Restriction of the growth of T7-like phages by plasmid prophage P1
Aims. Considerable part of T7 phage genome is responsible for interaction with the bacterial host, primarily for the avoidance of action of protective systems of cells, the restriction-modification complexes. Interactions of T7-like phages with RM systems of type I and II are relatively studied whil...
Gespeichert in:
| Veröffentlicht in: | Фактори експериментальної еволюції організмів |
|---|---|
| Datum: | 2014 |
| 1. Verfasser: | |
| Format: | Artikel |
| Sprache: | English |
| Veröffentlicht: |
Інститут молекулярної біології і генетики НАН України
2014
|
| Schlagworte: | |
| Online Zugang: | https://nasplib.isofts.kiev.ua/handle/123456789/178115 |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Zitieren: | Restriction of the growth of T7-like phages by plasmid prophage P1 / I.V. Faidiuk // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2014. — Т. 14. — С. 177-181. — Бібліогр.: 9 назв. — англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
nasplib_isofts_kiev_ua-123456789-178115 |
|---|---|
| record_format |
dspace |
| spelling |
Faidiuk, I.V. 2021-02-17T21:19:49Z 2021-02-17T21:19:49Z 2014 Restriction of the growth of T7-like phages by plasmid prophage P1 / I.V. Faidiuk // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2014. — Т. 14. — С. 177-181. — Бібліогр.: 9 назв. — англ. 2219-3782 https://nasplib.isofts.kiev.ua/handle/123456789/178115 578.4:578.81:579.842.1/.2 Aims. Considerable part of T7 phage genome is responsible for interaction with the bacterial host, primarily for the avoidance of action of protective systems of cells, the restriction-modification complexes. Interactions of T7-like phages with RM systems of type I and II are relatively studied while the question of impact by the type III systems on their growth remains unclear. Developing a relevant system would allow us to study the interaction of bacteriophages with host cells on the gene level including the interplay with prophage elements and RM-systems. Methods. Biological, genetics and molecular biology approaches combined with bioinformatic research were used. Results. The ability of P1 to infect and lysogenise Erwinia amylovora and Erwinia “horticola” cells as well as its maintainance as a single-copy plasmid in the cells of uncommon hosts was shown. A set of lysogenic strains was obtained. According to the level of restriction three types of phage-RM system interaction were discovered. Though polyvalent, phage FE44 undergoes abortive infection similar to other members of T7 phage group. Conclusions. The genes of restriction-modification complex EcoP1I are fully expressed regardless the bacterial host lysogeinzed by phage P1. Differences in interaction with cells are likely associated with the number of enzyme recognition sequences and the adsorption sites availability while gp 0.3 Ocr protein is not involved in this interaction. The constructed systems allow for the exploration of EcoР1I interaction with polyvalent phages able to grow both on E. coli and on such phytopathogens as E. “horticola” and E. amylovora. Key words: T7-like phages, Type III restriction-modification complexes, antirestriction, polyvalent bacteriophages, phytpathogens. en Інститут молекулярної біології і генетики НАН України Фактори експериментальної еволюції організмів Аналіз та оцінка генетичних ресурсів Restriction of the growth of T7-like phages by plasmid prophage P1 Article published earlier |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| title |
Restriction of the growth of T7-like phages by plasmid prophage P1 |
| spellingShingle |
Restriction of the growth of T7-like phages by plasmid prophage P1 Faidiuk, I.V. Аналіз та оцінка генетичних ресурсів |
| title_short |
Restriction of the growth of T7-like phages by plasmid prophage P1 |
| title_full |
Restriction of the growth of T7-like phages by plasmid prophage P1 |
| title_fullStr |
Restriction of the growth of T7-like phages by plasmid prophage P1 |
| title_full_unstemmed |
Restriction of the growth of T7-like phages by plasmid prophage P1 |
| title_sort |
restriction of the growth of t7-like phages by plasmid prophage p1 |
| author |
Faidiuk, I.V. |
| author_facet |
Faidiuk, I.V. |
| topic |
Аналіз та оцінка генетичних ресурсів |
| topic_facet |
Аналіз та оцінка генетичних ресурсів |
| publishDate |
2014 |
| language |
English |
| container_title |
Фактори експериментальної еволюції організмів |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| description |
Aims. Considerable part of T7 phage genome is responsible for interaction with the bacterial host, primarily for the avoidance of action of protective systems of cells, the restriction-modification complexes. Interactions of T7-like phages with RM systems of type I and II are relatively studied while the question of impact by the type III systems on their growth remains unclear. Developing a relevant system would allow us to study the interaction of bacteriophages with host cells on the gene level including the interplay with prophage elements and RM-systems. Methods. Biological, genetics and molecular biology approaches combined with bioinformatic research were used. Results. The ability of P1 to infect and lysogenise Erwinia amylovora and Erwinia “horticola” cells as well as its maintainance as a single-copy plasmid in the cells of uncommon hosts was shown. A set of lysogenic strains was obtained. According to the level of restriction three types of phage-RM system interaction were discovered. Though polyvalent, phage FE44 undergoes abortive infection similar to other members of T7 phage group. Conclusions. The genes of restriction-modification complex EcoP1I are fully expressed regardless the bacterial host lysogeinzed by phage P1. Differences in interaction with cells are likely associated with the number of enzyme recognition sequences and the adsorption sites availability while gp 0.3 Ocr protein is not involved in this interaction. The constructed systems allow for the exploration of EcoР1I interaction with polyvalent phages able to grow both on E. coli and on such phytopathogens as E. “horticola” and E. amylovora.
Key words: T7-like phages, Type III restriction-modification complexes, antirestriction, polyvalent bacteriophages, phytpathogens.
|
| issn |
2219-3782 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/178115 |
| citation_txt |
Restriction of the growth of T7-like phages by plasmid prophage P1 / I.V. Faidiuk // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2014. — Т. 14. — С. 177-181. — Бібліогр.: 9 назв. — англ. |
| work_keys_str_mv |
AT faidiukiv restrictionofthegrowthoft7likephagesbyplasmidprophagep1 |
| first_indexed |
2025-11-25T23:10:35Z |
| last_indexed |
2025-11-25T23:10:35Z |
| _version_ |
1850579182414725120 |
| fulltext |
177
УДК 578.4:578.81:579.842.1/.2
FAIDIUK I.V.
D.K. Zabolotny Institute of Microbiology and Virology of NAS of Ukraine,
Ukraine, 03680, Kyiv, Zabolotnoho str., 154, e-mail: i.v.faidiuk@gmail.com
RESTRICTION OF THE GROWTH OF T7-LIKE PHAGES BY PLASMID PROPHAGE P1
T7-like phages represent an example of the
saving utilization of the genome. Their DNA of
about 40 kb contains genes solely of phage origin
that are sufficient to perform the basic function of
bacteriophage, its reproduction. They are lytic
bacteriophages and hence do not waste coding
capabilities of their genome neither for lysogeny
establishment nor for transduction of bacterial
genes. Considerable part of T7 phage genome
though is responsible for the interaction with the
host, peculiarly for overcoming the cell barriers [1].
Thus, the structure of the attachment apparatus that
allows for adsorbtion to core of LPS is responsible
for the expansion of host range of T7-like phages
[1]. Some of them (eg FE44) are able to overcome
intergenera and interspecies barriers what
characterizes them as polyvalent [2]. The next step
of interaction consists in phage avoidance of action
of protective systems of bacterial cells, primarily
the restriction-modification (RM) complexes.
Interactions of T7 phage group members with RM
systems of type I and II are relatively studied while
the question of impact by the type III systems on
their growth remains opened [3].
The purpose of our work was to develop a
system allowing for studying on the gene level the
interaction of bacteriophages with cells both of
native and uncommon hosts; to explore the
interaction of the active phages with prophage
elements in these systems.
Materials and methods
The object of the study was a T7-like
polyvalent phage FE44 [2] obtained on different
host bacteria – E. coli C600 (FE44/C600) and E.
“horticola” 450 (FE44/450). Its genome was
recently sequenced, annotated and deposited to
GenBank database under accession number
KF700371. Other members of the group T3, T7 and
BA14 were used as controls. Podoviridae phage
E105 and Siphoviridae erwiniaphages 49, 59 [4]
and 59 mod/P1, obtained by passaging on the
lysogenic strain 450(P1) were used in experiments
performed on phytopathogens. Phage Р1Cmc1ts100
[5] carrying chloramphenicol resistance marker
(Tn9) and temperature-sensitive repressor protein
C1 was used for lyzogenization of the cells.
Three species of bacteria were used:
laboratory strains of E. coli (Eco) C600, C1a, S/6,
BE and 112(P1); the causative agent of fire blight
disease Erwinia amylovora (Eam) strains K8
(ATCC 29850), L4, L6, L7, K4, K5; Erwinia
"horticola" (Eho) 450, 60-1N, 60-3m, 43I, 43II,
120, 23a and artificially lysogenized strains 450(49)
and 60 (59, E105) – the phytopathogenic bacteria
causing black bacteriosis of apples and European
beech [6].
