6-Methylpurine derived sugar modified nucleosides: Synthesis and evaluation of their substrate activity with purine nucleoside phosphorylases

6-Methylpurine (MeP) is cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli PNP. The prototype MeP releasing prodrug, 9-(β-d-ribofuranosyl)-6-methylpurine, MeP-dR has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify non-toxic MeP prodrugs that could be used in conjunction with E. coli PNP. In this work, we report on the synthesis of 9-(6-deoxy-β-d-allofuranosyl)-6-methylpurine (3) and 9-(6-deoxy-5-C-methyl-β-d-ribo-hexofuranosyl)-6-methylpurine (4), and the evaluation of their substrate activity with several phosphorylases. The glycosyl donors; 1,2-di-O-acetyl-3,5-di-O-benzyl-α-d-allofuranose (10) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-methyl-β-d-ribohexofuran-ose (15) were prepared from 1,2:5,6-di-O-isopropylidine-α-d-glucofuranose in 9 and 11 steps, respectively. Coupling of 10 and 15 with silylated 6-methylpurine under Vorbrüggen glycosylation conditions followed conventional deprotection of the hydroxyl groups furnished 5′-C-methylated-6-methylpurine nucleosides 3 and 4, respectively. Unlike 9-(6-deoxy-α-l-talo-furanosyl)-6-methylpurine, which showed good substrate activity with E. coli PNP mutant (M64V), the β-d-allo-furanosyl derivative 3 and the 5′-di-C-methyl derivative 4 were poor substrates for all tested glycosidic bond cleavage enzymes.     

EGFR‐targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells

Tumor‐targeted Salmonella VNP20009 preferentially replicate within tumor tissue and partially suppress tumor growth in murine tumor models. These Salmonella have the ability to locally induce apoptosis when they are in direct contact with cancer cells but they lack significant bystander killing, which may correlate with their overall lack of antitumor activity in human clinical studies. In order to compensate for this deficiency without enhancing overall toxicity, we engineered the bacteria to express epidermal growth factor receptor (EGFR)‐targeted cytotoxic proteins that are released into the extracellular milieu. In this study, we demonstrate the ability of the Salmonella strain VNP20009 to produce three different forms of the Pseudomonas exotoxin A (ToxA) chimeric with a tumor growth factor alpha (TGFα) which results in its producing culture supernatants that are cytotoxic and induce apoptosis in EGFR positive cancer cells as measured by the tetrazolium dye reduction, and Rhodamine 123 and JC‐10 mitochondrial depolarization assays. In addition, exchange of the ToxA REDLK endoplasmic reticulum retention signal for KDEL and co‐expression of the ColE3 lysis protein resulted in an overall increased cytotoxicity compared to the wild type toxin. This approach has the potential to significantly enhance the antitumor activity of VNP20009 while maintaining its previously established safety profile. Biotechnol. Bioeng. 2016;113: 2698–2711. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.     

Efficacy against lung metastasis with a tumor-targeting mutant of Salmonella typhimurium in immunocompetent mice

Salmonella typhimurium double leu-arg auxotrophs have been shown to be highly effective as antitumor agents in nude mouse models of human metastatic cancer. In order to proceed to clinical development of the S. typhimurium double auxotroph, termed A1-R, it is necessary to evaluate antitumor efficacy in immunocompetent mice. In the present study, we have observed the efficacy of A1-R on the Lewis lung (LLC) carcinoma in vitro as well as in C57BL/6 (C57) immunocompetent mice. In vitro, A1-R treatment of LLC began to induce cell death within one hour. Various doses and schedules of A1-R were administered to C57 mice implanted with LLC, including bolus single intravenous injection; medium dose with weekly intravenous administration and metronomic treatment with small intravenous doses twice a week. Bolus treatment was toxic to the immunocompetent host in contrast to nude mice. Lower-dose weekly doses and metronomic doses were well-tolerated by the immunocompetent host. Weekly intravenous injection with 2 × 10⁷ bacteria and twice a week intravenous injection with 10⁷ bacteria significantly inhibited metastasis formation, while bolus injection was toxic. Intrathoracic administration was performed with 10⁸ A1-R bacteria injected into Lewis lung-bearing C57 mice weekly for three weeks. Lung metastasis was significantly inhibited by intrathoracic bacterial administration without toxicity. The results in this report, demonstrating the anti-metastatic efficacy of S. typhimurium A1-R in immunocompetent mice, indicate the clinical potential of bacterial therapy of cancer.Key words: Salmonella typhimurium, amino acid auxotroph, selective tumor targeting, lung, metastasis, RFP, GFP, fluorescence imaging, confocal microscopy     

