Antibiotic Resistance Cassette Research Articles

Lagging Strand-Biased Initiation of Red Recombination by Linear Double-Stranded DNAs

The recombination functions of the lambda bacteriophage, collectively called Red, can be exploited for simple and specific in vivo genetic manipulations of Escherichia coli, which is of great use in functional genomics, BAC (bacterial artificial chromosome) engineering, and gene therapy. Linear double-stranded DNAs (dsDNAs), such as PCR products flanked by short homologous sequences, can be used as recombination substrates with high efficiency. However, the molecular details of Red recombination must be more clearly understood to enable more effective substrate designs for complex applications. Here, we performed two recombination assays to show that Red recombination between linear dsDNAs and circular bacterial chromosomes takes place at the replication fork by single-strand annealing. By attaching nonhomologous segments to either end of an antibiotic resistance cassette, we measured the effects on recombination efficiency and found that nonhomology at one end was more inhibitory than that at the other end. We then designed linear dsDNAs that harbored three homologous regions and two antibiotic resistance genes to examine which of the two ends was more prone to initiating recombination. We found that the 3' single-stranded end complementary to the lagging-strand template of the bacterial chromosome preferentially annealed to the targets. Our results suggest that Red recombination occurs by an annealing and replication-dependent mechanism that involves the sequential exposure of homologous chromosomal regions as the replication fork advances, with the lagging-strand homologous region unwound first.

A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis

The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take approximately 1 month.

A family of serine proteases of Clavibacter michiganensis subsp. michiganensis: chpC plays a role in colonization of the host plant tomato

Genes for seven putative serine proteases (ChpA-ChpG) belonging to the trypsin subfamily and homologous to the virulence factor pat-1 were identified on the chromosome of Clavibacter michiganensis subsp. michiganensis (Cmm) NCPPB382. All proteases have signal peptides indicating export of these proteins. Their putative function is suggested by two motifs and an aspartate residue typical for serine proteases. Furthermore, six cysteine residues are located at conserved positions. The genes are clustered in a chromosomal region of about 50 kb with a significantly lower G + C content than common for Cmm. The genes chpA, chpB and chpD are pseudogenes as they contain frame shifts and/or in-frame stop codons. The genes chpC and chpG were inactivated by the insertion of an antibiotic resistance cassette. The chpG mutant was not impaired in virulence. However, in planta the titre of the chpC mutant was drastically reduced and only weak disease symptoms were observed. Complementation of the chpC mutant by the wild-type allele restored full virulence. ChpC is the first chromosomal gene of Cmm identified so far that affects the interaction of the pathogen with the host plant.

The Streptococcus pneumoniae Competence Regulatory System Influences Respiratory Tract Colonization

The Streptococcus pneumoniae ComDE two-component signaling system controls the development of genetic competence in the bacterium and affects virulence in models of pneumonia and bacteremia. We have investigated the impact of the competence pathway during colonization of the nasopharynx, the principal ecological niche of the pneumococcus. Previous work showed that deletion of the pneumococcal CiaRH signaling system inhibited colonization and increased expression of genes required for competence. We anticipated that signaling by the competence pathway might similarly reduce carriage. Consistent with this expectation, a comE deletion that blocked transformation increased colonization fitness such that the mutant outcompeted the wild type in an infant rat model of asymptomatic carriage. Deletion of comD-immediately upstream of comE and likewise required for competence-similarly increased colonization fitness if the orientation of the antibiotic resistance cassette inserted into the comD locus was such that it reduced transcription of comE. However, an alternative comD deletion mutation that caused an increase in comE transcription impaired colonization instead. Activation of the competence system through a comE(D143Y) mutation did not affect colonization, but an inability to secrete the competence-stimulating peptide due to deletion of comAB produced a density-dependent reduction in colonization fitness. These results suggest a model in which signaling by the unactivated form of ComE reduces colonization fitness compared to that of bacteria in which it is either activated or absent entirely, with the most substantial fitness gain accompanying deletion of comE. This observation demonstrates that the pneumococcus incurs a substantial fitness cost in order to retain a functional competence regulatory system.