For cells lysogenization a volume of 5 ml of
concentrated phage Р1Cmc1ts100 suspension near
108 PFU/ml was applied on the top layer of
semisolid agar containing cells of the corresponding
recipient: Eam, Eho and Eco. After drying the plates
were overturned and incubated at 28 ˚C for 28–30
hours. Bacterial cells picked out from the area of
application were carried into liquid LB medium and
incubated for 8–10 h at the same temperature. When
the early stationary phase of growth was reached the
bacteria were propagated to individual colonies on
selective LB-plates with chloramphenicol (Cm) in
concentration of 14 µg/ml.
Bioinformatic research was performed using
BLASTn and BLASTp common tools
(http://blast.ncbi.nlm.nih.gov/Blast.cgi), COBALT
(http://www.ncbi.nlm.nih.gov/tools/cobalt/) tool for
multiple protein alignment and APE plasmid editor
for virtual digestion
(http://biologylabs.utah.edu/jorgensen/wayned/ape/).
Results and discussion
The ocr antirestriction function. T7-like
phages are known to produce protein gp 0.3 (Ocr) to
protect against type I RM systems. Ocr protein of
T7 phage mimics the size and shape of curved DNA
molecule while that of T3 possesses additional
SAMase activity [1]. The role of gp 0.3 in T7-like
phages interaction with type III RM system is a
disputable issue: it can be argued that due to SAM-
ase activity it counteracts type III restriction-
modification complex which requires a cofactor
AdoMet to function [3, 7].
FE44 phage turned out to be convenient to
solve this issue since it is not able to express ocr
function. When titrated on bacterial hosts carrying
EcoB and EcoK restriction complexes (E. coli
BE[EcoB], S/6[EcoB], J53[EcoK]) its efficiency of
plating (EOP) decreased by value of 3–4 orders
comparing to r–m– hosts (C600, C1a). In control
experiments the EOP of T7 phage remained
178
constant or varied within one order of magnitude.
Interestingly, bioinformatic analysis revealed the
presence of gene 0.3 in FE44 genome. Predicted gp
0.3 protein features almost identical sequence to
that of BA14 with single amino acid replacement of
histidine in position 124 with proline found. The
difference may seem insignificant however ocr
expression is not observed. Thus, using FE44 the
impact of type III RM enzyme on phage DNA
excluding any interfering factors can be performed.
Construction of the bacterial strains systems
and restriction exploration. Phage P1 is known to
establish the type III RM-system EcoP1I in
prophage state both in the cells of traditional host E.
coli [5], and other enterobacteria (Klebsiella,
Pasteurella, Shigella) [8], P. atrosepticum [9]. To
study the interaction of EcoP1I with T7-like phages
the systems formed by isogenic pairs comprising
the parent and lysogen variant were constructed.
Sensitive bacterial strains were treated with
Р1Сmc1ts100 and lysogens were selected as clones
resistant to 14 µg/ml of chloramphenicol [5]. To
verify the introduction of prophage DNA into the
cell and its maintainance in it the electrophoretic
analysis of extrachromosomal DNA extracted from
the parent cells and CmR–strains of Eho, Eam and
Eco was performed.
As shown in fig. 1, compared to the parent
strains, resistant to Cm clones of Eho and Eam carry
additional extrachromosomal circular DNA. These
molecules of DNA coincide in size with that
extracted from E. coli С600(Р1), С1а(Р1) and of
control plasmid P1 of E. coli 112(Р1) strain; hence,
they appear to be a plasmid prophage P1. This
shows that phage P1 can be maintained in the cells
of uncommon host as a circular plasmid molecule of
approximately 94.8 kb similar to its maintainance in
native host E. coli [5].
To estimate the activity of RM-system in
constructed bacterial cells the efficiency of plating
(EOP) of phages on P1 lysogens was compared to
that on parent strain. FE44 was the single phage
able to grow on all used bacteria while T3 and T7
failed to infect most of the phytopathogenic strains
(table 1). Therefore specific erwiniaphages E105,
59 and 49 were as the control in case of Eho system.
