Differential expression of serine protease inhibitors 1 and 2 in Crassostrea corteziensis and C. virginica infected with Perkinsus marinus.

Proliferation of Perkinsus marinus (Dermo) in vitro is inhibited by the action of 2 serine protease inhibitors belonging to the I-84 family. We compared the levels of expression of serine protease inhibitors 1 and 2 (SPI-1 and SPI-2) in 2 oyster species (Crassostrea virginica and C. corteziensis) inoculated with the parasite P. marinus. C. virginica is well known to be susceptible to this parasite, whereas C. corteziensis is apparently more tolerant. Oysters were inoculated with trophozoites (1 × 106 trophozoites oyster-1) of P. marinus while control oysters were injected with saline solution. Oysters were maintained in a closed water system for 2 wk. The oysters were then sacrificed and parasite burden, histological damage, and gene expression were evaluated. The results showed that the challenged oysters presented a significant increase in parasite burden, which generated histological damage in digestive gland and gills. Quantitative PCR detected significant differences in SPI-1 and SPI-2 expression levels in the 2 oyster species, with C. corteziensis showing higher expression levels than C. virginica as a response to P. marinus inoculation. Our results provide valuable information for the understanding of the defense response in C. corteziensis and a possible explanation for its tolerance to the parasite.

Patterns in composition, abundance and scarring of whale sharks Rhincodon typus near Holbox Island, Mexico.

Photo-identification and conventional tagging were used to estimate population size and structure of the whale shark Rhincodon typus near Holbox Island, Mexico. From 2005 to 2008, photographs of spot patterns behind the last gill slit and in a lateral view on the left side of each animal were used to identify individuals. Additionally, 578 R. typus were tagged using conventional marker tags. Of these and the 350 R. typus that were identified from 1184 photographs, 65% were male; 27%, female and 8%, indeterminate sex. Photographed R. typus ranged in size from 2·5 to 9·5 m total length. Size was bimodal with a large peak at 6 m and a smaller peak at 7 m. Photo-identification showed that there was considerable loss of marker tags. Few of these remained on the animals for more than a year, so that interannual re-sights using tagging could not be used in population modelling. Forty six interannual re-sightings were found in the photographic library; the interval between these re-sightings was typically 1 year. It was estimated that the R. typus aggregation near Holbox Island ranged from 521 to 809 individuals, based on mark-recapture models. From 13 to 33% of R. typus photographed had scars that were attributable to boat strikes. This study provides a baseline for assessing the status of R. typus near Holbox Island. This information is useful to understand drivers of local population size and distribution and potential concerns about increasing effects of tourism on R. typus in this area and for designing better management programmes for R. typus conservation.

Lactobacillus fermentum BCS87 expresses mucus- and mucin-binding proteins on the cell surface.

AIMS: To identify and characterize adhesion-associated proteins in the potential probiotic Lactobacillus fermentum BCS87. METHODS AND RESULTS: Protein suspensions obtained from the treatment of Lact. fermentum BCS87 with 1 mol 1(-1) LiCl were analysed by Western blotting using HRP-labelled porcine mucus and mucin. Two adhesion-associated proteins with relative molecular weight of 29 and 32 kDa were identified. The N-terminal and internal peptides of the 32 kDa protein (32-Mmubp) were sequenced, and the corresponding gene (32-mmub) was found by inverse polymerase chain reaction. The complete nucleotide sequence of 32-mmub revealed an open reading frame of 903 bp encoding a primary protein of 300 amino acids and a mature protein of 272 residues. A basic local alignment search showed 47-99% identity to solute-binding components of ATP binding cassette transporter proteins in Lactobacillus, Streptococcus and Clostridium. An OpuAC-conserved domain was identified and phylogenetic relationship analysis confirmed that 32-Mmubp belongs to the OpuAC family. CONCLUSIONS: Adhesion of Lact. fermentum BCS87 appeared to be mediated by two surface-associated proteins. 32-Mmubp is a component of ABC transporter system that also functions as an adhesin. SIGNIFICANCE AND IMPACT OF THE STUDY: Characterization of 32-Mmubp and 32-mmub will contribute to understanding the host-bacteria interactions of Lact. fermentum with the intestinal tract of pigs.