Proteomic screening of anaerobically regulated promoters from Salmonella and its antitumor applications

Solid tumors often contain hypoxic and necrotic areas that can be targeted by attenuated Salmonella typhimurium VNP20009 (VNP). We sought to develop a hypoxia- inducible promoter system based on the tumor-specific delivered strain VNP to confine expression of therapeutic gene specifically or selectively within the tumor microenvironment. A hypoxia-inducible promoter - adhE promoter was screened from the hypoxia-regulated endogenous proteins of Salmonella through two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight/time-of-flight MS-based proteomics approaches. The efficiency and specificity of the selected adhE promoter were validated first in both bacteria and animal tumor models. The adhE promoter could specifically drive GFP gene expression under hypoxia, but not under normoxia. Furthermore, luciferase reporter expression controlled by the system was also confined to the tumors. Finally, we investigated the anticancer efficacy of VNP delivering human endostatin controlled by our adhE promoter system in both murine melanoma and Lewis lung carcinoma models. Our results demonstrated that by the dual effects of tumoricidal and anti-angiogenic activities, the recombinant Salmonella strain could generate enhanced antitumor effects compared with those of unarmed VNP treatment or untreated control. The recombinant VNP could retard tumor growth significantly and extend survival of tumor-bearing mice by inducing more apoptosis and more severe necrosis as well as inhibiting blood vessel density within tumors. Therefore, VNP carrying the endostatin gene under our tumor-targeted expression system holds promise for the treatment of solid tumors.     

大肠埃希菌嘌呤核苷磷酸化酶基因载体的构建及对胰腺癌细胞的作用

目的构建大肠埃希菌（Escherichia coli）嘌呤核苷磷酸化酶（purine nucleoside phosphorylase,PNP）基因表达载体,研究其生物活性,为肿瘤的基因治疗奠定基础。方法PCR扩增大肠埃希菌K12的PNP基因,T4连接酶将PNP连接人pMSCV逆转录病毒载体,构建重组逆转录病毒载体pMSCV／PNP。pM—SCV／PNP转化感受态大肠埃希菌XLI-Blue,提取pMSCV／PNP,酶切、PCR和测序鉴定。病毒包装细胞293产生重组逆转录病毒pMSCV／PNP,流式细胞仪测病毒滴度。pMSCV／PNP转染胰腺癌细胞BXPC-3,倒置荧光显微镜观察,FACS分离转染阳性细胞（GFP阳性）。RT—PCR检测PNPmRNA在胰腺癌细胞BXPC-3细胞中的表达,MTT法检测PNP基因的生物活性。结果PCR扩增出大肠埃希菌PNP基因（738bp）,酶切和PCR的电泳条带显示pMSCV／PNP,测序结果正常。293包装细胞产生高滴度（3．6×10^7U／m1）重组逆转录病毒pMSCV／PNP。RT—PCR实验结果表明,pMSCV／PNP转染的胰腺癌细胞BXPC-3表达PNPmRNA。前药6-甲基嘌呤-2’-脱氧核苷（MePdR）作用72h浓度达1．00mg／L,BXPC-3／PNP细胞存活率为10．09％,随着MePdR浓度加大,BXPC-3／PNP细胞存活率继续下降直至为0。结论构建了pMSCV／PNP载体,获得了表达大肠埃希菌PNP基因的BXPC-3细胞克隆,PNP／MePdR自杀基因系统对胰腺癌细胞BXPC-3有较强的抑杀作用。     

DESIGN AND EVALUATION OF 5′-MODIFIED NUCLEOSIDE ANALOGS AS PRODRUGS FOR AN E. COLI PURINE NUCLEOSIDE PHOSPHORYLASE MUTANT