Fig. 1. Electrophoregram of extrachromosomal DNA extracted from parent and CmR– strains of
E. amylovora, E. “horticola” and E. сoli. А. Eho:1 – 450, 2 – 50 (P1), 3 – 60-3m, 4 – 60-3m (P1), 5 – 60-
1N, 6 – 60-1N (P1), 8.–120 (P1); Eco: 7,9.– 12 (P1), 10.–C1a, 11.–C1a (P1), 12.– C600 (P1); B. 1 – Eco 112
(P1), 2 – Eho 120, 3 – 120 (P1), 4 – Eam L4 (P1)
Table 1. Sensitivity of Erwinia genus representatives to T7-like phages
E. “horticola” E. amylovora Strain
Phage
60 -
3m
60 -
1n
60(59,
E105) 450
450
(59)
43I
I 43I 23a 120 L4 К8 К4
T7 – – – – – – – – – – + N –
T3 +res – – – – +t +t +res +res + N + N –
FE44 + N + N + N + N + N +t – +res +res – + N + N
Note: “–” – here and in table 2 signifies insensibility to phage infection; “+res” – indicates on the restriction
of phage growth; “+res” – stands for normal infection development.
A
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4
В
179
Similar to other phages of T7 group
interaction of phage FE44 with EcoP1I-carrying
hosts results in the inevitable development of
abortive infection (fig. 2). In system formed with
E. coli C600 and C600(P1) and phage FE44/C600,
as well as T3 and T7 phages their EOP decreased
only by 1–2 orders of magnitude with each
subsequent propagation. Still the form and size of
plaques as well as the inability to restore normal
phage reproduction in subsequent passaging
indicated on the abi-infection. Interestingly,
restriction of FE44 growth was similar to T3 while
that of T7 was more stringent. This indicates that gp
0.3 is not involved in the interaction with type III
RM system. DNA of T7-like phages is not modified
by EcoP1I methylase, obviously for the reason of
multiplicity of EcoP1I recognition sites on their
DNA. Thus T7 DNA contains 126 and T3 has 154
recognition sequences while for FE44 this value
constitutes 156.
Analogous abortive infection (EOP on
lysogens about 10-3) was discovered for phage E105
interaction with P1 RM-system in systems of E.
“horticola” strain 60-1N and its lysogenic
derivatives.
Another kind of interaction was determined
in system formed by E. coli С1а and С1а(Р1)
strains and the used T7-like phages. The
development of phage FE44/C600, T3 and T7 gave
rise to abortive infection (Abi-phenotype) but the
bacteriophages were completely eliminated after the
first propagation on bacteria. Phage titers from 109–
1010 PFU/ml on the parent strain decreased to zero
on lysogenic. Similar results were obtained in E.
“horticola” system in case of phage FE44/450
propagation on pairs 450, 450(P1) and 60-1N, 60-
1N(Р1). Such efficient restriction by P1 is likely
associated not with the adsorption sites conversion
in lysogens.
Again abortive was the phage T3 infection in
lysogenic E. amylovora L4(P1) cells. This system
was shown to be inefficient for studying the details
of interaction between phage T3 and EcoP1I system
due to the low EOP of phage both on lysogenic and
parent strains.
In contrast to the mentioned cases phages 49
and 59 realize productive infection in Eho 450(P1)
lysogens. EOP of phages decreased by 6 orders of
magnitude after the first plating on P1 lysogens
lawn. However plaque size and phage titers
recovered in the following passages or when the
phage 59 modP1 was used. Thus, DNA of 49 and
59 phages is efficiently modified by
methyltransferase Mod of P1 RM-complex. Such
features the phage lambda behavior in the system of
E. coli P1 lysogens [5].
Conclusions
The constructed system of strains by P1 of E.
coli, E. “horticola” and E. amylovora allow for the
exploration of restriction-modification gene
complex EcoР1I interaction with T7-like phages as
well as with other polyvalent or specific phages.
The genes of EcoP1I RM system are fully
expressed regardless the bacterial host lysogeinzed
by phage P1. This proves the notion that RM-
systems represent certain universal mobile genetic
elements capable of functioning in any system and
outspreading due to residing on phage DNA.
According to the level of restriction three
types of phage-RM system interaction was
discovered. Differences in phage responses to the
presence of RM-system in the lysogenic host
correlate with the number of recognition sequences
on the DNA and the availability of adsorption sites
while gp 0.3 Ocr protein was proved not to be
involved in this interaction
Fig. 2. Phage FE44 plaques formed on lawns of E. coli C600 (А) and C600 (P1) (B)
A B
180
Table 2. Efficiency of plating of phages in the E. coli and E. ”horticola” systems
Bacteriophage
Strain
T7 T3 FE44a E105 59 59mod 49
Eco C600 1.0 1.0 1.0 – – – –
C600(P1) 5*10-2 0.18 0.16 – – – –
C1a 1.0 1.0 1.0 – – – –
C1a(P1) 0* 0* 0* – – – –
Eho 60-1N – – 1.0 1.0 x x x
Eho 60-1N – – 1.0 1.0 x x x
60-1N(P1)-1 – – 0* 7·10-3 x x x
60-1N(P1)-2 – – 0* 8·10-3 x x x
450 – – 1.0 – 1.0 1.0 1.0
450(P1)-2 – – 0* – 4.3·10-6 1.0 9.5·10-6
Note: “a” – On Eco strains the value is indicated for FE44/C600 phage and on Eho strains for FE44/450.
