The phages whose genome is dsDNA like lambda phage usually have a two-protein lysis system: this consists of endolysin, which destroys the bacterial cell wall, and holin, a hydrophobic p
Trang 1ion-dependent L-alanoyl-D-glutamate peptidase
encoded by bacteriophage T5
Galina V Mikoulinskaia1, Irina V Odinokova2, Andrei A Zimin3, Valentina Ya Lysanskaya3,
Sergei A Feofanov1and Olga A Stepnaya3
1 Branch of Shemyakin & Ovchinnikov’s Institute of Bioorganic Chemistry RAS, Pushchino, Moscow, Russia
2 Institute of Theoretical and Experimental Biophysics RAS, Pushchino, Moscow, Russia
3 Skryabin’s Institute of Biochemistry and Physiology of Microorganisms RAS, Pushchino, Moscow, Russia
Introduction
There are two ways by which phages lyse host cells
They can either cause ‘lysis from without’ – which
takes place when the cell is being infected – or
induce ‘lysis from within’, which occurs when the
phage progeny escape the host cell There can be at
least two evolutionary strategies for lysis from within
[1,2] The phages whose genome is dsDNA (like
lambda phage) usually have a two-protein lysis
system: this consists of endolysin, which destroys the
bacterial cell wall, and holin, a hydrophobic protein
providing access of endolysin to the substrate (pepti-doglycan of the cell wall) by forming lesions in the inner membrane Endolysins are proteins with vari-ous muralytic activities; as a rule, they are initially accumulated in the cytoplasm before lysis, which is eventually triggered by holin However, some endoly-sins have an N-terminal translocation domain, and will therefore accumulate in the periplasm; in these cases, holin is not necessary, and lysis will be medi-ated by the Sec system of the host cell [3]
Keywords
bacteriophage T5; endolysin; Gram-negative;
holin; L -alanoyl- D -glutamate peptidase
Correspondence
G V Mikoulinskaia, Branch of Shemyakin &
Ovchinnikov’s Institute of Bioorganic
Chemistry RAS, Prospekt Nauki, 6,
Pushchino, Moscow Region 142290, Russia
Fax: +7 4967 330527
Tel: +7 4967 731780
E-mail: mikulinskaya@fibkh.serpukhov.su
(Received 14 August 2009, revised
14 October 2009, accepted 16 October
2009)
doi:10.1111/j.1742-4658.2009.07443.x
Although bacteriophage T5 is known to have lytic proteins for cell wall hydrolysis and phage progeny escape, their activities are still unknown This is the first report on the cloning, expression and biochemical charac-terization of a bacteriophage T5 lytic hydrolase The endolysin-encoding lys gene of virulent coliphage T5 was cloned in Escherichia coli cells, and
an electrophoretically homogeneous product of this gene was obtained with
a high yield (78% of total activity) The protein purified was shown to be
an l-alanoyl-d-glutamate peptidase The enzyme demonstrated maximal activity in diluted buffers (25–50 mm) at pH 8.5 The enzyme was strongly inhibited by EDTA and BAPTA, and fully reactivated by calcium⁄ manga-nese chlorides It was found that, along with E coli peptidoglycan, peptidase of bacteriophage T5 can lyse peptidoglycans of other Gram-nega-tive microorganisms (Pectobacterium carotovorum, Pseudomonas putida, Proteus vulgaris, and Proteus mirabilis) This endolysin is the first example
of an l-alanoyl-d-glutamate peptidase in a virulent phage infecting Gram-negative bacteria There are, however, a great many sequences in databases that are highly similar to that of bacteriophage T5 hydrolase, indicating a wide distribution of endolytic l-alanoyl-d-glutamate peptidases The article discusses how an enzyme with such substrate specificity could be fixed in the process of evolution
Abbreviations
BAPTA, 1,2-bis-(O-aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid; DNF, 2,4-dinitrophenyl.
Trang 2Endolysins are divided into five classes according to
the type of bonds that they cleave in peptidoglycans
The classes are as follows: (a) lysozyme-like
mura-midases, which hydrolyze the glycoside bond between
N-acetylmuramic acid and N-acetylglucosamine; (b)
lytic transglycosylases, which attack the same bonds as
muramidases but additionally catalyze the
intramole-cular transfer of the O-muramylic residue to the
C6 hydroxyl; (c) N-acetyl-b-d-glucosaminidases, which
hydrolyze the glycoside bond between
N-acetylgluco-samine and N-acetylmuramic acid; (d)
N-acetylmura-myl-l-alanine amidases, which hydrolyze bonds
between N-acetylmuramic acid and l-alanine; and (e)
peptidases, which hydrolyze peptide bonds [4,5] Some
endolysins have multiple activities: for example,
endo-lysin of bacteriophage B30 is both a muramidase and
an endopeptidase [6]
Most endolysins have two functional domains: an
enzymatically active N-terminal domain, and a
C-ter-minal domain, which is responsible for recognition of
the substrate (peptidoglycan) and determines the
speci-ficity of the enzyme [4] The antibacterial effect of
end-olysins is not always associated with their enzymatic
activity For example, endolysin of bacteriophage T4
has four amphipathic C-terminal a-helices, whose basic
(positively charged) amino acids interact with
nega-tively charged components of the outer membrane,
and thus cause its degradation [7] Endolysins may be
acid or alkaline proteins with diverse pH and ionic
strength optima
The spectrum of antibacterial action of endolysins is
determined by the enzyme type, composition of the cell
wall components of the target bacterium, and
configu-ration of the substrate (peptidoglycan) The range of
bacteria sensitive to lysis may be wide, as in the case
of endolysin of Lactobacillus helveticus temperate
bac-teriophage u0303 [8] There are also counterexamples:
endolysin of bacteriophage u3626 specifically degrades
only cell walls of the bacterium Clostridium perfringens
[9] The selectivity of endolysin of bcateriophage C1
for streptococci allows one to forecast its use as a
agent against these bacteria colonizing the mucous
epithelium of the upper air passages [10] The
specific-ity of another phage endolysin for Bacillus anthracis
suggests that it has potential for application in diag-nostics [11] Also, endolysins may act synergistically with antibiotics [12] All this makes endolysins poten-tial candidates as antibacterial drugs The lysozyme of bacteriophage T4, for example, was successfully used
to protect potato from Bacillus subtilis [13]
The system of lytic proteins of bacteriophage T5 was first found in the process of sequencing of the early region of its genome, which is now deposited in the Gen-Bank database (GenGen-Bank accession no AY509815) [14] Analysis of the phage’s primary DNA sequence revealed holand lys genes, which turned out to be located in the same operon under a common promoter A search of protein databases for homologs of hol and lys gene products provided evidence that they are probably involved in the process of host cell lysis [14]
The objectives of the present work included preli-minary biochemical characterization of the novel end-olysin of bacteriophage T5, determination of the peptidoglycan cleavage site, and analysis of endolysin specificity for bacterial species
Results
The hol and lys genes are located in the early C region
of the bacteriophage T5 genome under a common promoter (Fig 1) Endolysin (GenBank accession no AAS19387; UniProt accession no Q6QGP7) is a polypeptide that consists of 137 amino acids (expected molecular mass of 15.266 kDa) and has a calculated
pK value of 8.32
Gene cloning and protein purification The lys gene was cloned into plasmid vector pET3a, and the electrophoretic analysis showed that the Escherichia coli clones selected induced synthesis of a protein product of the expected size (about 15 kDa) The sequence of the lys gene in the plasmid was checked
by sequencing both DNA strands Inside the cell, all of the protein product of lys gene was in a soluble state: after centrifugation of cellular homogenate, the pellet fraction did not contain the target protein, and this was confirmed electrophoretically (data not shown)
Fig 1 Organization of the bacteriophage T5 gene locus that carries genes coding for lytic proteins Flags indicate promoters; T-like signs indicate transcription terminators Arrows indicate open reading frames Numerals on the sides indicate the distance from the beginning of the genome in bp.
Trang 3Endolysin of bacteriophage T5 was extracted from
the cells of the producer strain and purified to an
elec-trophoretically homogeneous state by ion exchange
chromatography on two consecutive columns,
Toyo-pearl DEAE 650M and phosphocellulose (Fig 2;
Table 1) The activity of the final preparation was
calculated to be 8.38· 103units per mg of protein
Storage
It was shown that, when stored in Tris⁄ HCl buffer in
the presence of 1 mm EDTA at 4C, the protein
remained stable BSA (1 mgÆmL)1), 20% glycerol or
0.1% Triton X-100 had a positive effect on the protein
storage: the enzyme activity did not decrease over
several months
Optimal conditions for enzyme functioning
The activity of bacteriophage T5 endolysin was found
to depend strongly on the concentration of the buffer
(Fig 3A) The enzyme showed maximal activity in
diluted Tris⁄ HCl buffers (25–50 mm) With increasing
pH, the enzyme activity grew, and it reached its
maxi-mum at a pH of about 8.5 (Fig 3B)
Other components of the buffer also affected the
lytic ability of bacteriophage T5 endolysin We
mea-sured enzyme activity at two different pH values under
the chosen standard conditions, and compared it with the activity observed upon the addition of various sub-stances As can be seen in Fig 4, the enzyme is metal ion-dependent, because it was inhibited by EDTA This was confirmed by the experiments in which a pure peptidoglycan was used as the substrate High buffer concentrations and high divalent metal ion concentra-tions inhibited the enzyme activity Interestingly, the
Fig 2 SDS ⁄ PAGE analysis of fractions from bacteriophage T5
end-olysin purification steps Lanes: 1, molecular weight markers; 2,
crude extract; 3, chromatography on Toyopearl 650M; 4,
chroma-tography on phosphocellulose Wells 2–4 were loaded with 7 lg of
total protein each.
Table 1 Purification of recombinant endolysin of bacteriophage T5 Specific activity values represent the mean ± standard deviation (n ‡ 3).
Fraction volume (mL)
Protein concentration (mgÆmL)1)
Specific activity (UÆmg)1)
Total activity (U)
Purification factor (fold) Yield (%) Crude extract 10.0 6.0 1830 ± 58 109 800 1 100 Chromatography on Toyopearl 650M 33.0 0.8 4000 ± 105 105 600 2.2 96 Chromatography on phosphocellulose 6 1.7 8380 ± 140 85 480 4.6 78
Buffer concentration, mM
0 50 100 150 200 250
0 1 2 3 4
0 1 2 3 4
pH
5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
Fig 3 (A) The effect of buffer concentration on enzyme activity The activity was measured in Tris ⁄ HCl (pH 8.2) containing 0.1% Tri-ton X-100; the reaction was initiated by the addition of 0.015 lg of enzyme (B) Effect of pH on enzyme activity The activity was mea-sured in 50 m M Tris ⁄ HCl buffer containing 0.1% Triton X-100; the reaction was initiated by the addition of 0.015 lg of enzyme.
Trang 4activity depended on the type of buffer used: in the
potassium phosphate buffer, the enzyme worked much
worse than in the Tris⁄ HCl buffer, especially at high
pH values Perhaps the catalytic function of endolysin
requires the presence of ions of some metals whose
phosphate salts are poorly soluble We supposed that
the cations needed for endolysin functioning might
be Ca2+, Mg2+, or Zn2+: these metals form
low-solu-bility phosphate complexes, whose solulow-solu-bility drops
further upon alkalization of the medium
It is also significant that the enzyme is much more
active in the presence of 0.1% Triton X-100 Caldentey
and Bamford, [15] whose work suggested to us the idea
of using this soft nonionic detergent, considered that it
enhanced endolysin activity through action on the cell
wall (Triton X-100 probably makes peptidoglycan
more accessible or facilitates enzyme binding)
We also explored the effects of various cations on
the storage of endolysin The presence of 10 mm
Mg2+, Ca2+or Mn2+ in the storage medium was
shown to have no effect on the enzyme activity Other
divalent (Zn2+ and Cu2+) and trivalent (Fe3+ and
Cr3+) cations, at a concentration of 10 mm, were
found to completely inactivate the enzyme immediately
after addition to the storage medium
To determine which cations are necessary for the
enzyme to function, we used 0.1 mm metal chelators
with different affinity for metal ions: EDTA, which
binds many cations;
1,2-bis-(O-aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid (BAPTA), which selectively
binds Ca2+and, to a lesser extent, Mg2+, Zn2+[16], and
Fe2+[17]; 1,10-phenanthroline, which has high affinities for Zn2+, Ni2+, Co2+, and Cd2+, and which is rather effective in binding Mn2+, but is ineffective in binding
Mg2+and Ca2+[18,19]; and deferoxamine (desferriox-amine) and Tiron (4,5-dihydroxy-1,3-benzene-disulfonic acid), both of which are chelators of Fe3+
EDTA (a broad-spectrum chelator) and BAPTA (a specific chelator for Ca2+) completely inhibited the activity of endolysin at a concentration of 0.1 mm, whereas 0.1 mm 1,10-phenanthroline did not affect the enzyme activity – although the binding constants of phenanthroline for Zn2+and of BAPTA for Ca2+are very close [19] A 1-day incubation of the protein in
10 mm 1,10-phenanthroline did not decrease its activ-ity, in contrast to what was observed with 10 mm EDTA or BAPTA It should also be noted that, in the absence of chelators, 0.5 mm ZnCl2 completely inacti-vated the enzyme
We tried to restore activity of the enzyme inhibited with 0.1 mm EDTA or BAPTA by adding various salts
at concentrations of 0.1–1 mm (zinc, magnesium, man-ganese and calcium chlorides) Among those salts, only calcium and manganese chlorides were found to restore the enzyme activity completely (Table 2) Zinc chloride and, in the case of EDTA, magnesium chlo-ride at low (0.1–0.25 mm) concentrations partially restored enzyme activity, probably because of binding
of the chelator Increasing the concentration of magne-sium or zinc chloride to 0.5 mm resulted in inhibition
of the enzyme
There was no inhibition of enzyme activity in the presence of Tiron and deferoxamine; moreover, defe-roxamine even enhanced the activity by 25%, probably
by chelating some endogenous cations that could compete with Ca2+and thus affect enzyme activity
Classification of the enzyme by the type of bond hydrolyzed
To determine whether the enzyme studied was a glyco-syl hydrolase, peptidase, or amidase, we compared its hydrolytic action with that of egg white lysozyme To
do this, we analyzed the quantity of reducing and amino groups released after peptidoglycan hydrolysis (to exclude the effect of pre-existing groups, peptido-glycan was either acetylated or reduced prior to hydro-lysis) The results of the assay are presented in Table 3
Pre-acetylation of free amino groups in peptidogly-can practically eliminates staining for them in the sample hydrolyzed by egg white lysozyme This is not surprising, because this enzyme is a glycosyl hydrolase, not a peptidase In contrast, the samples treated with
Fig 4 The effect of reaction mixture composition on enzyme
activity Standard conditions: 50 m M Tris ⁄ HCl containing 0.1%
Triton X-100 K-P i is 50 m M potassium phosphate buffer containing
0.1% Triton X-100.
Trang 5bacteriophage T5 endolysin show the appearance of
free amino groups, suggesting that the enzyme has a
peptidase activity
The peptide subunit of E coli peptidoglycan is
l
-alanoyl-d-glutamyl-l-diaminopimelinoyl-d-alanoyl-d-alanine [20], and about one-third of the total number
of subunits are involved in the formation of
inter-subunit cross-bridges between diaminopimelic acid
and d-alanine [21] A TLC analysis of ether-extracted
2,4-dinitrophenyl (DNF)–amino acids (see
Experi-mental procedures) showed the presence of DNF– glutamic acid in the bacteriophage T5 hydrolysate (Fig 5) Accordingly, as revealed with an amino acid analyzer, the material remaining after extraction showed about 70% loss in the glutamic acid content (Table 4)
Thus, we can conclude that the enzyme studied hydrolyzes the bond between l-alanine and d-glu-tamic acid; that is, it is an l-alanoyl-d-glutamate peptidase
Table 3 Release of amino and reducing groups during enzymatic
hydrolysis of E coli peptidoglycan Values represent the mean ±
standard deviation (n ‡ 3).
Substrate Enzyme
Reducing groups (nmolÆmg)1)
Amino groups (nmolÆmg)1) Acetylated
peptidoglycan
T5 endolysin 23.06 ± 2.8 229.2 ± 13.3
Egg white lysozyme 36.09 ± 2.9 15.7 ± 0.6
Reduced
peptidoglycan
T5 endolysin 19.02 ± 1.6 550.6 ± 27.8
Egg white lysozyme 77.04 ± 5.9 382.0 ± 22.1
Fig 5 Analysis of amino acids released by peptidase of bacterio-phage T5 TLC was performed on Kieselgel 60 F254 plates as described in Experimental procedures Lanes: 1, DNF–alanine (Sigma); 2, DNF–glutamic acid (Sigma); 3, control peptidoglycan; 4, peptidoglycan hydrolyzed by bacteriophage T5 peptidase; 5, pepti-doglycan hydrolyzed by egg white lysozyme The thin lines at the bottom and the top indicate the initial and final eluent fronts, respectively.
Table 2 Restoration of lytic activity of the enzyme by cations after
its inhibition with EDTA or BAPTA The activity was measured
turbi-dimetrically (see Experimental procedures) Before measurements,
0.1 m M EDTA or BAPTA and enzyme (0.015 lg) were added to the
cell suspension The reaction was initiated by the addition of a
chlo-ride of the corresponding metal The activity was calculated as a
percentage of the initial activity, measured in the absence of
chela-tors Values represent the mean ± standard deviation (n ‡ 3) ND,
not determined.
Concentration
of the metal
ion (m M )
Relative activity (%)
EDTA BAPTA Control 0.000 2.8 ± 0.2 3.2 ± 0.2
Zn 2+ 0.110 51.5 ± 4.1 47.0 ± 3.2
0.125 53.7 ± 4.5 42.9 ± 3.0
0.250 31.32 ± 1.7 21.9 ± 1.8
0.500 10.8 ± 0.8 5.5 ± 0.5
Mg2+ 0.110 11.7 ± 0.8 ND
0.125 14.1 ± 1.0 1.1 ± 0.2
0.250 26.4 ± 2.1 4.2 ± 0.3
0.500 6.5 ± 0.3 3.8 ± 0.3
Mn 2+ 0.110 106.6 ± 2.9 105.6 ± 1.2
0.125 116.5 ± 5.4 106.9 ± 1.7
0.250 111.5 ± 4.9 122.9 ± 8.4
0.500 91.9 ± 5.7 101.1 ± 7.5
1.000 68.1 ± 5.7 66.2 ± 5.4
Ca 2+ 0.110 120.5 ± 13.6 92.1 ± 7.8
0.125 117.6 ± 8.3 103.0 ± 9.5
0.250 84.6 ± 6.9 101.6 ± 8.7
0.500 79.5 ± 6.0 95.8 ± 6.3
1.000 69.8 ± 10.8 90.8 ± 10.0
Table 4 Content of Glu relative to Ala in the samples of enzymati-cally hydrolyzed peptidoglycan of Ps putida The contents of individual amino acids were determined after acidic hydrolysis of peptidoglycan samples using an amino acid analyzer (see Experimental procedures).
Component
Peptidoglycan
Untreated
Treated with egg white lysozyme
Treated with bacteriophage T5 endolysin Ala 1.00 1.00 1.00 Glu 0.57 0.54 0.17
Trang 6Amino acid sequence analysis
A psi-blast analysis of bacteriophage T5
l-alanoyl-d-glutamate peptidase showed that there are many
similar proteins, of both phage and bacterial origin
(217 hits after the third iteration) The best hits with
low E-values ( 10)45–10)30) were sequences of
pro-teins from Gram-negative bacteria of the genera Yer-sinia, Shigella, Escherichia, Photorhabdus, Providencia, Agrobacterium, Proteus, Serratia, Hahella, Haemophi-lus, Chromobacterium, Vibrio, Vibrionales, Aliivibrio, Brevundimonas, Erwinia, and Bordetella, as well as bacteriophage proteins In the E-value region of 10)27–
10)20, one can find protein sequences of Gram-positive bacteria On the basis of multiple alignment of 53 sequences, which were selected among 95 of the best hits after removing repetitions, a phylogenetic tree was constructed under conditions of minimal evolution using the program mega v 4.0 (Fig 6) On this tree, the proteins of enterobacteria and close species, as well
as their bacteriophages, form a branch separated from the branches of Gram-positive bacteria and their bacteriophages Figure 7 shows an alignment of seven sequences constructed with the program clustalx The sequences belong to the branch of proteins that
Phage ST64T Phage PS3 Proteus mirabilis Yersinia bercovieri Phage RB43 Phage T5 Phage EPS7 Haemophilus influenzae Brevundimonas sp.
Phage phiEcoM Chloroherpeton thalassium Aliivibrio salmonicida Vibrio fischeri Phage Xp15
Bordetella bronchiseptica Bordetella parapertussis Photorhabdus asymbiotica Erwinia tasmaniensis Providencia stuartii Chromobacterium violaceum Yersinia enterocolitica
Yersinia mollaretii Yersinia pseudotuberculosis Yersinia pestis Pestoides F Proteus penneri Vibrio splendidus Vibrionales bacterium Phage PY100
Yersinia frederiksenii Agrobacterium tumefaciens
Hahella chejuensis Phage phiJL001 Phage RB49 Phage Phi1 Serratia proteamaculans Phage phiP27
Shigella boydii Escherichia coli Phage A500 Listeria innocua Phage A006 Phage P35 Phage SPO1 Exiguobacterium sibiricum Geobacillus sp.
Geobacillus kaustophilus Anoxybacillus flavithermus
Clostridium acetobutylicum Exiguobacterium sp.
Bacillus cereus Phage B025 Listeria monocytogenes Phage A118
100
100
100
96 100
100
99
99
87 99 99 95
88 90
88
87
85
84
79 83
79
78
73
29 70
53
50
29
37
36
36
26
22
20
20
19
17
4
9
4
2
2
40
37
17
47
15
41
0.1
Fig 6 Phylogenetic tree of amino acid sequences of bacterial and phage endolysins constructed by the minimum evolution method Numbers at the nodes represent the bootstrap values with 500 rep-lications The scale bar indicates an evolutionary distance of 0.1 amino acid substitutions per site Bootstrap values are indicated above or below the branches GenBank accession numbers are as follows: bacteriophage ST64T, NP_720320.1; bacteriophage PS3, CAA09701.1; P mirabilis, YP_002151708.1; Y bercovieri, ZP_ 00822433.1; bacteriophage RB43, YP_239135.1; bacteriophage T5, YP_006868.1; bacteriophage EPS7, YP_001836966.1; Haemophi-lus influenzae, YP_248988.1; Brevundimonas sp., YP_002588905.1; bacteriophage phiEcoM, YP_001595416.1; Chloroherpeton thalas-sium, YP_001995193.1; Aliivibrio salmonicida, YP_002262290.1; Vibrio fischeri, YP_205400.1; bacteriophage Xp15, YP_239293.1; Bordetella bronchiseptica, NP_889694.1; Bordetella parapertussis, NP_885037.1; Photorhabdus asymbiotica, CAR67777.1; Erwinia tas-maniensis, YP_001907932.1; Providencia stuartii, ZP_02961079.1; Chromobacterium violaceum, NP_903215.1; Yersinia enterocolitica, AAT90759.1; Yersinia mollaretii, ZP_00825275.1; Yersinia pseudotu-berculosis, YP_001399405.1; Yersinia pestis pestoides F, YP_ 001161675.1; Proteus penneri, ZP_03801884.1; Vibrio splendidus, ZP_00991905.1; Vibrionales bacterium, ZP_01814817.1; bacterio-phage PY100, CAJ28446.1; Yersinia frederiksenii, ZP_00828831.1; Agrobacterium tumefaciens, NP_353494.2; Hahella chejuensis, YP_ 435691.1; bacteriophage phiJL001, YP_224014.1; bacteriophage RB49, NP_891673.1; bacteriophage Phi1, YP_001469446.1; Serra-tia proteamaculans, YP_001471697.1; bacteriophage phiP27, NP_ 543082.1; Shigella boydii, YP_001880486.1; E coli, ZP_03049236 1; bacteriophage A500, YP_001468411.1; Listeria innocua, NP_ 469473.1; bacteriophage A006, YP_001468860.1; bacteriophage P35, YP_001468812.1; bacteriophage SPO1, YP_002300379.1; Exiguobacterium sibiricum, YP_001814297.1; Geobacillus sp., ZP_ 02914525.1; Geobacillus kaustophilus, YP_145852.1; Anoxybacillus flavithermus, YP_002315045.1; Clostridium acetobutylicum, NP_ 347645.1; Exiguobacterium sp., ZP_02992216.1; Bacillus cereus, YP_002446097.1; bacteriophage B025, YP_001468664.1; L mono-cytogenes, ZP_03669234.1; bacteriophage A118, NP_463486.1.
Trang 7are the closest to bacteriophage T5 peptidase; they are
encoded by the genomes of bacteriophages ST64T,
PS3, phiEcoM, and RB43, and the bacteria
Yer-sinia bercovieri and Proteus mirabilis In
multialign-ment, we did not use the bacteriophage EPS7 protein
sequence, which is the most similar to the sequence of
bacteriophage T5 peptidase, because it seems to be
shortened The high similarity of two bacterial proteins
(from Y bercovieri and P mirabilis) with the phage
proteins could indicate their coding by prophages As
a result of the analysis, we found the amino acid
sequence motifs HXXXXXXD and DXXH, which
are typical for peptidases of the C subfamily of
family M15 (http://merops.sanger.ac.uk/) The former
sequence is a part of the phage consensus sequence
SK(R)HI(L,M)T(S)GD(N)AI(V,L)DI(L,F)I(L,A,Y),P,
consisting of 13 amino acids, six of which are identical
and the others of which are highly conserved
Testing the lytic ability of endolysin on
heterologous microorganisms
To assess the spectrum of bacteriolytic action of
bacte-riophage T5 endolysin, we tested its ability to
hydro-lyze various substrates prepared from the cells of
selected bacteria according to a standard protocol
(Table 5) Among the bacteria selected were
Gram-positive and Gram-negative species, which differed in
the structure of peptidoglycan and the composition of
the cell wall
All of the tested Gram-negative bacteria were
sub-jected to rapid lysis Among the Gram-positive cells
examined, only B subtilis cells were lysed relatively well; however, the rate of their lysis was about 104-fold lower than that of Gram-negative cells In Listeria cells, the rate of lysis was an order of magnitude lower than that in bacilli (Table 5) The rest of the Gram-positive cells were not lysed
The specificity of bacteriophage T5 endolysin towards Gram-negative bacteria suggests that it might
be used as a selective antibacterial agent However, to make peptidoglycan accessible to the enzyme, it would
be necessary to perturb the outer cell membrane We therefore used polymyxin B, an antibiotic that can bind phospholipids of the outer membrane and destroy membrane integrity, as a putative destructive agent Figure 8 shows that, at a concentration of 40 lgÆmL)1, polymyxin B inhibited the growth of cells but did not lyse them (the attenuance of the cell mixture did not decrease) At the same time, endolysin with polymyxin
B lysed the cells completely The nontreated and end-olysin-treated E coli cells grew well on a nutrient agar medium, forming a dense bacterial lawn (Fig 8A,D); treatment of cells with polymyxin B resulted in difficul-ties in cell growth (Fig 8B); and combined endoly-sin⁄ polymyxin B treatment led to complete lysis of all living cells – there was no further growth (Fig 8C)
Discussion
In this study, we cloned the gene of a novel enzyme and then purified and characterized this enzyme, which turned out to be endolysin of bacteriophage T5, a component of the phage cell lysis system Our
experi-Fig 7 Multiple alignment of protein
sequences of bacteriophages T5, ST64T,
PS3d, RB43, and phiEcoM, and the bacteria
Y bercovieri and P mirabilis, constructed
using the program CLUSTAL X Amino acids
common to all the sequences are marked in
gray; conservative amino acids, which
par-ticipate in metal ion binding and catalysis,
are marked in black.
