Absence Of Kanamycin Research Articles

A ratiometric electrochemical aptasensor for sensitive detection of kanamycin in food based on entropy-driven strand displacement reaction

Kanamycin (KAN) has the potential to cause significant harm to human health, so the KAN residue present in food should be subject to strict regulation and control. Therefore, we constructed an enzyme-free ratiometric electrochemical aptasensor based on entropy-driven strand displacement reaction (ESDR) for sensitive detection of KAN in food. In the presence of KAN, it binds specifically to the aptamer, causing KAN-sensitive aptamer-tDNA (A-T) complex dissociation and the release of the tDNA, which triggers ESDR and results in an IFc/IMB maximum. In the absence of KAN, the release of the tDNA is not possible, leading to minimal IFc/IMB value. The aptasensor has a LOD of 74 fg/mL, enabling trace detection of KAN. In addition, the aptasensor demonstrated high specificity for KAN and remarkable reproducibility (RSD: 2.455%), which can be utilised for evaluating KAN concentrations in real samples. Moreover, the recovery rate of KAN in food samples ranged from 93.4% to 105.4%, suggesting that the ratiometric electrochemical aptasensor holds great potential in food safety detection.

Construction of Liquid Crystal-Based Sensors Using Enzyme-Linked Dual-Functional Nucleic Acid on Magnetic Beads.

The development of liquid crystal (LC)-based sensors with superior performances such as high portability, excellent stability, great convenience, and remarkable sensitivity is highly demanded. This work proposes a new strategy for constructing the LC-based sensor using enzyme-linked dual-functional nucleic acid (d-FNA) on magnetic beads (MBs). The detection of kanamycin (KA) is demonstrated as a model. Acetylcholinesterase (AChE) is assembled onto the KA aptamer-modified MBs with a d-FNA strand that consists of an AChE aptamer and the complementary sequence of a KA aptamer. As the specific recognition of KA by its aptamer triggers the release of AChE from the MBs, the myristoylcholine (Myr) solution after incubation with the MBs causes the black image of the LCs due to the formation of the Myr monolayer at the aqueous/LC interface. Otherwise, in the absence of KA, AChE is still decorated on the MBs and causes the hydrolysis of Myr. Therefore, a bright image of LCs is obtained. The detection of KA is successfully achieved with a lower detection limit of 48.1 pg/mL. In addition, a thin polydimethylsiloxane (PDMS) layer-coated glass and a portable optical device are used to improve the stability and portability of the LC-based sensor to advance potential commercial applications. Furthermore, the detection of KA in milk with a portable device is demonstrated, showing the potential of the proposed enzyme-linked LC-based sensor.

A competitive colorimetric aptasensor for simple and sensitive detection of kanamycin based on terminal deoxynucleotidyl transferase-mediated signal amplification strategy

We developed a rapid and sensitive colorimetric biosensor based on competitive recognition between kanamycin (KAN), magnetic beads-kanamycin (MBs-KAN) and aptamer and terminal deoxynucleotidyl transferase (TdT)-mediated signal amplification strategy. In the absence of KAN, aptamers recognize MBs-KAN. TdT can amplify oligonucleotides to the 3′-OH ends of aptamers, with biotin-dUTP being embedded in the long single stranded DNA (ssDNA). Then the assay produced visual readout due to the horseradish peroxidase (HRP)-catalyzed color change of the substrate after the linkage between biotin and streptavidin (SA)-HRP. In the presence of KAN, however, aptamers tend to bind free KAN rather than MBs-KAN. In this case, aptamers are isolated by magnetic separation, resulting in the failure of signal amplification and catalytic signals. This competitive colorimetric sensor showed excellent selectivity toward KAN with the limit of detection (LOD) as low as 9 pM. And recovery values were between 93.8 and 107.8% when spiked KAN in milk and honey samples.

