Development and use of an efficient system for random mariner transposon mutagenesis to identify novel genetic determinants of biofilm formation in the core Enterococcus faecalis genome

Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, most of the genetic analysis of these organisms has focused on mobile genetic elements, and existing tools for manipulation and analysis of the core E. faecalis chromosome are limited. We are interested in a comprehensive analysis of the genetic determinants for biofilm formation encoded within the core E. faecalis genome. To identify such determinants, we developed a substantially improved system for transposon mutagenesis in E. faecalis based on a mini-mariner transposable element. Mutagenesis of wild-type E. faecalis with this element yielded predominantly mutants carrying a single copy of the transposable element, and insertions were distributed around the entire chromosome in an apparently random fashion. We constructed a library of E. faecalis transposon insertion mutants and screened this library to identify mutants exhibiting a defect in biofilm formation. Biofilm-defective mutants were found to carry transposon insertions both in genes that were previously known to play a role in biofilm formation and in new genes lacking any known function; for several genes identified in the screen, complementation analysis confirmed a direct role in biofilm formation. These results provide significant new information about the genetics of enterococcal biofilm formation and demonstrate the general utility of our transposon system for functional genomic analysis of E. faecalis.     

Construction of Signature-tagged Mutant Library in Mesorhizobium loti as a Powerful Tool for Functional Genomics

Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29 330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G+C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology.Key words: Mesorhizobium loti, signature-tagged mutagenesis, mutant library, reverse genetics     

Identification of candidate gammaherpesvirus 68 genes required for virus replication by signature-tagged transposon mutagenesis

Current methods for determining the role of a given gene product in the gammaherpesvirus 68 (gammaHV68) life cycle require generation of a specific mutation by either homologous recombination in mammalian cells or bacterial artificial chromosome-mediated mutagenesis in Escherichia coli. The mutant virus is then compared to wild-type virus, and the role of the gene in the viral life cycle is deduced from its phenotype. This process is both time-consuming and labor intensive. Here we present the use of random, transposon-mediated signature-tagged mutagenesis for the identification of candidate viral genes involved in virus replication. Pools of viral mutants, each containing a random insertion of a transposon, were generated with a transposon donor library in which each transposon contains a unique sequence identifier. These pools were transfected into mammalian cells, and the ability of each mutant to replicate was assessed by comparing the presence of virus in the output pool to that present in the input pool of viral genomes. With this approach we could rapidly screen up to 96 individual mutants simultaneously. The location of the transposon insertion was determined by sequencing individual clones with a common primer specific for the transposon end. Here we present the characterization of 53 distinct viral mutants that correspond to insertions in 29 open reading frames within the gammaHV68 genome. To confirm the results of the signature-tagged mutagenesis screen, we quantitated the ability of each mutant to replicate compared to wild-type gammaHV68. From these analyses we identified 16 gammaHV68 open reading frames that, when disrupted by transposon insertions, score as essential for virus replication, and six other open reading frames whose disruption led to significant attenuation of virus replication. In addition, transposon insertion in five other gammaHV68 open reading frames did not affect virus replication. Notably, all but one of the candidate essential replication genes identified in this screen have been shown to be essential for the replication of at least one other herpesvirus.     

Blue ghosts: a new method for isolating amber mutants defective in essential genes of Escherichia coli.

We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli. Using this approach, we have isolated three amber mutants defective in the rho gene. An extension of the technique allows the detection of ochre mutants and transposon insertions in essential genes.     

Transposon-tagging identifies novel pathogenicity genes in Fusarium graminearum

With the increase of sequenced fungal genomes, high-throughput methods for functional analyses of genes are needed. We assessed the potential of a new transposon mutagenesis tool deploying a Fusarium oxysporum miniature inverted-repeat transposable element mimp1, mobilized by the transposase of impala, a Tc1-like transposon, to obtain knock-out mutants in Fusarium graminearum. We localized 91 mimp1 insertions which showed good distribution over the entire genome. The main exception was a major hotspot on chromosome 2 where independent insertions occurred at exactly the same nucleotide position. Furthermore insertions in promoter regions were over-represented. Screening 331 mutants for sexual development, radial growth and pathogenicity on wheat resulted in 19 mutants (5.7%) with altered phenotypes. Complementation with the original gene restored the wild-type phenotype in two selected mutants demonstrating the high tagging efficiency. This is the first report of a MITE transposon tagging system as an efficient mutagenesis tool in F. graminearum.     

Integration Profiling of Gene Function With Dense Maps of Transposon Integration

Understanding how complex networks of genes integrate to produce dividing cells is an important goal that is limited by the difficulty in defining the function of individual genes. Current resources for the systematic identification of gene function such as siRNA libraries and collections of deletion strains are costly and organism specific. We describe here integration profiling, a novel approach to identify the function of eukaryotic genes based upon dense maps of transposon integration. As a proof of concept, we used the transposon Hermes to generate a library of 360,513 insertions in the genome of Schizosaccharomyces pombe. On average, we obtained one insertion for every 29 bp of the genome. Hermes integrated more often into nucleosome free sites and 33% of the insertions occurred in ORFs. We found that ORFs with low integration densities successfully identified the genes that are essential for cell division. Importantly, the nonessential ORFs with intermediate levels of insertion correlated with the nonessential genes that have functions required for colonies to reach full size. This finding indicates that integration profiles can measure the contribution of nonessential genes to cell division. While integration profiling succeeded in identifying genes necessary for propagation, it also has the potential to identify genes important for many other functions such as DNA repair, stress response, and meiosis.     

Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis

Pathogenic Yersinia species are associated with both localized and systemic infections in mammalian hosts. In this study, signature-tagged transposon mutagenesis was used to identify Yersinia enterocolitica genes required for survival in a mouse model of infection. Approximately 2000 transposon insertion mutants were screened for attenuation. This led to the identification of 55 mutants defective for survival in the animal host, as judged by their ability to compete with the wild-type strain in mixed infections. A total of 28 mutants had transposon insertions in the virulence plasmid, validating the screen. Two of the plasmid mutants with severe virulence defects had insertions in an uncharacterized region. Several of the chromosomal insertions were in a gene cluster involved in O-antigen biosynthesis. Other chromosomal insertions identified genes not previously demonstrated as being required for in vivo survival of Y. enterocolitica. These include genes involved in the synthesis of outer membrane components, stress response and nutrient acquisition. One severely attenuated mutant had an insertion in a homologue of the pspC gene (phage shock protein C) of Escherichia coli. The phage shock protein operon has no known biochemical or physiological function in E. coli, but is apparently essential for the survival of Y. enterocolitica during infection.     

Mu-seq: Sequence-Based Mapping and Identification of Transposon Induced Mutations

Mutations tagged by transposon insertions can be readily mapped and identified in organisms with sequenced genomes. Collections of such mutants allow a systematic analysis of gene function, and can be sequence-indexed to build invaluable resources. Here we present Mu-seq (Mutant-seq), a high-throughput NextGen sequencing method for harnessing high-copy transposons. We illustrate the efficacy of Mu-seq by applying it to the Robertson’s Mutator system in a large population of maize plants. A single Mu-seq library, for example, constructed from 576 different families (2304 plants), enabled 4, 723 novel, germinal, transposon insertions to be detected, identified, and mapped with single base-pair resolution. In addition to the specificity, efficiency, and reproducibility of Mu-seq, a key feature of this method is its adjustable scale that can accomodate simultaneous profiling of transposons in thousands of individuals. We also describe a Mu-seq bioinformatics framework tailored to high-throughput, genome-wide, and population-wide analysis of transposon insertions.     

Identification of Streptococcus agalactiae virulence genes in the neonatal rat sepsis model using signature-tagged mutagenesis

Group B streptococcal (GBS) infections are the most common cause of bacterial sepsis in the immediate newborn period. Apart from the capsule, the factors required for survival of GBS in the host are not well defined. In this study, signature-tagged transposon mutagenesis (STM) was used to identify genes required for growth and survival of GBS in a neonatal rat sepsis infection model. Approximately 1600 transposon mutants were screened in pools of 80 mutants, and approximately 120 mutants defective for survival in the animal host were identified. We successfully cloned and sequenced DNA flanking the transposon insertions from 92 of the mutants. Fifty per cent of the mutants had transposon insertions in genes with homologues in the public databases, whereas the remaining 50% had transposon insertions in genes with unknown function. A significant proportion of the avirulent mutants had transposon insertions in genes encoding transport-associated or regulatory proteins or in genes involved in cell surface metabolism, emphasizing the significance of these functions for in vivo survival of GBS. Overall, STM analysis revealed GBS genomic loci that encode a wide variety of functional gene classes, underscoring the diversity of bacterial processes required for the infection process. Currently, the function of the genes identified during the screening can only be inferred by homology to previously described genes. However, a number of the genes identified in this study have been shown to correlate with virulence in other pathogens. A virulence of a subset of mutants identified during the screening was confirmed by performing competitive index assays and lethal dose assays. This represents the first report of a genome-wide scan for virulence factors in GBS. The identified genes will further our understanding of the pathogenesis of GBS infections and may represent targets for intervention or lead to the development of novel therapies.     

A system of transposon mutagenesis for bacteriophage T4

We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24-deleted phage. The transposition occurred at a frequency of only 10(-7) per progeny phage, even though a dam- host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon-specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.     

Assignment of 44 Ds Insertions to the Linkage Map of Arabidopsis

Transposon tagging is a useful tool for biological studies. Transposon insertions have been used to obtain new mutants which are extremely helpful in understanding gene function. These insertions immediately provide a means to isolate the corresponding genes. Transposon tagging has also been used to clone genes previously defined by point mutations. In addition, transposon insertions into cloned genes that lack mutations can be generated to facilitate functional analysis. The maize Ac/Ds transposon elements are known to transpose to local sites with high frequencies and have been shown to function in several dicots. To generate a collection of Ds elements for the purpose of targeted insertional mutagenesis of mapped genes in Arabidopsis, we have mapped 44 Ds insertions by simple sequence length polymorphism (SSLP). Because the Arabidopsis genome project is advancing rapidly, many genes will be discovered whose functions are unknown. The mapped 44 Ds insertions will be a useful resource for post-genome analysis of gene functions in Arabidopsis.     

Site-selected insertion of the transposon Tc1 into a Caenorhabditis elegans myosin light chain gene.

We used the polymerase chain reaction to detect insertions of the transposon Tc1 into mlc-2, one of two Caenorhabditis elegans regulatory myosin light chain genes. Our goals were to develop a general method to identify mutations in any sequenced gene and to establish the phenotype of mlc-2 loss-of-function mutants. The sensitivity of the polymerase chain reaction allowed us to identify nematode populations containing rare Tc1 insertions into mcl-2. mlc-2::Tc1 mutants were subsequently isolated from these populations by a sib selection procedure. We isolated three mutants with Tc1 insertions within the mlc-2 third exon and a fourth strain with Tc1 inserted in nearby noncoding DNA. To demonstrate the generality of our procedure, we isolated two additional mutants with Tc1 insertions within hlh-1, the C. elegans MyoD homolog. All of these mutants are essentially wild type when homozygous. Despite the fact that certain of these mutants have Tc1 inserted within exons of the target gene, these mutations may not be true null alleles. All three of the mlc-2 mutants contain mlc-2 mRNA in which all or part of Tc1 is spliced from the pre-mRNA, leaving small in-frame insertions or deletions in the mature message. There is a remarkable plasticity in the sites used to splice Tc1 from these mlc-2 pre-mRNAs; certain splice sites used in the mutants are very different from typical eukaryotic splice sites.     

Mos as a tool for genome-wide insertional mutagenesis in Caenorhabditis elegans: results of a pilot study

The sequence of the Caenorhabditis elegans genome contains approximately 19 000 genes. Available mutants currently exist for <20% of these genes. The existence of a Mos-based inducible transposon system in C.elegans could theoretically serve as a tool to saturate the genome with insertions. We report here the results of a pilot study aimed at assaying this strategy. We generated 914 independent random Mos insertions and determined their location by inverse PCR. The distribution of the insertions throughout the genome does not reveal any gross distortion, with the exception of a major hotspot on chromosome I (rDNA locus). Transposons are evenly distributed between the genic and intergenic regions. Within genes, transposons insert preferentially into the introns. We derived the consensus target site for Mos in C.elegans (ATATAT), which is common to Tc1, another mariner element. Finally, we assayed the mutagenic properties of insertions located in exons by comparing the phenotype of homozygous strains to that of known mutations or RNAi of the same gene. This pilot experiment shows that a Mos-based approach is a viable strategy that can contribute to the constitution of genome-wide collections of identified C.elegans mutants.     

A transposon insertion single-gene knockout library and new ordered cosmid library for the model organism Streptomyces coelicolor A3(2)

A simple and high-throughput transposon mediated mutagenesis system employing in vitro shuttle transposon mutagenesis has been used to systematically mutagenise the Streptomyces coelicolor genome. To achieve the highest coverage, a new ordered cosmid library was also constructed. Individual cosmids from both the existing and new libraries were disrupted using the Tn5-based mini-transposon Tn5062. A total of 35,358 insertions were sequenced resulting in the disruption of 6,482 genes (83% of the predicted open reading frames). Complete information for both the newly generated cosmids as well as all the insertions has been uploaded onto a central database, StrepDB ( http://strepdb.streptomyces.org.uk/ ). All insertions, new cosmids and a range of transposon exchange cassettes are available for study of individual gene function.     

Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.     

Isolation and characterization of FecA- and FeoB-mediated iron acquisition systems of the spirochete Leptospira biflexa by random insertional mutagenesis

The specific mechanisms by which Leptospira spp. acquire iron from their ecological niches are unknown. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. In this study, we have developed a system for random transposon mutagenesis of Leptospira biflexa using a mariner transposon, Himar1. To demonstrate the validity of Himar1 in vivo transposon mutagenesis in L. biflexa, a screen of mutants for clones impaired in amino acid biosynthesis was first performed, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, 2,000 L. biflexa transposon mutants were screened onto media with and without hemin, thus allowing the identification of five hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Three mutants had distinct insertions in a gene encoding a protein which shares homology with the TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified two mutants with a Himar1 insertion into a feoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Interestingly, the growth inhibition exhibited by the fecA and feoB mutants was relieved by deferoxamine, suggesting the presence of a ferric hydroxamate transporter. These results confirm the importance of iron for the growth of Leptospira and its ability to use multiple iron sources.     

A genome-wide search for synaptic vesicle cycle proteins in Drosophila

The recent completion of the Drosophila genome sequence opens new avenues for neurobiology research. We screened the fly genome sequence for homologs of mammalian genes implicated directly or indirectly in exocytosis and endocytosis of synaptic vesicles. We identified fly homologs for 93% of the vertebrate genes that were screened. These are on average 60% identical and 74% similar to their vertebrate counterparts. This high degree of conservation suggests that little protein diversification has been tolerated in the evolution of synaptic transmission. Finally, and perhaps most exciting for Drosophila neurobiologists, the genomic sequence allows us to identify P element transposon insertions in or near genes, thereby allowing rapid isolation of mutations in genes of interest. Analysis of the phenotypes of these mutants should accelerate our understanding of the role of numerous proteins implicated in synaptic transmission.     

Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.

The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity.