Isolation, characterization and analysis of bacteriophages from the

RESEARCH ARTICLE

Isolation, characterization and analysis of

bacteriophages from the haloalkaline lake

Elmenteita, Kenya

Juliah Khayeli Akhwale

1,2

*, Manfred Rohde

3☯

, Christine Rohde

1☯

, Boyke Bunk

1☯

Cathrin Spro

1☯

, Hamadi Iddi Boga

4☯

, Hans-Peter Klenk

5☯

, Johannes Wittmann

1☯

1 Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig,

Germany, 2 Department of Zoology, Jomo Kenyatta University of Agriculture and Technology, Nairobi,

Kenya, 3 Helmholtz Centre for Infection Research, Central Facility for Microscopy, Braunschweig, Germany,

4 Taita Taveta University College, Voi, Kenya, 5 School of Natural and Environmental Sciences, Newcastle

University, Newcastle upon Tyne, United Kingdom

☯ These authors contributed equally to this work.

* [email protected]

Abstract

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 morpho-

types 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 con-

dition 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 nucleo-

tide 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.

Introduction

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 combina-

tion of these conditions, viruses of archaea are well represented [3]. In these extreme environ-

ments, viruses are the only known predators of prokaryotes. Virus particles in hot springs have

PLOS ONE | https://doi.org/10.1371/journal.pone.0215734 April 25, 2019 1 / 19

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OPEN ACCESS

Citation: Akhwale JK, Rohde M, Rohde C, Bunk B,

Spro¨er C, Boga HI, et al. (2019) Isolation,

characterization and analysis of bacteriophages

from the haloalkaline lake Elmenteita, Kenya. PLoS

ONE 14(4): e0215734. https://doi.org/10.1371/

journal.pone.0215734

Editor: Rui Lu, Louisiana State University, UNITED

STATES

Received: August 27, 2018

Accepted: April 8, 2019

Published: April 25, 2019

access article distributed under the terms of the

Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevant data are

within the manuscript and its Supporting

Information files.

Funding: This work was supported by The German

Academic Exchange Service (DAAD) within a PhD

scholarship award for J.K. Akhwale (REF NO. A./

12/91556) and was complemented by in-house

resources of the DSMZ Department of

Microbiology to cover material costs. The funders

had no role in study design, data collection and

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 par-

ticular 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].

Natural phage communities are reservoirs of considerable uncharacterized genetic diversity

on Earth [12] and provide a valuable resource to development of modern biotechnology [13].

Complete phage genomes facilitate studies of phage evolutionary history and relationships,

biodiversity, biogeography and identification of novel phage taxa [14][15][16]. Insight into

understanding of phage biology can be exploited to generate a broad application spectrum like

novel nanotechnologies, bacterial detection strategies and biological control of pathogenic bac-

teria on an industrial scale [17][18]. Despite their importance and ubiquitous abundance, far

too little is known about their diversity in natural ecosystems [19][20][21][22]. The best stud-

ied groups of phages are those examples infecting bacterial pathogens. Most studies on natural

populations of phages and their host relationship have been performed in terrestrial, marine,

and freshwater environments and very few in unusual or extreme habitats [23][24][25] where

over 90% of the earth’s bacterial diversity is thought to reside [26][25]. Furthermore, most

known phages are from North America and Europe, while little is known of phages in the envi-

ronment of vast regions such as Africa and South America [27].

Soda lakes are strongly alkaline lakes, typically with a pH of 8.5 to >12, high concentrations

of carbonate ions and with salinities ranging from brackish to hypersaline [28]. The groups of

microbes able to grow under alkaline conditions in the presence of high salt are referred to as

haloalkaliphiles [29]. They possess special adaptation mechanisms to survive and grow under

high salinity and alkaline pH. These properties of dual extremity of halophiles and alkaliphiles

make them interesting from both fundamental research and biotechnological points of view

[30]. Kenya’s Great Rift Valley contains this type of Lakes namely Elmenteita, Magadi, Bogoria,

Nakuru and Sonachi [31]. Studies on diversity and isolation of bacterial species from Lake

Elmenteita have been highly documented [32][33][34]. However, viruses from these environ-

ments are particularly under-studied at present. Hence, rich reservoirs of enormous genetic

and biological diversity therefore remain to be explored and analyzed. Previous studies on the

Soda lakes include isolation of phages from Lake Magadi by Jamison et al [35] and Muruga

et al [36]. Moulton et al [37] also isolated and studied a phage infecting an alkaliphilic Vibrio

metschnikovii from Lake Magadi. Peduzzi et al [38] carried out an electron microscopic study

of cyanophages that affect African flamingo population in Lake Nakuru.

As a step towards better understanding of the diversity and biology of phages and their

hosts in haloalkaline Lake Elmenteita, phages were isolated from sediments and overlying

water using indigenous bacteria as hosts. The phages were characterized by their morphology,

host range analysis, structural protein profile analysis, restriction endonuclease patterns analy-

sis and genome size estimation by pulsed-field gel electrophoresis (PFGE). A further goal of

this research was to sequence, annotate and analyse the genome of some phages from the

haloalkaline Lake Elmenteita using various available bioinformatics tools.

The study site, Lake Elmenteita, is situated at 0˚ 27´ S 36˚ 15´ E on the floor of the Kenyan

Rift Valley at 1776 m above sea level and has no direct outlet [39]. The region is characterized

by a hot, dry and semi-arid climate with a mean annual rainfall of about 700 mm [40]. Due to

Isolation, characterization and analysis of bacteriophages

PLOS ONE | https://doi.org/10.1371/journal.pone.0215734 April 25, 2019 2 / 19

analysis, decision to publish, or preparation of the

manuscript.

Competing interests: The authors have declared

that no competing interests exist.

the high temperatures there are very high evaporation rates during the drier seasons, leading

to a seasonal reduction in the total surface area. The size of Lake Elmenteita is roughly 20 km

and the depth rarely exceeds 1.0 m [33]. The alkalinity of the water is high with a high concen-

tration of carbonates (1200 mg Na

-1

), chlorides and sulphates [32]. The water tempera-

ture ranges between 30 and 40˚C and the pH is above 9.