Highly sensitive electrochemiluminescence aptasensor based on a g-C3N4-COOH/ZnSe nanocomposite for kanamycin detection

In this paper, carboxylated C3N4 (g-C3N4-COOH) was prepared by a new method, and then combined with ZnSe quantum dots (QDs) through amide reaction to form g-C3N4-COOH/ZnSe nanocomposite and used for detecting kanamycin. The prepared g-C3N4-COOH/ZnSe nanocomposite was used as the subatrate of aptasensor. Then, the g-C3N4-COOH/ZnSe nanocomposite-modified electrode was incubated with aptamer DNA (NH2-DNA), after cholamine blocked the unbond sites of the electrode surface, the ferrocene-labled quenching probe (Fc-DNA) as electrochemiluminescence (ECL) signal quencher was incubated on the electrode surface through base complementation with aptamer DNA. In the absence of kanamycin, the ECL signal was quenched effectively by Fc-DNA. On the contrary, kanamycin was specifically bond with aptamer DNA, and Fc-DNA was detached from the aptasensor electrode surface because of the deformation of aptamer DNA, generating an effectively enhanced ECL signal. The aptasensor exhibited a wide detection range for detecting kanamycin (1.0 nM − 100 μM) with a low limit of detection (0.7982 nM). The prepared aptasensor displayed a stable and selective detection for kanamycin, and also provided a promising strategy for a variety of other protein target.

Fluorescence resonance energy transfer-based aptasensor for sensitive detection of kanamycin in food

Kanamycin (KAN) is widely used in animal husbandry to treat bacterial infections. However, excessive KAN may cause residues and be transmitted to humans and the environment, causing serious adverse effects on humans. Herein, a simple fluorescence resonance energy transfer (FRET)-based aptasensor has been developed for sensitive detection of KAN in food. In the absence of KAN, UCNPs-aptamer hybridized with BHQ3-cDNA and quenched fluorescence was observed due to the FRET effect between BHQ3 and UCNPs. In the presence of KAN, double strands separated and the fluorescence intensity was recovered. Additionally, a linear relation (R2 = 0.9926) was found in the range of 0.05–50 μM and the recovered fluorescence intensity at 654 nm with a detection limit of 18.9 nM. The method was verified by standard recovery method and HPLC with satisfactory recovery rate (87.0–109.6%) and accuracy (P > 0.05). These results showed the proposed method could be successfully applied to detect KAN in food samples.

Development of Structure-Switching Aptamers for Kanamycin Detection Based on Fluorescence Resonance Energy Transfer.

The structure-switching aptamers are designed for the simple and rapid detection of kanamycin based on the signal transduction principle of fluorescence resonance energy transfer (FRET). The structure switch is composed of kanamycin-binding aptamers and the complementary strands, respectively labeled with fluorophore and quencher, denoted as FDNA and QDNA. In the absence of kanamycin, FDNA and QDNA form the double helix structure through the complementary pairing of bases. The fluorophore and the quencher are brought into close proximity, which results in the fluorescence quenching because of the FRET mechanism. In the presence of kanamycin, the FDNA specifically bind to the target due to the high affinity of aptamers, and the QDNA are dissociated. The specific recognition between aptamers and kanamycin will obstruct the formation of structure switch and reduce the efficiency of FRET between FDNA and QDNA, thus leading to the fluorescence enhancement. Therefore, based on the structure-switching aptamers, a simple fluorescent assay for rapid detection of kanamycin was developed. Under optimal conditions, there was a good linear relationship between kanamycin concentration and the fluorescence signal recovery. The linear range of this method in milk samples was 100–600 nM with the detection limit of 13.52 nM (3σ), which is well below the maximum residue limit (MRL) of kanamycin in milk. This method shows excellent selectivity for kanamycin over the other common antibiotics. The structure-switching aptamers have been successfully applied to the detection of kanamycin spiked in milk samples with the satisfying recoveries between 101.3 and 109.1%, which is well-consistent with the results from LC-MS/MS. Due to the outstanding advantages of facile operation, rapid detection, high sensitivity, excellent specificity, and low cost, the application and extension of this strategy for rapid determination of antibiotics in food samples may greatly improve the efficiency in food safety and quality supervision.

A "turn-on" fluorometric assay for kanamycin detection by using silver nanoclusters and surface plasmon enhanced energy transfer.

A rapid method is described for the determination of the antibiotic kanamycin. It integrates a kanamycin-binding aptamer and surface plasmon enhanced energy transfer (SPEET) between DNA-templated silver nanoclusters (AgNCs) and gold nanoparticles (AuNPs). The AgNCs and AuNPs were selected as energy donor and energy acceptor, respectively. The aptamer was designed to regulate the energy transfer between AgNCs and AuNPs. The aptamer was adsorbed on the AuNPs. Upon addition of kanamycin, the aptamer-kanamycin complex is formed, and this results in the aggregation of the AuNPs in high salt concentration, the formation of a blue coloration, and in the suppression of the SPEET process. The fluorescence of the AgNCs (with excitation/emission peaks at 560/600nm) is quenched by the aptamer protected AuNPs in absence of kanamycin. The fluorescence on addition of kanamycin increases linearly in the 5 to 50nM concentration range, with a lower detection limit of 1.0 nM (at S/N = 3). The assay can be performed within 30min. It was successfully applied to the determination of kanamycin in spiked milk samples, and recoveries ranged between 90.2 and 95.4%. Conceivably, the strategy has a wide potential for screening by simply changing the aptamer. Graphical abstract Schematic presentation of the aptamer regulated surface plasmon enhance energy transfer (SPEET) between silver nanoclusters (Ag NCs) and gold nanoparticles (Au NCs) in high salt concentration buffer, and of the procedure for the detection of kanamycin.