First Report of a 16SrI-B Group Phytoplasma Associated with a Yellows-Type Disease Affecting Tomato Plants in the Baja California Peninsula of Mexico.

Since 2000, a phytoplasma-like disease (locally known as "permanent yellowing") was observed on tomatoes (Lycopersicon esculentum Mill.) grown in the Valle de San Quintín in northern Baja California Peninsula. Affected plants showed general chlorosis, severe stunting, upwardly rolled leaves, bronzing of mature leaves, purple discoloration of veins, "little leaf", abnormal floral structures, and excessive branching of axillary shoots. Total DNA extracted from symptomatic and asymptomatic plants was used in nested (n)-PCR assays driven by phytoplasma-universal primer pair P1/P7 (3), followed by primer pair R16F2n/R16R2 (1) targeting the 16S ribosomal RNA gene of the putative phytoplasma. PCR conditions (direct and nested) were conducted as previously described (l,3). Restriction fragment length polymorphism (RFLP) patterns of nPCR-amplified products (≈ 1.25-kbp 16S rDNA fragments) digested with enzymes AluI, MseI, HhaI, and HpaII showed that 85% (17 of 20) of PCR-positive tomato samples had restriction patterns typical of phytoplasmas belonging to the aster yellows group, subgroup B (16SrI-B) "Candidatus Phytoplasma asteris" (2). Only 10% (2 of 20) of the samples were associated with a phytoplasma related to the 16SrXIII-A Mexican periwinkle virescence group (formerly group 16SrI, subgroup I). None of the symptomless plants tested positive. Subsequently, these results were confirmed by nPCR using 16SrI specific primer pair P1/Aint (4) and specific primers rp(I-B)F1/rp(I-B)R1 that amplify the ribosomal protein (rp) gene operon of aster yellows phytoplasma subgroup B (16SrI-B[rp-B]) (1). The presence of the phytoplasmas in symptomatic plants was confirmed by scanning electron microscopy. Characteristic yellow symptoms could be experimentally reproduced by graft inoculation of tomato seedlings (cv. Maya) with tissue of field-infected plants. Symptoms similar to those of field-grown diseased plants were observed consistently in most of the plants, and when graft transmitted from tomato to periwinkle (Catharantus roseus (L.) G. Don), symptoms of virescencent, small flowers were observed. In contrast, no symptoms were observed on plants grafted with tissues from healthy plants. In Baja California, it appears that at least two distinct phytoplasmas are involved in the disease complex. To our knowledge, this is the first molecular evidence of the presence of a phytoplasma associated with yellows-type diseases in the major tomato cultivation areas of the peninsula. References: (1) I.-M. Lee et al. Phytopathology 93:1368, 2003. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (3) B. Schneider et al. Page 369 in: Molecular and Diagnostic Procedures in Mycoplasmology. Academic Press, San Diego, CA, 1995. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

Bacterial Canker Caused by Clavibacter michiganensis subsp. michiganensis on Tomato in the Baja California Peninsula of Mexico.

During the 2005 tomato-growing (Lycopersicon esculentum Mill.) season, an apparent bacterial disease with cankers on the stems and bird's eye lesions on the fruit appeared in commercial fields and greenhouses in the San Quintin and San Simon areas (a 60-mile long coastal plain) near the central region of the Baja California Peninsula of Mexico. The disease was found in midseason, especially when plants were flowering, and the mature and ripe stage. Incidence ranged from 4 to 46%, which represented an important loss in field and greenhouse production. Symptomatic plants showed reddish brown cavities in the stem, discoloration, and water soaking of vascular tissue. Diseased tissues were washed with phosphate buffer and placed on semiselective Clavibacter medium (SCM) (1), and a gram-positive, nonmotile, nonspore-forming, aerobic, curved rod bacterium was consistently isolated and morphologically characterized. Twenty-eight isolates were identified as Clavibacter michiganensis subsp. michiganensis by polymerase chain reaction (PCR) technology with primers CMM5/CMM6 to amplify a fragment of approximately 6.2 kb (2). The isolates were also identified by REP-PCR (repetitive extragenic palindromic PCR) genomic fingerprinting techniques (3) with REP and BOX primer sets (4). Pathogenicity tests consisting of three replicates of 4-week-old tomato seedlings (cv. Tequila) were performed by spraying (twice, 2 days apart) inocula at 108 CFU/ml. Control seedlings were sprayed with sterile water. Inoculated plants previously covered in polyethylene bags were incubated in a growth chamber at 25°C for 48 h. Within 3 weeks, symptoms of reddish-brown cavities, water-soaked lesions, and asymmetrical wilting appeared on inoculated plants and were similar to those symptoms observed in the field. No symptoms were observed on control plants. Confirmation of the causal agent was done by culturing the bacteria on SCM and PCR analysis. Occurrence of the disease in San Quintin Valley is relevant because the disease is one of the five most serious tomato diseases in the peninsula. Moreover, the potential spread of the pathogen by tomato seedlings represents a permanent risk to other pathogen-free areas in the peninsula. Although bacterial canker has been observed in Baja California (Punta Colonet, Vicente Guerrero, San Quintin, and San Simon), to our knowledge, this is the first confirmation of C. michiganensis subsp. michiganensis in Baja, Mexico. References: (1). C. Alarcon et al. Phytopathology 88:306, 1998. (2) J. Dreier et al. Phytopathology 85:462, 1995. (3) F. J. Louws et al. Phytopathology 88:862, 1998. (4) J. Versalovic et al. Methods Mol. Cell. Biol. 5:25, 1994.

Ostreid herpesvirus 1 (OsHV-1) detection among three successive generations of Pacific oysters (Crassostrea gigas).

Ostreid Herpesvirus 1 (OsHV-1) was likely detected in Pacific oysters, Crassostrea gigas, at different stages of development. Viral infections were associated with high mortality rates in the spat and larvae. Furthermore, the persistance of OsHV-1 in asymptomatic adults was demonstrated by detection of viral DNA and proteins. In the present study, three successive generations of C. gigas (G0 and G1 parental oysters, G1 and G2 larvae) were screened for OsHV-1 by PCR. Viral DNA was detected in 2-day-old larvae, indicating that infection may take place at very early stages. Although results strengthen the hypothesis of a vertical transmission, it was not possible to predict the issue of a particular type of cross. Indeed, the detection of viral DNA in parental oysters did not systematically correspond to a productive infection or result in a successful transmission to the progeny. However, the infective status of the parents appeared to have an influence on both the infection and the survival rates of the progeny. Crosses involving an OsHV-1 infected male and a non-infected female resulted in hatching and larval survival rates statistically lower than those observed in the other types of cross. These results suggest that OsHV-1-infected females may transmit to their offspring some kind of protection or resistance against viral infection.

A New Begomovirus Causes Tomato Leaf Curl Disease in Baja California Sur, Mexico.

More than 10,000 ha of tomatoes are grown in the field and greenhouses on the Baja California Peninsula of Mexico. Information about the etiology of geminivirus-like diseases affecting tomato crops in all horticultural regions in the area has been difficult to obtain and assess. From 2001 through 2003, stunting, foliar discoloration, reduced leaf size, and leaf crumpling symptoms were observed and analyzed in one large area of tomato plantings in El Carrizal (near the city of La Paz in Baja California Sur). This leaf curl disease resembled that caused by Chino del tomate virus and has been observed at levels of incidence ranging from 60 to 90%. DNA isolated from symptomatic plants was analyzed using DNA hybridizaton and polymerase chain reaction (PCR) amplification of the 5' regions of the replication and coat protein genes, including the intergenic region (3). Comparisons of the nucleotide sequence (GenBank Accession No. AY339619) with corresponding sequences in GenBank resulted in 84.2% identity with Tomato mild mottle virus and 61.7% with Tomato severe leaf curl virus; both isolates originate from Central America. The relatively low nucleotide sequence identities from its closest relatives suggested that the virus may be a new begomovirus species of unambiguous American ancestry. In a phylogenetic analysis using PAUP 4.0 software, the Baja California isolate clustered in a separate group from other Mexican sequences. Moreover, the iteron (iterative sequences motifs associated in virus replication) arrangements (1) are unique among known New World begomoviruses, but identical to analogous elements from a tobacco-infecting begomovirus from China. On the other hand, it is well known that there are interactions between geminiviruses in mixed infections in some horticultural areas of Mexico (2). To determine the identity of the putative geminivirus involved in the disease, we used selected restriction enzyme (EcoRI, HindIII and XbaI) analysis and PCR with specific primers. No evidence of mixed infections with other geminiviruses was obtained. DNA fragments of the expected size (1.1 kb) showed different digestion patterns compared with other well-characterized geminiviruses isolated from Mexico such as Chino del tomate virus, Pepper huasteco yellow vein virus, Tomato leaf curl Sinaloa virus, and Pepper golden mosaic virus. Epidemiological, experimental, and natural host range studies indicated that the Baja California isolate has a relatively narrow host range infecting tomatoes, peppers (Capsicum annuum L.), and Peruvian apple (Nicandra physalodes L.). Reproduction of characteristic leaf curling symptoms in tomato seedlings infected with viruliferous whiteflies (Bemisia tabaci Genn.) and inoculated biolistically using infectious DNA (0.5 µg/ml) as inoculum were obtained. Koch's postulates were completed using PCR and DNA hybridization to confirm virus identity. These results confirm that the Baja California isolate is different from other begomoviruses isolated from Mexico. The virus is tentatively named Tomato chino Baja California virus (ToChBCV), genus Begomovirus, family Geminiviridae. References: (1) G. R. Arguello-Astorga et al. Arch. Virol. 146:1465, 2001. (2) J. Mendez-Lozano et al. Phytopathology 93:270, 2003. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Polymorphism at the ITS and NTS Loci of Perkinsus marinus isolated from cultivated oyster Crassostrea corteziensis in Nayarit, Mexico and phylogentic relationship to P. marinus along the Atlantic Coast.

Prevalence of the protozoan Perkinsus spp. in the gills of the pleasure oyster Crassostrea corteziensis from two estuaries in Nayarit, Mexico, was measured. The protozoan was identified by PCR amplification of the internal transcribed spacer (ITS) region of the rDNA of Perkinsus spp. The pathogen was found in 92% of oysters from Boca de Camichín and 77% of oysters from Pozo Chino. ITS sequences characterized from C. corteziensis showed 96-100% similarity to Perkinsus marinus. The most frequent ITS sequence (GenBank JQ266236) had 100% identity with the ITS locus of P. marinus from New Jersey, Maryland, South Carolina and Texas, and the second most frequent observed sequence (GenBank JQ266240) was 100% identical to ITS sequences of P. marinus from New Jersey, South Carolina, Louisiana, and Bahía Kino, Sonora, Mexico. The 14 sequences from the non-transcribed spacer (NTS) showed 98% similarity to P. marinus from Texas. The most frequent polymorphism identified was at nucleotide 446 of the ITS region; however, the NTS showed the highest nucleotide diversity, thereby suggesting that this region is suitable for genotype identification. Moreover, the most conserved ITS marker is better for species-specific diagnosis. Both the ITS and NTS sequences of P. marinus obtained from C. corteziensis were grouped in two clades, identifying two allelic variants of P. marinus.

Diagnosis of Ostreid herpesvirus 1 in fixed paraffin-embedded archival samples using PCR and in situ hybridisation.

In 1994, some of the high mortality episodes that affected oysters cultured in France were associated with herpesviral infections. Through histology analysis, however, viral presence could only be suspected and confirmation of histological diagnosis by transmission electron microscopy was performed in only a few cases. Subsequently, the characterisation and genome sequencing of Ostreid herpesvirus 1 (OsHV-1) made possible the development of specific molecular detection (PCR and in situ hybridisation (ISH)). Using both molecular tools, attempts were made to screen for OsHV-1 a number of fixed, paraffin-embedded oyster samples collected and processed in 1994. The aim was to compare these techniques and to estimate the accuracy of histology-based indication of viral infection. Existing DNA extraction protocols were adapted for oyster samples and two pairs of specific primers targeting small fragments (less than 200bp) were designed (C(9)/C(10) and B(4)/B(3)). The poor consistency observed between the results of PCR with both primer pairs was confirmed by statistical analysis. C(9)/C(10), which targets a repeated region of the OsHV-1 genome, appears to be the primer of choice for viral detection in archival samples. In situ hybridisation may furnish complementary information concerning the localisation of viral foci. Under certain conditions, retrospective examination of archival samples by molecular techniques may therefore provide valuable epidemiological data.

First Report of a Geminivirus Associated with Leaf Curl in Baja California Peninsula Tomato Fields.

Since November 2001, geminivirus-like symptoms (stunting, reduced leaf size, and leaf curling "chino") have been observed in tomato (Lycopersicon esculentum Mill.) plantings in Baja California Sur, Mexico. Samples of symptomatic plants were collected from commercial fields and analyzed by traditional and molecular methods for the presence of geminiviruses. Inocula prepared from infected plants were experimentally transmitted to tomato seedlings and Datura stramonium by mechanical inoculation and whitefly transmission. Leaf curling and interveinal chlorosis symptoms similar to those found in the field were observed in inoculated tomato and D. stramonium. DNA from infected plants was extracted and analyzed by polymerase chain reaction (PCR) and electrophoresis using degenerate primers PALIv1978/PARIc494 (1). PCR fragments of the expected size (1.1 kb) for the common region (CR) were obtained from 28 of 64 plants, cloned and sequenced (GenBank Accession No. AY336088). Comparisons of CR sequences with the NCBI database by using BLAST and MegAlign (DNASTAR, London) indicated that the Baja Californian isolates were New World bipartite begomoviruses sharing the highest nucleotide sequence identity (93%) with a partially characterized geminivirus (Tomato severe leaf curl virus (ToSLCV); GenBank Accession No. AF130415) from Guatemala. References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Adhesive properties of a LamB-like outer-membrane protein and its contribution to Aeromonas veronii adhesion.

AIMS: To identify and characterize nonfimbrial proteins from Aeromonas veronii involved in the attachment to epithelial cells in vitro. METHODS AND RESULTS: Two Aer. veronii mucin- and lactoferrin-binding proteins with molecular masses of 37 and 48 kDa were identified by Western blot analysis. According to its N-terminal amino acid sequence, the 48-kDa protein was identified as Omp48, an outer-membrane protein similar to LamB of Escherichia coli. LamB is a well-known porin involved in maltose transport across the outer membrane in E. coli. In a microtitre plate assay, Omp48 bound to the immobilized extracellular matrix proteins collagen and fibronectin, and the mucin- and lactoferrin-binding activity was confirmed. Adhesion of Omp48 to mucin, lactoferrin and collagen was diminished by preincubation with homologous glycoproteins or other carbohydrates, suggesting a putative Omp48 lectin-like binding domain. Anti-Omp48 antiserum significantly inhibited the Aer. veronii adhesion to confluent HeLa cell monolayers and pretreatment of cells with purified Omp48 elicited competitive inhibition of adhesion. Similarly, cross-inhibition of Aer. hydrophila and Aer. caviae adhesion was achieved with the same treatments, indicating the existence of a conserved surface protein among these species. CONCLUSIONS: Taken together, these data indicate that Omp48 is involved in Aer. veronii adhesion to epithelial cells and might be an alternative adhesion factor of this micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: The adhesive potential of Aeromonas spp. is correlated with pathogenicity; however, the adhesion mechanism is complex and not well understood. This study provides evidence of a putative adhesion factor that might be contributing to pathogenicity of Aer. veronii and could be used for vaccine development.

Yeasts isolated from the intestine of rainbow trout adhere to and grow in intestinal mucus.

The yeast strains Saccharomyces cerevisiae CBS 7764 and Debaryomyces hansenii Hf1 (CBS 8339), isolated from the intestine of rainbow trout, were studied with respect to adhesion to and growth in fish intestinal mucus. The level of adhesion was dependent on the physiologic state of the yeast culture. Growing cells of both strains adhered more strongly than nongrowing cells. This correlates with a previously shown shift in cell surface hydrophobicity of these yeasts. In addition, forces other than hydrophobic interactions may participate, as all strains tested adhered more strongly to the membrane lipid phosphatidylserine than to phosphatidylcholine and phosphatidylethanolamine. Debaryomyces hansenii Hf1 also adhered to the most hydrophobic of the neutral lipids present in mucus, while no adhesion was observed to the other neutral lipids or to the hydrophilic silica gel, again suggesting hydrophobic interactions. Finally, the fish-isolated yeasts grew rapidly in isolated fish intestinal mucus as the sole source of energy and nutrients.

Differences in the susceptibility of American white shrimp larval substages (Litopenaeus vannamei) to four vibrio species.

The rapid expansion of commercial culture of penaeid shrimp is threatened by Vibrio diseases affecting survival and growth. These opportunistic microorganisms are considered part of the normal ecosystem of penaeid shrimp and cause diseases only under conditions that favor them over the host. Shrimp larvae show different susceptibility to these pathogenic agents. In the present work, we report on a comparative study of the susceptibility of all American white shrimp (Litopenaeus vannamei) larval substages to four potentially pathogenic Vibrio species (V. harveyi, V. parahaemolyticus, V. alginolyticus, and V. penaeicida). Strains of these bacterial species were used to infect nauplii, protozoea I-III, mysis I-III, and postlarvae 1 by immersion challenge at 10(3), 10(5), or 10(7) cfu mL(-1) for 30 min. V. alginolyticus infection had no significant effect on survival rate, compared to control, in all shrimp larvae and at all doses tested. Shrimp larvae infected with V. alginolyticus showed a high survival rate compared to other Vibrio species at the three dose levels. V. penaeicida produced a significant mortality effect (P < 0.01) in all shrimp substages and only in postlarvae 1 at low infection dose (10(3) cfu mL(-1)). V. harveyi and V. parahaemolyticus induced significant mortality rates (P < 0.01) only at high doses in shrimp larvae. In summary, shrimp larvae demonstrated an age susceptibility that depends on the Vibrio species and dose level.

Adhesion of yeast isolated from fish gut to crude intestinal mucus of rainbow trout, Salmo gairdneri.

Two yeast strains, Saccharomyces cerevisiae CBS 7764 and Debaryomyces hansenii Hfl CBS 8339, with a high capacity to colonize fish intestine were used in this study. The ability to adhere to crude mucus obtained from fish intestine was demonstrated for both strains. Scatchard analysis of the binding indicated a positive cooperativity for D. hansenii Hfl and absence of cooperativity for S. cerevisiae CBS 7764. In neither of the strains was adhesion extensively affected by reducing the hydrophobic interaction with p-nitrophenol, or by enhancing the hydrophobic interaction with ammonium sulfate. The adhesion was heat sensitive but resistant to trypsin treatment. We conclude that adhesion is mediated partly by specific mechanisms and partly by cell surface hydrophobicity.

The expression of potential colonization factors of yeasts isolated from fish during different growth conditions.

Three strains, Rhodotorula rubra, Rhodotorula glutinis, and Candida zeylanoides, isolated from fish, were tested for the expression of putative tissue colonization factors. All strains were able to bind collagen type I, fibronectin, and laminin to various degrees after growing on various solid and broth media, while the binding to collagen type IV was sparse under all conditions tested. For the three strains tested, a very low cell surface hydrophobicity was shown for growth on various solid and broth media. Mostly, the strains also expressed a negatively charged surface. Extracellular protease activity using different substrates was shown for all three strains. Furthermore, two properties related to iron scavenging, i.e., binding of lactoferrin and production of siderophores, were also tested. For the three strains a capacity to bind lactoferrin as well as a capacity to excrete siderophores were demonstrated. Since these different properties have been correlated to virulence and to the capacity of colonization in other organisms, we address the question of whether the expression of these properties in yeasts could contribute to colonization in fish.

Binding of collagen, fibronectin, lactoferrin, laminin, vitronectin and heparan sulphate to Staphylococcus aureus strain V8 at various growth phases and under nutrient stress conditions.

We have examined how Staphylococcus aureus strain V8 cells interact with 125I-labelled extracellular matrix (ECM) and serum proteins (collagen type I and IV), fibronectin, lactoferrin, laminin, vitronectin, and heparan sulphate at various phases of the growth cycle. Maximal binding of these glycoproteins and heparan sulphate to the bacteria occurred after 17 to 20 h in the late stationary phase except for fibronectin-binding, which was maximal after 12 to 14 h. Binding of the glycoproteins and heparan sulphate to S. aureus V8 under nutrient stress conditions exhibited complex patterns based on different starving conditions and various binding ligands. In general, bacteria starved in distilled water and 0.02 M potassium phosphate buffer (pH 7.2) at room temperature showed high susceptibility to all binding ligands within the first 18 h, followed by entering a lower binding period (except for collagen-binding which still remained high). The binding was not correlated to cell surface charge or hydrophobicity of the bacteria. Furthermore, extracellular and cell-associated proteolytic activity of starved cells against ECM and serum proteins was found to be greater than for non-starved cells. Thus, S. aureus could sustain its ability to bind various connective tissue and cell surface components during a long period of time even in the absence of energy-yielding substrates.

Cyanobacterial diversity in extreme environments in Baja California, Mexico: a polyphasic study.

Cyanobacterial diversity from two geographical areas of Baja California Sur, Mexico, were studied: Bahia Concepcion, and Ensenada de Aripez. The sites included hypersaline ecosystems, sea bottom, hydrothermal springs, and a shrimp farm. In this report we describe four new morphotypes, two are marine epilithic from Bahia Concepcion, Dermocarpa sp. and Hyella sp. The third, Geitlerinema sp., occurs in thermal springs and in shrimp ponds, and the fourth, Tychonema sp., is from a shrimp pond. The partial sequences of the 16S rRNA genes and the phylogenetic relationship of four cyanobacterial strains (Synechococcus cf. elongatus, Leptolyngbya cf. thermalis, Leptolyngbya sp., and Geitlerinema sp.) are also presented. Polyphasic studies that include the combination of light microscopy, cultures and the comparative analysis of 16S rRNA gene sequences provide the most powerful approach currently available to establish the diversity of these oxygenic photosynthetic microorganisms in culture and in nature.

Molecular cloning, sequencing and characterization of omp48, the gene encoding for an antigenic outer membrane protein from Aeromonas veronii.

AIMS: To clone, sequence and characterize the gene encoding the Omp48, a major outer membrane protein from Aeromonas veronii. METHODS AND RESULTS: A genomic library of Aer. veronii was constructed and screened to detect omp48 gene sequences, but no positive clones were identified, even under low stringency conditions. The cloned gene probably was toxic to the host Escherichia coli strain, so the cloning of omp48 was achieved by inverse PCR. The nucleotide sequence of omp48 consisted of an open reading frame of 1278 base pairs. The predicted primary protein is composed of 426 amino acids, with a 25-amino-acid signal peptide and common Ala-X-Ala cleavage site. The mature protein is composed of 401 amino acids with a molecular mass of 44,256 Da. CONCLUSIONS: The omp48 gene from Aer. veronii was cloned, sequenced and characterized in detail. BLAST analysis of Omp48 protein showed sequence similarity (over 50%) to the LamB porin family from other pathogenic Gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial diseases are a major economic problem for the fish farming industry. Outer membrane proteins are potentially important vaccine components. The characterization of omp48 gene will allow further investigation of the potential of Omp48 as recombinant or DNA vaccine component to prevent Aer. veronii and related species infections in reared fish.