It is expected that the genomic sequences will give insight into genome architecture and

content in terms of gene function as well as the level of their similarity compared to currently

available phages. To our knowledge, these experiments represent the first report of isolation

and characterization of bacteriophages from the haloalkaline Lake Elmenteita.

Materials and methods

Research authorization in Kenya was given by the National Commission for Science, Technol-

ogy and Innovation (NACOSTI), Kenya Wildlife Service (KWS) and National Environmental

Management Authority (NEMA).

Isolation and characterization of bacterial host strains

Sediment sample plus the overlying water were collected (March, 2013) into sterile jars, capped

on site and preserved in cooled boxes for transportation to the molecular laboratory in Jomo

Kenyatta University of Agriculture and Technology (JKUAT). In the laboratory the samples

were packaged for transfer to Leibniz Institute—DSMZ in Braunschweig, Germany and stored

at 8˚C.

Approximately 2 g of sediment was used to make a mastermix using filter (0.20 μm pore

size) sterilised water (10 ml) from the lake and the solution serially diluted using the same

water. Aliquots (100 μl) of serial dilutions were plated onto solid LB medium adjusted to

approximately pH 9.5 with Sodium Sesquicarbonate (4.3 g NaHCO

/5.2 g NaCO

/100 ml dis-

tilled water). The plates were incubated at 28˚C for 3 days. Colonies appearing on the plates

were purified by three consecutive single colony passages. Isolated bacterial strains were used

as hosts for the detection of lytic bacteriophages from the same lake. Susceptible strains were

stocked in LB broth (pH 9.5) with 15% glycerol (v/v) at -20˚C.

Growth of the strains on different media (LB, Nutrient agar and Horikoshi 1) at 28˚C was

assessed. Growth was also assessed at temperature 20–45˚C (in increments of 5˚C), pH values

from 5.0–13.0 (in increments of 1.0 pH unit) using LB as the basal medium. The colony fea-

tures were observed under a binocular microscope [41]. Cell morphology (size, shape, arrange-

ment) was determined by phase-contrast microscopy (magnification, 400×) after 3 days of

incubation at 28˚C. Gram stain was performed using the KOH test [42]. Bacterial hosts’

genomic DNA extraction, PCR-mediated amplification of 16S rDNA gene using universal bac-

terial primer sets 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-TACGGYTAC

CTTGTTACGACTT-3’), and purification of PCR products was carried out as previously

described by Kim et al [43]. Identification of phylogenetically closest taxa and calculation of

pairwise 16S rRNA gene sequence similarity was performed using the EzTaxon server (http://

www.eztaxon.org) [44]. The genomic homogeneity of the strains was also examined in com-

parison with their close relatives by Matrix assisted laser desorption/ionization time of flight

(MALDI-TOF) mass spectra (MS) analysis [45].

Isolation and characterization of bacteriophages

Bacteriophage propagation and purification. LB medium supplemented with 2mM

CaCl

(Sigma-Aldrich, St. Louis, MO) adjusted to approximately pH 9.5 using 1M Sodium-

Sesquicarbonate (4.3 g NaHCO

, 5.2 g NaCO

, 100 ml distilled water; 1 ml in 10 ml medium)

Isolation, characterization and analysis of bacteriophages

PLOS ONE | https://doi.org/10.1371/journal.pone.0215734 April 25, 2019 3 / 19

was used. Approximately 1 g of sediment sample was suspended in 9 ml LB broth in a sterile

15 ml centrifuge tube and mixed thoroughly on an overhead shaker for 1 hour at room tem-

perature. The sample was thereafter centrifuged at 7,500 r.p.m. for 15 minutes then the super-

natant further filtered through a 0.45 μm pore size syringe filter (Millipore corp, Billerica,

MA). The supernatant (5 ml) was added to equal amount of double strength LB broth and

inoculated with an early log-phase (0.1 ml) host culture. After overnight enrichment at 28˚C

with gentle shaking, the culture was centrifuged at 7,500 r.p.m. for 15 minutes [46]. This

enrichment procedure was repeated thrice. The supernatant obtained from the final enrich-

ment step was filter sterilized through a 0.45 μm pore size syringe filter and checked for the

presence of phages by the soft agar overlay method. The soft agar was prepared by adding

100 μl phage lysate to 200 μl of an overnight culture of indicator strain and mixed with 5 ml of

liquid soft agar at 45˚C. This mixture was spread on solid LB medium, incubated overnight at

28˚C and checked for the presence of plaques [47]. Uninfected host strain was used as negative

control for checking bacteriocin reactions to confirm the validity of plaques [48]. Underlay

procedure for phage purification [46] was followed. Phage particle from a well isolated plaque

was streaked on solid LB medium as though attempting to obtain single colony isolates from a

bacterial culture, followed by overlay containing host cells poured over the surface of the plate

and incubated after setting. The procedure was repeated three times.

To recover phages, phages were collected from plates with confluent lysis and eluted by

transferring agar overlayer aseptically into 10 ml of mid-log host cell culture in LB broth and

incubated at 28˚C with gentle shaking (overnight). The phage supernatant was collected by

centrifugation at 7,500 r.p.m. for 15 minutes, filtered (0.45 μm) and the phage stock stored at

4˚C. The titer of the stock was determined by the soft agar overlay method. 1 ml of the phage

lysate was transferred aseptically to 10 ml of the mid-log host cell culture in LB broth and incu-

bated at 28˚C with gentle shaking until clearing was observed (overnight). The phage superna-

tant was collected by centrifugation at 7,500 r.p.m. for 15 minutes (Sorvall RC6, F10S-6×500y

rotor). The fresh lysate (10 ml) was added to 200 ml of mid-log host cell culture and repeated

as above. Phages were concentrated by centrifugation at 12,000 r.p.m. for 2 hours (Sorvall

RC6, F21S-8 × 50 rotor). Phages were purified using CsCl density-gradient ultracentifugation.