Our studies have led to the identification of an E. coli PNP mutant (M64V) that is able to cleave numerous 5'-modified nucleoside analogs with much greater efficiency than the wild-type enzyme. The biological activity of the three best substrates of this mutant (9-[6-deoxy-α-L-talofuranosyl]-6-methylpurine (methyl(talo)-MeP-R), 9-[6-deoxy-α-L-talofuranosyl]-2-F-adenine, and 9-[α-L-lyxofuranosyl]-2-F-adenine) were evaluated so that we can optimally utilize these compounds. Our results indicated that the mechanism of toxicity of methyl(talo)-MeP-R to mice was due to its cleavage to MeP by a bacterial enzyme, and that the toxicity of the two F-Ade analogs was due to their cleavage to F-Ade by mammalian methylthioadenosine phosphorylase.     

Design and evaluation of 5'-modified nucleoside analogs as prodrugs for an E. coli purine nucleoside phosphorylase mutant

Our studies have led to the identification of an E. coli PNP mutant (M64V) that is able to cleave numerous 5'-modified nucleoside analogs with much greater efficiency than the wild-type enzyme. The biological activity of the three best substrates of this mutant (9-[6-deoxy-alpha-L-talofuranosyl]-6-methylpurine (methyl(talo)-MeP-R), 9-[6-deoxy-alpha-L-talofuranosyl]-2-F-adenine, and 9-[alpha-L-lyxofuranosyl]-2-F-adenine) were evaluated so that we can optimally utilize these compounds. Our results indicated that the mechanism of toxicity of methyl(talo)-MeP-R to mice was due to its cleavage to MeP by a bacterial enzyme, and that the toxicity of the two F-Ade analogs was due to their cleavage to F-Ade by mammalian methylthioadenosine phosphorylase.     

Characterization of para-Nitrophenol-Degrading Bacterial Communities in River Water by Using Functional Markers and Stable Isotope Probing

Microbial degradation is a major determinant of the fate of pollutants in the environment. para-Nitrophenol (PNP) is an EPA-listed priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [¹³C₆]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified Pseudomonas syringae to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved pnpA from Pseudomonas spp. However, analysis of maleylacetate reductase (encoded by mar), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that mar has limited use as a specific marker of PNP biodegradation. Both the pnpA and mar genes were detected in a PNP-degrading isolate, P. syringae AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of Pseudomonas spp. are representative of environmental PNP-degrading populations.     

Efficacy against lung metastasis with a tumor-targeting mutant of Salmonella typhimurium in immunocompetent mice

Salmonella typhimurium double leu-arg auxotrophs have been shown to be highly effective as antitumor agents in nude mouse models of human metastatic cancer. In order to proceed to clinical development of the S. typhimurium double auxotroph, termed A1-R, it is necessary to evaluate antitumor efficacy in immunocompetent mice. In the present study, we have observed the efficacy of A1-R on the Lewis lung (LLC) carcinoma in vitro as well as in C57BL/6 (C57) immunocompetent mice. In vitro, A1-R treatment of LLC began to induce cell death within one hour. Various doses and schedules of A1-R were administered to C57 mice implanted with LLC, including bolus single intravenous injection; medium dose with weekly intravenous administration and metronomic treatment with small intravenous doses twice a week. Bolus treatment was toxic to the immunocompetent host, in contrast to nude mice. Lower-dose weekly doses and metronomic doses were well tolerated by the immunocompetent host. Weekly intravenous injection with 2 x 10⁷ bacteria and twice a week intravenous injection with 10⁷ bacteria significantly inhibited metastasis formation, while bolus injection was toxic. Intra-thoracic administration was carried out with 10⁸ bacteria A1-R injected into Lewis lung-bearing C57 mice weekly for three weeks. Lung metastasis was significantly inhibited by intrathoracic bacterial administration, without toxicity. The results in this report, demonstrating the anti-metastatic efficacy of S. typhimurium A1-R in immunocompetent mice, indicate the clinical potential of bacterial therapy of cancer.     

