Characterization of Motility and Surface Attachment in Thirteen Members of the Roseobacter Clade

The Roseobacter clade is an abundant and biogeochemically relevant group of marine bacteria. Physiological and ecological traits identified in specific representatives of the clade are often universally attributed to all Roseobacter group members, however, culture-dependent studies utilizing phylogenetically distinct members are rare. Other attributes often associated with this clade include motility, biofilm formation and surface attachment, chemotaxis and quorum sensing. This study compared a collection of 13 diverse Roseobacter strains both pheno- and genotypically on the basis of these traits. Motility was determined for seven previously uncharacterized strains, with five of the strains demonstrating motility. Microscopic analysis using both phase contrast and transmission electron microscopy supported this finding. A crystal violet assay was used to assess biofilm formation on plastic and glass surfaces with a range of surface properties and yielded a wide array of phenotypic responses. Taking into account the variety of surface types and media types tested approximately half (54%) of the strains showed pronounced biofilm formation and all motile strains were capable of forming biofilms. Degenerate primer sets were designed to probe strains for which no genome sequence is currently available for genes involved in flagellar synthesis and chemotaxis. Two strains that demonstrated no signs of motility in the laboratory were found to possess a necessary gene for flagellar formation and a flagellar-associated chemotaxis gene. Genome analysis including other sequenced Roseobacter strains revealed that flagellar, chemotaxis and quorum sensing operons are abundant in members of this lineage, with 89% possessing flagellar and chemotaxis operons and 78% possessing genes believed to be involved in quorum sensing. This study underscores the diversity of this clade and emphasizes the difficulty of assigning phenotypic capabilities to all lineage members

Papaya Ringspot Virus Resistance of Transgenic Rainbow and SunUp is Affected by Gene Dosage, Plant Development, and Coat Protein Homology

R1 plants of the transgenic papaya line 55-1, which expresses a single coat protein (CP) gene of the mild strain of the papaya ringspot virus (PRSV) HA from Hawaii, were previously shown to be resistant only to PRSV isolates from Hawaii. Two transgenic papaya cultivars were subsequently derived from line 55-1. UH SunUp (SunUp) is homozygous for the CP gene insertion and UH Rainbow (Rainbow) is hemizygous for the CP gene because it is a F1 hybrid of a cross between SunUp and the nontransgenic papaya cultivar Kapoho. To determine the various parameters that affect the resistance of SunUp and Rainbow, plants at different developmental stages (younger and older) were inoculated with PRSV isolates from Hawaii, Brazil, Jamaica, and Thailand. Hawaiian isolates shared nucleotide sequence identities of 96.7-99.8% to the CP transgene, and the other isolates shared sequence identities of 89.5-92.5%. Resistance was affected by CP gene dosage, plant developmental stage, and CP sequence identity of the challenge isolate. Young and older hemizygous Rainbow plants were resistant to the homologous PRSV HA (99.8% homology to CP transgene), while only older Rainbow plants were resistant to the other Hawaiian isolates (96.7% homology). However, all inoculated Rainbow plants were susceptible to PRSV isolates collected from Jamaica, Brazil, and Thailand. In contrast, SunUp was resistant to all PRSV isolates, except the one from Thailand, regardless of the plant developmental stage. Resistance to the Thailand isolate, which shares 89.5% homology to the transgene, was observed only with SunUp plants inoculated at an older stage. Steady state RNA analysis and nuclear run-on experiments suggested that resistance of the transgenic papaya is RNA-mediated via post-transcriptional gene silencin

Analysis of the Size of the Carcinoembryonic Antigen (CEA) Gene Family

Five members of the human CEA gene family [human pregnancy-specific β1-glycoprotein (PSβG), hsCGM1, 2, 3 and 4] have been isolated and identified through sequencing the exons containing their N-terminal domains. Sequence comparisons with published data for CEA and related molecules reveal the existence of highly-conserved gene subgroups within the CEA family. Together with published data eleven CEA family members have so far been determined. Apart from the highly conserved coding sequences, these genes also show strong sequence conservation in their introns, indicating a duplication of whole gene units during the evolution of the CEA gene family

Long-Range Chromosomal Mapping of the Carcinoembryonic Antigen (CEA) Gene Family Cluster

A long-range physical map of the carcinoembryonic antigen (CEA) gene family cluster, which is located on the long arm of chromosome 19, has been constructed. This was achieved by hybridization analysis of large DNA fragments separated by pulse-field gel electrophoresis and of DNA from human/rodent somatic cell hybrids, as well as the assembly of ordered sets of cosmids for this gene region into contigs. The different approaches yielded very similar results and indicate that the entire gene family is contained within a region located at position 19q13.1–q13.2 between the CYP2A and the D19S15/D19S8 markers. The physical linkage of nine genes belonging to the CEA subgroup and their location with respect to the pregnancy-specific glycoprotein (PSG) subgroup genes have been determined, and the latter are located closer to the telomere. From large groups of ordered cosmid clones, the identity of all known CEA subgroup genes has been confirmed either by hybridization using gene-specific probes or by DNA sequencing. These studies have identified a new member of the CEA subgroup (CGM8), which probably represents a pseudogene due to the existence of two stop codons, one in the leader and one in the N-terminal domain exons. The gene order and orientation, which were determined by hybridization with probes from the 5′ and 3′ regions of the genes, are as follows: cen/3′-CGM7-5′/3′-CGM2-5′/5′-CEA-3′/5′-NCA-3′/5′- CGM1-3′/3′-BGP-5′/3′-CGM9-5′/3′-CGM6-5′/5′-CGM8-3′/PSGcluster/qter

Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root-specific rolD promoter from Agrobacterium rhizogenes

The nitrous oxide (N2O) reduction pathway from a soil bacterium, Pseudomonas stutzeri, was engineered in plants to reduce N2O emissions. As a proof of principle, transgenic plants expressing nitrous oxide reductase (N2OR) from P. stutzeri, encoded by the nosZ gene, and other transgenic plants expressing N2OR along with the more complete operon from P. stutzeri, encoded by nosFLZDY, were generated. Gene constructs were engineered under the control of a root-specific promoter and with a secretion signal peptide. Expression and rhizosecretion of the transgene protein were achieved, and N2OR from transgenic Nicotiana tabacum proved functional using the methyl viologen assay. Transgenic plant line 1.10 showed the highest specific activity of 16.7 µmol N2O reduced min−1 g−1 root protein. Another event, plant line 1.9, also demonstrated high specific activity of N2OR, 13.2 µmol N2O reduced min−1 g−1 root protein. The availability now of these transgenic seed stocks may enable canopy studies in field test plots to monitor whole rhizosphere N flux. By incorporating one bacterial gene into genetically modified organism (GMO) crops (e.g., cotton, corn, and soybean) in this way, it may be possible to reduce the atmospheric concentration of N2O that has continued to increase linearly (about 0.26% year−1) over the past half-century

Gene regulation in parthenocarpic tomato fruit

Parthenocarpy is potentially a desirable trait for many commercially grown fruits if undesirable changes to structure, flavour, or nutrition can be avoided. Parthenocarpic transgenic tomato plants (cv MicroTom) were obtained by the regulation of genes for auxin synthesis (iaaM) or responsiveness (rolB) driven by DefH9 or the INNER NO OUTER (INO) promoter from Arabidopsis thaliana. Fruits at a breaker stage were analysed at a transcriptomic and metabolomic level using microarrays, real-time reverse transcription-polymerase chain reaction (RT-PCR) and a Pegasus III TOF (time of flight) mass spectrometer. Although differences were observed in the shape of fully ripe fruits, no clear correlation could be made between the number of seeds, transgene, and fruit size. Expression of auxin synthesis or responsiveness genes by both of these promoters produced seedless parthenocarpic fruits. Eighty-three percent of the genes measured showed no significant differences in expression due to parthenocarpy. The remaining 17% with significant variation (P <0.05) (1748 genes) were studied by assigning a predicted function (when known) based on BLAST to the TAIR database. Among them several genes belong to cell wall, hormone metabolism and response (auxin in particular), and metabolism of sugars and lipids. Up-regulation of lipid transfer proteins and differential expression of several indole-3-acetic acid (IAA)- and ethylene-associated genes were observed in transgenic parthenocarpic fruits. Despite differences in several fatty acids, amino acids, and other metabolites, the fundamental metabolic profile remains unchanged. This work showed that parthenocarpy with ovule-specific alteration of auxin synthesis or response driven by the INO promoter could be effectively applied where such changes are commercially desirable