The phage pellet was re-suspended in 1 ml of TE buffer (20 mM Tris [pH 7.5], 50 mM NaCl)

[49]. 1.5 ml concentrated phage suspension was overlaid onto a four-step Cesium Chloride

(CsCl) gradient containing 0.7 ml each of 1.7 g/ml, 1.5 g/ml, 1.4 g/ml and 1.3 g/ml CsCl (Opti-

cal grade, Gibco) in a 4.3 ml ultracentrifuge tube (Beckman Coulter). Phages were centrifuged

for 2 h at 20˚C and 22,000 r.p.m. in ultracentrifuge (Beckman Coulter, Optima L-XP; SW 60

Ti 12E873 rotor). Phage-containing bands (white-to-grey) were extracted by puncturing the

wall of the ultracentrifuge tube using a needle, and the CsCl removed by dialysis (visking dialy-

sis tubing: Type (inch) 8/32, wall thickness (mm) 0.050, width (mm) 10, Ø (mm) 6.3; ROTH)

for 15 h with two changes of TE buffer (10 mM Tris [pH 7.5], 50 mM NaCl) [49].

Negative staining and electron microscopy of bacteriophages. Thin carbon support

films were prepared by sublimation of a carbon thread onto a freshly cleaved mica surface.

Phages were adsorbed onto the carbon film and negatively stained with 2% (w/v) aqueous ura-

nyl acetate, pH 5.0 [50]. Samples were examined in a TEM 910 transmission electron micro-

scope (Carl Zeiss, Oberkochen) at an acceleration voltage of 80 kV. Images were taken at

calibrated magnifications using a line replica. Images were recorded digitally with a Slow-Scan

CCD-Camera (ProScan, 1024×1024, Scheuring, Germany) with ITEM-Software (Olympus

Soft Imaging Solutions, Mu¨nster, Germany). The phenotypic diversity of the bacteriophages

was determined using the morphological criteria outlined by the International Committee of

Taxonomy of Viruses [27].

Isolation, characterization and analysis of bacteriophages

PLOS ONE | https://doi.org/10.1371/journal.pone.0215734 April 25, 2019 4 / 19

Thermal and pH stability tests. The thermal stability of the phages was examined by pre-

incubating phage suspensions at different temperatures (20, 25, 30, 35, 40, 45 and 50˚C respec-

tively) at pH 7.0 for 6 hours. After the incubation, phage suspensions were immediately cooled

in ice water and the surviving phages were tittered by the double agar layer method. The pH

stability of phages was examined by pre-incubating the phage suspensions of different pH lev-

els (2, 4, 6, 8, 10 and 12 respectively) at 25˚C for 6 hours. The surviving phages were immedi-

ately counted by the double agar layer method [51].

Host range analysis of bacteriophages. To evaluate the lytic spectrum of the obtained

bacteriophages, all the susceptible bacterial strains isolated in this study were used. Double

layer agar plates with different bacterial strains were prepared. The lysis spectrum of isolated

phages was determined by spotting 10 μl of phage lysate on each agar plate with different bac-

terial strains. The plates were incubated at 28˚C overnight and examined for clearing zones

[52]. Observed inhibition of growth as marked by clearing where the lysate was spotted, was

denoted as susceptibility of the bacteria.

Stuctural protein profiles. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

(SDS-PAGE) was performed by the method of Laemmli [53]. A sample of 50 μl purified phage

particles (5×10

pfu/ml) was dissolved in 50 μl loading buffer (50 μl Mercaptoethanol, 950 μl

Laemmli sample buffer (2×) for SDS-PAGE; SERVA electrophoresis). After heating at 95˚C

for 5 min, the samples were subjected to electrophoresis in 12% SDS-PAGE gel along with pro-

tein marker (PageRuler Broad Range Unstained protein ladder; Thermo Scientific) with Tris-

glycine as running buffer. After electrophoresis, proteins were visualized by staining with Coo-

massie Brilliant Blue R250 dye (Sigma).

Bacteriophage genomic and phylogenetic analysis

DNA extraction. DNA was extracted from CsCl purified high-titre stocks of phage using

phage DNA isolation kit (Norgen Biotek Corp., Thorold, ON, Canada) according to the manu-

facturer’s instructions. The purity and the concentration of the DNA were determined using

spectrophotometer (Invitrogen Qubit).

Genome estimation. Pulsed field gel electrophoresis (PFGE) was used to estimate the

genomes sizes for the 14 phage isolates according to the protocol published by Lingohr et al

[54]. Plugs were prepared according to procedure and gel run at 5 V/cm for 24 h at 14˚C with

initial switch at 5s and final switch 15s.

Restriction digestion patterns. For comparison of DNA fragment patterns, phage geno-

mic DNA was digested with different restriction endonucleases according to the instructions

of the manufacturer (Fermentas life sciences, UK). A total of five restriction endonucleases

namely; DraI, EcoRI, HindIII, KpnI and PstI were used. Restriction fragments were separated

by electrophoresis (1h, 90V) on 1.0% agarose (Sigma, USA) gel stained with ethidium bro-

mide. DNA molecular weight marker (mi-1Kb DNA Marker; Metabion, Germany) was used

for size determination of DNA fragments [55].