Multiplex electrochemical aptasensor for detecting multiple antibiotics residues based on carbon fiber and mesoporous carbon-gold nanoparticles

We proposed a multiplex electrochemical aptasensor based on a screen-printed carbon electrode (SPCE), which was modified by carbon nanofibers (CNFs) and mesoporous carbon-gold nanoparticles (OMC-AuNPs) for the ultrasensitive detection of kanamycin (KAN) and streptomycin (STR). The designed aptasensor had some characteristics of a high electrochemical conductivity and a high specific surface area because of rod-like OMC-AuNPs and CNFs. Moreover, CNFs and OMC-AuNPs could be homogeneously and firmly adhered to the surface of SPCE, and complimentary strands of aptamers could also be well immobilized on the surface of the electrode modified with CNFs and OMC-AuNPs. In the absence of KAN and STR, the aptamers bound to their complimentary strands. Upon addition of KAN and STR, the aptamers bound to their targets, which led to the complementary strands released from the aptamers and more changes of current peaks because of the aptamers labelled with CdS and PbS. Under the optimal conditions, this aptasensor showed a high stability and a selectivity toward KAN and STR with limits of detection (LODs) as low as 87.3 and 45.0 pM, respectively. The applicability of the developed aptasensor was successfully assessed by detection of KAN and STR in a spiked milk sample without any interference from the sample matrix. It is expected that the proposed aptasensor can be easily detect residues of other antibiotics in milk by replacing different aptamers.

Novel aptasensor for the ultrasensitive detection of kanamycin based on grapheneoxide quantum-dot-linked single-stranded DNA-binding protein

We coupled single-stranded DNA-binding protein (SSB) with graphene oxide quantum dots (GOQDs) to form QDs–SSB, as a new type of fluorescent probe. Considering the superiority of FRET and the urgency of realizing kanamycin (KAN) sensitive detection, we combined this fluorescent probe with Apt-BHQ1, and applied the FRET principle to achieve ultra-trace detection of KAN. The superiority of detection depends on: SSB has a strong affinity for aptamer (apt) in free state but not in collapsed state; The aptamer is in a free state when not bound to the target and is folded when combined with the target. In the absence of KAN, the fluorescence of the QDs–SSB was quenched by Apt-BHQ1 via FRET. However, when the KAN was added to the QDs–SSB/apt-BHQ1 system, the combination of aptamer with KAN caused the aptamer to assume a folded state and lose its ability to bind with SSB, thereby reducing FRET and the fluorescence of QDs–SSB recovery. The detection limit of this aptasensor was low to 6 pg/mL with a linear range of 0.01–90 ng/mL. Moreover, the method had high selectivity, stability, and anti-interference. Thus, this is a promising method that can be widely used for biological detection.

Nitrogen fertilization induced changes in ammonia oxidation are attributable mostly to bacteria rather than archaea in greenhouse-based high N input vegetable soil

Little is known about the effects of nitrogen (N) fertilization rates on ammonia-oxidizing bacteria (AOB) and archaea (AOA) and their differential contribution to ammonia oxidation, particularly in greenhouse-based high N input vegetable soils. Potential ammonia oxidation (PAO) of these vegetable soils was evaluated under five levels of N fertilization (with urea) in the presence or absence of the bacterial protein synthesis inhibitors kanamycin and gentamicin. Abundance and community composition of AOB and AOA were evaluated by quantitative polymerase chain reaction (PCR) and clone libraries. The five annual N fertilization rates studied were 100% (N870: 300, 270 and 300 kg N ha−1 for tomato, cucumber and celery, respectively), 80% (N696), 60% (N522), 40% (N348) and 0% (N0) of the conventional N rate. The PAO decreased significantly with increasing N fertilization rates irrespective of the presence of kanamycin plus gentamicin. PAO was significantly lower in the presence than in the absence of kanamycin plus gentamicin, and was decreased by 71.9% under N0, 77.2% under N348, 54.9% under N522, 49.9% under N696, and 51.6% under N870. The abundance of bacterial and archaeal amoA genes was significantly decreased at the highest N fertilization rate. The clone sequences of AOB and AOA were mostly affiliated with the genus Nitrosospira and group 1.1b thaumarchaeota. Changes in community composition were more pronounced in AOB than in AOA after long-term (6-year) N fertilization. Our results suggest that AOB may play a more important role than AOA in NH3 oxidation in such high N input vegetable soils.