Transferable, multiple antibiotic and mercury resistance in Atlantic Canadian isolates of Aeromonas salmonicida subsp. salmonicida is associated with carriage of an IncA/C plasmid similar to the Salmonella enterica plasmid pSN254

The aim of this study was to examine the molecular basis for multiple antibiotic and mercury resistance in Canadian isolates of Aeromonas salmonicida subsp. salmonicida. Phenotypic and genotypic methods were employed to identify plasmid-associated antibiotic and mercury resistance genes and to determine the organization of those genes in multidrug-resistant (MDR) A. salmonicida isolates. The MDR phenotype was transferable via conjugation using Escherichia coli, Aeromonas hydrophila and Edwardseilla tarda as recipients. Antibiotic and mercury resistance genes were carried by a conjugative IncA/C plasmid. Three distinct antibiotic resistance cassettes were characterized; first a class I integron containing an aadA7 gene encoding for an aminoglycoside-3'-adenyltransferase, the second cassette showed 99.9% nucleotide sequence homology to a cassette previously identified in the Salmonella enterica IncA/C plasmid pSN254, containing floR, tetA, sulII and strA/strB sequences. The third cassette showed 100% nucleotide sequence similarity to a transposon-like element, containing a bla(CMY-2) beta-lactamase in association with sugE and blc sequences. This element is known to be widely distributed among clinical and food-borne Salmonella and other Enterobacteriaceae throughout Asia and the United States. Mercury resistance was linked to the presence of a mer operon that showed 100% nucleotide sequence homology to the mer operon carried by plasmid pSN254. Each MDR A. salmonicida isolate carried the same plasmid, which was related to plasmid pSN254. This is the first report of plasmid-mediated florfenicol-resistant A. salmonicida in North America. In addition, it is the first report of a plasmid-associated AmpC beta-lactamase sequence in a member of the Aeromonadaceae.

Lipopolysaccharide O-Antigen Chain Length Regulation in Pseudomonas aeruginosa Serogroup O11 Strain PA103

The Wzz proteins are important for determining the length of the O-antigen side chain attached to lipopolysaccharide (LPS). Several bacteria, including Pseudomonas aeruginosa strain PAO1 (serogroup O5), produce two such proteins responsible for the preference of two different chain lengths on the surface. Our group has previously identified one wzz gene (wzz1) within the O-antigen locus of P. aeruginosa strain PA103 (serogroup O11). In this study we have identified the second wzz gene (wzz2), located in the same region of the genome and with 92% similarity to PAO1's wzz2 gene. Mutations were generated in both wzz genes by interruption with antibiotic resistance cassettes, and the effects of these mutations were characterized. Wild-type PA103 prefers two O-antigen chain lengths, referred to as long and very long. The expression of the long O-antigen chain length was reduced in the wzz1 mutant, indicating the Wzz1 protein is important for this chain length preference. The wzz2 mutant, on the other hand, was missing O-antigens of the very long chain length, indicating the Wzz2 protein is responsible for the production of very long O-antigen. The effects of the wzz mutations on virulence were also investigated. In both serum sensitivity assays and a mouse pneumonia model of infection, the wzz1 mutants exhibited greater defects in virulence compared to either wild-type PA103 or the wzz2 mutant, indicating the long chain length plays a greater role during these infectious processes.

Construction of Targeted Insertion Mutations in Francisella Tularensis Subsp. Novicida

Francisella tularensis is one of the most deadly bacterial agents, yet most of the genetic determinants of pathogenesis are still unknown. We have developed an efficient targeted mutagenesis strategy in the model organism F. tularensis subsp. novicida by utilizing universal priming of optimized antibiotic resistance cassettes and splicing by overlap extension (SOE). This process enables fast and efficient construction of targeted insertion mutations in F. tularensis subsp. novicida that have characteristics of nonpolar mutations; optimized targeted mutagenesis strategies will promote the study of this mysterious bacterium and facilitate vaccine development against tularemia. Moreover the general strategy of gene disruption by PCR-based antibiotic resistance cassette insertion is broadly applicable to many bacterial species.

Search for Genes Essential for Pneumococcal Transformation: the RadA DNA Repair Protein Plays a Role in Genomic Recombination of Donor DNA

We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counterselection of mutants in 21 different genes during the challenge. Eight of the counterselected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants of seven of the eight remaining genes, i.e., spr0459-spr0460, spr0777, spr0838, spr1259-spr1260, and spr1357, resulted in reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation with chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and in DNA damage repair in S. pneumoniae.