PacBio library preparation and sequencing. Three bacteriophages of this study;

vB-VmeM-32, vB_EauS-123 and vB_BhaS-171, were randomly chosen for complete genome

study. SMRTbell template libraries were prepared according to the instructions from Pacific

Biosciences, Menlo Park, CA, USA, following the Procedure and Checklist Greater than 10 kb

Template Preparation and Sequencing using a multiplex workflow with symmetric barcoded

adapter of 16 nucleotides (F1 to F3), each for one of the phages. Briefly, for preparation of

10kb libraries ~ 4μg genomic DNA isolated from phages were sheared applying g-tubes from

Covaris (Woburn, MA) according to the manufacturer´s instructions. DNAs were end-

repaired and ligated overnight to hairpin adapters applying components from the DNA/

Isolation, characterization and analysis of bacteriophages

PLOS ONE | https://doi.org/10.1371/journal.pone.0215734 April 25, 2019 5 / 19

Polymerase Binding Kit P5 from Pacific Biosciences, Menlo Park, CA, USA, respectively. Reac-

tions were carried out according to the manufacturer´s instructions. DNAs from phages were

combined equimolar. SMRTbell template was exonuclease treated for removal of incompletely

formed reaction products. Conditions for annealing of sequencing primers and binding of

polymerase to purified SMRTbell template were assessed with the Calculator in RS Remote

(Pacific Biosciences, Menlo Park, CA, USA). SMRT sequencing was carried out on the PacBio

RSII (Pacific Biosciences, Menlo Park, CA, USA) taking one 180-minutes movie.

Demultiplexing, genome assembly and annotation. Data from one SMRT Cell was

demultiplexed according to barcodes F1 to F3 using the “RS_Subreads.1” protocol included in

SMRTPortal version 2.2.0. Hereby, the “barcoding” option was activated and “symmetric” bar-

coding was selected in the barcode option pulldown menu. A FASTA-file containing all bar-

codes was uploaded prior analysis to the “Reference” section of SMRTPortal and selected

within the protocol. Output of demultiplexing workflow (barcoded-fastqs.tgz) was used to

create whitelists of polymerase reads for each barcode (compare https://github.com/

PacificBiosciences/Bioinformatics-Training/wiki/HGAP-Whitelisting-Tutorial). Hereby, a

bash script named “Barcode_HGAP.sh “assisted in creating the necessary folder structure,

generating the whitelist.txt files as well as the settings.xml file for each subsequent genome

assembly. Whitelisted SMRT sequencing data from each phage was assembled independently

using the “RS_HGAP_Assembly.3”protocol in SMRTPipe with minimum subread lengths of 1

kbp and an estimated genome size of 50 kbp with exception of phage vB_VmeM-32 (200 kbp).

Each phage assembly revealed the fully resolved chromosomes as one contig. The assemblies

where either linearized due to recognition of distinct start and end points in the phage assem-

blies or circularized removing artificial redundancies at the ends of the contigs. Validity of the

assemblies was checked using SMRTView and IGV [56]. Finally, the genomes were annotated

using Prokka 1.8 [57] with subsequent manual curation in Artemis [58].

Two criteria were used to define potential protein coding genes; they had to contain greater

than 25 codons and employ ATG, GTG or TTG as initiation codons. Genome size, G+C %

content, coding density, total number of genes and additional elements such as inspection of

the sequence to search start and termination codons was determined using ARTEMIS tool for

sequence visualization [59]. The intergenic genome regions of the phage were searched for

transcriptional regulation elements. A search for tRNA genes was done with the tRNAscan-SE

program v1.2.1 [60] and ARAGORN v1.2.36 [61]. Homology assignments were based on

amino acid sequence alignment searches (BlastP) and were accepted only if the statistical sig-

nificance of the sequence similarities (E value) was less than 1x10

-5

, the percentage query cover

was 60% and the percentage identity between the aligned sequences was 35%.

Termini phylogenetic analysis. Sequences for termini phylogenetic analysis were chosen

by large terminase gene products of BlastP. The sequences were aligned with other phage

sequences with known DNA packaging strategies from a reduced set used by Fouts et al [62]

using the program ClustalW [63] with default parameters in MEGA v.7 (Pairwise alignment: gap

opening penalty = 10, gap extension penalty = 0.1. Multiple alignment: gap opening penalty = 10,

gap extension penalty = 0.2. Protein weight matrix = Gonnet. Delay divergent cutoff = 30%)

[64]. Phylogenetic tree was inferred using the Maximum—Likelihood method [65] based on the

Poisson correction model [66]. Bootstrapping was set to 1000 replicates and the tree rooted.

Results

Isolation and characterization of bacterial host strains

Nine bacterial isolates from Lake Elmenteita were found to be susceptible to phages. They all,

apart from Vibrio metschnikovii, were Gram-positive, grew well on alkaline nutrient (DSMZ

Isolation, characterization and analysis of bacteriophages

PLOS ONE | https://doi.org/10.1371/journal.pone.0215734 April 25, 2019 6 / 19

medium 31), basal media for alkaliphilic micro-organisms; Horikoshi-1 (DSMZ medium

1081) and LB (DSMZ medium 381) media, over a temperature range of 25–45˚C (optimum,

30–35˚C) and pH range of 7.0–12.0 (optimum, pH 10.0–12.0). The comparative analysis of

partial (approximately 900 bp) 16S rRNA gene sequences revealed that they all, apart from

Vibrio metschnikovii, belong to the order Bacillales. The level of similarity between the isolates

and their closest known relatives was between 98–100%. This was supported by MALDI-TOF

protein spectra analysis. The bacteria showed different morphologies as indicated in Table 1.

A summary of selected physiological properties to further characterize the isolates, as indi-

cated by API 20NE and API ZYM (bioMe

rieux) identification systems are presented in

S1 Table.

Isolation and characterization of bacteriophages

A total of 17 seemingly morphologically different phages were isolated following enrichment

of sediment. Transmission electron microscope revealed tailed forms of bacteriophages with

variety of structural features, to be present in this lake. According to Ackermann’s classifica-

tion [67], they were all tailed phages similar to those belonging to order Caudovirales and con-

sists of three families; Myoviridae, Siphoviridae and Podoviridae (Fig 1).