Trang 8ments have shown that the protein is a metal
ion-dependent l-alanoyl-d-glutamate peptidase The
enzyme is strongly inhibited by EDTA and BAPTA,
and completely reactivated by Ca2+ and Mn2+
Considering BAPTA as a specific chelator of Ca2+, we
calculated the concentration of free Ca2+ in the
med-ium, using the program cabuf, and concluded that, to
cleave peptidoglycan, the enzyme would require free
Ca2+ at a concentration of 0.05–0.4 mm Probably,
in vitro, Ca2+can be replaced with Mn2+
It is known that bacteriophage T5 requires 0.1 mm
Ca2+ to produce phage progeny in E coli cells [22]
Calcium ions play a key role at certain stages of phage
development: upon infection, when the phage DNA
penetrates into cells [22]; and during the synthesis of
phage RNA [23] and proteins [24] Interestingly, the
enzyme that is directly involved in the final stage of
development (lysis) is also activated by the same
con-centrations of Ca2+ There is another example of such
activation: aminopeptidase A (EC 3.4.11.7), a Zn2+
-containing enzyme (gluzincin) of metallopeptidase
family M1, is also activated by Ca2+and can be
reac-tivated by Ca2+ and Mn2+ [25] The Ca2+, which
binds to the enzyme through aspartic acids [26,27],
contributes to its substrate specificity: it forms a bridge
between an aspartic acid of the enzyme and an acidic
N-terminal amino acid of the substrate [26] The
affin-ity of bacteriophage T5 endolysin for d-glutamic acid
of peptidoglycan and the ability of endolysin to be
activated by Ca2+may be related as well On the basis
of data on enzyme metal ion dependence, we can
include l-alanoyl-d-glutamate peptidase of
bacterio-phage T5 in the sub-subclass of metalloendopeptidases
[EC 3.4.24 (probable); the enzyme can be listed in EC
after publication of evidence that it catalyzes this
reac-tion)] The analysis of the primary amino acid sequence of bacteriophage T5 peptidase revealed con-served amino acids (His66, Asp73, Asp130, and His133) that are typical for metallopeptidases of the M15 family and take part in the binding of the metal ion in the process of catalysis [28]
This is the first example of an l-alanoyl-d-glutamate peptidase to be found in a virulent phage infecting Gram-negative bacteria Enzymes of this class, Ply118 and Ply500, were first found in two temperate phages, A118 and A500, which infect Gram-positive rods of the Listeria genus [29] Since then, there has been only one l-alanoyl-d-glutamate peptidase – from the Gram-positive bacteria B subtilis – whose existence has been proved biochemically [30] It is interesting that Listeria and Bacillus are not close relatives of E coli, but their peptidoglycan is also of the A1c type Ply118 and Ply500 show rather high substrate specificities: apart from Listeria, they affect only three species of the Bacillus genus (which also have peptidoglycan of the A1c type) [29] l-Alanoyl-d-glutamate peptidase of bacteriophage T5 is similar to the N-terminal, enzy-matically active domain of Ply118 (25% identity) The C-terminal domain of Ply118 is responsible for sub-strate recognition [31] The enzyme of bacteriophage T5 is much shorter than Ply118 (137 and 281 amino acids, respectively); evidently, in bacteriophage T5 end-olysin, the functions of substrate binding and catalysis
Table 5 The effect of bacteriophage T5 peptidase on heterologous
microorganisms The rate of cell lysis was measured
turbidimetri-cally (see Experimental procedures), using autoclaved bacterial cells
as a susbstrate Values represent the mean ± standard deviation
(n ‡ 3).
Organism Relative rate of lysis (UÆmg)1enzyme)
E coli K-12 (1.12 ± 0.12) · 10 4
Pe carotovorum (1.01 ± 0.15) · 10 4
Ps putida (1.35 ± 0.18) · 10 4
P vulgaris (1.19 ± 0.20) · 10 4
P mirabilis (1.04 ± 0.15) · 10 4
B subtilis 0.48 ± 0.05
L monocytogenes 0.016 ± 0.001
S aureus 0.0
C xerosis 0.0
M luteus 0.0
Fig 8 Analysis of E coli cell viability after endolysin action Frag-ments of plates containing cells preincubated with: (A) pure endoly-sin (40 lgÆmL)1); (B) polymyxin B (40 lgÆmL)1); (C) polymyxin B (40 lgÆmL)1) and pure endolysin (40 lgÆmL)1); and (D) control cells.
Trang 9are carried out by a single domain Interestingly, a
recent crystallographic examination of the Ply500
enzyme [28] showed the presence of Zn2+in the active
center – a phenomenon that is typical for a number of
proteins of the peptidase M15 family [32] The
inhibi-tion of bacteriophage T5 peptidase by the specific
Ca2+ chelator BAPTA and by ZnCl2 does not agree
with those data, and nor does the absence of an
inhibi-tory effect of 1,10-phenanthroline, which has a high
affinity for zinc However, the above-mentioned Zn2+
-containing Ca2+-activated aminopeptidase A is
inhib-ited by Zn2+ too [33], and such a feature can also be
found among other Zn2+-containing metalloenzymes
[34] In addition, the failure to observe inhibition with
a particular chelating agent need not be an absolute
gauge of the absence of Zn2+ [19] Perhaps the
question of which cation is bound at the active center
of bacteriophage T5 endolysin will be answered
after crystallographic examination Nevertheless, the
stimulatory effect of Ca2+on the enzyme is evident
The blast analysis shows that there are more than
100 proteins – both from phages and from bacteria
(mainly Gram-negative bacteria) – that are much
more similar to bacteriophage T5 endolysin than
Ply500 and Ply118 Probably, all of them are
l-ala-noyl-d-glutamate peptidases and have a common
ori-gin, and bacteriophage T5 peptidase is a typical
enzyme of the bacterial cell lysis system It is
possi-ble that such a wide distribution of endolytic
l-ala-noyl-d-glutamate peptidases is related to the frequent
occurrence of the l-alanine–d-glutamate bond in
pep-tidoglycan
The hypothesis that phage l-alanoyl-d-glutamate
peptidases have a common origin is supported by the
results of comparison of phage holin sequences In
most cases, holin sequences demonstrate little
similar-ity to each other, and these proteins are considered to
have evolved independently of endolysins [35]
How-ever, holin of bacteriophage T5 shows significant
similarity to holins of bacteriophages RB43 (E =
3· 10)16) and RB49 (E = 9· 10)21), as does
bacte-riophage T5 l-alanoyl-d-glutamate peptidase towards
RB43 and RB49 putative endolysins (E = 7· 10)19
and E = 2· 10)16, respectively) It is interesting that
bacteriophages RB43 and RB49 belong to the group
of pseudo T-even phages Holin of bacteriophage T4,
a product of gene t, also resembles holin of
bacterio-phage T5 (E = 6· 10)13) However, endolysin E of
bacteriophage T4 is a muramidase, and has no relation
to bacteriophage T5 peptidase It is possible that there
might have been horizontal gene transfer between
pseudo-T-even phages and bacteriophage T5 It should
be noted that in bacteriophages RB43, RB49, and T4,
endolysin and holin are located in different genome regions – not in the same operon under a common promoter, as in bacteriophage T5 It can be supposed, therefore, that the variant present in bacteriophage T5 (colocalization of the lytic system genes) brings more evolutionary advantages in terms of their coordinated expression
In addition to holin and endolysin, some dsDNA phages have another pair of proteins that are involved
in cell lysis and provide a competitive advantage to the phage under unfavorable growth conditions (analogs
of the products of the Rz and Rz1 genes of bacterio-phage k) [36] One of these proteins is a lipoprotein, and the other is a transmembrane protein; defects in their genes result in Mg2+-dependent disorders in the process of lysis Analogs of these proteins have recently been found in quite a large number of phages, including T5, T4, RB32, RB43, RB49, and RB69 [36] All of these proteins are located separately from holin and endolysin The products of genes T5p045 (Rz ana-log) and T5p044 (Rz1 anaana-log) show a slight resem-blance to the products of genes PseT.3 and PseT.2 of bacteriophages T4 and RB32 Interestingly, the operon
in which genes T5p045 and T5p044 are located in is the upstream neighbor of the holin–endolysin gene locus (Fig 1), but the coding sequences of these genes are included in the early transcript At the same time, the bacteriophage T5 holin–endolysin promoter is probably late, although it is located in the early gen-ome region It contains more GC pairs, particularly in the region +10 to +20, which is typical for late promoters of bacteriophage T5 [37]; in addition, the conservative region )33 contains a TTnAnA sequence (typical for late bacteriophage T5 promoters) and does not contain TTGCTn, which is a sign of early promot-ers [38] It should be noted that holin T and endolysin
E of bacteriophage T4 are late proteins, although gene
eis located in the early region [39]
To assess the spectrum of bacteriolytic action of bacteriophage T5 endolysin, we tested the bacteria of Gram-positive and Gram-negative species, which differ
in the structure of peptidoglycan and the composition
of the cell wall For example, Gram-negative cells con-tains peptidoglycan of the A1c type (not amidated, with c-mesodiaminopimelic acid in the third position);
in B subtilis, the peptidoglycan is of the same type but amidated; Listeria monocytogenes also has peptido-glycan of the A1c type, but its cell wall structure has some features (for instance, teichoic acid of the Liste-ria cell wall contains two substituents, N-acetylglucos-amine and dirhamnosyl, whereas teichoic acids of the other Gram-positive bacteria include only one type of substituting group) [40]; Staphylococcus aureus has an
Trang 10A3a-type peptidoglycan (with l-lysine in the third
position); the type of peptidoglycan of Micrococcus
luteus is A2 (l-lysine in the third position; teichuronic
acids instead of teichoic acids); and the cell walls of
corynebacteria contain lipids, like those of
Gram-nega-tive microorganisms [20] The action of bacteriophage
T5 endolysin seemed to be directed towards
peptido-glycan type A1c, although the cell wall composition of
Gram-positive bacteria (L monocytogenes and B
sub-tilis) protected them from rapid lysis Thus, our
preli-minary results suggest that the peptidase of
bacteriophage T5 is specific for the cell walls of
Gram-negative microorganisms, containing A1c
peptidogly-can and lacking either teichoic or teichuronic acids,
which are typical of the cell walls of the Gram-positive
bacteria
Some Gram-negative microorganisms are pathogenic
for animals and plants (Pseudomonas, Klebsiella,
Pro-teus, and Agrobacterium), making bacteriophage T5
peptidase a potential candidate for the development of
drugs that could be of use in biotechnology and plant
cultivation
Successful application of bacteriophage T5 endolysin
as a selective lytic agent against Gram-negative cells
requires the presence of factors that disturb outer
membrane permeability There are a number of these:
EDTA, sodium tripolyphosphate, heat, pH [41],
ultra-sound [42], peptidolipids [43], and polymyxin B [44] In
this work, we have demonstrated that E coli cell lysis
by bacteriophage T5 endolysin is possible after
poly-myxin B treatment This constitutes an example
prov-ing the effectiveness of this approach, and other
permeabilizing agents could also be applicable
Experimental procedures
Materials
E coli strains B, Z85 and BL21(DE3) and bacteriophage
T5+ were taken from the collection of the Laboratory of
Molecular Microbiology of the Institute of Biochemistry
and Physiology of Microorganisms (IBPM RAS) Strains
of E coli K12, B subtilis, M luteus, Pseudomonas putida,
S aureus, Pectobacterium carotovorum, Proteus vulgaris,
P mirabilis, L monocytogenes and Corynebacterium xerosis
were obtained from the All-Russian Collection of
Micro-organisms (IBPM RAS) Plasmid pET3a was provided
by Novagen (Madison, WI, USA) Bacteria and phages
were grown either in liquid LB broth or on agarized LB
medium Selection of clones was performed on plates with
ampicillin (50 lgÆmL)1) Crystalline egg white lysozyme was
purchased from Serva (Heidelberg, Germany) Restriction
endonucleases were from Fermentas (Vilnius, Lithuania)
All other chemicals were purchased, unless otherwise stated, from either ICN (Irvine, CA, USA) or Sigma (St Louis,
MO, USA)
Cloning of the lys gene
The lys gene of bacteriophage T5 was amplified by PCR with primers LysF (5¢-gtcgagacATATGAGTTTTAAAT TTGGT-3¢) and LysR (5¢-ctggatccATTAAACTAGTTCG ACATG-3¢), which contain sites hydrolyzed by restriction endonucleases NdeI and BamHI The PCR fragment was cloned into plasmid vector pET3a (into the region con-trolled by the promoter of gene 10 of bacteriophage T7), using standard molecular biology techniques The clones carrying the insert were selected after treatment with restric-tion endonucleases and electrophoresis in 1% agarose The construct obtained was named pT5lys Plasmid pT5lys was further used to transform cells of E coli strain BL21(DE3) The synthesis of endolysin was induced with 0.5 mm isopro-pyl-thio-b-d-galactoside at a culture density corresponding
to attenuance D550 nm= 1.0; the cells were harvested by centrifugation (6000 g, 10 min) 2.5 h later
Isolation and purification of endolysin
Cells of E coli BL21(DE3) from 200 mL of culture (1.1 g) carrying plasmid pT5lys were suspended in 10 mL of
25 mm Tris⁄ HCl (pH 8.0), containing 40 mm NaCl and
1 mm EDTA, and disrupted by sonication for 1 min (two
30 s treatments at a power of 75 W) The suspension was centrifuged at 20 000 g for 30 min The supernatant (9.5 mL) was passed through an 11.4 mL column with Toyopearl DEAE 650M (TosoHaas, Stuttgart, Germany) and then applied to a 10 mL phosphocellulose column equilibrated with the same buffer Proteins were eluted by a linear gradient of sodium chloride (0.05–0.50 m) in 25 mm Tris⁄ HCl buffer (pH 8.0) containing 1 mm EDTA (total volume, 100 mL) Fractions (2 mL) were analyzed by PAGE in a 15% polyacrylamide gel The target protein was eluted with 0.3 m NaCl
Enzyme activity assay
The substrate for enzyme activity assay was prepared as follows An overnight culture of E coli B cells was treated with chloroform (added to 5% of total volume) for 15 min, and the cells were then were washed twice with water and stored frozen Just before the measurement, the cells were suspended in a reaction buffer (50 mm Tris⁄ HCl, pH 8.2, containing 0.1% Triton X-100) The activity was deter-mined spectrophotometrically, by the decrease of attenu-ance at 450 nm, in 1 cm acrylic cuvettes at room temperature An activity unit was defined as the quantity of enzyme that provides the rate of attenuance decrease of 1.0