Vessel destruction by tumor-targeting Salmonella typhimurium A1-R is enhanced by high tumor vascularity

Our laboratory has previously developed a tumor-targeting double-auxotrophic mutant of Salmonella typhimurium termed A1-R. The present report demonstrates that S. typhimurium A1-R destroys tumor blood vessels and this is enhanced in tumors with high vascularity. Red fluorescent protein (RFP)-expressing Lewis lung cancer cells (LLC-RFP) were transplanted subcutaneously in the ear, back skin and footpad of nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice, which selectively express GFP in nascent blood vessels. Color-coded in vivo imaging demonstrated that the LLC-RFP ear tumor had the highest cell density and the footpad tumor had the least. The ear tumor had more abundant blood vessels than that on the back or footpad. The tumor-bearing mice were treated with A1-R bacteria via tail-vein injection. Tumors in the ear were the earliest responders to bacterial therapy and hemorrhaged severely the day after A1-R administration. Tumors growing in the back were the second fastest responders to bacterial treatment and appeared necrotic 3 days after A1-R administration. Tumors growing in the footpad had the least vascularity and were the last responders to A1-R. Therefore, tumor vascularity correlated positively with tumor efficacy of A1-R. The present study suggests that bacteria efficacy on tumors involves vessel destruction which depends on the extent of vascularity of the tumor.Key words: tumor targeting bacteria, Salmonella typhimurium A1-R, Lewis lung carcinoma, RFP, GFP, nestin, nude mice     

Genetic Characterization of a Stable F' lac Plasmid

A mutant F′ plasmid has been isolated in a strain of Salmonella typhimurium harboring F_ts114lac. This mutant, designated FlacS, exhibits unique genetic stability in strains of S. typhimurium and Escherichia coli. It shows no thermolability and is lost at frequencies of 20 to 100 times less than the wild-type F′lac (F42) in the same genetic backgrounds. The FlacS is also insensitive to conventional plasmid curing agents, whereas both F_ts114lac and F42 are readily cured. The nature of the mutation(s) conferring stability to the FlacS is unclear, but plasmid linkage has been established. The high frequency of conjugal transfer of the FlacS and its behavior in recombination-deficient strains of S. typhimurium and E. coli argue against its stability being due to stable chromosomal integration. The FlacS is also capable of transferring chromosomal markers in S. typhimurium and E. coli mating systems. No major differences in chromosomal mobilization have been observed among F42, F_ts114lac, and FlacS donors of either genus.     

Purine Nucleoside Phosphorylase Targeted by Annexin V to Breast Cancer Vasculature for Enzyme Prodrug Therapy

Background and Purpose

The targeting of therapeutics is a promising approach for the development of new cancer treatments that seek to reduce the devastating side effects caused by the systemic administration of current drugs. This study evaluates a fusion protein developed as an enzyme prodrug therapy targeted to the tumor vasculature. Cytotoxicity would be localized to the site of the tumor using a protein fusion of purine nucleoside phosphorylase (PNP) and annexin V. Annexin V acts as the tumor-targeting component of the fusion protein as it has been shown to bind to phosphatidylserine expressed externally on cancer cells and the endothelial cells of the tumor vasculature, but not normal vascular endothelial cells. The enzymatic component of the fusion, PNP, converts the FDA-approved cancer therapeutic, fludarabine, into a more cytotoxic form. The purpose of this study is to determine if this system has a good potential as a targeted therapy for breast cancer.

Methods

A fusion of E. coli purine nucleoside phosphorylase and human annexin V was produced in E. coli and purified. Using human breast cancer cell lines MCF-7 and MDA-MB-231 and non-confluent human endothelial cells grown in vitro, the binding strength of the fusion protein and the cytotoxicity of the enzyme prodrug system were determined. Endothelial cells that are not confluent expose phosphatidylserine and therefore mimic the tumor vasculature.

Results

The purified recombinant fusion protein had good enzymatic activity and strong binding to the three cell lines. There was significant cell killing (p<0.001) by the enzyme prodrug treatment for all three cell lines, with greater than 80% cytotoxicity obtained after 6 days of treatment.