Among them, nine (vB_BpsS-36, vB_EauS-123a, vB_EauS-123b, vB_BboS-125, vB_BhoS-

126, vB_BcoS-136, vB_BpsS-140, vB_BhaS-171 and vB_EalS-137b) were siphoviruses, seven

(vB_EauM-23, vB_VmeM-32, vB_BpsM-61, vB_EalM-132, vB_EalM-137, vB_EalM-137a and

vB_VmeM-196) myoviruses and one (vB_BhoP-126) podovirus. The capsid diameters ranged

between 47–130 nm and tail lengths measured from the bottom of the neck to the base plate

ranged between 37–546 nm. The bacteriophages were further named according to the recom-

mendations outlined by Kropinski et al [68]. Results are summarized in Table 2.

Thermal and pH stability tests

After 6 h of incubation under different thermal conditions, at 20˚C generally the phages had

lost infectivity as no plaques formed. Plaque forming units however increased exponentially

from 25˚C with maximum at 35˚C. The infectivity of the phages was highest at 30–35˚C, but

was lost with increasing temperature with non at 50˚C as they had lost their infection capabil-

ity. After 6 h of incubation under different pH conditions, at pH 2 and 4 no plaques were

observed as the infectivity had been hindered by the low pH. Plaques formed from pH 7 and

increased with increasing pH values with maximum at pH 10. Infection capability of the

phages was highest between pH 10–12 (Fig 2).

Table 1. Summary of bacterial hosts’ characteristics. Hosts and their characteristic morphologies as observed under phase contrast microscope (×400).

Host reference No. Name Morphology

1 HS32 Vibrio metschnikovii vibrio

2 HS61 Bacillus pseudofirmus short rods

3 HS123 Exiguobacterium aurantiacum cocci

4 HS125 Bacillus bogoriensis rods

5 HS126 Bacillus horikoshii long rods

6 HS132 Exiguobacterium alkaliphilum cocci

7 HS136 Bacillus cohnii short rods

8 HS140 Bacillus pseudalcaliphilus long-rods

9 HS171 Bacillus halmapulus long rods

https://doi.org/10.1371/journal.pone.0215734.t001

Isolation, characterization and analysis of bacteriophages

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Fig 1. Transmission electron micrographs. Micrographs of bacteriophages from Lake Elmenteita showing different morphotypes. CsCl-purified bacteriophage

preparations were negatively stained with 2% (w/v) aqueous uranyl acetate (pH 5.0). Samples were examined in a TEM 910 transmission electron microscope (Carl

Zeiss, Oberkochen) at an acceleration voltage of 80 kV.

https://doi.org/10.1371/journal.pone.0215734.g001

Table 2. Bacteriophage morphology and naming. Structural characterization, classification and naming of phages isolated from Lake Elmenteita.

Host Family Name Phage size (nm)

Head diameter Tail length Total size

1 Bacillus pseudofirmus myovirus vB_BpsM-61 66 192 258

2 Exiguobacterium aurantiacum myovirus vB_EauM-23 60 125 185

3 Exiguobacterium aurantiacum siphovirus vB_EauS-123 49 138 187

4 Exiguobacterium aurantiacum siphovirus



vB_EauS-123b - - -

5 Bacillus bogoriensis siphovirus vB_BboS-125 61 179 240

6 Bacillus horikoshii siphovirus vB_BhoS-126 57 119 177

7 Bacillus horikoshii podovirus vB_BhoP-126 47 37 85

8 Exiguobacterium alkaliphilum myovirus vB_EalM-132 85 161+160 406

9 Bacillus cohnii siphovirus vB_BcoS-136 59 145 204

10 Exiguobacterium alkaliphilum myovirus



vB_EalM-137a - - -

11 Exiguobacterium alkaliphilum siphovirus



vB_EalS-137b - - -

12 Exiguobacterium alkaliphilum myovirus vB_EalM-137 62 214 276

13 Bacillus pseudalcaliphilus siphovirus vB_BpsS-140 83 546+179 809

14 Bacillus halmapulus siphovirus vB_BhaS-171 58 117 175

15 Bacillus pseudalcaliphilus siphovirus vB_BpsS-36 57 110 167

16 Vibrio metschnikovii myovirus vB_VmeM-32 130 109 239

17 Vibrio metschnikovii myovirus vB_VmeM-196 77 159 236



The phage dimension was not determined as the culture was not clonal

https://doi.org/10.1371/journal.pone.0215734.t002

Isolation, characterization and analysis of bacteriophages

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Host range analysis revealed that the bacteriophages exhibited high specificity for their host

bacteria and did not infect other bacteria strain tested (S2 Table).

Structural protein profiles

The molecular weights of the structural polypeptides ranged from 10 to 100 kDa. While the

minor bands varied in position, most phages had the major band at 20 kDa (Fig 3).

Genome size estimation

The genome sizes of all the 12 phages ranged between ~30 to 200 kb. Bacteriophages

vB_VmeM-32, vB_EalM-132, vB_BcoS-136 and vB_VmeM-196 had the largest genomes of

this study ranging between ~140 to 200 kb. The rest of the phages had genome sizes ranging

between ~30 to 60 kb (Fig 4).

Restriction digestion patterns

The patterns of restriction digest profiles for each phage were different (S1 Fig). Endonuclease

EcoRI was able to digest all genomes. DraI also digested all the genomes but vB_BpsS-140.

Most phages showed insensitivity to restriction endonucleases PstI (all but vB_EauM-23,

vB_BboS-125 and vB_BpsS-36) and BamH1 (all but vB_BpsM-61) (S3 Table). Restriction

digests further confirm that the phages are double stranded DNA viruses.

Fig 2. Thermal and pH stability experiments. Infection capability of the phages was highest at 30–35˚C and more

infective in alkali condition of between pH 10–12.

https://doi.org/10.1371/journal.pone.0215734.g002

Isolation, characterization and analysis of bacteriophages

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Genome characteristics and annotation

The genomes of phages vB_VmeM-32, vB_EauS-123 and vB_BhaS-171 have genome sizes of

199,912 bp, 30,925 bp, and 38,975 bp, respectively. A total of 260 open reading frames (ORFs)

were predicted for phage vB_VmeM-32 while 56 ORFs were predicted for vB_EauS-123 and

67 ORFs for vB_BhaS-171. Phages vB_VmeM-32 and vB_EauS-123 encoded 6 transcriptional

terminators each and vB_BhaS-171 encoded 5 transcriptional terminators. 3 tRNA genes

(Met

cat

, Arg

tct

and Asn

gtt

) were detected in the genome of phage vB-VmeM-32 clustered at

region 27879–28124 bp, while vB_EauS-123 and vB_BhaS-171 did not encode any tRNA gene.

