The discovery that viruses may be the most abundant organisms in natural waters, surpassing the number of bacteria by an order of magnitude, has inspired a resurgence of interest in viruses in the aquatic environment. Surprisingly little was known of the interaction of viruses and their hosts in nature. In the decade since the reports of extraordinarily large virus populations were published, enumeration of viruses in aquatic environments has demonstrated that the virioplankton are dynamic components of the plankton, changing dramatically in number with geographical location and season. The evidence to date suggests that virioplankton communities are composed principally of bacteriophages and, to a lesser extent, eukaryotic algal viruses. The influence of viral infection and lysis on bacterial and phytoplankton host communities was measurable after new methods were developed and prior knowledge of bacteriophage biology was incorporated into concepts of parasite and host community interactions. The new methods have yielded data showing that viral infection can have a significant impact on bacteria and unicellular algae populations and supporting the hypothesis that viruses play a significant role in microbial food webs. Besides predation limiting bacteria and phytoplankton populations, the specific nature of virus-host interaction raises the intriguing possibility that viral infection influences the structure and diversity of aquatic microbial communities. Novel applications of molecular genetic techniques have provided good evidence that viral infection can significantly influence the composition and diversity of aquatic microbial communities.

/pdf/virioplankton-viruses-in-aquatic-ecosystems-30m49tuwrn.pdf

Virioplankton: viruses in aquatic ecosystems

Vibrios are ubiquitous and abundant in the aquatic environment. A high abundance of vibrios is also detected in tissues and/or organs of various marine algae and animals, e.g., abalones, bivalves, corals, fish, shrimp, sponges, squid, and zooplankton. Vibrios harbour a wealth of diverse genomes as revealed by different genomic techniques including amplified fragment length polymorphism, multilocus sequence typing, repetetive extragenic palindrome PCR, ribotyping, and whole-genome sequencing. The 74 species of this group are distributed among four different families, i.e., Enterovibrionaceae, Photobacteriaceae, Salinivibrionaceae, and Vibrionaceae. Two new genera, i.e., Enterovibrio norvegicus and Grimontia hollisae, and 20 novel species, i.e., Enterovibrio coralii, Photobacterium eurosenbergii, V. brasiliensis, V. chagasii, V. coralliillyticus, V. crassostreae, V. fortis, V. gallicus, V. hepatarius, V. hispanicus, V. kanaloaei, V. neonatus, V. neptunius, V. pomeroyi, V. pacinii, V. rotiferianus, V. superstes, V. tasmaniensis, V. ezurae, and V. xuii, have been described in the last few years. Comparative genome analyses have already revealed a variety of genomic events, including mutations, chromosomal rearrangements, loss of genes by decay or deletion, and gene acquisitions through duplication or horizontal transfer (e.g., in the acquisition of bacteriophages, pathogenicity islands, and super-integrons), that are probably important driving forces in the evolution and speciation of vibrios. Whole-genome sequencing and comparative genomics through the application of, e.g., microarrays will facilitate the investigation of the gene repertoire at the species level. Based on such new genomic information, the taxonomy and the species concept for vibrios will be reviewed in the next years.

Biodiversity of Vibrios

The sixth pandemic of cholera and, presumably, the earlier pandemics were caused by the classical biotype of Vibrio cholerae O1, which was progressively replaced by the El Tor biotype representing the seventh cholera pandemic. Although the classical biotype of V. cholerae O1 is extinct, even in southern Bangladesh, the last of the niches where this biotype prevailed, we have identified new varieties of V. cholerae O1, of the El Tor biotype with attributes of the classical biotype, from hospitalized patients with acute diarrhea in Bangladesh. Twenty-four strains of V. cholerae O1 isolated between 1991 and 1994 from hospitalized patients with acute diarrhea in Matlab, a rural area of Bangladesh, were examined for the phenotypic and genotypic traits that distinguish the two biotypes of V. cholerae O1. Standard reference strains of V. cholerae O1 belonging to the classical and El Tor biotypes were used as controls in all of the tests. The phenotypic traits commonly used to distinguish between the El Tor and classical biotypes, including polymyxin B sensitivity, chicken cell agglutination, type of tcpA and rstR genes, and restriction patterns of conserved rRNA genes (ribotypes), differentiated the 24 strains of toxigenic V. cholerae O1 into three types designated the Matlab types. Although all of the strains belonged to ribotypes that have been previously found among El Tor vibrios, type I strains had more traits of the classical biotype while type II and III strains appeared to be more like the El Tor biotype but had some classical biotype properties. These results suggest that, although the classical and El Tor biotypes have different lineages, there are possible naturally occurring genetic hybrids between the classical and El Tor biotypes that can cause cholera and thus provide new insight into the epidemiology of cholera in Bangladesh. Furthermore, the existence of such novel strains may have implications for the development of a cholera vaccine.

