-resistantEscherichia coliandKlebsiella pneumoniaefrom Dublin

Twenty-two gentamicin-resistant urinary isolates of Escherichia coli and five gentamicin-resistant urinary isolates of Klebsiella pneumoniae from a Dublin hospital were examined for resistance to the veterinary aminoglycoside antibiotic apramycin. Five isolates of E. coli and one isolate of K. pneumoniae were found to be resistant. The apramycin-resistant isolates, which were also resistant to the veterinary anthelmintic agent hygromycin B, hybridized with a DNA probe for the gene encoding the enzyme 3-N-aminoglycoside acetyltransferase type IV (AAC(3)IV). Resistance to apramycin and hygromycin B was co-transferable in four of the five isolates of E. coli and the isolate of K. pneumoniae. In one isolate of E. coli apramycin resistance was not transferable. On the basis of their restriction enzyme digestion profiles and the antimicrobial resistance traits encoded, the transferable plasmids encoding resistance to apramycin and hygromycin B comprised three distinct types. Genetic linkage between the gene encoding AAC(3)IV and genes encoding resistance to ampicillin and either tetracycline or trimethoprim, means that the relatively widespread use of these antimicrobial agents provides a selective pressure for the persistence of resistance to apramycin and gentamicin even in the absence of bacterial exposure to aminoglycosides.

Characterization of the synthesis and expression of the GTA-kinase from transformed and normal rodent cells

The murine transformed cell line YC-8 and β-adrenergic receptor agonist (isoproternol) treated rat and mouse parotid gland acinar cells ectopically express cell surface β1–4 galactosyltransferase during active proliferation. This activity is dependent upon the expression of the GTA-kinase (p58) in these cells. Using total RNA, cDNA clones for the protein coding region of the kinase were isolated by reverse transcriptase - PCR cloning. DNA sequence analysis failed to show sequence differences with the normal homolog from mouse cells although Southern blot analysis of YC-8, and a second cell line KI81, indicated changes in the restriction enzyme digestion profile relative to murine cell lines which do not express cell surface galactosyltransferase. The rat cDNA clone from isoproterenol-treated salivary glands showed a high degree of protein and nucleic acid sequence homology to the GTA-kinase from both murine and human sources. Northern blot analysis of YC-8 and a control cell line LSTRA revealed the synthesis of a major 3.0 kb mRNA from both cell lines plus the unique expression of a 4.5 kb mRNA in the YC-8 cells. Reverse transcriptase-PCR of LSTRA and YC-8 confirmed the increased steady state levels of the GTA-kinase mRNA in YC-8. In the mouse, induction of cell proliferation by isoproterenol resulted in a 50-fold increase in steady state mRNA levels for the kinase over the low level of expression in quiescent cells. Expression of the rat 3′ untranslated region in rat parotid cells in vitro led to an increased rate of DNA synthesis, cell number and ectopic expression of cell surface galactosyltransferase in the sense orientation. Antisense expression or vector alone did not alter growth characteristics of acinar cells. A polyclonal antibody monospecific to a murine amino terminal peptide sequence revealed a uniform distribution of GTA-kinase over the cytoplasm of acinar and duct cells of control mouse parotid glands. However, upon growth stimulation, kinase was detected primarily in a perinuclear and nuclear immunostaining pattern. Western blot analysis confirmed a translocation from a cytoplasmic localization in both LSTRA and quiescent salivary cells to a membrane-associated localization in YC-8 and proliferating salivary cells.

Genotypic Diversity among Bacillus cereus and Bacillus thuringiensis Strains

Twenty-four strains of Bacillus cereus were analyzed by pulsed-field gel electrophoresis (PFGE) and compared with 12 Bacillus thuringiensis strains. In addition, the 36 strains were examined for variation in 15 chromosomal genes encoding enzymes (by multilocus enzyme electrophoresis [MEE]). The genome of each strain had a distinct NotI restriction enzyme digestion profile by PFGE, and the 36 strains could be assigned to 27 multilocus genotypes by MEE. However, neither PFGE nor MEE analysis could distinguish between the two species. Two of the B. cereus strains contained extrachromosomal DNA that hybridized to a cryIA insecticidal toxin probe, and seven strains contained DNA with homology to a Tn4430 transposon probe derived from B. thuringiensis. The results strongly indicate that B. cereus and B. thuringiensis should be regarded as one species.