See summary in Table 3.

Fig 3. SDS-polyacrylamide gel electrophoretic profiles of phage structural proteins. (M) broad range Page Ruler

protein molecular weight marker (Thermoscientific), (1) vB_BpsM-61, (2) vB_EauM-23, (3) vB_EauS-123, (4)

vB_BboS-125, (5) vB_BhoP-126, (6) vB_BhoP-126, (7) vB_EalM-132, (8) vB_BcoS-136, (9) vB_EalM-137, (10)

vB_BpsS-140, (11) vB_BhaS-171, (12) vB_BpsS-36, (13) vB_VmeM-32, (14) vB_VmeM-196, Numbers to the left

indicate band size in kDa.

https://doi.org/10.1371/journal.pone.0215734.g003

Fig 4. Pulsed-field gel of the 13 phage genomes. Lane, (1) vB_EauM-23, (2) vB_VmeM-32, (3) vB_BpsS-36, (4)

vB_BpsM-61, (5) vB_EauS-123, (6) vB_BboS-125, (7) vB_BhoP-126, (8) vB_EalM-132, (9) vB_BcoS-136, (10)

vB_EalM-137, (11) vB_BpsS-140, (12) vB_BhaS-171, (13) vB_VmeM-196, (M) Low range PFGE DNA marker in Kb

(Biolabs, England).

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Isolation, characterization and analysis of bacteriophages

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Most of the ORFs of phages vB_EauS-123 and vB_BhaS-171 are located on the reverse

(minus) strand while vB-VmeM-32 has all its genes transcribed on the forward (plus) strand

(Fig 5).

Based on sequence similarity (E value < 10

−5

), 135 out of 260 (52%), 40 out of 66 (61%) and

24 out of 55 (44%) of the protein-coding genes for phages vB-VmeM-32, vB_EauS-123 and

vB_BhaS-171, respectively, share significant sequence similarity to known protein sequences

contained in the GenBank non-redundant protein database. Genome wide comparison of the

phages with other genomes in the non-redundant NCBI database showed no significant

sequence similarity hence novel. Further analysis of vB_VmeM-32 genome revealed genes

with suggested functions like putative N-acetylmuramoyl-L-alanine amidase (VmeM-

32_00065) for host cell lysis and a putative DNA polymerase (VmeM-32_00094) for replica-

tion. Gene for a putative helicase (VmeM-32_00016), exonuclease protein (VmeM-32_00081)

and endonuclease protein (VmeM-32_00138) were also identified. We identified structural

genes with conserved domains in phage vB_EauS-123 that showed no similarities to other

phages (EauS-123_00048, EauS-123_00051). Further analysis of this genome revealed few

more genes for proteins with suggested functions like a putative N-acetylmuramoyl-L-alanine

amidase (EauS-123_00053) for host cell lysis, a putative recombinase (EauS-123_00055), a

putative phage regulatory protein (EauS-123_00008), a putative Holliday junction resolvase

(EauS-123_00012), a putative dUTPase (EauS-123_00020) and two proteins for replication,

containing a DnaC (EauS-123_00004) and DnaD domain (EauS-123_00003) respectively. The

rest of genome did not show any similarity to any other genes with known functions so far.

Most of the few similarities vB_BhaS-171 shared with other phages were assigned to temperate

phage 11143 that was induced from Bacillus cereus strain NCTC11143 [69]. These included

genes of the cluster for DNA packaging and head morphogenesis (BhaS-171_00005 and BhaS-

171_00012), e.g. genes for two terminase subunits and a portal protein, and a gene for a puta-

tive helicase (BhaS-171_00053). Generally, vB_BhaS-171 had a typical gene cluster for head

and tail proteins, though most of those genes were annotated based on conserved domains at

amino acid level and not based on similarities to other known viruses. Downstream the lysis

cluster, we also identified genes for an FtsK/SpoIIIE-like protein and a putative replication/

Table 3. General features of genomes vB_VmeM-32, vB_EauS-123 and vB_BhaS-171. A summary of genome characteristics.

Phage Genome size (bp) G +C % content Coding % CDS tRNAs Transcriptional Terminators Start Codon

ATG GTG TTG

1 vB-VmeM-32 199, 912 36.25 91.2 260 3 48 246 3 11

2 vB_EauS-123 30, 925 47.73 91.5 56 - 6 52 2 2

3 vB_BhaS-171 38, 975 40.82 91.6 67 - 5 50 8 9

https://doi.org/10.1371/journal.pone.0215734.t003

Fig 5. Genome maps of bacteriophages vB-VmeM-32, vB_EauS-123 and vB_BhaS-171 respectively, drawn to

scale. First track show forward transcribed ORFs and second tracks show reverse transcribed ORFs respectively. Third

track shows terminators (red). Moving inward, the track show the %GC content (purple = low %GC) and innermost of

the genome map GC skew ([G-C]/[G+C]).

https://doi.org/10.1371/journal.pone.0215734.g005

Isolation, characterization and analysis of bacteriophages

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relaxation protein similar to vB_BpsS-36. The replication cluster revealed some genes similar

to deep-sea thermophilic bacteriophage GVE2 [69][48], e.g. for a helicase (BhaS-171_00053), a

Ssb protein (BhaS-171_00061) and an endonuclease (BhaS-171_00057). Additionally, this

phage harbored a gene with a conserved domain for an NTP-PPase and a cytosine-C5 specific

DNA methylase. A comprehensive list of protein coding genes carried by the phages along

with the corresponding positions, sizes, and sequence homologies are presented in S4, S5 and

S6 Tables.