New Variants of Vibrio cholerae O1 Biotype El Tor with Attributes of the Classical Biotype from Hospitalized Patients with Acute Diarrhea in Bangladesh

Alkaline phosphatase (AP) catalyzes the hydrolytic cleavage of phosphate monoesters under alkaline conditions and plays important roles in microbial ecology and molecular biology applications. Here, we report on the first isolation and biochemical characterization of a thermolabile AP from a metagenome. The gene encoding a novel AP was isolated from a metagenomic library constructed with ocean-tidal flat sediments from the west coast of Korea. The metagenome-derived AP (mAP) gene composed of 1,824 nucleotides encodes a polypeptide with a calculated molecular mass of 64 kDa. The deduced amino acid sequence of mAP showed a high degree of similarity to other members of the AP family. Phylogenetic analysis revealed that the mAP is shown to be a member of a recently identified family of PhoX that is distinct from the well-studied classical PhoA family. When the open reading frame encoding mAP was cloned and expressed in recombinant Escherichia coli, the mature mAP was secreted to the periplasm and lacks an 81-amino-acid N-terminal Tat signal peptide. Mature mAP was purified to homogeneity as a monomeric enzyme with a molecular mass of 56 kDa. The purified mAP displayed typical features of a psychrophilic enzyme: high catalytic activity at low temperature and a remarkable thermal instability. The optimal temperature for the enzymatic activity of mAP was 37°C and complete thermal inactivation of the enzyme was observed at 65°C within 15 min. mAP was activated by Ca2+ and exhibited maximal activity at pH 9.0. Except for phytic acid and glucose 1-phosphate, mAP showed phosphatase activity against various phosphorylated substrates indicating that it had low substrate specificity. In addition, the mAP was able to remove terminal phosphates from cohesive and blunt ends of linearized plasmid DNA, exhibiting comparable efficiency to commercially available APs that have been used in molecular biology. The presented mAP enzyme is the first thermolabile AP found in cold-adapted marine metagenomes and may be useful for efficient dephosphorylation of linearized DNA.

/pdf/a-novel-psychrophilic-alkaline-phosphatase-from-the-26uy4m7o9f.pdf

A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments

The use of molecular techniques to characterize the microbial communities in contaminated soil and water

Vibrio cholerae phage φ l49 codes tRNAs specific for twelve different amino acids. These tRNA genes are contained in two different HindIII fragments 11 and 3.4 kb in size, of the phage genome. The 3.4 kb HindIII fragment was cloned inEscherichia coli using pBR328 as vector. The recombinant plasmid pNR347 produced nine of the twelve tRNA species (arginine, proline, serine, tyrosine, histidine, lysine, leucine, tryptophan and aspartic acid) encoded in the phage genome.

Structural organization of the transfer RNA gene clusters of cholera phage φ l49

Background In recent years, non-O1, O139 serogroups of Vibrio cholerae have become a major source of pathogenic infection. However, the origin and acquisition of their virulence properties remain under explored. In this regard bacteriophages of Vibrio cholerae are well known to be the carriers of pathogenic traits across various strains. So, any possible association of vibriophages and non-O1, O139 serogroups would provide a deeper insight of their pathogenic threats. Material/methods Ten non-O1, O139 clinical isolates of Vibrio cholerae were induced by mitomycin C. Virulence profiles of those isolates were determined by multiplex PCR. BglII, KpnI and HaeII were used to generate the restriction profile of isolated bacteriophage. Two of the phage harboring strains was ribotyped by Southern hybridization. Results In the present study, ten non-O1, O139 diarrheal isolates of Vibrio cholerae were examined for their ability to produce infectious phage particles out of which two strains, PG128 and PG130 were found to be positive. The host range and restriction profile of phage particles were identical to a biotype converting temperate vibriophage PS166. Both PG128 and PG130 carried unique ribotype pattern and lacked the major virulence determinants. But PG128 was found to carry hlyA, mshA, rtxC and toxR, a set of accessory virulence determinants. Conclusions The evidences present here provide definite clues for a possible phage mediated emergence of newer Vibrio choleare pathogens.

Identification and characterization of phage PS166 lysogens from non-O1, O139 strains of Vibrio cholerae.

Background Temperate phages of Vibrio cholerae are the major force in the emergence of newer pathogenic clones. PS166, a temperate vibriophage, has the ability to convert pathogenic biotypes of the V. Cholerae O1 serogroup. During the conversion process, PS166 also tends to produce a group of lysogens neither reported nor characterized so far. Characterization of those lysogens may provide insight into the emergence of newer pathogenic clones of V. Cholerae. Material/methods The V. cholerae eltor strain MAK757 was used to generate PS166 lysogens which were serotyped by using antisera specific to V. Cholerae O1 and O139 strains. Multiplex PCR was used to compare the status of various pathogenic markers of those lysogens. The outer membrane proteins (OMP) of the lysogens were determined by SDS-PAGE. Results Temperate phage PS166 infection of V. cholerae eltor MAK757 gave rise to a new kind of lysogens termed unserotypable due to their lack of reactivity to both O1 and O139 antisera. Moreover, these lysogens displayed a new phage sensitivity profile different from both classical and parental eltor biotypes. The genetic profiles of the CTX element and VPI region of these lysogens were found to be unaltered, together with the biotype-specific markers mshA and hlyA. Though the genetic loci of major outer membrane proteins did not change after lysogenization, the outer membrane protein profile of these lysogens showed the absence of a 40-kDa band corresponding to the toxR-controlled porin, ompT. Conclusions We provide evidence for the vibriophage PS166-mediated lysogenic conversion of pathogenic cholera strains.

Ranajit K. Ghosh

Papers

Structural organization of the transfer RNA gene clusters of cholera phage φ l49

Identification and characterization of phage PS166 lysogens from non-O1, O139 strains of Vibrio cholerae.

Isolation and characterization of unserotypable lysogens of Vibrio choleraephage PS166.

The cloning and expression of transfer RNA gene cluster of Vibrio eltor phage e4.