The composition of extracellular polymeric substances (EPS) from Shewanella sp. HRCR-1 biofilms was investigated using infrared spectroscopy and proteomics to provide insight into potential ecophysiological functions and redox activity of the EPS. Both bound and loosely associated EPS were extracted from Shewanella sp. HRCR-1 biofilms prepared using a hollow-fibre membrane biofilm reactor. Fourier transform infrared spectra revealed the presence of proteins, polysaccharides, nucleic acids, membrane lipids and fatty acids in the EPS fractions. Using a global proteomic approach, a total of 58 extracellular and outer membrane proteins were identified in the EPS. These included homologues of multiple Shewanella oneidensis MR-1 proteins that potentially contribute to key physiological biofilm processes, such as biofilm-promoting protein BpfA, surface-associated serine protease, nucleotidases (CpdB and UshA), an extracellular lipase, and oligopeptidases (PtrB and a M13 family oligopeptidase lipoprotein). In addition, 20 redox proteins were found in extracted EPS. Among the detected redox proteins were the homologues of two S. oneidensis MR-1 c-type cytochromes, MtrC and OmcA, which have been implicated in extracellular electron transfer. Given their detection in the EPS of Shewanella sp. HRCR-1 biofilms, c-type cytochromes may contribute to the possible redox activity of the biofilm matrix and play important roles in extracellular electron transfer reactions.

Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis throughout the world. This pathogen has two type III secretion systems (TTSS) encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that deliver virulence factors (effectors) to the host cell cytoplasm and are required for virulence. While many effectors have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this proteomic study, we identified effector proteins secreted into defined minimal medium designed to induce expression of the SPI-2 TTSS and its effectors. We compared the secretomes of the parent strain to those of strains missing essential (ssaK::cat) or regulatory (⌬ssaL) components of the SPI-2 TTSS. We identified 20 known SPI-2 effectors. Excluding the translocon components SseBCD, all SPI-2 effectors were biased for identification in the ⌬ssaL mutant, substantiating the regulatory role of SsaL in TTS. To identify novel effector proteins, we coupled our secretome data with a machine learning algorithm (SIEVE, SVM-based identification and evaluation of virulence effectors) and selected 12 candidate proteins for further characterization. Using CyaA reporter fusions, we identified six novel type III effectors and two additional proteins that were secreted into J774 macrophages independently of a TTSS. To assess their roles in virulence, we constructed nonpolar deletions and performed a competitive index analysis from intraperitoneally infected 129/SvJ mice. Six mutants were significantly attenuated for spleen colonization. Our results also suggest that non-type III secretion mechanisms are required for full Salmonella virulence.Salmonella enterica serovars are intracellular pathogens that can cause gastroenteritis and typhoid fever. In the developing world, they are a leading cause of morbidity and mortality resulting from dehydration and untreated sepsis (21)(22)43). Salmonella actively secretes effector proteins into the host cell cytoplasm to create a replicative niche and inhibit the immune system. Many of these effectors are delivered by one of two type III secretion systems (TTSS), which are encoded on Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2, respectively) (24). The SPI-1 TTSS facilitates host cell entry and inflammation, whereas SPI-2 mediates intracellular survival (19,51). Both SPI-1 and SPI-2 are required in a mouse model of persistent infection (35). While over 30 TTSS effectors have been identified to date (5,25, 42,(58)(59), the list is thought to be an underestimate of the true effector repertoire because several virulence phenotypes are dependent on TTS but are not linked to any known effectors (34,57).A proteomic study of Escherichia coli O157:H7 identified over 31 new type III effectors. This analysis took advantage of a sepL mutant that secreted effector proteins into culture medium in vitro (60). E. coli SepL interacts with Tir (63), a type III effector that inserts into the host plasma membrane and functi...

A linkage map for European hazelnut (Corylus avellana L.) was constructed using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers and the 2-way pseudotestcross approach. A full-sib population of 144 seedlings from the cross OSU 252.146 x OSU 414.062 was used. RAPD markers in testcross configuration, segregating 1:1, were used to construct separate maps for each parent. Fifty additional RAPD loci were assigned to linkage groups as accessory markers whose exact location could not be determined. Markers in intercross configuration, segregating 3:1, were used to pair groups in one parent with their homologues in the other. Eleven groups were identified for each parent, corresponding to the haploid chromosome number of hazelnut (n = x = 11). Thirty of the 31 SSR loci were able to be assigned to a linkage group. The maternal map included 249 RAPD and 20 SSR markers and spanned a distance of 661 cM. The paternal map included 271 RAPD and 28 SSR markers and spanned a distance of 812 cM. The maps are quite dense, with an average of 2.6 cM between adjacent markers. The S-locus, which controls pollen-stigma incompatibility, was placed on chromosome 5S where 6 markers linked within a distance of 10 cM were identified. A locus for resistance to eastern filbert blight, caused by Anisogramma anomala, was placed on chromosome 6R for which two additional markers tightly linked to the dominant allele were identified and sequenced. These maps will serve as a starting point for future studies of the hazelnut genome, including map-based cloning of important genes. The inclusion of SSR loci on the map will make it useful in other populations.

Interspecific hybridization can lead to the extinction of native populations and increased aggressiveness in hybrid forms relative to their parental lineages. However, interbreeding among subspecies is less often recognized as a serious threat to native species. Phragmites australis offers an excellent opportunity to investigate intraspecific hybridization since both native and introduced lineages occur in North America. Introduced Phragmites is a highly successful estuarine plant invader throughout North America, but native Phragmites populations are declining in the eastern US. Despite range overlaps, hybridization has not yet been detected between the native and introduced lineages in the wild, suggesting that phenological or physiological barriers preclude cross-pollination. We demonstrate, for the first time, that native and introduced populations of Phragmites can hybridize. There is substantial overlap in flowering period between native and introduced populations from the same geographic locations. We manually cross-pollinated native individuals with pollen from introduced Phragmites and recovered viable offspring. We then used microsatellite markers to prove that alleles unique to the pollen parent were transferred to progeny. Our results imply a mechanism for the further decline of native Phragmites in North America and a potential for the formation of aggressive hybrid offspring.

Vibrio vulnificus is a bacterial contaminant of shellfish and causes highly lethal sepsis and destructive wound infections. A definitive identification of virulence factors using the molecular version of Koch's postulates has been hindered because of difficulties in performing molecular genetic analysis of this opportunistic pathogen. For example, conjugation is required to introduce plasmid DNA, and allelic exchange suicide vectors that rely on sucrose sensitivity for counterselection are not efficient. We therefore incorporated USER friendly cloning techniques into pCVD442-based allelic exchange suicide vectors and other expression vectors to enable the rapid and efficient capture of PCR amplicons. Upstream and downstream DNA sequences flanking genes targeted for deletion were cloned together in a single step. Based on results from Vibrio cholerae, we determined that V. vulnificus becomes naturally transformable with linear DNA during growth on chitin in the form of crab shells. By combining USER friendly cloning and chitin-based transformation, we rapidly and efficiently produced targeted deletions in V. vulnificus, bypassing the need for two-step, suicide vector-mediated allelic exchange. These methods were used to examine the roles of two flagellin loci (flaCDE and flaFBA), the motAB genes, and the cheY-3 gene in motility and to create deletions of rtxC, rtxA1, and fadR. Additionally, chitin-based transformation was useful in moving antibiotic resistance-labeled mutations between V. vulnificus strains by simply coculturing the strains on crab shells. The methods and genetic tools that we developed should be of general use to those performing molecular genetic analysis and manipulation of other gram-negative bacteria.Vibrio vulnificus is a halophilic bacterium present naturally in estuarine waters and often contaminates oysters and other shellfish (for a review, see reference 15). V. vulnificus is an opportunistic pathogen of humans, causing primary septicemia and wound infection in susceptible individuals, and is the leading cause of reported seafood-related deaths in the United States. In susceptible humans, V. vulnificus causes a rapid, fulminating disease process resulting in extensive tissue damage. Mortality rates for susceptible individuals who develop fulminating primary septicemia are greater than 50% (17). Skin infections can lead to severe cellulitis, necrotizing fasciitis, and myositis requiring surgical debridement of infected tissues or amputation of the limb (4, 29, 42). Therapeutic intervention is often difficult since death can occur in less than 24 h after contact with the bacteria. In a mouse model of infection, V. vulnificus replicates extremely rapidly in host tissues (40, 41) and kills host cells including neutrophils (41).Over 20 years of genetic analysis, only a few virulence factors have been identified and confirmed by using the molecular version of Koch's postulates (15). Among the confirmed virulence factors are capsular polysaccharide (49), acquisition of iron (34, 52), type IV p...