Research Article: Isolation, characterization and analysis of bacteriophages from the haloalkaline lake Elmenteita, Kenya

Date Published: April 25, 2019

Publisher: Public Library of Science

Author(s): Juliah Khayeli Akhwale, Manfred Rohde, Christine Rohde, Boyke Bunk, Cathrin Spröer, Hamadi Iddi Boga, Hans-Peter Klenk, Johannes Wittmann, Rui Lu.


As a step towards better understanding of diversity and biology of phages and their hosts in haloalkaline Lake Elmenteita, phages were isolated from sediment samples and overlying water using indigenous bacteria as hosts. 17 seemingly different phages of diverse morphotypes with different dimensions and partly exhibiting remarkably unusual ultrastructures were revealed by transmission electron microscopy. 12 clonal phage isolates were further characterized. Infection capability of the phages was optimum at 30–35°C and in alkali condition with optimum at pH 10–12. Structural protein profiles and restriction fragment length polymorphism analyses patterns were distinct for each of the phage type. Complete nucleotide sequences of phages vB-VmeM-32, vB_EauS-123 and vB_BhaS-171 genomes varied in size from 30,926–199,912 bp and G + C content of between 36.25–47.73%. A range of 56–260 potential open reading frames were identified and annotated. The results showed that the 12 phages were distinct from each other and confirmed the presence and diversity of phages in extreme environment of haloalkaline Lake Elmenteita. The phages were deposited at the German Collection of Microorganisms and Cell Cultures and three of their genomes uploaded to NCBI GenBank.

Partial Text

Viruses that infect bacteria called bacteriophages (commonly referred to as phages) are known to exist in essentially every possible niche where bacteria reside [1] and profoundly influence ecosystems by infecting and subsequently killing their hosts, thereby impacting the cycling of carbon and nutrients [2]. In environments of extreme temperature, pH, salinity, or a combination of these conditions, viruses of archaea are well represented [3]. In these extreme environments, viruses are the only known predators of prokaryotes. Virus particles in hot springs have been observed by electron microscopy [4] and also cultured on bacteria and archaea isolated from these ecosystems [5][6][7][8]. Witte et al [9] isolated a novel archaeal virus, ɸCh1 from a haloalkalophilic archaeon Natronobacterium magadii upon spontaneous lysis. Danovaro et al [10] evaluated the selectivity of viral infections on deepsea floor by using several independent approaches, including an innovative molecular method based on the quantification of archaeal versus bacterial genes released by viral lysis. Since many viruses are strain-specific, when a particular microbial strain becomes dominant in a system, the number of its viral predators increases exponentially and kill it off leaving a niche for another microbial strain to grow into, that will subsequently be killed off by another viral type. This “kill-the-winner” hypothesis explains much of the observed microbial diversity and changes in community structure [11].

Research authorization in Kenya was given by the National Commission for Science, Technology and Innovation (NACOSTI), Kenya Wildlife Service (KWS) and National Environmental Management Authority (NEMA).

Nine bacterial host strains obtained in this study from the haloalkaline lake Elmenteita, showed physiological characteristics similar to previously isolated bacteria from this lake [70] which include growth in alkaliphilic conditions and temperatures above 30°C, with order Bacillales being the most abundant and easily isolated bacteria [33][34].

The effective use of bacteriophage in all applications must be preceded by detailed understanding of the bacteriophages themselves and analysis of their physiologic characteristics. Isolation, characterization and comparative analysis of phages were the main accomplishments of this study, as an outcome the phages turned out to be different in identity. The taxonomic grouping based upon ultrastructural characteristics, structural proteins, restriction endonuclease patterns and genome size analysis is therefore an effective approach to the classification of the phages. Although we investigated only a small part of the viral community, we established that there is great morphological and genetic variation in the bacteriophages, which leads to high levels of species and strain diversity. Molecular studies of the phages based on GC-ratios, and DNA-DNA similarity between the phages is necessary to confirm the taxonomic status of the groups and provide more information into interaction of phages and hosts. Genome sequencing and computational analysis of the three phages revealed basic and important information about the DNA structure, genome organization and layout and phage relatedness. Further investigations of phage ecology are also recommended in order to gain a more complete understanding of microbial interactions in Lake Elmenteita.

The bacteriophages were accessed to the German Collection of Microorganisms and Cell Cultures (DSMZ) under the following Accession numbers: vB_EauM-23 (DSM 29710), vB_VmeM-32 (DSM 29703), vB_BpsS-36 (DSM 29701), vB_BpsM-61 (DSM 29705), vB_EauS-123 (DSM 29709), vB_BboS-125 (DSM 29706), vB_BhoS-126a (DSM 29707), vB_BhoP-126b (DSM 29708), vB_BcoS-136 (DSM 29699), vB_BpsS-140 (DSM 29700), vB_BhaS-171 (DSM 29702), vB_PmeM-196 (DSM 29704) and the genome sequences deposited at NCBI GenBank under the accession numbers vB_VmeM-32 (KU160494), vB_EauS-123 (KU160495) and vB_BhaS-171 (KU160496).