Conclusion

These results suggest that this treatment could be useful as a targeted therapy for breast cancer.     

Antitumour activity of Salmonella typhimurium VNP20047 in B16(F10) murine melanoma model

A tumour therapy is proposed based on attenuated Salmonella typhimurium VNP20047 expressing the Escherichia coli cytosine deaminase gene. VNP20047 was administered intravenously to B16(F10) melanoma-bearing C57BL/6 mice. VNP20047 proliferated within tumours and livers regardless of the initial inoculum dose. After 10 days the number of bacteria increased in livers up to 4.2 x 10(6) cfu/g and decreased in tumours down to 5.9 x 10(6) cfu/g. VNP20047 at 1 x 10(5) cfu/mouse, when combined with 5-fluorocytosine, inhibited tumour growth by 85% without prolonging animal survival. Histology studies revealed severe lesions in tumours and livers. These data suggest that S. typhimurium VNP20047 induced inflammatory responses, even though the strain was attenuated.     

MECP2 Isoform-Specific Vectors with Regulated Expression for Rett Syndrome Gene Therapy

Background

Rett Syndrome (RTT) is an Autism Spectrum Disorder and the leading cause of mental retardation in females. RTT is caused by mutations in the Methyl CpG-Binding Protein-2 (MECP2) gene and has no treatment. Our objective is to develop viral vectors for MECP2 gene transfer into Neural Stem Cells (NSC) and neurons suitable for gene therapy of Rett Syndrome.

Methodology/Principal Findings

We generated self-inactivating (SIN) retroviral vectors with the ubiquitous EF1α promoter avoiding known silencer elements to escape stem-cell-specific viral silencing. High efficiency NSC infection resulted in long-term EGFP expression in transduced NSC and after differentiation into neurons. Infection with Myc-tagged MECP2-isoform-specific (E1 and E2) vectors directed MeCP2 to heterochromatin of transduced NSC and neurons. In contrast, vectors with an internal mouse Mecp2 promoter (MeP) directed restricted expression only in neurons and glia and not NSC, recapitulating the endogenous expression pattern required to avoid detrimental consequences of MECP2 ectopic expression. In differentiated NSC from adult heterozygous Mecp2^tm1.1Bird+/− female mice, 48% of neurons expressed endogenous MeCP2 due to random inactivation of the X-linked Mecp2 gene. Retroviral MECP2 transduction with EF1α and MeP vectors rescued expression in 95–100% of neurons resulting in increased dendrite branching function in vitro. Insulated MECP2 isoform-specific lentiviral vectors show long-term expression in NSC and their differentiated neuronal progeny, and directly infect dissociated murine cortical neurons with high efficiency.

Conclusions/Significance

MeP vectors recapitulate the endogenous expression pattern of MeCP2 in neurons and glia. They have utility to study MeCP2 isoform-specific functions in vitro, and are effective gene therapy vectors for rescuing dendritic maturation of neurons in an ex vivo model of RTT.     

Role of anaerobiosis in virulence of Salmonella typhimuirium

Intestinal pathogens are exposed to various stress conditions during their infectious cycle. Anaerobiosis, one of such hostile condition, is offered by the host within gut and intestinal lumen, where survival, multiplication and entry into intestinal epithelial cells is priority for the invading pathogen. In the present study, a virulent strain of S. typhimurium (1402/84) was grown under anaerobic conditions and its virulence characteristics such as host cell binding, penetration and intracellular survival were compared with aerobic S. typhimurium. Anaerobically grown S. typhimurium showed significantly higher binding to immobilized mice enterocytes and intestinal mucus as compared to bacteria grown aerobically. Anaerobic bacteria also showed an early penetration of mucus and subsequent binding to underlying immobilized enterocytes, in vitro. Anaerobic S. typhimurium exhibited increased intracellular survival within spleen macrophages of mice and caused significantly higher fluid accumulation in ligated rabbit ileal loops as compared to aerobic bacteria. LD₅₀ of anaerobic S. typhimurium was also observed to be 2 fold lower when compared to aerobic bacteria. Cell surface hydrophobicity of anaerobic S. typhimurium was also found to be significantly higher than aerobic bacteria. Thus, it appears that exposure of S. typhimurium to anaerobiosis results in its enhanced virulence, adhesion and penetration of host cells.     

