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.

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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.

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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

An unusual filamentous bacteriophage, VSK, containing single-stranded, circular DNA as its genome was isolated from Vibrio cholerae 0139 strains P07 and B04. Unlike other single-stranded DNA phages, VSK can integrate its genome into the chromosome of the host and enter into a lysogenic state. The double-stranded replicative form (RF) of the single-stranded phage DNA was isolated. A restriction map of the VSK RF DNA was constructed using HaeII, AvaII, ClaI and XbaI. By Southern blot analysis of the chromosomal DNA of the lysogen using labeled phage DNA as probe, the attachment site (attP) on the viral genome was also identified.

Integration of the DNA of a novel filamentous bacteriophage VSK from Vibrio cholerae 0139 into the host chromosomal DNA

Identification of the Gene for the Monomeric Alkaline Phosphatase of Vibrio Cholerae Serogroup O1 Strain

Tandem repeats of cholera toxin gene in Vibrio cholerae 0139

Summary
Biophysical characteristics of Vibrio eltor phage e4, a key phage in the Vibrio cholerae typing scheme were studied. This icosahedral phage was found to contain 12 structural polypeptides with mol. wt. ranging from 25000 to 120000. One of these polypeptides of mol. wt. 50000 accounted for most of the structural proteins present and was probably the major phage capsid protein. The phage genome comprised a single linear, double-stranded DNA molecule, 69.2 kbp in length (45.6 × 106 mol. wt.) as determined by electron microscopy and restriction fragment analyses. The G + C content was 34.6%. Electron microscopy data indicated that unlike the DNAs of other cholera phages, phage e4 DNA is not circularly permuted. Adsorption under normal conditions was biphasic with rate constants of 1.02 × 10-9/ml/min up to 60% adsorption and 3 × 10-10/ml/min thereafter. Intracellular phage multiplication was characterized by a latent period of 27 min. The burst size was approximately 100 phage particles per infected cells.

Ranajit K. Ghosh

Papers

Integration of the DNA of a novel filamentous bacteriophage VSK from Vibrio cholerae 0139 into the host chromosomal DNA

Identification of the Gene for the Monomeric Alkaline Phosphatase of Vibrio Cholerae Serogroup O1 Strain

Tandem repeats of cholera toxin gene in Vibrio cholerae 0139

Characterization of Vibrio eltor typing phages: properties of the Eltor phage e4.

Localization of transfer RNA genes on the physical map of Vibrio eltor phage e4 genome