Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis

Herpesviruses are important pathogens in animals and humans. The large DNA genomes of several herpesviruses have been sequenced, but the function of the majority of putative genes is elusive. Determining which genes are essential for their replication is important for identifying potential chemotherapy targets, designing herpesvirus vectors, and generating attenuated vaccines. For this purpose, we recently reported that herpesvirus genomes can be maintained as infectious bacterial artificial chromosomes (BAC) in Escherichia coli. Here we describe a one-step procedure for random-insertion mutagenesis of a herpesvirus BAC using a Tn1721-based transposon system. Transposon insertion sites were determined by direct sequencing, and infectious virus was recovered by transfecting cultured cells with the mutant genomes. Lethal mutations were rescued by cotransfecting cells containing noninfectious genomes with the corresponding wild-type subgenomic fragments. We also constructed revertant genomes by allelic exchange in bacteria. These methods, which are generally applicable to any cloned herpesvirus genome, will facilitate analysis of gene function for this virus family.     

Use of signature-tagged transposon mutagenesis to identify Vibrio cholerae genes critical for colonization

The pathogenesis of cholera begins with colonization of the host intestine by Vibrio cholerae . The toxin co-regulated pilus (TCP), a fimbrial structure produced by V . cholerae , is absolutely required for colonization (i.e. the persistence, survival and growth of V . cholerae in the upper intestinal milieu), but many other aspects of the colonization process are not well understood. In this study, we use signature-tagged transposon mutagenesis (STM) to conduct a screen for random insertion mutations that affect colonization in the suckling mouse model for cholera. Of approximately 1100 mutants screened, five mutants (approximately 0.5%) with transposon insertions in TCP biogenesis genes were isolated, validating the use of STM to identify attenuated mutants. Insertions in lipopolysaccharide, biotin and purine biosynthetic genes were also found to cause colonization defects. Similar results were observed for mutations in homologues of pta and ptfA , two genes involved in phosphate transfer. Finally, our screen identified several novel genes, disruption of which also caused colonization defects in the mouse model. These results demonstrate that STM is a powerful method for isolating colonization-defective mutants of V . cholerae .     

Global transposon mutagenesis and essential gene analysis of Helicobacter pylori

We have constructed a genome-saturating mutant library of the human gastric pathogen Helicobacter pylori. Microarray tracking of transposon mutants (MATT) allowed us to map the position of 5,363 transposon mutants in our library. While we generally found insertions well distributed throughout the genome, 344 genes had no detectable transposon insertions, and this list is predicted to be highly enriched for essential genes. Comparison to the essential gene set of other bacteria revealed a surprisingly limited overlap with all organisms tested (11%), while 55% were essential in some organisms but not others. We independently verified the essentiality of several gene products, including an HtrA family serine protease, a hypothetical protein with putative phospholipase D activity, and a riboflavin specific deaminase. A limited screen for motility mutants allowed us to estimate that 4.5% of the genome is dedicated to this virulence-associated phenotype.     

Identification of essential genes in the human fungal pathogen Aspergillus fumigatus by transposon mutagenesis

The opportunistic pathogen Aspergillus fumigatus is the most frequent cause of deadly airborne fungal infections in developed countries. In order to identify novel antifungal-drug targets, we investigated the genome of A. fumigatus for genes that are necessary for efficient fungal growth. An artificial A. fumigatus diploid strain with one copy of an engineered impala160 transposon from Fusarium oxysporum integrated into its genome was used to generate a library of diploid strains by random in vivo transposon mutagenesis. Among 2,386 heterozygous diploid strains screened by parasexual genetics, 1.2% had a copy of the transposable element integrated into a locus essential for A. fumigatus growth. Comparison of genomic sequences flanking impala160 in these mutants with that of the genome of A. fumigatus allowed the characterization of 20 previously uncharacterized A. fumigatus genes. Among these, homologues of genes essential for Saccharomyces cerevisiae growth have been identified, as well as genes that do not have homologues in other fungal species. These results confirm that heterologous transposition using the transposable element impala is a powerful tool for functional genomics in ascomycota, and they pave the way for defining the complete set of essential genes in A. fumigatus, the first step toward target-based development of new antifungal drugs.     

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.     

Mapping of neutralizing epitopes on Renibacterium salmoninarum p57 by use of transposon mutagenesis and synthetic peptides

Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish.     

Bends in SV40 DNA: use of mutagenesis to identify the critical bases involved.

Five fragments of DNA exhibiting sequence directed bends were isolated from the Simian Virus 40 genome using a two-dimensional polyacrylamide gel fractionation. The bend sites were mapped for each fragment using the circular permutation test. All five sites have multiple, short runs of A residues with helical spacing typical of other bent fragments. Base pairs important for the bends were determined for one fragment by utilizing a random, single base pair mutagenesis. Of 28 mutants with decreased or increased bends, 14 had alterations that could be interpreted to affect the spaced runs of A residues, supporting their role in bends as predicted by the ApA wedge model. One major mutation was not explainable by existing models. The remaining minor mutations may only be due to small, local DNA conformational changes in the surrounding B-DNA.     

Using disruptive insertional mutagenesis to identify the in situ structure‐function landscape of the Shigella translocator protein IpaB

Bacterial type III secretion systems (T3SS) are used to inject proteins into mammalian cells to subvert cellular functions. The Shigella T3SS apparatus (T3SA) is comprised of a basal body, cytoplasmic sorting platform and exposed needle with needle “tip complex” (TC). TC maturation occurs when the translocator protein IpaB is recruited to the needle tip where both IpaD and IpaB control secretion induction. IpaB insertion into the host membrane is the first step of translocon pore formation and secretion induction. We employed disruptive insertional mutagenesis, using bacteriophage T4 lysozyme (T4L), within predicted IpaB loops to show how topological features affect TC functions (secretion control, translocon formation and effector secretion). Insertions within the N‐terminal half of IpaB were most likely to result in a loss of steady‐state secretion control, however, all but the two that were not recognized by the T3SA retained nearly wild‐type hemolysis (translocon formation) and invasiveness levels (effector secretion). In contrast, all but one insertion in the C‐terminal half of IpaB maintained secretion control but were impaired for hemolysis and invasion. These nature of the data suggest the latter mutants are defective in a post‐secretion event, most likely due to impaired interactions with the second translocator protein IpaC. Intriguingly, only two insertion mutants displayed readily detectable T4L on the bacterial surface. The data create a picture in which the makeup and structure of a functional T3SA TC is highly amenable to physical perturbation, indicating that the tertiary structure of IpaB within the TC is more plastic than previously realized.     

Mechanism and regulation of kinesin‐5, an essential motor for the mitotic spindle

Mitotic cell division is the most fundamental task of all living cells. Cells have intricate and tightly regulated machinery to ensure that mitosis occurs with appropriate frequency and high fidelity. A core element of this machinery is the kinesin‐5 motor protein, which plays essential roles in spindle formation and maintenance. In this review, we discuss how the structural and mechanical properties of kinesin‐5 motors uniquely suit them to their mitotic role. We describe some of the small molecule inhibitors and regulatory proteins that act on kinesin‐5, and discuss how these regulators may influence the process of cell division. Finally, we touch on some more recently described functions of kinesin‐5 motors in non‐dividing cells. Throughout, we highlight a number of open questions that impede our understanding of both this motor's function and the potential utility of kinesin‐5 inhibitors.     

Characterization of inhibitor binding to human kinesin spindle protein by site-directed mutagenesis

A number of inhibitors of kinesin spindle protein (KSP) have been described, which are known from X-ray crystallography studies to bind to an induced fit pocket defined by the L5 loop. We describe the characterization of eight mutant forms of KSP in which six residues that line this pocket have been altered. Mutants were analyzed by measuring rates of enzyme catalysis, in the presence and absence of six KSP inhibitors of four diverse structural classes and of varied ATP-competition status. Our analysis was in agreement with the model of binding established by the structural studies and suggests that binding energy is well distributed across functional groups in these molecules. The majority of the mutants retained significant enzymatic activity while diminishing inhibitor binding, indicating potential for the development of drug resistance. These data provide detailed information on interactions between inhibitor and binding pocket at the functional group level and enable the development of novel KSP inhibitors.     

Use of Illumina sequencing to identify transposon insertions underlying mutant phenotypes in high‐copy Mutator lines of maize

High‐copy transposons have been effectively exploited as mutagens in a variety of organisms. However, their utility for phenotype‐driven forward genetics has been hampered by the difficulty of identifying the specific insertions responsible for phenotypes of interest. We describe a new method that can substantially increase the throughput of linking a disrupted gene to a known phenotype in high‐copy Mutator (Mu) transposon lines in maize. The approach uses the Illumina platform to obtain sequences flanking Mu elements in pooled, bar‐coded DNA samples. Insertion sites are compared among individuals of suitable genotype to identify those that are linked to the mutation of interest. DNA is prepared for sequencing by mechanical shearing, adapter ligation, and selection of DNA fragments harboring Mu flanking sequences by hybridization to a biotinylated oligonucleotide corresponding to the Mu terminal inverted repeat. This method yields dense clusters of sequence reads that tile approximately 400 bp flanking each side of each heritable insertion. The utility of the approach is demonstrated by identifying the causal insertions in four genes whose disruption blocks chloroplast biogenesis at various steps: thylakoid protein targeting (cpSecE), chloroplast gene expression (polynucleotide phosphorylase and PTAC12), and prosthetic group attachment (HCF208/CCB2). This method adds to the tools available for phenotype‐driven Mu tagging in maize, and could be adapted for use with other high‐copy transposons. A by‐product of the approach is the identification of numerous heritable insertions that are unrelated to the targeted phenotype, which can contribute to community insertion resources.     

Functional regions in the essential light chain of smooth muscle myosin as revealed by the mutagenesis approach.

The endogenous essential light chain (LC17) of myosin from intestine smooth muscle was replaced with mutated essential light chains prepared using recombinant techniques. Complete exchange was observed with histidine-tagged derivatives of LC17a, LC17b and E122A-LC17a (LC17a and LC17b are the usual constituants of smooth muscle myosin), with small changes in the ATPase activity of reconstituted myosins. Much less exchange was observed with the light-chain derivative lacking the last 12 amino acid residues, demonstrating the importance of this segment, which may act as one arm of a pair of pincers to bind the myosin heavy chain.     

Characterization of the mgl operon of Escherichia coli by transposon mutagenesis and molecular cloning

We used transposon insertion mutagenesis, molecular cloning, and a novel procedure for in vitro construction of polar and nonpolar insertion mutations to characterize the genetic organization and gene products of the beta-methylgalactoside (Mgl) transport system, which utilizes the galactose-binding protein. The data indicate that the mgl operon contained three genes, which were transcribed in the order mglB, mglA, and mglC. The first gene coded for the 31,000 Mr galactose-binding protein, which was synthesized as a 3,000-dalton-larger precursor form. The mglA product was a 50,000 Mr protein which was tightly associated with the membrane, and the mglC product was a 38,000 Mr protein which was apparently loosely associated with the membrane and was probably located on the internal face of the cytoplasmic membrane. Identification of gene products was facilitated by in vitro insertion of a fragment of Tn5 containing the gene conferring kanamycin resistance into a restriction site in the operon. The fragment proved to have a polar effect on the expression of promoter-distal genes only when inserted in one of the two possible orientations. The three identified gene products were necessary and apparently sufficient for transport activity, but only the binding protein was required for chemotaxis towards galactose. The transport system appeared to contain the minimum number of components for a binding protein-related system: a periplasmic recognition component, a transmembrane protein, and a peripheral membrane protein that may be involved in energy linkage.     

The use of Pseudomonas sp. M plasmid pM3 for the transposon mutagenesis of Erwinia

The transposon containing derivatives pMTF9 (Tn9), pMTF10 (Tn10) and pMTF59 (Tn5, Tn9) of the Pseudomonas sp. M conjugative plasmid pM3 demonstrating temperature-dependent instability in Erwinia cells incubated at 37 degrees C have been isolated. The obtained plasmids have been shown to be usable for transposon-mediated mutability in the bacterial cells of Erwinia generum incubated at 37 degrees C.     

State uncertainty models and mark‐resight models for understanding non‐breeding site use by Piping Plovers

Conservation of beach‐nesting medium‐distance migrants has focused on breeding areas because protection of nests is more tractable than protection of non‐breeding habitat. As breeding ground management has encountered diminishing returns, interest in understanding threats in non‐breeding areas has increased. However, robust estimates of non‐breeding demographic rates and abundance are generally lacking, hindering the study of limiting factors. Estimating such rates is made more difficult by complex population dynamics at non‐breeding sites. In South Carolina, endangered Piping Plovers Charadrius melodus start arriving in July and some depart prior to December (the autumn‐only population) while others remain through at least March (the wintering population). State uncertainty capture‐mark‐recapture models provide a means for estimating vital rates for such co‐occurring populations. We estimated the proportion of the population entering the study area per survey (entry probability) and proportion remaining per survey (persistence rate) for both populations during autumn, and abundance of the wintering population, at four sites in South Carolina in 2006/7 and 2007/8, taking advantage of birds previously colour‐ringed on the breeding grounds. We made fairly precise estimates of entry and persistence rates with small sample sizes. Cumulative entry probability was ~50% by the end of July and reached 95% for both populations by October. Estimated stopover duration for birds in the autumn‐only population was 35 days in year 1 and 42 days in year 2. We estimated a wintering super‐population size of 71 ± 16 se birds in the first year and 75 ± 16 in the second. If ringing programmes on the breeding grounds continue, standardized resighting surveys in the non‐breeding period and mark‐recapture models can provide robust estimates of entry and persistence rates and abundance. Habitat protection intended to benefit non‐breeding Piping Plovers at our coastal sites should be in effect by late summer, as many birds are resident from July to the end of winter.     

Humans shape the year‐round distribution and habitat use of an opportunistic scavenger

Research focused on evaluating how human food subsidies influence the foraging ecology of scavenger species is scarce but essential for elucidating their role in shaping behavioral patterns, population dynamics, and potential impacts on ecosystems. We evaluate the potential role of humans in shaping the year‐round distribution and habitat use of individuals from a typical scavenger species, the yellow‐legged gull (Larus michahellis), breeding at southwestern Spain. To do this, we combined long‐term, nearly continuous GPS‐tracking data with spatially explicit information on habitat types and distribution of human facilities, as proxied by satellite imagery of artificial night lights. Overall, individuals were mainly associated with freshwater habitats (mean proportion, 95% CI: 40.6%, 36.9%–44.4%) followed by the marine‐related systems (40.3, 37.7%–42.8%), human‐related habitats (13.5%, 13.2%–13.8%), and terrestrial systems (5.5%, 4.6%–6.5%). However, these relative contributions to the overall habitat usage largely changed throughout the annual cycle as a likely response to ecological/physiological constraints imposed by varying energy budgets and environmental constraints resulting from fluctuations in the availability of food resources. Moreover, the tight overlap between the year‐round spatial distribution of gulls and that of human facilities suggested that the different resources individuals relied on were likely of anthropogenic origin. We therefore provide evidence supporting the high dependence of this species on human‐related food resources throughout the annual cycle. Owing to the ability of individuals to disperse and reach transboundary areas of Spain, Portugal, or Morocco, international joint efforts aimed at restricting the availability of human food resources would be required to manage this overabundant species and the associated consequences for biodiversity conservation (e.g., competitive exclusion of co‐occurring species) and human interests (e.g., airports or disease transmission).     

Raman spectroscopy and PCA‐SVM as a non‐invasive diagnostic tool to identify and classify qualitatively glycated hemoglobin levels in vivo

In this study we identify and classify high and low levels of glycated hemoglobin (HbA1c) in healthy volunteers (HV) and diabetic patients (DP). Overall, 86 subjects were evaluated. The Raman spectrum was measured in three anatomical regions of the body: index fingertip, right ear lobe, and forehead. The measurements were performed to compare the difference between the HV and DP (22 well controlled diabetic patients (WCDP) (HbA1c <6.5%), and 49 not controlled diabetic patients (NCDP) (HbA1c ≥6.5%)). Multivariable methods such as principal components analysis (PCA) combined with support vector machine (SVM) were used to develop effective diagnostic algorithms for classification among these groups. The forehead of HV versus WCDP showed the highest sensitivity (100%) and specificity (100%). Sensitivity (100%) and specificity (60%), were highest in the forehead of WCDP, versus NCDP. In HV versus NCDP, the fingertip had the highest sensitivity (100%) and specificity (80%). The efficacy of the diagnostic algorithm by receiver operating characteristic (ROC) curve was confirmed. Overall, our study demonstrated that the

combination of Raman spectroscopy and PCA‐SVM are feasible non‐invasive diagnostic tool in diabetes to classify qualitatively high and low levels of HbA1c in vivo.     

Binding of adenosine diphosphoribosyltransferase to the termini and internal regions of linear DNAs

Binding mechanisms of ADPR-transferase to restricted double-stranded DNA fragments of SV40 and pBR322 DNA were determined by nuclease protection techniques. Top and bottom strands of double-stranded DNA were identified by specific labeling with 32P. Protection against specific exonucleases identified binding of ADPR-transferase to DNA termini, whereas binding to internal regions of linear DNAs was probed by protection against endonucleases. ADPR-transferase protein protected against exonucleolytic attack from lambda exo and exoIII in all DNA fragments tested, demonstrating that the enzyme protein binds indiscriminately to all DNA termini. Extending earlier results [Sastry, S.S., & Kun, E. (1988) J. Biol. Chem. 263, 1505-1512], the modifying effect of the binding of ADPR-transferase to DNA induced changes in DNA conformation, as evident from altered pause sites that appeared following digestion of DNA fragments by lambda exonuclease in the presence of ADPR-transferase. In contrast to the nonselective binding of ADPR-transferase to DNA termini, ADPR-transferase conferred protection endonuclease attack (DNase I and micrococcal nuclease) only to the 209-bp EcoRI-PstI SV40 DNA fragment. These results indicate that binding of ADPR-transferase to relatively rare internal regions of restricted DNA fragments exhibits some degree of specificity. Specificity of binding appears to be related to the coincidental relative A+T-rich regions in DNA, and to DNA bending, both identified in the 209-bp SV40 DNA fragment. Synthetic polydeoxyribonucleotides containing dA-dT bind ADPR-transferase stronger than polydeoxyribonucleotides containing dG-dC. It was deduced from endonuclease protection patterns that binding of the enzyme protein leaves no defined footprints on the 209-bp SV40 DNA fragment, but there is significant modification of DNA structure following binding of the enzyme protein. Methylation protection assays and the prevention of the binding of ADPR-transferase to T4 DNA by its glucosylation indicate that the enzyme binds in the major groove of DNA. The 36-kDa A peptide fragment of ADPR-transferase [Buki, K. G., & Kun, E. (1988) Biochemistry 27, 5990-5995] exhibits the same protection against endonucleolytic enzymes as the intact ADPR-transferase molecule.     

Transfection by DNAs of avian erythroblastosis virus and avian myelocytomatosis virus strain MC29.

Chicken embryo fibroblasts and NIH 3T3 mouse cells were transformable by DNAs of chicken cells infected with avian myelocytomatosis virus strain MC29 or with avian erythroblastosis virus. Transfection of chicken cells appeared to require replication of MC29 or avian erythroblastosis virus in the presence of a nontransforming helper virus. In contrast, NIH 3T3 cells transformed by MC29 or avian erythroblastosis virus DNA contained only replication-defective transforming virus genomes.     

Using non‐linear mixed effects models to identify patterns of chick growth in House Sparrows Passer domesticus

For many animals, adult size is an important determinant of fitness. Thus, after a period of food restriction, offspring often grow quickly to approach an optimal size. Offspring can approach an optimal size by increasing mass faster than the peak growth of offspring that do not delay development (compensatory growth) or by extending the period of rapid growth to reach an optimal size (catch‐up growth). Unfortunately, the most common statistical techniques make it difficult to differentiate alternative growth patterns among developing offspring. Here, I show how random effect estimates can be used to uncover important variation in growth in a short‐lived passerine, the House Sparrow Passer domesticus. Specifically, I show that much of the variation in offspring growth can be explained by differences in the timing of peak growth and in final adult size, both within a single population and within treatments of an experimental manipulation. These results suggest that much of the variation in offspring growth may be explained by factors other than growth rate. I also show that offspring that delay development either maintain slow but steady growth across development and reach a small adult size, or extend the period of rapid growth to reach an optimal size, indicative of catch‐up growth. This pattern of extending the period of rapid growth may allow offspring to minimize the cellular damage caused by compensatory growth but still maximize size‐related fitness benefits (e.g. increased survival and fecundity) prior to fledging.