Evolutionary implications of potential frameshift hot-spots, methylation sites, and codon usage differences between genes and between organisms in Escherichia coli and Salmonella typhimurium

The published nucleotide sequences of the E. coli and S. typhimurium trp A and trp B genes show a high degree of similarity between homologous genes of the two organisms, and an even greater degree of similarity between the amino acid sequences of the gene products. In spite of this, analysis of the nucleotide sequences reveals that there are marked differences between E. coli and S. typhimurium genes with respect to potential frameshift mutation hot-spots and dam and mec, mutationally important, methylation sites. Such existing differences may well lead to divergent evolution of these two, presently closely related, bacteria. Codon usage patterns in the trp A and trp B genes of E. coli and S. typhimurium, and the lac I gene of E. coli, have been re-analysed in terms of AT-rich, GC-rich, neutral, or unique codons and marked preferences found. In some cases particular amino acids are most often specified by AT-rich, in others by the GC-rich, alternative codons. In still other cases the codon preference depends on the gene studied. These patterns can be interpreted in terms of enteric bacterial evolution, via hybridizations, from ancestral bacteria with AT- or GC-rich DNA.     

Fine mechanisms of the interaction of silver nanoparticles with the cells of Salmonella typhimurium and Staphylococcus aureus

Silver nanoparticles possess antibacterial effect for various bacteria; however mechanisms of the interaction between Ag-NPs and bacterial cells remain unclear. The aim of our study was to obtain direct evidence of Ag-NPs penetration into cells of Gram-negative bacterium S. typhimurium and Gram-positive bacterium S. aureus, and to study cell responses to Ag-NPs. The Ag-NPs (most 8–10 nm) were obtained by gas-jet method. S. typhimurium (7.81 × 10⁷ CFU), or S. aureus (8.96 × 10⁷ CFU) were treated by Ag-NPs (0.05 mg/l of silver) in orbital shaker at 190 rpm, 37 °C. Bacteria were sampled at 0.5, 1, 1.5, 2, 5 and 23 h of the incubation for transmission electron microscopy of ultrathin sections. The Ag-NPs adsorbed on outer membrane of S. typhimurium and cell wall of S. auereus; penetrated and accumulated in cells without aggregation and damaging of neighboring cytoplasm. In cells of S. aureus Ag-NPs bound with DNA fibers. Cell responses to Ag-NPs differed morphologically in S. typhimurium and S. aureus, and mainly were presented by damage of cell structures. The cytoplasm of S. aureus became amorphous, while S. typhimurium showed lumping and lysis of cytoplasm which led to formation of “empty” cells. Other difference was fast change of cell shape in S. typhimurium, and late deformation of S. aureus cells. The obtained results showed how different could be responses induced by the same NPs in relatively simple prokaryotic cells. Evidently, Ag-NPs directly interact with macromolecular structures of living cells and are exert an active influence on their metabolism.     

Correlation between the toxicity of platinum drugs to L1210 leukaemia cells and their mutagenic properties.

There is a good correlation between the growth inhibitory properties of a series of Pt(II) derivatives on cultured L1210 mouse leukaemia cells and their efficiency of reversion to his⁺ of S. typhimurium TA100, or with their forward mutagenic effects on prophage λ. Reactivity towards DNA may thus explain the antitumor properties of these drugs.     

Gene Transfer between Related Bacteria by Electrotransformation: Mapping Salmonella typhi Genes in Salmonella typhimurium

Transfer of newly isolated mutations into a fresh background is an essential step of genetic analysis and strain construction. Gene transfer is hampered in Salmonella typhi and in other pathogenic bacteria by the lack of a generalized transduction system. We show here that this problem can be partially circumvented by using electrotransformation as a means for delivering S. typhi DNA into suitable S. typhi or Salmonella typhimurium recipients. Transferred DNA can recombine with the homologous region in the host chromosome. In one application of the method, mutations isolated in S. typhi were genetically mapped in S. typhimurium.