DNA Polymerase V Allows Bypass of Toxic Guanine Oxidation Products in Vivo

Reactive oxygen and nitrogen radicals produced during metabolic processes, such as respiration and inflammation, combine with DNA to form many lesions primarily at guanine sites. Understanding the roles of the polymerases responsible for the processing of these products to mutations could illuminate molecular mechanisms that correlate oxidative stress with cancer. Using M13 viral genomes engineered to contain single DNA lesions and Escherichia coli strains with specific polymerase (pol) knockouts, we show that pol V is required for efficient bypass of structurally diverse, highly mutagenic guanine oxidation products in vivo. We also find that pol IV participates in the bypass of two spiroiminodihydantoin lesions. Furthermore, we report that one lesion, 5-guanidino-4-nitroimidazole, is a substrate for multiple SOS polymerases, whereby pol II is necessary for error-free replication and pol V for error-prone replication past this lesion. The results spotlight a major role for pol V and minor roles for pol II and pol IV in the mechanism of guanine oxidation mutagenesis.

Fusion PCR-targeted tylCV gene deletion of Streptomyces fradiae for producing desmycosin, the direct precursor of tilmicosin

Tilmicosin is a semisynthetic macrolide antibiotic derived from tylosin. The disruption of tylCV gene from tylosin producer— Streptomyces fradiae would result in the accumulation of desmycosin, the direct precursor of tilmicosin. TylCV gene disruption cassette was constructed by fusion PCR. The tylCV replacement strain of S. fradiae was obtained through intergeneric conjugation between S. fradiae and E. coli. The antibiotic resistance cassette is flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT RK2 to generate unmarked, nonpolar mutation. After optimization of the culture conditions, the yield of desmycosin reached 7.3 g l −l in 5 l jar fermentor, which could be scale-up for industrial fermentation to produce large amount of desmycosin for semisynthesis of tilmicosin directly.

HtrA in Porphyromonas gingivalis can regulate growth and gingipain activity under stressful environmental conditions

In several micro-organisms, HtrA, a serine periplasmic protease, is considered an important virulence factor that plays a regulatory role in oxidative and temperature stress. The authors have previously shown that the vimA gene product is an important virulence regulator in Porphyromonas gingivalis. Further, purified recombinant VimA physically interacted with the major gingipains and the HtrA from P. gingivalis. To further evaluate a role for HtrA in the pathogenicity of this organism, a 1.5 kb fragment containing the htrA gene was PCR-amplified from the chromosomal DNA of P. gingivalis W83. This gene was insertionally inactivated using the ermF-ermAM antibiotic-resistance cassette and used to create an htrA-deficient mutant by allelic exchange. In one randomly chosen isogenic mutant designated P. gingivalis FLL203, there was increased sensitivity to hydrogen peroxide. Growth of this mutant at an elevated temperature was more inhibited compared to the wild-type. Further, in contrast to the wild-type, there was a significant decrease in Arg-gingipain activity after heat shock in FLL203. However, the gingipain activity in the mutant returned to normal levels after a further 30 min incubation at room temperature. Collectively, these data suggest that HtrA may play a similar role in oxidative and temperature stress in P. gingivalis as observed in other organisms.

Gentamicin and Other Cassettes for Chromosomal Gene Replacement in Escherichia Coli

Small genetic elements conferring resistance to antibiotics are widely used for strain construction in bacteria. Here, we describe a gentamicin resistance cassette with properties optimized for efficient gene replacement in Escherichia coli. This cassette offers advantages in versatility and ease of use over previously described versions. The Red recombination system of bacteriophage λ, in the absence of other components of the phage lytic cycle, promotes efficient homologous recombination between the chromosome and linear DNAs in some bacteria (1–4). We and others have generally found that minimal antibiotic resistance cassettes, consisting of one or two genes from a transposon along with closely linked regulatory sequences, work well as selectable chromosomal markers in such crosses. Table 1 shows combinations of priming sequences and templates, which we have used for gene replacements (for an example, see Reference 5). A colony of a bacterial strain bearing the transposon can serve as template in PCR with primers that amplify the cassette and have 40-base flanks targeting the cassette to a site in the chromosome of a recipient. The PCR product in each case is small (1–2 kb) and is introduced via electroporation into a Red-expressing recipient strain, in which it generates a recombinant that can be easily selected. That is, the recombinant forms a reasonably large colony on a plate of LB agar containing a high enough concentration of an antibiotic to suppress colony formation by 108 nonrecombinants, following overnight incubation at 37°C. Cassettes of this type conferring resistance to chloramphenicol, kanamycin, and tetracycline have been used widely. The bla gene of Tn3 also works well for this purpose, but is perhaps less useful due to its widespread use as a selectable marker (ampicillin resistance) on plasmid cloning vectors. The aadA1 (spectinomycin/streptomycin resistance) gene of Tn21 and the aacC1 (gentamicin resistance) gene of Tn1696 have been developed as useful markers for other applications (6–9). We tested the suitability of cassettes, containing them for Red-mediated gene replacement, and found that both worked well. However, the gentamicin cassette suffered from two disadvantages. First, we found that cassettes lacking Tn1696 sequences downstream from the aacC1 gene conferred only weak gentamicin resistance in single copy in the chromosome. Second, the upstream regulatory sequences of aadA1 and aacC1 are nearly identical. A strain bearing aadA1 would thus contain a homology target for RedGentamicin and other cassettes for chromosomal gene replacement in Escherichia coli

Detection of Iss and Bor on the surface of Escherichia coli

To confirm the presence of Iss and Bor on the outer membrane of Escherichia coli using Western blots of outer membrane protein (OMP) preparations and fluorescence microscopy, and explore the use of fluorescence microscopy for the detection of avian pathogenic E. coli (APEC) and diagnosis of avian colibacillosis. Knockout mutants of iss and bor were created using a one-step recombination of target genes with PCR-generated antibiotic resistance cassettes. Anti-Iss monoclonal antibodies (Mabs) that cross-react with Bor protein were used to study the mutants relative to the wild-type organism. These Mabs were used as reagents to study OMP preparations of the mutants with Western blotting and intact E. coli cells with fluorescence microscopy. Iss and Bor were detected in Western blots of OMP preparations of the wild type. Also, Iss was detected on Deltabor mutants, and Bor was detected on Deltaiss mutants. Iss and Bor were also detected on the surface of the intact, wild-type cells and mutants using fluorescence microscopy. These results demonstrate that Bor and Iss are exposed on E. coli's outer membrane where they may be recognized by the host's immune system. To our knowledge, this is the first report confirming Iss' location in the outer membrane of an E. coli isolate. Such surface exposure has implications for the use of these Mabs for APEC detection and colibacillosis control.

Different versions of Pseudomonas syringae pv. tomato DC3000 exist due to the activity of an effector transposon

SUMMARY The plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000 is a key model organism to study plant-pathogen interactions. We realized that two versions of this strain, which carry plasmids of different sizes, exist in our strain collections. The difference was located to a 9.4-kb deletion within the larger of the two endogenous plasmids encompassing the partitioning genes parA and parB and a putative mobile element encoding the type III effector hopAM1-2 (formerly avrPpiB2). Both plasmid variants are lost in similar frequency, indicating that the partitioning genes are not essential for stability of the plasmid. In addition, the deletion derivative strain DC3001 exhibited the same virulence towards Arabidopsis as strain DC3000. The deletion site in DC3001 is located immediately adjacent to a putative transposon that carries the effector hopX1 (formerly avrPphE), suggesting that the deletion originated from an aberrant transposition event of this element. By tagging the hopX1 transposon with an antibiotic resistance cassette on a suicide plasmid it was shown that the element is functional and produces a target site duplication of 5 bp. The plasmid also integrated into the chromosome in several cases, possibly mediated by one-ended transposition of the hopX1 transposon. This is the first report that describes an active effector-transposon. Comparison of DC3000 strains from several sources revealed that strains exist with differences in the endogenous plasmid composition.

Developing liveShigellavaccines using λ Red recombineering

Live attenuated Shigella vaccines have shown promise in inducing protective immune responses in human clinical trials and as carriers of heterologous antigens from other mucosal pathogens. In the past, construction of Shigella vaccine strains relied on classical allelic exchange systems to genetically engineer the bacterial genome. These systems require extensive in vitro engineering of long homologous sequences to create recombinant replication-defective plasmids or phage. Alternatively, the lambda red recombination system from bacteriophage facilitates recombination with as little as 40 bp of homologous DNA. The process, referred to as recombineering, typically uses an inducible lambda red operon on a temperature-sensitive plasmid and optimal transformation conditions to integrate linear antibiotic resistance cassettes flanked by homologous sequences into a bacterial genome. Recent advances in recombineering have enabled modification of genomic DNA from bacterial pathogens including Salmonella, Yersinia, enteropathogenic Escherichia coli, or enterohemorrhagic E. coli and Shigella. These advances in recombineering have been used to systematically delete virulence-associated genes from Shigella, creating a number of isogenic strains from multiple Shigella serotypes. These strains have been characterized for attenuation using both in vivo and in vitro assays. Based on this data, prototypic Shigella vaccine strains containing multiple deletions in virulence-associated genes have been generated.