A selective and sensitive fluorescent aptasensor for detection of kanamycin based on catalytic recycling activity of exonuclease III and gold nanoparticles

Kanamycin is an aminoglycoside antibiotic used in human and veterinary medicine. Sensitive and selective methods to detect kanamycin residues for food safety and clinical diagnosis are of great interest. In this study a fluorescent aptasensor was designed to detect kanamycin based on exonuclease III activity (Exo III), gold nanoparticles (AuNPs) and FAM-Labeled complimentary strand of aptamer (CS).In the absence of kanamycin, aptamer binds to its CS to form a double-stranded DNA (dsDNA) with 3′-overhang end at aptamer and 3′-blunt end at CS. The formed dsDNA leaves the surface of AuNPs. Upon addition of Exo III, aptamer is recycled from dsDNA and the cycle goes on, leading to a very strong fluorescence emission. In the presence of kanamycin, aptamer binds to its target and CS remains on the surface of AuNPs, resulting in a weak fluorescence emission.The designed aptasensor showed high selectivity toward aminoglycoside antibiotics, including kanamycin and gentamicin, with a limit of detection (LOD) as low as 321pM for kanamycin.This aptasensor was successfully used to detect kanamycin in milk and serum.

An aptamer-based signal-on bio-assay for sensitive and selective detection of Kanamycin A by using gold nanoparticles

In this study, a simple and sensitive aptamer-based fluorescence method for the detection of Kanamycin A by using gold nanoparticles (AuNPs) has been developed. In this assay, AuNPs were utilized as DNA nanocarrier as well as efficient fluorescence quencher. In the absence of Kanamycin A, dye-labeled aptamer could be adsorbed onto the surface of AuNPs and the fluorescence signal was quenched. In the presence of Kanamycin A, the specific binding between dye-labeled aptamer and its target induced the formation of rigid structure, which led to dye-labeled aptamer releasing from the surface of AuNPs and the fluorescence intensity was recovered consequently. Under optimum conditions, calibration modeling showed that the analytical linear range covered from 0.8nM to 350nM and the detection limit of 0.3nM was realized successfully. This proposed bio-assay also showed high selectivity over other antibiotics. Meanwhile, this strategy was further used to determine the concentrations of Kanamycin A in milk sample with satisfying results.

RNA-seq analysis of the effect of kanamycin and the ABC transporter AtWBC19 on Arabidopsis thaliana seedlings reveals changes in metal content.

Plants are exposed to antibiotics produced by soil microorganisms, but little is known about their responses at the transcriptional level. Likewise, few endogenous mechanisms of antibiotic resistance have been reported. The Arabidopsis thaliana ATP Binding Cassette (ABC) transporter AtWBC19 (ABCG19) is known to confer kanamycin resistance, but the exact mechanism of resistance is not well understood. Here we examined the transcriptomes of control seedlings and wbc19 mutant seedlings using RNA-seq analysis. Exposure to kanamycin indicated changes in the organization of the photosynthetic apparatus, metabolic fluxes and metal uptake. Elemental analysis showed a 60% and 80% reduction of iron uptake in control and wbc19 mutant seedlings respectively, upon exposure to kanamycin. The drop in iron content was accompanied by the upregulation of the gene encoding for FERRIC REDUCTION OXIDASE 6 (FRO6) in mutant seedlings but not by the differential expression of other transport genes known to be induced by iron deficiency. In addition, wbc19 mutants displayed a distinct expression profile in the absence of kanamycin. Most notably the expression of several zinc ion binding proteins, including ZINC TRANSPORTER 1 PRECURSOR (ZIP1) was increased, suggesting abnormal zinc uptake. Elemental analysis confirmed a 50% decrease of zinc content in wbc19 mutants. Thus, the antibiotic resistance gene WBC19 appears to also have a role in zinc uptake.

