Plasmids isolated from marine sediment microbial communities contain replication and incompatibility regions unrelated to those of known plasmid groups.

Two hundred ninety-seven bacteria carrying plasmids that range in size from 5 to 250 kb were identified from more than 1,000 aerobic heterotrophic bacteria isolated from coastal California marine sediments. While some isolates contained numerous (three to five) small (5- to 10-kb) plasmids, the majority of the natural isolates typically contained one large (40- to 100-kb) plasmid. By the method of plasmid isolation used in this study, the frequency of plasmid incidence ranged from 24 to 28% depending on the samples examined. Diversity of the plasmids occurring in the marine sediment bacterial populations was examined at the molecular level by hybridization with 14 different DNA probes specific for the incompatibility and replication (inc/rep) regions of a number of well-characterized plasmid incompatibility groups (repB/O, FIA, FII, FIB, HI1, HI2, I1, L/M, X, N, P, Q, W, and U). Interestingly, we found no DNA homology between the plasmids isolated from the culturable bacterial population of marine sediments and the replicon probes specific for numerous incompatibility groups developed by Couturier et al. (M. F. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Our findings suggest that plasmids in marine sediment microbial communities contain novel, as-yet-uncharacterized, incompatibility and replication regions and that the present replicon typing system, based primarily on plasmids derived from clinical isolates, may not be representative of the plasmid diversity occurring in some marine environments. Since the vast majority of marine bacteria are not culturable under laboratory conditions, we also screened microbial community DNA for the presence of broad- and narrow-host-range plasmid replication sequences. Although the replication origin of the conjugally promiscuous broad-host-range plasmid RK2 (incP) was not detectable in any of the plasmid-containing culturable marine isolates, DNA extracted from the microbial community and amplified by PCR yielded a positive signal for RK2 oriV replication sequences. The strength of the signal suggests the presence of a low level of the incP replicon within the marine microbial community. In contrast, replication sequences specific for the narrow-host-range plasmid F were not detectable in DNA extracted from marine sediment microbial communities. With the possible exception of mercuric chloride, phenotypic analysis of the 297 plasmid-bearing isolates did not demonstrate a correlation between plasmid content and antibiotic or heavy metal resistance traits.     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Impact of a Genetically Engineered Bacterium with Enhanced Alkaline Phosphatase Activity on Marine Phytoplankton Communities

An indigenous marine Achromobacter sp. was isolated from coastal Georgia seawater and modified in the laboratory by introduction of a plasmid with a phoA hybrid gene that directed constitutive overproduction of alkaline phosphatase. The effects of this "indigenous" genetically engineered microorganism (GEM) on phosphorus cycling were determined in seawater microcosms following the addition of a model dissolved organic phosphorus compound, glycerol 3-phosphate, at a concentration of 1 or 10 (mu)M. Within 48 h, a 2- to 10-fold increase in the concentration of inorganic phosphate occurred in microcosms containing the GEM (added at an initial density equivalent to 8% of the total bacterial population) relative to controls containing only natural microbial populations, natural populations with the unmodified Achromobacter sp., or natural populations with the Achromobacter sp. containing the plasmid but not the phoA gene. Secondary effects of the GEM on the phytoplankton community were observed after several days, evident as sustained increases in phytoplankton biomass (up to 14-fold) over that in controls. Even in the absence of added glycerol 3-phosphate, a numerically stable GEM population (averaging 3 to 5% of culturable bacteria) was established within 2 to 3 weeks of introduction into seawater. Moreover, alkaline phosphatase activity in microcosms with the GEM was substantially higher than that in controls for up to 25 days, and microcosms containing the GEM maintained the potential for net phosphate accumulation above control levels for longer than 1 month.     

Adaptation of model genetically engineered microorganisms to lake water: growth rate enhancements and plasmid loss.

When a genetically engineered microorganism (GEM) is released into a natural ecosystem, its survival, and hence its potential environmental impact, depends on its genetic stability and potential for growth under highly oligotrophic conditions. In this study, we compared plasmid stability and potential for growth on low concentrations of organic nutrients of strains of Pseudomonas putida serving as model GEMs. Plasmid-free and plasmid-bearing (NAH7) prototrophic isogenic strains and two amino-acid auxotrophs, all containing antibiotic resistance markers, were held physically separate from but in chemical contact with lake water containing the natural bacterium-sized microbial populations. Cells were reisolated at intervals over a 2-month period to determine the percent retaining the plasmid and the specific growth rate on various media. Plasmid stability in lake water was strongly strain specific; the NAH7 plasmid was stably maintained by the prototrophic strain for the duration of the test but was lost within 24 h by both of the auxotrophs. Specific growth rates of reisolates, compared with those of the corresponding non-lake water-exposed strains (i.e., parental strains), were not different when measured in rich medium (Luria-Bertani broth). However, specific growth rates were 42, 55, and 63% higher in reisolates of auxotrophs and the plasmid-free prototroph, respectively, when measured in 10-fold-diluted medium after exposure of 15 days or longer to lake water. Moreover, lake water-exposed strains grew actively when reintroduced into sterile lake water (28- to 33-fold increase in numbers over 7 days), while the corresponding unadapted parental strains exhibited no growth over the same period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ecological response of nitrification to oil spills and its impact on the nitrogen cycle

Marine oil spills are catastrophic events that cause massive damage to ecosystems at all trophic levels. While most of the research has focused on carbon-degrading microorganisms, the potential impacts of hydrocarbons on microbes responsible for nitrification have received far less attention. Nitrifiers are sensitive to hydrocarbon toxicity: ammonia-oxidizing bacteria and archaea being 100 and 1000 times more sensitive than typical heterotrophs respectively. Field studies have demonstrated the response of nitrifiers to hydrocarbons is highly variable and the loss of nitrification activity in coastal ecosystems can be restored within 1–2 years, which is much shorter than the typical recovery time of whole ecosystems (e.g., up to 20 years). Since the denitrification process is mainly driven by heterotrophs, which are more resistant to hydrocarbon toxicity than nitrifiers, the inhibition of nitrification may slow down the nitrogen turnover and increase ammonia availability, which supports the growth of oil-degrading heterotrophs and possibly various phototrophs. A better understanding of the ecological response of nitrification is paramount in predicting impacts of oil spills on the nitrogen cycle under oil spill conditions, and in improving current bioremediation practices.     

Rapid Identification of Vibrio parahaemolyticus by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Vibrio parahaemolyticus is a pathogenic marine bacterium that is the main causative agent of bacterial seafood-borne gastroenteritis in the United States. An increase in the frequency of V. parahaemolyticus-related infections during the last decade has been attributed to the emergence of an O3:K6 pandemic clone in 1995. The diversity of the O3:K6 pandemic clone and its serovariants has been examined using multiple molecular techniques including multilocus sequence analysis, pulsed-field gel electrophoresis, and group-specific PCR analysis. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for rapidly distinguishing between related bacterial species. In the current study, we demonstrate the development of a whole-cell MALDI-TOF MS method for the distinction of V. parahaemolyticus from other Vibrio spp. We identified 30 peaks that were present only in the spectra of the V. parahaemolyticus strains examined in this study that may be developed as MALDI-TOF MS biomarkers for identification of V. parahaemolyticus. We detected variation in the MALDI-TOF spectra of V. parahaemolyticus strains isolated from different geographical locations and at different times. The MALDI-TOF MS spectra of the V. parahaemolyticus strains examined were distinct from those of the other Vibrio species examined including the closely related V. alginolyticus, V. harveyi, and V. campbellii. The results of this study demonstrate the first use of whole-cell MALDI-TOF MS analysis for the rapid identification of V. parahaemolyticus.Recent food-borne illness outbreaks have emphasized the need for rapid, robust, and low-cost methods for microbial identification. Vibrio parahaemolyticus is one of several Vibrio species that cause human infection and occur in coastal estuarine and marine environments worldwide. V. parahaemolyticus causes gastroenteritis, wound infections, and septicemia upon exposure to contaminated water or contaminated undercooked seafood. In the United States, V. parahaemolyticus is the leading causative agent of bacterial seafood-borne gastroenteritis (8). Gastroenteritis-associated V. parahaemolyticus strains typically possess one or both of the thermostable direct hemolysin genes (tdh and trh); however, recent studies have indicated the presence of additional virulence-associated genes including two type III secretion systems (6, 7, 26, 28, 33). Following the emergence of the V. parahaemolyticus O3:K6 pandemic clone in 1995, there has been a rise in the number of reported V. parahaemolyticus-associated infections each year, making this species a pathogen of increasing concern (8, 11). The V. parahaemolyticus pandemic clone was first isolated from outbreaks in Asia in 1995 with the O3:K6 serotype and has since emerged with additional serotypes (30). The worldwide spread of the V. parahaemolyticus O3:K6 clone is a recognized international public health issue that requires the use of standardized methods for global monitoring and surveillance such as pulsed-field gel electrophoresis (PFGE) (22, 34).Initial isolation of V. parahaemolyticus is often conducted by culturing strains on thiosulfate citrate bile salts sucrose (TCBS) growth medium (15, 23). TCBS is used to selectively enrich for Vibrio spp. from cooccurring non-Vibrio strains; however, TCBS cannot differentiate V. parahaemolyticus from closely related species such as Vibrio harveyi and Vibrio campbellii. Additional molecular analyses are required to positively distinguish V. parahaemolyticus from other, closely related Vibrio species. These methods include group-specific PCR (4), multiplex PCR (38), multilocus sequence analysis (MLSA) (9, 17), comparative gene arrays (43), and whole-genome arrays (18). Often, several of these techniques are employed to distinguish V. parahaemolyticus from closely related Vibrio spp. and to provide greater resolution for discriminating among the pandemic clones (17, 18, 27). The development of a rapid method to distinguish V. parahaemolyticus from other Vibrio species including Vibrio pathogens would greatly aid the identification of strains involved in disease outbreaks when time is critical.Recent studies have shown that whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the rapid identification of bacteria including Streptococcus spp. (44), Salmonella strains (14), Mycobacterium spp. (35), Arthrobacter spp. (42), Listeria spp. (2), Burkholderia spp. (41), and other diverse nonfermenting clinical bacteria (12, 29). These studies have demonstrated the use of whole-cell MALDI-TOF MS analysis to generate highly reproducible and unique profiles to differentiate these bacterial strains at the species and subspecies levels. Whole-cell MALDI-TOF MS involves growing bacteria under standardized conditions and preparing cells for analysis by washing them to remove residual medium components, followed by resuspension of cells in a matrix that allows protein ionization. The cell-matrix suspension is then spotted onto a MALDI plate, each spot is ionized with a laser, and the ionizable proteins migrate based on their size resulting in the different peak sizes (kDa) in the MALDI-TOF MS spectra. Bacteria are typically grown overnight; however, the specific growth conditions and medium type must be determined and replicated to avoid condition-dependent differences in MADLI-TOF MS spectra (42). The method for preparation of the cells consists of only a few steps, and the protein ionization and generation of the spectra take several seconds. Whole-cell MALDI-TOF MS analysis can thus quickly provide accurate and reproducible generation of bacterial fingerprints that may be analyzed for the presence of biomarker peaks representative of a species or clonal group (2, 25, 35, 41, 44).In the current study, we have developed a method for whole-cell MALDI-TOF MS identification of V. parahaemolyticus. MALDI-TOF MS analysis was used to differentiate V. parahaemolyticus from nine other Vibrio spp. (V. campbellii, V. cholerae, V. fischeri, V. fluvialis, V. harveyi, V. vulnificus, V. alginolyticus, V. mimicus, and V. mediterranei) and to identify potential V. parahaemolyticus-specific biomarker peaks. The objectives of this study were to determine whether MALDI-TOF MS analysis is reliable for (i) distinguishing V. parahaemolyticus from closely related Vibrio spp. and (ii) detecting variation among the V. parahaemolyticus pandemic clones. Furthermore, we analyzed whether strains that have undergone single gene deletions will have unique fingerprints resulting from changes in their ionizable proteins. This is the first study to use whole-cell MALDI-TOF MS analysis to generate reproducible and unique fingerprints that may be used to rapidly identify Vibrio spp. and to distinguish V. parahaemolyticus from related vibrios.     

Approaches to investigating the ecology of plasmids in marine bacterial communities

To better understand prokaryotic gene flux in marine ecosystems and to determine whether or not environmental parameters can effect the composition and structure of plasmid populations in marine bacterial communities, information on the distribution, diversity, and ecological traits of marine plasmids is necessary. This mini-review highlights recent insights gained into the molecular diversity and ecology of plasmids occurring in marine microbial communities.     

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.