Broad host range vectors for stable genomic library construction

We describe the construction of 36 stable vectors for genomic library construction in gram-negative species. These vectors contain the pBBR1 replicon that has been shown to stably replicate in every gram-negative species tested. The plasmids also contain bidirectional, rho-independent transcriptional terminators flanking the multiple cloning site, which allows for greater insert stability, and thus, greater genomic representation. Each vector varies in its antibiotic resistance cassette, mobilization function, and promoter used to express insert sequences. These vectors should prove useful in the screening of highly representational genomic libraries in a broad variety of gram-negative species.

Genetic Manipulation of Treponema denticola

The oral anaerobic spirochete, Treponema denticola, has been implicated in the etiology of human periodontal diseases; however, the molecular basis for the virulence of these organisms is still unclear. Potential pathogenic factors expressed by T. denticola have recently begun to be identified through the development of gene transfer approaches in this organism following electroporetic transformation. Several antibiotic resistance markers have been developed for use in the construction of monospecific mutants in these organisms. In addition, these antibiotic resistance cassettes have been more recently utilized to construct shuttle plasmids for complementation analysis of the mutants. These plasmids were also used to express heterologous spirochete genes in T. denticola. The transformation of other spirochetes such as T. phagedenis with these plasmids further suggests that it should be possible to develop similar gene transfer systems in other cultivable treponemes.

A Novel Method for Somatic Cell Nuclear Transfer to Mouse Embryonic Stem Cells

Nuclear reprogramming by somatic cell nuclear transfer (SCNT) provides a practical approach for generating autologous pluripotent cells from adult somatic cells. It has been shown that murine somatic cells can also be reprogrammed to a pluripotent-like state by fusion with embryonic stem (ES) cells. Typically, the first step in SCNT involves enucleation of the recipient cell. However, recent evidence suggests that enucleated diploid ES cells may lack reprogramming capabilities. Here we have developed methods whereby larger tetraploid ES cells are first generated by fusion of two mouse ES cell lines transfected with plasmids carrying different antibiotic-resistance cassettes, followed by double antibiotic selection. Tetraploid ES cells grown on tissue culture disks or wells can be efficiently enucleated (up to 99%) using a combination of cytochalasin B treatment and centrifugation, with cytoplasts generated from these cells larger than those obtained from normal diploid ES cells. Also, we show that the enucleation rate is dependent on centrifugation time and cell ploidy. Further, we demonstrate that normal diploid ES cells can be fused to tetraploid ES cells to form heterokaryons, and that selective differential centrifugation conditions can be applied where the tetraploid nucleus is removed while the diploid donor nucleus is retained. This technology opens new avenues for generating autologous, diploid pluripotent cells, and provides a dynamic model for studying nuclear reprogramming in ES cells.

Haemophilus influenzae luxS mutants form a biofilm and have increased virulence

To gain insight into the role of luxS Hi in disease pathogenesis, we inactivated that gene in several non-typeable Haemophilus influenzae isolates with an antibiotic resistance cassette. Gene inactivation was confirmed by PCR and by Southern blot analysis in each strain. Culture filtrates from luxS Hi mutants contained a decreased amount of autoinducer-2 (AI-2) activity in comparison to the wild-type isolates using the Vibrio harveyi BB170 bioassay. Culture filtrates from Escherichia coli strain DH5α expressing a cloned luxS Hi contained 350-fold more AI-2 activity per cell than E. coli DH5α containing the vector alone. The growth rate in several liquid media, and the cell density after overnight growth were not significantly different between the parents and the luxS Hi mutants. Two clinical H. influenzae and their luxS Hi mutants produced an identical biofilm in a flow system. Invasion of human cells by the luxS Hi mutants, in comparison to the wild-type parents was strain-dependent, and cell type-dependent, but the luxS Hi mutants tended to be more invasive. The luxS Hi mutant of an otitis media isolate, strain R3157 appeared more virulent in the chinchilla model of otitis media: there were more bacteria in the middle ear, a greater inflammatory response and more goblet cell hyperplasia 10 days after the inoculation. We conclude that the H. influenzae homologue of luxS modulates certain virulence traits.

An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants

BackgroundTraditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process.ResultsWe describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family.ConclusionWhile maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria.