Apical secretion and association of the Drosophila yellow gene product with developing larval cuticle structures during embryogenesis.

Summary The yellow (y) gene of Drosophila melanogaster is required for the pigmentation of larval and adult cuticle structures. The deduced y protein sequence includes two putative N-linked glycosylation sites and a putative signal peptide, suggesting that it might be a secreted molecule. Consistent with the characteristics of a secreted protein, our in vitro translation studies using RNA synthesised from the y cDNA demonstrate that the nascent y polypeptide is a preprotein that cotranslationally translocates into the endoplasmic reticulum (ER) membrane and becomes glycosylated. The N-terminal peptide is cleaved from the preprotein between the two alanine residues at positions 21 and 22, to release the final product into the lumen of the ER. Antibodies raised against the y polypeptide detect the protein starting at 13 h post-fertilization in epidermal cells and in the cuticle structures secreted by them that later become pigmented; in addition, yellow protein is detected in the cuticle structures associated with Keilin's organs. The embryonic -galactosidase staining pattern of a transgene, bearing a construct in which expression of the lacZ gene is driven by the y promoter, is also described and is similar to that of the y protein. Our results indicate that the y gene product is an apically secreted protein which becomes an immobilised structural component of the pigmented cuticle.     

Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants

One hundred and forty-eight temperature-sensitive cell division cycle (cdc) mutants of Saccharomyces cerevisiae have been isolated and characterized. Complementation studies ordered these recessive mutations into 32 groups and tetrad analysis revealed that each of these groups defines a single nuclear gene. Fourteen of these genes have been located on the yeast genetic map. Functionally related cistrons are not tightly clustered.

Mutations in different cistrons frequently produce different cellular and nuclear morphologies in the mutant cells following incubation at the restrictive temperature, but all the mutations in the same cistron produce essentially the same morphology. The products of these genes appear, therefore, each to function individually in a discrete step of the cell cycle and they define collectively a large number of different steps.

The mutants were examined by time-lapse photomicroscopy to determine the number of cell cycles completed at the restrictive temperature before arrest. For most mutants, cells early in the cell cycle at the time of the temperature shift (before the execution point) arrest in the first cell cycle while those later in the cycle (after the execution point) arrest in the second cell cycle. Execution points for allelic mutations that exhibit first or second cycle arrest are rather similar and appear to be cistron-specific. Other mutants traverse several cycles before arrest, and its suggested that the latter type of response may reveal gene products that are temperature-sensitive for synthesis, whereas the former may be temperature-sensitive for function.

The gene products that are defined by the cdc cistrons are essential for the completion of the cell cycle in haploids of a and α mating type and in a/α diploid cells. The same genes, therefore, control the cell cycle in each of these stages of the life cycle.

     

Biofilm Formation by Psychrobacter arcticus and the Role of a Large Adhesin in Attachment to Surfaces

Psychrobacter arcticus strain 273-4, an isolate from a Siberian permafrost core, is capable of forming biofilms when grown in minimal medium under laboratory conditions. Biofilms form at 4 to 22°C when acetate is supplied as the lone carbon source and with 1 to 7% sea salt. P. arcticus is also capable of colonizing quartz sand. Transposon mutagenesis identified a gene important for biofilm formation by P. arcticus. Four transposon mutants were mapped to a 20.1-kbp gene, which is predicted to encode a protein of 6,715 amino acids (Psyc_1601). We refer to this open reading frame as cat1, for cold attachment gene 1. The cat1 mutants are unable to form biofilms at levels equivalent to that of the wild type, and there is no impact on the planktonic growth characteristics of the strains, indicating a specific role in biofilm formation. Through time course studies of the static microtiter plate assay, we determined that cat1 mutants are unable to form biofilms equivalent to that of the wild type under all conditions tested. In flow cell experiments, cat1 mutants initially are unable to attach to the surface. Over time, however, they form microcolonies, an architecture very different from that produced by wild-type biofilms. Our results demonstrate that Cat1 is involved in the initial stages of bacterial attachment to surfaces.     

Identification of the genetic determinants of Salmonella enterica serotype Typhimurium that may regulate the expression of the type 1 fimbriae in response to solid agar and static broth culture conditions

Background

Type 1 fimbriae are the most commonly found fimbrial appendages on the outer membrane of Salmonella enterica serotype Typhimurium. Previous investigations indicate that static broth culture favours S. Typhimurium to produce type 1 fimbriae, while non-fimbriate bacteria are obtained by growth on solid agar media. The phenotypic expression of type 1 fimbriae in S. Typhimurium is the result of the interaction and cooperation of several genes in the fim gene cluster. Other gene products that may also participate in the regulation of type 1 fimbrial expression remain uncharacterized.

Results

In the present study, transposon insertion mutagenesis was performed on S. Typhimurium to generate a library to screen for those mutants that would exhibit different type 1 fimbrial phenotypes than the parental strain. Eight-two mutants were obtained from 7,239 clones screened using the yeast agglutination test. Forty-four mutants produced type 1 fimbriae on both solid agar and static broth media, while none of the other 38 mutants formed type 1 fimbriae in either culture condition. The flanking sequences of the transposons from 54 mutants were cloned and sequenced. These mutants can be classified according to the functions or putative functions of the open reading frames disrupted by the transposon. Our current results indicate that the genetic determinants such as those involved in the fimbrial biogenesis and regulation, global regulators, transporter proteins, prophage-derived proteins, and enzymes of different functions, to name a few, may play a role in the regulation of type 1 fimbrial expression in response to solid agar and static broth culture conditions. A complementation test revealed that transforming a recombinant plasmid possessing the coding sequence of a NAD(P)H-flavin reductase gene ubiB restored an ubiB mutant to exhibit the type 1 fimbrial phenotype as its parental strain.

Conclusion

Genetic determinants other than the fim genes may involve in the regulation of type 1 fimbrial expression in S. Typhimurium. How each gene product may influence type 1 fimbrial expression is an interesting research topic which warrants further investigation.     

Xylella fastidiosa Afimbrial Adhesins Mediate Cell Transmission to Plants by Leafhopper Vectors

The interactions between the economically important plant-pathogenic bacterium Xylella fastidiosa and its leafhopper vectors are poorly characterized. We used different approaches to determine how X. fastidiosa cells interact with the cuticular surface of the foreguts of vectors. We demonstrate that X. fastidiosa binds to different polysaccharides with various affinities and that these interactions are mediated by cell surface carbohydrate-binding proteins. In addition, competition assays showed that N-acetylglucosamine inhibits bacterial adhesion to vector foregut extracts and intact wings, demonstrating that attachment to leafhopper surfaces is affected in the presence of specific polysaccharides. In vitro experiments with several X. fastidiosa knockout mutants indicated that hemagglutinin-like proteins are associated with cell adhesion to polysaccharides. These results were confirmed with biological experiments in which hemagglutinin-like protein mutants were transmitted to plants by vectors at lower rates than that of the wild type. Furthermore, although these mutants were defective in adhesion to the cuticle of vectors, their growth rate once attached to leafhoppers was similar to that of the wild type, suggesting that these proteins are important for initial adhesion of X. fastidiosa to leafhoppers. We propose that X. fastidiosa colonization of leafhopper vectors is a complex, stepwise process similar to the formation of biofilms on surfaces.     

Temperature-sensitive mutants blocked in the folding or subunit assembly of the bacteriophage P22 tail spike protein: III. Inactive polypeptide chains synthesized at 39 °C

Salmonella typhimurium cells infected by temperature-sensitive mutants in gene 9 of bacteriophage P22 at the restrictive temperature (39 °C) fail to accumulate functional tail spike protein. We report here studies of the inactive mutant tail spike polypeptide chains synthesized at 39 °C by temperature-sensitive mutants at 15 different sites of gene 9. For all 15 mutants, the gene 9 polypeptide chains were synthesized at 39 °C at rates similar to wild type. The mutant polypeptide chains were stable within the infected cells.The inactive polypeptide chains were tested for three functions displayed by the mature tail spike protein: irreversible binding to phage heads, endorhamnosidase activity, and reaction with anti-tail antibody. The 15 mutant proteins that accumulated at 39 °C lacked all three functions. Since the amino acid substitutions do not affect these functions of the mature protein, the mutant polypeptide chains synthesized at 39 °C have a conformation very different from the wild type, and different from the same proteins when matured at 30 °C. The fact that amino acid substitutions throughout the 76,000 M_r polypeptide chain prevent all three functions suggests that the mutations prevent the correct folding of the gene 9 polypeptide chain at restrictive temperature. Thus, these mutations identify sites in the polypeptide chain critical for protein maturation.Many of the mutant proteins could be activated in the absence of new protein synthesis by shifting infected cells from restrictive to permissive temperature before cell lysis. For these mutants, the immature chains accumulating at high temperature must be reversibly related to intermediates in protein folding or subunit assembly.     

Thermolability of the stringent factor in rel mutants of Escherichia coli

The stringent factor extracted from several independently isolated rel^- mutants is more thermolabile than the stringent factor extracted from the parental rel⁺ strain. This thermolability is characteristically different in each of the mutants. This strongly suggests that the stringent factor is the product of the rel gene.     

Leaf cuticle ultrastructure of Pseudofrenelopsis dalatzensis (Chow et Tsao) Cao ex Zhou (Cheirolepidiaceae) from the Lower Cretaceous Dalazi Formation of Jilin,China

The cheirolepidiaceous conifer Pseudofrenelopsis dalatzensis from the Lower Cretaceous Dalazi Formation, Jilin Province, Northeast China is studied in detail based on abundant material. The gross morphology of vegetative shoots shows a greater variation than previously known. Additional information of microstructure is that the adaxial cuticle from the free part of leaves is also papillate as the abaxial cuticle. The excellent preservation of cuticles allows for the first time in this family to four distinct types of cuticle in ultrastructural level: subsidiary cells, guard cells, epidermal cells with and without papillae. Based on 30 measurements the statistic approach on each type of cell cuticle, i.e. not only the total thickness of the cuticle but also details and proportions of all different layers. A key is provided using a combination of 7 significant ultrastructural characters. These results seem to be very promising for the identity of each taxon within the Cheirolepidiaceae family. The cuticular ultrastructure of this family is characterized by having a cuticle proper A composed of A1 wavy polylamellate layer and A2 granular layer, above the cuticular layer B with B1 fibrillate and B2 granular layers. The importance of ultrastructural characters at different levels of taxonomy and palaeoecology is also discussed.     

Dependence of cytoplasmic on mitochondrial protein synthesis inK. lactis CBS 2360

Summary A fewery ^R mutants independently isolated fromK. lactis CBS 2360 display a conditional lethal phenotype at the temperature of 36° C. In addition to drug resistance, also conditional lethality shows a non-Mendelian pattern of inheritance and is affected by exposure of the cells to Ethidium Bromide, indicating that in this yeast mitochondrial DNA controls cell viability. The results obtained from biochemical analysis suggest that the cellular functions in which are involved the gene products of the mitochondrial mutants analized are cytoplasmic protein and RNA syntheses.     

A Synthetic Community Approach Reveals Plant Genotypes Affecting the Phyllosphere Microbiota

The identity of plant host genetic factors controlling the composition of the plant microbiota and the extent to which plant genes affect associated microbial populations is currently unknown. Here, we use a candidate gene approach to investigate host effects on the phyllosphere community composition and abundance. To reduce the environmental factors that might mask genetic factors, the model plant Arabidopsis thaliana was used in a gnotobiotic system and inoculated with a reduced complexity synthetic bacterial community composed of seven strains representing the most abundant phyla in the phyllosphere. From a panel of 55 plant mutants with alterations in the surface structure, cell wall, defense signaling, secondary metabolism, and pathogen recognition, a small number of single host mutations displayed an altered microbiota composition and/or abundance. Host alleles that resulted in the strongest perturbation of the microbiota relative to the wild-type were lacs2 and pec1. These mutants affect cuticle formation and led to changes in community composition and an increased bacterial abundance relative to the wild-type plants, suggesting that different bacteria can benefit from a modified cuticle to different extents. Moreover, we identified ein2, which is involved in ethylene signaling, as a host factor modulating the community''s composition. Finally, we found that different Arabidopsis accessions exhibited different communities, indicating that plant host genetic factors shape the associated microbiota, thus harboring significant potential for the identification of novel plant factors affecting the microbiota of the communities.     

Range of potential functions of the Drosophila melanogaster hdc gene

The Drosophila melanogaster hdc gene controls trachea branching, which starts during embryo development. Expression in imaginal disks and reproductive organs suggests additional functions for the hdc gene. The gene was demonstrated to have a maternal effect, which was denied previously. Analysis of cell proliferation in imaginal disks with hdc mutations showed that the gene does not possess tumor suppressor properties at the levels of mosaic cuticle clones of adults and transplanted imaginal disks. Transplanted imaginal disks homozygous, but not heterozygous, for an hdc mutation were found to affect oogenesis in the recipient females, implicating the hdc activity in exchanging signals between different organs. Amino acid sequence analysis of the HDC protein revealed a region homologous to the human HRS proteins, which directly interact with the NF2 tumor suppressor on experimental evidence.     

Metamorphic changes of wild type and mutant Drosophila tissues induced by 20-hydroxy ecdysone in vitro

Wild type (Oregon R) and non-pupariating as well as late-pupariating mutant larval tissues were cultured in vitro up to 5 weeks with and without 20-hydroxy ecdysone (1 μg/ml). The following responses were elicited by the hormone: in the case of wild type tissues detachment of the larval epidermis and muscles from the cuticle; puparial tanning and sclerotization of the larval cuticle; dissociation of the fat body into single cells; inhibition of the movement of the hind intestine. Most of these responses developed within 1 week of culturing. Of the 4 mutants tested, 3 behaved like the wild type. In cultures of ?(1)npr-1, however, puparial tanning, disc evagination, and inhibition of the movement of the hind intestine was abnormally weak and the dissociation of fat body was not observed at all. Detachment of the epidermis and muscles as well as formation of the pupal cuticle by disc tissue occurred normally. The results are discussed with respect to the ecdysteroid-induced metamorphosis of the tissues and the autonomy of mutant gene action.     

Adhesion of Conidia of Drechmeria coniospora to Caenorhabditis elegans Wild Type and Mutants

Adhesion of conidia of the endoparasitic fungus Drechmeria coniospora to the cuticles of the wild type and four different head defective mutants of Caenorhabditis elegans, and subsequent infection, was studied. The conidia adhered around the sensory structures in the head region, vulva, and occasionally to other parts of the cuticle in both mutant and wild type hosts. Infection took place after adhesion to the head region by penetration through the cuticle, and, following adhesion around the vulva, through the natural orifice. Infection was not observed after adhesion to other parts of the cuticle. Adhesion was reduced after treatment of the nematodes with Pronase E. Adhesion returned towards normal again within 2 hours, indicating that the proteinaceous material emanating from the sensory structures was rapidly replaced.     

The CURLY LEAF Interacting Protein BLISTER Controls Expression of Polycomb-Group Target Genes and Cellular Differentiation of Arabidopsis thaliana

Polycomb-group (Pc-G) proteins are important regulators of many developmental processes in plants and animals and repress gene expression by imparting histone H3 lysine 27 trimethylation (H3K27me3). Here, we present the identification of the novel, plant-specific Arabidopsis thaliana protein BLISTER (BLI), which interacts with the Pc-G histone methyltransferase CURLY LEAF (CLF). We map the interaction of BLI with CLF to a predicted coiled-coil domain in BLI that shares similarity with STRUCTURAL MAINTENANCE OF CHROMOSOMES proteins. BLI colocalizes with CLF in the nucleus, shows an overlapping expression pattern with CLF throughout plant development that is strongest in dividing cells, and represses a subset of Pc-G target genes. Loss of BLI results in a pleiotropic developmental mutant phenotype, indicating that BLI prevents premature differentiation. Furthermore, bli mutants exhibit severe epidermal defects, including loss of cell adhesion, outgrowth of cells, and increased cotyledon cell size. As these phenotypes have not been observed in Pc-G mutants, we propose that BLI has functions related to Pc-G proteins but can also act independently in Arabidopsis development.     

Combined Extracellular Matrix Cross-linking Activity of the Peroxidase MLT-7 and the Dual Oxidase BLI-3 Is Critical for Post-embryonic Viability in Caenorhabditis elegans

The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as molting. A targeted RNA interference screen using a cuticle collagen marker has been employed to identify components of the cuticle biosynthetic pathway. We have characterized an essential peroxidase, MoLT-7 (MLT-7), that is responsible for proper cuticle molting and re-synthesis. MLT-7 is an active, inhibitable peroxidase that is expressed in the cuticle-synthesizing hypodermis coincident with each larval molt. mlt-7 mutants show a range of body morphology defects, most notably molt, dumpy, and early larval stage arrest phenotypes that can all be complemented with a wild type copy of mlt-7. The cuticles of these mutants lacks di-tyrosine cross-links, becomes permeable to dye and accessible to tyrosine iodination, and have aberrant collagen protein expression patterns. Overexpression of MLT-7 causes mutant phenotypes further supporting its proposed enzymatic role. In combination with BLI-3, an H₂O₂-generating NADPH dual oxidase, MLT-7 is essential for post-embryonic development. Disruption of mlt-7, and particularly bli-3, via RNA interference also causes dramatic changes to the in vivo cross-linking patterns of the cuticle collagens DPY-13 and COL-12. This points toward a functionally cooperative relationship for these two hypodermally expressed proteins that is essential for collagen cross-linking and proper extracellular matrix formation.