A phylogenetic tree for large terminase subunit generated using Maximum—Likelihood

method revealed that phage vB_VmeM-32 cluster together with T4-like phages with a low

bootstrap value of 43%, while Bacillus phages vB_BhaS-171 and vB_EauS-123 clustered with

T5-like phages with low bootstrap values of 34% and 30% respectively (Fig 6).

Discussion

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].

Besides genomics, the most important criterion for phage taxonomy is ultrastructure [67].

The phenotypic diversity of the 17 bacteriophages was examined by electron microscopy. The

phages were identified using morphological criteria outlined by the International Committee

of Taxonomy of Viruses and the species concept of Ackermann et al [67]. All the bacterio-

phages belong to the order Caudovirales characterized by tailed phages. The order has three

common virus types; myoviruses, siphoviruses and podoviruses. Siphovirus and myovirus

Fig 6. Phylogenetic analysis of large terminase subunits compared to phages with known DNA packaging

strategies. The maximum—Likelihood tree was inferred based on ClustalW alignment of large terminase subunits

amino acid sequences. The tree was rooted via midpoint rooting. The numbers at the nodes represent bootstrap values

based on 1,000 resamplings.

https://doi.org/10.1371/journal.pone.0215734.g006

Isolation, characterization and analysis of bacteriophages

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phages were prevalent morphotypes compared to podovirus. Most of the phages have morpho-

logical features that have been described previously from a marine environment by Sime-

ngando et al [71]. Few showed unique structures that have not been reported previously for

haloalkaliphilic viruses. The unique structural features, different phage dimensions and plaque

morphology all indicate diversity within the various families. The stability of phages under dif-

ferent thermal and pH conditions was investigated based on infectivity of phages after treat-

ment. Infection capability of the phages was highest at 30–35˚C. Phages were more infective in

alkali than acidic environment. Optimal temperature and pH for bacteriophage growth and

plaque formation was similar to that of bacterial host strains.

The bacteriophage exhibited high specificity for their hosts. This characteristic has been

previously reported for marine phages by Børsheim [72]. Because of their specificity, the

phages can therefore be applied to map the distribution of bacteria. Phage mapping is a very

sensitive tool for tracing specific groups of bacteria, compared to using taxonomy of bacteria

[72].

Protein profiles assessment exhibited variations. The molecular weights of the structural

polypeptides as indicated by SDS-PAGE, ranged from 10 to 100 kDa. While the minor bands

varied in position, most phages had the major band at 20 kDa, which might represent the

major capsid protein. The minor bands were varied and might be responsible for host-specific-

ity or the characteristics specific to a particular phage [73]. Resistance to restriction enzymes as

indicated by the phages of this study to endonucleases Pstl and BamH1 is common and has

been reported previously [74][75]. Several explanations have been proposed for this anti-

restriction mechanisms. Among these explanations is elimination of restriction sites as an evo-

lutionary response of phages to pressures from their host restriction enzymes [76], integration

of unusual bases in the viral DNA such as hydroxymethyl uracil or hydroxymethyl cytosine

that make DNA somewhat refractory to endonuclease cleavage. Alternatively, phage genomes

may encode methyltransferases that modify specific nucleotides within the recognition site of

one or more of the restriction endonucleases [77][78].

28% of the vB_VmeM-32 genes would result in proteins of less than 100 amino acid resi-

dues. 58.5% of putative vB_VmeM-32 genes resulted in BLASTp hits with identities to various

biological groups (Phage and Bacteria) but few lacked any database matches (Hypothetical).

The phage genes were further subdivided on the basis of their best blastp hit (BLASTp values E

>10

−5

). A high degree of similarity in most protein coding genes was with a Schizo T-even

Aeromonas phage Aeh1, an Aeromonas hydrophila phage isolated from a sewage treatment

plant in Wisconsin [79]. SchizoT-evens phages comprise the subgroup of T4-types that have

diverged significantly from the T-evens and infect host distant from E. coli e.g., Aeromonas

and Vibrio [80]. Genome sequencing and assembly of Phage vB_BhaS-171 and phage

vB_EauS-123 and comparison with other phage genomes via BLASTN showed they share only

few similarities with other phages.

The large terminase sub-unit is considered the most universally conserved gene sequence

in phages hence used to construct phylogeny to decipher evolutionary relationships among

phages belonging to different families [79][81]. Casjens and Gilcrease [82] have also shown the

phylogeny of large terminase sub-unit proteins is correlated with the virus DNA packaging

strategy. Since the sequence of Vibrio phage vB-VmeM-32 clustered phylogenetically with the

T4-like phages which are known to package DNA by a headful packaging mechanism, we

therefore conclude that phage vB-VmeM-32 also package DNA by the same headful packaging

mechanism. Phage vB_VmeM-32 can therefore be classified as a new member of the T4-like

phages, subgroup Schizo T-evens, and infecting bacteria of the genus Vibrio. vB_EauS-123 and

vB_BhaS-171 clustered with T5-like phages which show long exact direct repeat ends mecha-

nism of DNA packaging.

Isolation, characterization and analysis of bacteriophages

PLOS ONE | https://doi.org/10.1371/journal.pone.0215734 April 25, 2019 13 / 19

Conclusion

The effective use of bacteriophage in all applications must be preceded by detailed understand-

ing 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 group-

ing 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 sequenc-

ing 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.

Nucleotide sequence accession numbers

The bacteriophages were accessed to the German Collection of Microorganisms and Cell Cul-

tures (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 depos-

ited at NCBI GenBank under the accession numbers vB_VmeM-32 (KU160494), vB_EauS-

123 (KU160495) and vB_BhaS-171 (KU160496).

Supporting information

S1 Fig. Restriction profiles. Restriction profiles of the phages after digestion of DNA with

restriction enzymes, overnight at 37˚C and electrophoresed on 1% agarose gel. Different

restriction enzymes were used which cut wherever the recognition sequence was present.

(A) DraI, (B) KpnI, (C) PstI (D) HindIII (E) EcoRI and (F) BamH1 all from Fermentas. Lane

(1) vB_BpsM-61, (2) vB_EauM-23, (3) vB_EauS-123, (4) vB_BboS-125, (5) vB_BhoP-126, (6)

vB_BhoP-126, (7) vB_EalM-132, (8) vB_BcoS-136, (9) vB_EalM-137, (10) vB_BpsS-140, (11)

vB_BhaS-171, (12) vB_BpsS-36, (13) vB_VmeM-32, (14) vB_VmeM-196, (M) 1kb DNA

marker (Metabione). Numbers to the right indicate band size in kb.

(TIF)

S1 Table. Physiological properties. Selected phenotypic characteristics of host bacteria as

indicated by API identification system.

(DOCX)

S2 Table. Host range analysis of bacteriophages. Evaluation of the lytic spectrum of the

phages against bacterial strains isolated in this study.

(DOCX)

S3 Table. Grouping of restriction endonucleases by cutting pattern. Non cutters produced

only one high molecular weight band by gel electrophoresis. Poor cutters produced few bands,

Isolation, characterization and analysis of bacteriophages

PLOS ONE | https://doi.org/10.1371/journal.pone.0215734 April 25, 2019 14 / 19

good cutters produced five or more bands and complete cutters caused complete digestion of

DNA.

(DOCX)

S4 Table. Overview of bacteriophage vB_VmeM-32 ORFS and summary of homology

searches. ORFs are arranged according to their position (Start-End) in the genome. Significant

database matches are given in the column marked Putative homolog. Tools used to search for

similarity are blastn (nucleotide Blast search) or blastp (protein Blast search). Scores and E-val-

ues obtained in the Blast searches are given in the last three columns. Homology assignments

were accepted only if the statistical significance of the sequence similarities (E value) was less

than 1x10

-5

, the percentage query cover was 60% and the percentage identity between the

aligned sequences was 35%.

(DOCX)

S5 Table. Overview of bacteriophage vB_EauS-123 ORFS and summary of homology

searches. ORFs are arranged according to their position (Start-End) in the genome. Significant

database matches are given in the column marked Putative homolog. Tools used to search for

similarity are blastn (nucleotide Blast search) or blastp (protein Blast search). Scores and E-val-

ues obtained in the Blast searches are given in the last three columns. Homology assignments

were accepted only if the statistical significance of the sequence similarities (E value) was less

than 1x10

-5

, the percentage query cover was 60% and the percentage identity between the

aligned sequences was 35%.

(DOCX)

S6 Table. Overview of bacteriophage vB_BhaS-171 ORFS and summary of homology

searches. ORFs are arranged according to their position (Start-End) in the genome. Significant

database matches are given in the column marked Putative homolog. Tools used to search for

similarity are blastn (nucleotide Blast search) or blastp (protein Blast search). Scores and E-val-

ues obtained in the Blast searches are given in the last three columns. Homology assignments

were accepted only if the statistical significance of the sequence similarities (E value) was less

than 1x10

-5

, the percentage query cover was 60% and the percentage identity between the

aligned sequences was 35%.

(DOCX)

Acknowledgments

We would like to gratefully acknowledge the help of Bettina Henze, Simone Severitt, Nicole

Heyer and Sabrina Willems for technical assistance (DSMZ, Braunschweig). The work was

done at the Leibniz Institute-DSMZ (German Collection of Microorganisms and Cell Cul-

tures) Braunschweig.

Author Contributions

Conceptualization: Juliah Khayeli Akhwale, Manfred Rohde, Christine Rohde, Hamadi Iddi

Boga, Hans-Peter Klenk, Johannes Wittmann.

Data curation: Juliah Khayeli Akhwale, Manfred Rohde, Christine Rohde, Boyke Bunk, Cath-

rin Spro¨er, Hamadi Iddi Boga, Hans-Peter Klenk, Johannes Wittmann.

Formal analysis: Juliah Khayeli Akhwale, Manfred Rohde, Christine Rohde, Boyke Bunk,

Cathrin Spro¨er, Hamadi Iddi Boga, Hans-Peter Klenk, Johannes Wittmann.

Funding acquisition: Juliah Khayeli Akhwale, Hamadi Iddi Boga.

Isolation, characterization and analysis of bacteriophages

PLOS ONE | https://doi.org/10.1371/journal.pone.0215734 April 25, 2019 15 / 19

Investigation: Juliah Khayeli Akhwale, Manfred Rohde, Christine Rohde, Hamadi Iddi Boga,

Hans-Peter Klenk, Johannes Wittmann.

Methodology: Juliah Khayeli Akhwale, Manfred Rohde, Christine Rohde, Boyke Bunk,

Johannes Wittmann.

Project administration: Manfred Rohde, Hamadi Iddi Boga, Johannes Wittmann.

Resources: Juliah Khayeli Akhwale, Cathrin Spro¨er, Hamadi Iddi Boga, Hans-Peter Klenk,

Johannes Wittmann.

Software: Juliah Khayeli Akhwale, Manfred Rohde, Boyke Bunk, Johannes Wittmann.

Supervision: Manfred Rohde, Christine Rohde, Boyke Bunk, Cathrin Spro¨er, Hamadi Iddi

Boga, Hans-Peter Klenk, Johannes Wittmann.

Validation: Juliah Khayeli Akhwale, Manfred Rohde, Cathrin Spro¨er, Hamadi Iddi Boga,

Hans-Peter Klenk, Johannes Wittmann.

Visualization: Juliah Khayeli Akhwale, Johannes Wittmann.

Writing – original draft: Juliah Khayeli Akhwale, Manfred Rohde, Christine Rohde, Boyke

Bunk, Hamadi Iddi Boga, Hans-Peter Klenk, Johannes Wittmann.

Writing – review & editing: Juliah Khayeli Akhwale, Boyke Bunk, Johannes Wittmann.

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