Two Genetically Diverse Groups of Strains Are Included in Xanthomonas campestris pv. vesicatoria

Two genetically diverse groups of strains were identified among cultures of Xanthomonas campestris pv. vesicatoria isolated from plants with bacterial spot of pepper and tomato. Group A strains do not pit pectate gels or hydrolyze starch, whereas group B strains are strongly positive for these reactions. Group A strains cause a hypersensitive reaction in plants of tomato breeding line Hawaii 7998, but group B strains do not. Other differences between the two groups of strains were discovered in tests for utilization of carbon compounds, serology, fatty acid profiles, silver-stained protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and DNA restriction enzyme digestion profiles. The levels of DNA homology between strains belonging to the same group were more than 74%, but the levels of DNA homology between strains belonging to different groups were less than 46%. The two groups of strains have different genetic backgrounds, but cause essentially the same disease of tomato and pepper.

On the mechanism of cytoplasmic male sterility in the 447 line of Vicia faba

The trait of cytoplasmic male sterility, expressed in plants bearing the 447 cytoplasm of Vicia faba, is uniquely and positively correlated with the presence of a linear double-stranded RNA molecule (dsRNA) 16.7 kb in size. Restriction enzyme digestion profiles of mitochondrial DNA isolated from fertile and cytoplasmic malesterile (CMS) lines do show a limited number of specific differences in fragment intensities and mobilities. However, mitochondria isolated from the progeny of the cross CMS × Restorer line contain DNA with an identical restriction profile as the male-sterile parent: moreover, subsequent generations are completely and permanently fertile, even upon segregation of the nuclear restoration gene. Southern hybridizations, using cDNA clones as probes, reveal homology between the CMS-associated dsRNA and the nuclear genome of both sterile and fertile lines. The regions cloned, representing approximately 22% of the total dsRNA sequence, show no homology to organelle DNA. We have not been able to stably transmit the dsRNA to fertile lines of V. faba or any other plant species, using a variety of standard virological techniques.

Plasmid-associated virulence of Salmonella typhimurium.

We investigated the role of the 100-kilobase (kb) plasmid of Salmonella typhimurium in the virulence of this organism for mice. Three strains, LT2-Z, SR-11, and SL1344, which possessed 100-kb plasmids with identical restriction enzyme digestion profiles, were cured of their respective 100-kb plasmids after Tnmini-tet was used to label plasmids. Curing wild-type virulent strains SR-11 and SL1344 raised peroral 50% lethal doses from 3 x 10(5) and 6 x 10(4) CFU, respectively, to greater than 10(8) CFU. Both wild-type strains had intraperitoneal 50% lethal doses of less than 50 CFU, whereas the intraperitoneal 50% lethal doses for cured SR-11 and SL1344 were less than 50 and 400 CFU, respectively. Reintroduction of the Tnmini-tet-labeled, 100-kb plasmid restored wild-type virulence. Invasion from Peyer's patches to mesenteric lymph nodes and spleens after peroral inoculation was the stage of pathogenesis most affected by curing S. typhimurium of the 100-kb plasmid. Wild-type S. typhimurium replicated in spleens of mice inoculated intravenously to a greater extent than did plasmid-cured derivatives. Wild-type and cured strains equally adhered to and invaded Henle-407, HEp-2, and CHO cells; furthermore, the presence of the 100-kb plasmid was not necessary for replication of S. typhimurium within CHO cells. The 100-kb plasmid had no effect on phagocytosis and killing of S. typhimurium by murine peritoneal macrophages in vitro and in vivo. Similarly, wild-type and plasmid-cured strains were resistant to killing by 90% normal human, rabbit, and guinea pig sera. All wild-type and plasmid-cured S. typhimurium strains possessed complete lipopolysaccharide, as determined by silver staining solubilized cells in sodium dodecyl sulfate-polyacrylamide gels. We have confirmed the role of the 100-kb plasmid of S. typhimurium in virulence, primarily in invasion to mesenteric lymph nodes and spleens after peroral inoculation of mice. Involvement of the 100-kb plasmid in infection of mesenteric lymph nodes and spleens suggests a role for the plasmid in the complex interaction of S. typhimurium with cells of the reticuloendothelial system.