An oligonucleotide-based switch-on luminescent probe for the detection of kanamycin in aqueous solution

Abstract We report herein the first oligonucleotide-based luminescent method for the sensitive and selective detection of kanamycin in aqueous solution. The assay utilizes a square-planar luminescent platinum(II) complex and the kanamycin aptamer. The luminescence signal of the platinum(II) complex with the oligonucleotide is weak in the absence of kanamycin. Upon the addition of kanamycin, the aptamer changes from a random-coiled structure into a specific conformation containing a hairpin region. This facilitates intercalation of the platinum(II) complex into the bound aptamer, thereby resulting in an enhanced luminescence signal of the system in response to kanamycin. The assay is sensitive and selective towards kanamycin over other antibiotics. The assay could be used to detect trace levels of kanamycin in fish extracts with a comparable sensitivity to aqueous buffer solution.

Agrobacterium-mediated genetic transformation of pigeon pea [Cajanus cajan (L.) Millsp.] for resistance to legume pod borer Helicoverpa armigera

Agrobacterium-mediated genetic transformation was performed using embryonic axes explants of pigeon pea. Both legume pod borer resistant gene (cry1Ac) and plant selectable marker neomycine phosphor transferase (nptII) genes under the constitutive expression of the cauliflower mosaic virus 35S promoter (CaMV35S) assembled in pPZP211 binary vector were used for the experiments. An optimum average of 44.61% successfully hardened dot blot Southern hybridization positive plants were obtained on co-cultivation media supplemented with 200 μM acetosyringone without L-cysteine. The increased transformation efficiency from a baseline of 11.53% without acetosyringone to 44.61% with acetosyringone was further declined with the addition of different concentrations of L-cysteine to co-cultivation media. Transgenic shoots were selected on 50 and 75 mg L−1 kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 20 g L−1 sucrose and 0.5 mg L−1 indole butyric acid in the absence of kanamycin. Furthermore, 100% seed setting was found among all the transgenic events. The plants obtained were subjected to multi- and nochoice tests to determine the behavioral responses and mortality through Helicoverpa armigera bioassays on the leaf and relate their relationship with the expression of cry1Ac protein which was found to be less in leaf as compared to the floral buds, anther, pod, and seed.

Biodegradation of chicken feathers waste directed by Bacillus subtilis recombinant cells: Scaling up in a laboratory scale fermentor

Biodegradation of chicken feathers waste directed by Bacillus subtilis DB 100 (p5.2) cells was successfully carried out in 14L Bio Flo 110 laboratory scale fermentor. Seven liters of feathers-based modified basal medium II, feathers-based tap water and feathers-based distilled water separately in the fermentor were inoculated with activated bacterial cells. The fermentation processes were conducted at 37°C, 700 rpm agitation speed and 0.7 vvm air flow rate in the absence of kanamycin. Highest net levels of released feathers hydrolysis end products [soluble proteins and NH(2)-free amino groups] and keratinolytic alkaline protease activity in the fermentor were greatly comparable to those of shake flasks. Interestingly, the plasmid (p5.2) inside the recombinant B. subtilis cells growing in the fermentor displayed 100% stability till the fifth day of incubation and this presents a great challenge. Data certainly would encourage the transfer to larger scale fermentors to carry out feathers biodegradation process.

Key determinants affecting sheep wool biodegradation directed by a keratinase-producing Bacillus subtilis recombinant strain

OVAT (one variable at a time) approach was applied in this study to screen the most important physicochemical key determinants involved in the process of sheep wool biodegradation. The process was directed by a keratinase-producing Bacillus subtilis DB 100 (p5.2) recombinant strain. Data indicate that, sheep wool could be degraded efficiently in cultures incubated at 30°C, with initial pH of 7 with agitation at 150 rpm. Two times autoclaved alkali treated and undefatted chopped sheep wool is more accessible to biodegradation. B. subtilis recombinant cells could utilize sheep wool as a sole source of carbon and nitrogen. Sheep wool-based modified basal medium II, lacking NH₄Cl and yeast extract, could greatly support the growth of these bacterial cells. Sheep wool biodegradation was conducted efficiently in the absence of kanamycin consequently; high stability of the recombinant plasmid (p5.2) represents a great challenge upon scaling up this process. Three key determinants (sheep wool concentration, incubation time and inoculum size) imposing considerable constraints on the process are highlighted. Sheep wool-based tap water medium and sheep wool-based distilled water medium were formulated in this study. High levels of released end products, produced from sheep wool biodegradation are achieved upon using these two sheep wool-based water media. Data indicate that, sheep wool hydrolysate is rich in some amino acids, such as tyrosine, phenylalanine, lysine, proline, isoleucine, leucine, valine, aspartic acid and glutamic acid. Moreover, the resulting sheep wool hydrolysate contains soluble proteins of high and intermediate molecular weights. The present study demonstrates a feasible, cheap, reproducible, efficient and rapid biotechnological approach towards utilization of raw sheep wool waste through a recombinant bacterium.