“*0” – stands for the absence of individual plaques while zones of lysis are evident “x” – experiments not
performed.
References
1. Molineux I.J. The T7 group // The Bacteriophages, 2nd edition, edited by R. Calendar. – Oxford University Press. –
2006. – P. 277–301.
2. Товкач Ф.И. Молекулярно-биологические свойства вирулентного бактериофага FE44 // Доповіді
Національної академії наук України. – 2002. – № 6. – С. 175–178.
3. Moffatt B.A., Studier F.W. Entry of bacteriophage T7 DNA into the cell and escape from host restriction // J.
Bacteriol. – 1988. – 170, N 5. – P. 2095–2105.
4. Товкач Ф.И., Шевченко Т.В., Горб Т.Е., Муквич Н.С., Романюк Л.В. Сравнительное изучение умеренных
ервиниофагов 49 и 59 // Микробиол. журн. – 2002. – 64, № 2. – Р. 65–81.
5. Rosner J.L. Formation, induction, and curing of bacteriophage P1 lysogens// Virology. – 1972. – 48, N 3. – P. 679–
689.
6. Товкач Ф.И., Мороз С.Н., Король Н.А., Файдюк Ю.В., Кушкина А.И. Поливалентность бактериофагов,
изолированных из плодовых деревьев, пораженных бактериальным ожогом // Микробиол. журн. – 2013. –
75, № 2. – С. 80–88.
7. Kruger D.H., Reuter M., Hansen S., Schroeder C. Influence of phage T3 and T7 gene functions on a type III
(EcoP1) DNA Restriction-Modification system in vivo // Mol Gen Genet. – 1982. – 185. – P. 457–461.
8. Goldberg R.B., Bender R.A., Streicher S.L. Direct selection for P1-sensitive mutants of enteric bacteria // J.
Bacteriol. –1974. – 118, N 3. – P. 810–814.
9. Бурова Л.М., Товкач Ф.И. Экспрессия генов профага Р1 Escherichia coli в клетках фитопатогенных эрвиний
// Микробиол. журн. – 2006. – 68, № 2. – С. 39–47.
FAIDIUK I.V.
D.K. Zabolotny Institute of Microbiology and Virology of NAS of Ukraine,
Ukraine, 03680, Kyiv, Zabolotnoho str., 154, e-mail: i.v.faidiuk@gmail.com
RESTRICTION OF THE GROWTH OF T7-LIKE PHAGES BY PLASMID PROPHAGE P1
Aims. Considerable part of T7 phage genome is responsible for interaction with the bacterial host, primarily
for the avoidance of action of protective systems of cells, the restriction-modification complexes.
Interactions of T7-like phages with RM systems of type I and II are relatively studied while the question of
impact by the type III systems on their growth remains unclear. Developing a relevant system would allow
us to study the interaction of bacteriophages with host cells on the gene level including the interplay with
prophage elements and RM-systems. Methods. Biological, genetics and molecular biology approaches
combined with bioinformatic research were used. Results. The ability of P1 to infect and lysogenise Erwinia
amylovora and Erwinia “horticola” cells as well as its maintainance as a single-copy plasmid in the cells of
uncommon hosts was shown. A set of lysogenic strains was obtained. According to the level of restriction
three types of phage-RM system interaction were discovered. Though polyvalent, phage FE44 undergoes
abortive infection similar to other members of T7 phage group. Conclusions. The genes of restriction-
modification complex EcoP1I are fully expressed regardless the bacterial host lysogeinzed by phage P1.
181
Differences in interaction with cells are likely associated with the number of enzyme recognition sequences
and the adsorption sites availability while gp 0.3 Ocr protein is not involved in this interaction. The
constructed systems allow for the exploration of EcoР1I interaction with polyvalent phages able to grow both
on E. coli and on such phytopathogens as E. “horticola” and E. amylovora.
Key words: T7-like phages, Type III restriction-modification complexes, antirestriction, polyvalent
bacteriophages, phytpathogens.
|