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Sequence and comparative genomic analysis of lactococcal bacteriophages jj50, 712 and P008: evolutionary insights into the 936 phage species

Jennifer Mahony, Hélène Deveau, Stephen Mc Grath, Marco Ventura, Carlos Canchaya, Sylvain Moineau, Gerald F. Fitzgerald, Douwe Van Sinderen
DOI: http://dx.doi.org/10.1111/j.1574-6968.2006.00372.x 253-261 First published online: 1 August 2006

Abstract

The complete genome sequences of three lactococcal 936-type bacteriophages, 712, jj50 and P008, were determined. Comparative genomic analysis of these phages with the previously sequenced 936-type phages, sk1 and bIL170, reveals a strict conservation of the overall genetic organization of this geographically diverse phage group. Genetic divergence was mainly observed in the early expressed region of the phage genomes, where a number of deletions, exchanges and insertions appear to have occurred. These genetic differences may be responsible for the observed differential sensitivity to the lactococcal DNA injection blocking protein, Sie2009, and the abortive infection system, AbiA.

Keywords
  • bacteriophage
  • Lactococcus
  • comparative genomics
  • abortive infection
  • superinfection exclusion

Introduction

Bacteriophages infecting Lactococcus lactis disrupt dairy fermentation processes, which may result in considerable economic losses. These bacteriophages are classified into several genetically distinct groups, the three most prevalent of which are the c2, 936 and P335 species. This practical relevance has resulted in these three species receiving more focused research attention, particularly at the level of sequence and functional analyses. Members of the c2 and 936 phage groups are virulent phages, while members of the P335 group may be virulent or temperate. More recent phage population studies have indicated that 936-type phages account for most of the isolates obtained from industrial settings (Josephsenet al,1994; Moineauet al,1996; Bissonnetteet al,2000). Yet, only two complete phage genomes from the 936 group are currently available in GenBank, namely sk1 (Chandryet al,1997) and bIL170 (Crutz-Le Coqet al,2002).

Some members of the 936 phage type, such as sk1, jj50 and P008, appear to infect a wider range of lactococcal strains unlike many others, including φjw31, jw32 and the prototype phage, φ936 (Dupontet al,2005). Host range studies in conjunction with phylogenetic analysis of the receptor-binding proteins (Dupontet al,2004b) of many phages of this group have permitted a tripartite subgrouping of the 936 phages, namely the sk1-type, the bIL170-type and the 712-type (Dupontet al,2005), although a recent study showed additional RBP subgroupings within this lactococcal phage group (Tremblayet al,2006).

Nonetheless, this general divide is also observed when testing the sensitivity of lactococcal phages against antiviral defence mechanisms naturally present in some L. lactis strains. For example, Sie2009 is a membrane-associated DNA injection blocking protein that provides resistance to the expressing lactococcal host against some 936 phages capable of infecting Lactococcus lactis ssp. cremoris strains but not those infecting Lactococcus lactis ssp. lactis strains (McGrathet al,2002). The ability of the sk1-like and bIL170-like phages to infect L. lactis ssp. cremoris and lactis strains, respectively, is due, at least in part, to the variable C-terminal domain of the receptor-binding proteins as deduced by Dupont (2004b).

Another type of phage defence mechanism found in L. lactis is called abortive infection or Abi. These systems, also called phage exclusion, block phage multiplication and cause cell death upon phage infection. This outcome decreases the number of progeny virions and limits their spread, allowing the bacterial population to survive (Chopinet al,2005). AbiA is an abortive infection system that is known to affect the early stages of the phage lytic cycle (Dinsmore & Klaenhammer, 1994). The protein exhibits increased hydrophobicity at its amino terminus, in a manner comparable to Sie2009. Its early-acting nature, coupled with the similarity in predicted membrane topology, encouraged a study of the effect of the lactococcal abortive infection system, AbiA (Dinsmore & Klaenhammer, 1994). The sensitivity pattern of the assayed phages to these resistance mechanisms appeared to coincide with the host specificity of the phages identified by Dupont (2004b).

This observation and the limited number of complete genomic sequences for 936-like phages prompted the sequencing of the lactococcal phages 712, jj50 and P008. These three virulent lactococcal phages of the 936 group have been isolated from geographically distinct regions throughout Europe, some of which have evolved the ability to overcome phage-defence mechanisms to a varying extent. This article increases our knowledge on this significant phage group and provides insights into their adaptive genome evolution.

Materials and methods

Phages and bacterial strains

The lactococcal strains, plasmids and 936-type phages used in this study are listed in Table 1. Lactococcal host strains were grown at 30°C in M17 broth (Oxoid Ltdet al, Hampshire, England) supplemented with 0.5% glucose. Chloramphenicol and erythromycin were added at a final concentration of 5μgmL−1 where appropriate.

View this table:
Table 1

Lactococcal stains, plasmids and bacteriophages used in this study

Relevant featuresReference/Source
Lactococcus lactis strains
ssp. cremoris NZ9000Host to sk1, jj50, 712 & p2Kuipers (1998)
ssp. lactis IL1403Host to P008, bIL170, P113G, P272 & bIL66Chopin (1984)
ssp. cremoris W34Host to jw31 & jw32Josephsen (1994)
Plasmids
pNZ44sie2009pNZ44 containing sie2009, CmrMcGrath (2002)
pCI816pSA3 containing 6.2kb StuI fragment from pCI829. Emr AbiA+Coffey (1991)
Bacteriophages
sk1Propagated on NZ9000Chandry (1997)
jj50Propagated on NZ9000Josephsen & Vogensen (1989)
712Propagated on NZ9000Rince (2000)
p2Propagated on NZ9000Higgins (1988)
P008Propagated on IL1403Loof (1983)
bIL170Propagated on IL1403Crutz-Le Coq (2002)
bIL66Propagated on IL1403Bidnenko (1995)
P113GPropagated on IL1403Loof & Teuber (1986)
P272Propagated on IL1403Loof & Teuber (1986)
jw31Propagated on W34Josephsen (1999)
jw32Propagated on W34Josephsen (1999)

Sie2009 and AbiA activity spectra determination

Plaque assays were performed using the protocol described by Lillehaug (1997). The effectiveness of AbiA was examined as previously described by Coffey (1991), while Sie2009 was assayed against lactococcal phages as previously described by McGrath (2002).

DNA sequencing and sequence analysis

DNA of phages 712 and jj50 was isolated as described previously (Moineauet al,1994). The DNA was mechanically sheared and the resultant fragments were shotgun-cloned in pGem-Teasy (Promega, Madison, WI) to generate a plasmid library in Escherichia coli DH10b. The cloned fragments were subsequently sequenced, providing a fivefold sequence coverage (MWG Biotech AG, Ebersberg, Germany), representing c. 60% of the phage genomes. Sequence assembly was performed using the Seqman programe, version 5.05, of the DNAStar software package (DNAStar Incet al, Madison WI). Alignment to the publicly available sequences of bIL170 and sk1 permitted size estimation of the remaining gaps, which, in the case of small gaps, were closed by primer walking directly on phage DNA. Larger gaps were PCR-amplified and sequenced by primer walking. DNA of phage P008 was isolated with the Maxi Lambda DNA purification kit (Qiagen) according to the modifications described previously by Deveau (2002). This DNA was sequenced on both strands by primer walking using an ABI Prism 3700 apparatus from the genomic platform at the research center of the Centre Hospitalier de l'Université Laval. To determine the cos region, phage DNA was ligated and then, the region was amplified by PCR and the purified PCR products were sequenced. Phage terminal ends were also defined by sequence drop-off using unligated phage DNA as a template.

Assignment of ORF was firstly based on minimal size lengths (at least 30 amino acids) and secondly, the putative start codon (ATG, TTG or GTG) should be preceded by a potential ribosomal-binding site or translationally coupled to a preceding ORF. Automatic ORF prediction was carried out using Artemis software or OrfFinder available on the NCBI web site, followed by a manual check of identified ORFs. Computer-assisted DNA analyses were performed using a Genetics Computer Group sequence analysis software package, version 10.3, including GenBank release 146.0, GenPept release 146.0, UniProt release 4.3, Swiss-Prot release 46.3, TrEMBL release 29.3, NREF release 1.64, PROSITE release 18.45, Pfam release 14.0, REBASE release 503 and ClustalW web site (http://www.ebi.ac.uk/clustalw/). psi-blast and Advanced Blast Search 2.1 were also used for sequence comparisons with databases (Altschulet al,1997).

Nucleotide sequence accession numbers

The complete genomic sequences of lactococcal phages analysed in this study are available under the following GenBank accession numbers: phage bIL170 (NC001909), sk1 (NC001835), 712 (DQ227763), jj50 (DQ227764) and P008 (DQ054536).

Results and discussion

Activity Spectra of Sie2009 and AbiA

McGrath (2002) previously established that expression of Sie2009 confers a phage-resistant phenotype on the lactococcal host against 936-like phages sk1, jj50 and 712, while no such effect was observed against phage P008. In this study, Sie2009 was shown not to affect proliferation of phage bIL170, bIL66, P272 or P113G (Table 2). Interestingly, bacteriophages (P008, bIL170, bIL66, P272, P113G) infecting L. lactis ssp. lactis IL1403 were unaffected by Sie2009 while those infecting L. lactis ssp. cremoris (sk1, jj50, 712, p2, jw32) were more sensitive to the effects of this phage-encoded resistance system (Table 2). This suggests that Sie2009 is specific in its activity to a subgroup of the 936-type phages and perhaps exclusively active against a subgroup of phages infecting specific L. lactis ssp. cremoris strains. This may be further evidenced by the activity of Sie2009 against φjw32 and p2, which both infect L. lactis ssp. cremoris strains (Table 2). The apparent inactivity of this system against φjw31 and the previous observation of inactivity against another 936-type phage, 18-16, may point to the phage-specific activity of this system. It cannot be precluded that Sie2009 may interact with the host receptor for the sk1-like phages or another host-encoded factor in order to exert its effect. The host receptors for the sk1- and bIL170-like phages are different, which may explicate the selective action of Sie2009 (Dupontet al,2004a). However, it is interesting to note that in both cases, a putative glycosyltransferase and a membrane-associated protein of the operon encoding cell wall polysaccharide biosynthesis are apparently necessary for phage adsorption (Dupontet al,2004a).

View this table:
Table 2

Phage characteristics and responses to Sie2009 and AbiA

GenomeEOP
PhageHostLength
(kb)% G+C
content# ORFs
predictedSie2009AbiA
sk1NZ90002845134.5154<10−910−3
jj50NZ90002745334.9449<10−910−2
712NZ90003051033.8955<10−9<10−9
P008IL14032853834.685811
bIL170IL14033175434.356411
bIL66IL140311
P272IL140311
P113GIL140311
p2NZ90000.510−3
jw31W3411
jw32W3410−21
  • Efficiencies of plaquing (EOP) values are the mean values of five independent assays.

AbiA exhibited an activity pattern similar to that of Sie2009, albeit to various extents, against sk1, jj50 and 712 (Table 2). Interestingly, this abortive infection system was unable to prevent proliferation of bIL170 and P008 or bIL66, P272, P113G, jw31 and jw32. The variation in sensitivity observed between P008/bIL170 and sk1/jj50/712 to Sie2009 and AbiA provoked an interest in studying the genetic diversity within the 936 phage group.

Phage genome sequence analysis

The complete nucleotide sequences of the bacteriophages jj50, 712 and P008 were determined by a dual strategy of shotgun cloning and primer walking. The genomes of jj50, P008 and 712 were found to be 27453, 28538 and 30510bp in length, respectively. The genomes of phages jj50, P008 and 712 contain 49, 58 and 55 ORFs, respectively, with the same overall genetic organization as previously determined for both sk1 and bIL170 (Chandryet al,1997; Crutz-Le Coqet al,2002). Table 3 outlines the identified ORFs of P008 and its homologues in the other completely sequenced 936-type phages. Each of the functional modules appears to be maintained in these newly described phages (Fig. 1).

View this table:
Table 3

Coordinates of phage P008 ORFs and representative ORFs in other 936-type phages

Homologous ORF (% aa identity)
ORFStartStopPredicted
protein Size
(kDa)sk1bIL170712jj50Putative function
Late expressed region
125477819.90ORF 1 (96)L1 (98)ORF 1 (98)ORF 1 (97)Terminase small subunit
2779240163.01ORF 2 (98)L2 (96)ORF 2 (98)ORF 2 (99)Terminase large subunit
32391267511.18ORF 3 (97)L3 (97)ORF 3 (98)ORF 3 (98)HNH endonuclease
42688382443.32ORF 4 (95)L4 (97)ORF 4 (96)ORF 4 (96)Portal protein
53805434119.90ORF 5 (98)L5 (96)ORF 5 (99)ORF 5 (98)Protease
64334551543.73ORF 6 (99)L6 (97)ORF 6 (99)ORF 6 (99)Structural protein
74559551535.16Non-annotated (99)L7 (98)Structural protein
85536579910.01ORF 7 (100)L8 (100)ORF7 (99)ORF 7 (98)Structural protein
95799611311.87ORF 8 (94)L9 (94)ORF8 (96)ORF 8 (97)Structural protein
106110645112.88ORF 9 (96)L10 (98)ORF 9 (94)ORF 9 (99)Structural protein
116442680713.65ORF 10 (97)L11 (96)ORF 10 (97)ORF 10 (97)Major tail structural protein
126836727315.82L12 (80)ORF 12 (40)Putative collar structure
137297820232.35ORF 11 (97)L13 (97)ORF 11 (87)ORF 11 (94)
148257853210.61ORF 12 (98)L14 (100)ORF 13(100)ORF 12 (100)
158552906419.86ORF 13 (97)L15 (98)ORF 14 (97)ORF 13 (99)
1690641181496.21ORF 14 (71)L16 (94)ORF 15 (78)ORF 14 (80)Tail tape measure
17118141271034.53ORF 15 (91)L17 (97)ORF 16 (88)ORF 15 (95)
18127101383742.82ORF 16 (94)L18 (98)ORF 17 (95)ORF 16 (95)
19138271412011.31ORF 17 (97)L19 (100)ORF 18 (98)ORF 17 (94)
20141101491328.65ORF 18 (72)L20 (95)ORF 19 (60)ORF 18 (56)Receptor-binding protein
21149351528813.34ORF 19 (96)L21 (94)ORF 20 (96)ORF 19 (95)Holin
22152851598626.26ORF 20 (77)L22 (97)ORF 21 (70)ORF 20 (70)Lysin
Early expressed region
23166301609419.93Non-annotated (93)E36 (91)ORF 23 (74)
24169771672010.12E35 (99)
25172671699810.32ORF 21 (93)E33 (96)ORF 24 (90)ORF 21 (94)
26177871744013.59ORF 23 (67)E31 (98)ORF 26 (82)ORF 22 (67)
2717954177876.86ORF 24 (40)E30 (96)ORF 27 (41)ORF 23 (41)
2818142179547.23E29 (95)
2918389181359.98ORF 25 (75)E28 (98)ORF 28 (73)ORF 24 (74)
3018656184538.22ORF 26 (55)E27 (96)ORF 25 (56)
3118860187175.59E25 (95)
32192971885717.57
33196041929912.10ORF 26 (85)E24 (83)ORF 25 (84)
3419732196044.41ORF 27 (56)E23 (59)ORF 26 (60)
35201061979211.86ORF 31 (80)E17 (86)ORF 30 (78)
36205972017216.56E20 (73)ORF 31 (50)HNH endonuclease
37211852067620.06ORF 32 (91)E15 (92)ORF 32 (94)ORF 31 (92)
3821397211828.30ORF 33 (97)E14 (97)ORF 33 (93)ORF 32 (97)
39217692141012.99ORF 34 (94)E13 (92)ORF 34 (94)ORF 33 (94)
40223482177322.13ORF 35 (54)E12 (50)ORF 35 (88)ORF 34 (59)sak
41226502232412.71ORF 36 (63)ORF 35 (55)
42230442263715.71ORF 38 (80)E10 (88)ORF 38 (81)ORF 37 (82)DNA polymerase subunit
43231472285711.87ORF 39 (97)E9 (94)ORF 38 (94)
44235242314414.67ORF 40 (52)E8 (83)ORF 39 (72)ORF 39 (59)
45238402357710.36ORF 41 (89)E7 (94)ORF 40 (87)ORF 40 (87)
4624021238128.03E6 (100)ORF 41 (44)
47249102401434.24ORF 43 (41)E5 (40)ORF 42 (96)ORF 42 (44)
4825115249546.16E4 bIL66M1 (95)ORF 45 (77)
4925386251628.62
5026031259124.52E2 (100)ORF 46 (98)
5126169263155.53ORF 0 (89)
5226610264436.83ORF 50 (87)E1 (83)ORF 49 (88)ORF 45 (84)
Middle expressed region
5326761268894.81ORF 51 (97)M1 (97)ORF 51 (93)ORF 46 (98)
5426894270255.12ORF 52 (95)M2 (97)ORF 52(100)ORF 47 (95)
55270222750418.04ORF 53 (95)M3 (96)ORF 53 (97)ORF 48 (94)Holliday junction endonuclease
5627501276686.14ORF 54 (98)M4 (96)ORF 54 (91)ORF 49 (98)
5727882280044.26
5828298284264.82
  • sak, Gene identified to be involved in AbiK-sensitivity (Bouchard & Moineau, 2004); E4bIL66M1, Early expressed ORF E4 of the mutant derivative of the 936-type phage, bIL66 (Bidnenkoet al,1995).

  • Note: ORFs 1A, 25, 44, 45 and 50 of φ712 possess no homologous counterparts in the other sequenced phages. ORF 22 of φ712 possesses 39% aa identity to E37 of bIL170, which is an HNH endonuclease not identified on the table above.

Figure 1

Schematic representation of the genomic organization of the 936-type phages, jj50, sk1 (Chandryet al,1997), 712, P008 and bIL170 (Crutz-Le Coqet al,2002), highlighting functional modules within the temporally transcribed regions. Genomic regions connected by a grey block are indicative of those possessing at least 60% identity at the amino acid level. The putative origins of replication (ori) of the phages are indicated below the relevant region of the individual phages. ORFs of the same colour represent those with at least 80% identity at the amino acid level.

Owing to the genetic similarities, it seems obvious that phages bIL170, 712 and P008 have evolved from the same ancestral core as sk1 and jj50 but with greater divergence occurring throughout their evolution. It is hypothesized that the ancestors of the phages used in this study and other 936-like phages were, at some point, capable of infecting the same host strain. This would have provided the opportunity for genetic recombination to occur, resulting in diversification of this phage species. The frequency of homologous recombination of 936-like phages was observed to be relatively high in vitro and is proposed to mimic the natural situation (Crutz-Le Coqet al,2006).

Notwithstanding the high degree of similarity of these phages, it should be highlighted that some features of these phages vary and fall into subgroups within this closely related phage group. The most noteworthy of these are the proposed origin of replication, the cos terminal regions and the presence/absence of a homologue of the putative collar protein of bIL170 (L12).

Replication origin

Previously, the origin of replication of phage sk1 was experimentally determined to be within orf47 (Chandryet al,1997). Phage bIL170 does not possess a homologue of this gene. However, an additional orf (orf0) was found in this phage at a corresponding position, with a high A+T content (71mol%) and a short conserved region between the two phages (Crutz-Le Coqet al,2002). Consequently, orf0bIL170 was assigned as the putative replication origin of bIL170 (Crutz-Le Coqet al,2002). Phage P008 possesses a homologue (orf51P008) to orf 0bIL170 and consequently, both phages possibly have a similar origin of replication. Sequence analysis revealed that jj50 carries an ORF with 93% homology to ORF47sk1 and it is, therefore, assumed that this phage has the same replication origin (Fig. 1). The genome of 712 does not possess a homologue of either orf47sk1 or orf0bIL170. However, phage 712 possesses a homologue of e2bIL170, which is absent in sk1 and jj50. This gene is located just downstream of orf0 on the genome of bIL170 and its homologous equivalent in 712 possesses an A+T composition similar to that of orf0bIL170 (71.4%). This orf of phage 712, and possibly the A+T-rich flanking regions, could be implied as being necessary as the replication origin. In summary, three types of putative origin of replication are found within these five 936-like phages, namely sk1/jj50, bIL170/P008 and 712.

Genomic termini

With respect to the terminal genomic regions, Parreira (1996) observed a maintenance of the cos site, two of the four putative terminase binding sites (R1 and R3) and five AATCT sequences postulated to be involved in DNA bending and the 10bp inverted repeats positioned on either side of the cos site between four 936-like phages, namely bIL66, P008, sk1 and bIL41. A similar observation was made for phages jj50, 712 and bIL170. Additionally, the deletion of R2 observed in P008 and bIL66 is also found in bIL170, thus compounding the relatedness of these phages (Fig. 2).

Figure 2

Comparison of the cos terminal regions of the 936-type phages. Shaded are the cos sites, inverted repeats (IR), direct repeats (D1–5) and the putative terminase binding sites (R1–4).

Putative collar structure

The middle and late genes of all five phages are well conserved and overall, there is little divergence in these regions (Fig. 1), with the exception of the putative collar protein (located between the capsid and the tail) of bIL170 (L12), which has truncated homologues in 712 and P008 but no homologous counterpart in either sk1 or jj50. A collar structure with protruding whiskers has been observed for φP008 in previous reports, although with limited ability to demonstrate the whisker structure due to the staining technique (Loofet al,1983; Jarviset al,1991). This collar structure was also evidenced in φbIL170 while electron micrographs of φsk1 demonstrate no such structure (Dupontet al,2004b). Under the conditions used in this study, electron micrographs of φjj50, 712 and P008 are comparable to those of φsk1, revealing no discernible collar structure. Previous studies also demonstrate that φP113G and P272 do not possess a collar/whisker structure, while both structures are clearly visible in another 936-type phage, φ853 (Jarvis & Meyer, 1986; Loof & Teuber, 1986). Heteroduplex analysis of the genomes of φP008 and P272/P113G revealed that P113G differs from P008 only in the region encoding the collar/whisker structure (Loof & Teuber, 1986). However, a deletion within the gene coding for L12 of phage bIL41 has shown that it is a structural component that does not influence phage propagation or tail assembly but is hypothesized to play a nonessential role in host-range determination/extension (Crutz-Le Coqet al,2006). The presence of a host-recognition domain (found in other phages eg. the P335 phages bIL309 and BK5-T) provides a persuasive argument to support the theory of its involvement in additional roles apart from its structural function.

Early expressed region

The greatest level of divergence between the five phages compared in this study was apparent in the early expressed region. Crutz-Le Coq (2002) observed a similar finding when genomic comparisons of sk1 and bIL170 were made. Phages sk1 and jj50 share the greatest homology, with the majority of the proteins possessing over 90% aa identity. The numerous insertions/deletions, termed indels, observed in the early expressed region of the phages may have arisen through the acquisition of mobile DNA as suggested by the presence of the homing endonucleases observed in the early expressed regions of bIL170, P008 and 712. It is believed that these ORFs have arisen independently in bIL170, although the conservation of the relative positioning of these ORFs may indicate a similar evolutionary pathway of bIL170, P008 and 712.

Relationship between genome analysis and sensitivity to AbiA or Sie2009

Sie2009 is highly effective against phages sk1, jj50, 712 (EOP<10−9) while AbiA also confers resistance but to a lesser extent against the same three phages. Of note, despite numerous trials, we failed to obtain insensitive phage mutants derived from these three phages (data not shown), possibly indicating that ORFs involved in sensitivity (or required for activation) to this defence system may be absent on the genomes of the phages (P008 and bIL170) insensitive to these systems. This ORF, if present in phages sensitive to the phage defence systems, would be proposed to reside in the early expressed region as the middle and late expressed regions are well conserved. An early expressed gene of the P335 phage, φ31(ORF245), was identified as the ORF involved in sensitivity to AbiA (Dinsmore & Klaenhammer, 1997). No such gene was identified in φsk1 although 12% of the genome, covering part of the early expressed region, could not be cloned in this study and it was postulated that if φsk1 possesses such a gene, it would be in this region (Dinsmore & Klaenhammer, 1997).

While this is currently conjecture, the information provided by the genome sequences represents an excellent basis and comprehensive approach for studies relating to the sensitivity of this industrially problematic group of phages to a spectrum of phage defence mechanisms. This work also represents the first broad-ranging study of the genetic diversity within this group of phages of distinct geographical backgrounds. Given the recent resurgence of interest in phage biology, most notably the recent studies of the receptor binding and structural proteins of the 936-type phages (Crutz-Le Coqet al,2006; Spinelliet al,2006), the sequences embody a valuable resource to evolutionary and biological studies of dairy phages.

Acknowledgements

The authors wish to thank J. Josephsen (The Royal Veterinary and Agricultural University, Denmark) for kindly providing lactococcal phages and strains used as part of this study. We also thank Dr Ackermann for his excellent assistance with the electron micrographic work. J. Mahony is in receipt of research funding by the Irish Research Council for Science Engineering and Technology under the Embark Initiative. D. van Sinderen is a recipient of a Science Foundation of Ireland Investigatorship award (01/IN1/B198). H. Deveau is a recipient of a graduate student scholarship from the Fonds Québécois de Recherche sur la Nature et les Technologies (FQRNT). This study was funded, in part, by a strategic grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to S. Moineau.

References

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