Kanamycin-resistant alfalfa has a point mutation in the 16S plastid rRNA.

Genes conferring resistance to kanamycin are frequently used to obtain transgenic plants as spontaneous resistance to kanamycin is not known to exist in higher plants. Nevertheless, mutations conferring kanamycin resistance have been identified in Chlamydomonas reinhardtii, raising the question as to why kanamycin-resistant mutants have not been found in higher plants. While attempting plastid transformation of alfalfa, we obtained non-transgenic but kanamycin-resistant somatic embryos following 2 months of culture in the presence of 50 mg l(-1) kanamycin. Sequencing of the plastid DNA region corresponding to the decoding site of the 16S rRNA in ten independent resistant events revealed an A to C transversion at position 1357 of the 16S plastid rDNA, the same site at which an A to G conversion confers kanamycin resistance to C. reinhardtii by reducing the ability of the antibiotic to bind to its target site. All plants derived from the resistant embryos through additional cycles of somatic embryogenesis in the absence of kanamycin retained the mutant phenotype, suggesting that the mutation was homoplastomic. Resistant plants produced 85% less biomass than controls; their leaves were chlorotic during early development and over time slowly turned green. The absence of kanamycin- resistant mutants in higher plants might be explained by the requirement for a regeneration system capable of resulting in homoplastomic individuals, or it may be the result of the detrimental effect of the mutation on the phenotype.

Antibiotic-induced DNA methylation changes in calluses of Arabidopsis thaliana

This study was carried out to determine the involvement of the antibiotic kanamycin, commonly used as a selective agent in transformation protocols, in the phenomenon of somaclonal variation. Both genetic and epigenetic events were looked for. Two complementary approaches were used to evaluate global methylation changes in the genome of callus derived from the leaves of Arabidopsis thaliana L.; immunolabelling using monoclonal-antibodies raised against 5-methylcytosine in order to define the relative abundance of methylated cytosine; the analysis of methylation-sensitive polymorphism technique (MSAP) to assess methylation changes at CCGG sequences. In addition, the same samples were analysed using AFLPs in order to determine the extent of genomic change with respect to callus grown in the absence of kanamycin and plants grown from seed. The use of kanamycin as a selective agent caused extensive methylation changes in the genome with both hyper- and hypomethylation events seen. However, the net result was genome-wide hypomethylation: this effect was dosage dependent; the higher the dose, the greater the effect. At the same time, sequence mutation was detected.

A system for generalized mutagenesis of Haemophilus ducreyi

The lack of a generalized mutagenesis system for Haemophilus ducreyi has hampered efforts to identify virulence factors expressed by this sexually transmitted pathogen. To address this issue, the transposable element Tn1545-delta 3, which encodes resistance to kanamycin, was evaluated for its ability to insert randomly into the H. ducreyi chromosome and produce stable, isogenic mutants. Electroporation of H. ducreyi with 1 microgram of plasmid pMS1 carrying Tn1545-delta 3 resulted in the production of 10(4) kanamycin-resistant transformants; Southern blot analysis of a number of these transformants indicated that insertion of the transposon into the chromosome occurred at a number of different sites. This pMS1-based transposon delivery system was used to produce an H. ducreyi mutant that expressed an altered lipooligosaccharide (LOS). Passage of this mutant in vitro in the presence or absence of kanamycin did not affect the stability of the transposon insertion. To confirm that the observed mutant phenotype was the result of the transposon insertion, a chromosomal fragment containing Tn1545-delta 3 was cloned from this H. ducreyi LOS mutant. Electroporation of the wild-type H. ducreyi strain with this DNA fragment yielded numerous kanamycin-resistant transformants, the majority of which had the same altered LOS phenotype as the original mutant. Southern blot analysis confirmed the occurrence of proper allelic exchange in the LOS-deficient transformants obtained in this backcross experiment. The ability of Tn1545-delta 3 to produce insertion mutations in H. ducreyi should facilitate genetic analysis of this pathogen.